Yin/Yang associated differential responses to Psoralea corylifolia Linn. in rat models: an integrated metabolomics and transcriptomics study

Yin/Yang associated differential responses to Psoralea corylifolia Linn. in rat models: an integrated metabolomics and transcriptomics study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Yin/Yang associated differential responses to Psoralea corylifolia Linn. in rat models: an integrated metabolomics and transcriptomics study Ming-Liang Zhang, Xu Zhao, Wei-Xia Li, Xiao-Yan Wang, Ming Niu, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2301287/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Aug, 2023 Read the published version in Chinese Medicine → Version 1 posted 5 You are reading this latest preprint version Abstract Aim Psoralea corylifolia Linn. (BGZ) is a commonly used traditional Chinese medicine (TCM) for the treatment of kidney-yang deficiency syndrome (Yang syn ) with good curative effect and security. However, BGZ was also reported to induce liver injury in recent years. According to TCM theory, taking BGZ may induce a series of adverse reactions in patients with kidney-yin deficiency syndrome (Yin syn ), which suggests that BGZ-induced liver damage may be related to its unreasonable clinical use. The study aimed to investigate the differential responses to BGZ in Yang syn and Yin syn rat models and identify the corresponding characteristic biomarkers. Materials and methods The corresponding animal models of Yang syn and Yin syn were induced by hydrocortisone and thyroxine + reserpine respectively. Body weight, organ index, serum biochemistry, and Hematoxylin and Eosin (HE) staining were used to evaluate the liver toxicity effect of BGZ on rats with Yang syn and Yin syn . Transcriptomics and metabonomics were used to screen the representative biomarkers (including metabolites and differentially expressed genes (DEGs)) changed by BGZ in Yang syn and Yin syn rats, respectively. Results The level changes of liver organ index, ALT, and AST suggested that BGZ has liver-protective and liver-damaging effects on Yang syn and Yin syn rats, respectively, and the results also were confirmed by the pathological changes of liver tissue. The results showed that 102 DEGs and 27 metabolites were significantly regulated related to BGZ’s protective effect on Yang syn , which is mainly associated with the glycerophospholipid metabolism, arachidonic acid metabolism, pantothenate, and CoA biosynthesis pathways. While 28 DEGs and 31 metabolites, related to the pathway of pantothenate and CoA biosynthesis, were significantly regulated for the BGZ-induced liver injury in Yin syn . Furthermore, 4 DEGs (Aldh1b1, Slc25a25, Pim3, Oaf) and 4 metabolites (phosphatidate, phosphatidylcholine, N-Acetylleucine, biliverdin) in the Yang syn group and 1 DEGs (Lgals5) and 1 metabolite (5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxylate) in Yin syn group were significantly correlated to the ALT and AST levels of BGZ treated and untreated groups (ROC ≥ 0.9). Conclusions Yin syn and Yang syn are the predisposed syndrome for BGZ to exert liver damage and liver protection respectively, which are mainly related to the regulation of amino acid metabolism, lipid metabolism, energy metabolism, and metabolism of cofactors and vitamins. The results further suggest that attention should be paid to the selection of predisposed populations when using drugs related to the regulation of energy metabolism, and the Yin syn /Yang syn animal models based on the theory of TCM syndromes may be a feasible method for identifying the susceptible population to receive TCM. Psoralea corylifolia Linn. liver injury predisposed individual metabolomics transcriptomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction In recent years, the proportion of drug-induced liver injury (DILI) caused by traditional Chinese herbs and dietary supplements has increased year by year (Hoofnagle and Björnsson, 2019 ; Shen et al., 2019 ). This is particularly noticeable for non-toxic traditional Chinese medicine (TCM) such as Polygonum multiflorum Thunb. (Zhang et al., 2020 ) and Psoralea corylifolia Linn. (BGZ) (Wang et al., 2020 ), which have been used for thousands of years in China. This increase is causing great confusion for doctors and patients. DILI usually involves two factors, the “drug” and the “host” (Chen et al., 2015 ). For drug factors, Chen et al. constructed a “role-of-two” model (Chen et al., 2013 ) and modified the version used (Chen et al., 2016 ). The model enhanced the ability to predict whether a drug will cause DILI, but it was unable to fully explain individual differences in drug usage. Therefore, it cannot be used to predict individuals who are predisposed to DILI. Host factors are also important in understanding the susceptibility to DILI. Host factors that are generally recognized by modern research include hosts who carry specific HLA genes or the immune homeostasis of the host (Chen et al., 2016 ). This is consistent with the concept of TCM syndrome, which summarizes the pathological changes of the body at a certain stage of disease development based on TCM theory. TCM treatment requires “therapy based on syndrome differentiation.” Symptomatic treatment can result in a better therapeutic effect, otherwise, it can aggravate the disease process and induce new diseases. Therefore, it may be feasible to explore the predisposition of individuals to TCM liver injury based on TCM syndrome theory. BGZ has been used for many years in China to treat symptoms such as impotence, nocturnal emission, enuresis, frequent urination, cold aching in the lower back and knees, kidney deficiency, and premature ejaculation (Chinese Pharmacopoeia Commission ( 2020 )). BGZ has definite pharmacological effects, such as anti-tumor, anti-oxidation, antibacterial, anti-inflammatory, anti-depression, estrogen level regulation, bone growth promotion, nerve protection, and influence on the liver (Zhang et al., 2016 ). Its active ingredients, psoralen and 5-methoxypsoralen, have become commonly used in the clinical treatment of vitiligo and psoriasis (Gupta and Anderson, 1987 ; McNeely and Goa, 1998 ). In addition, BGZ and its related compounds are also commonly used in health food and dietary supplements. However, in recent years, BGZ and its related compound preparations Zhuanggu Guanjie Pill and Xianling Gubao Capsules were reported to induce DILI in China (Deng et al., 1996 , Li et al., 2020 ; Liu et al., 2019 ; Wang et al., 2020 ). The BGZ-induced liver injury also occurred in South Korea (Nam et al., 2005 ). Although some studies have confirmed that BGZ and some of its components have hepatotoxicity (Li et al., 2017 ; Wang et al., 2020 ; Zhang et al., 2022 ), these studies only focused on the drug itself and did not consider the host factors that induced DILI. Accordingly, by integrating the basic disease characteristics of the population treated with BGZ, our previous study found that most BGZ-induced DILI patients had osteoporosis, psoriasis, osteoarthritis, and other basic diseases related to immune activation (Ge et al., 2021 ), and such diseases were found to be closely related to the imbalance of kidney-yang deficiency syndrome (Yang syn ) and kidney-yin deficiency syndrome (Yin syn ) according to TCM diagnosis (Dermatology Branch of China Association of Chinese Medicine. 2017, Dermatology Branch of China Association of Chinese Medicine. 2018, Ge et al. 2020 ). The ancient TCM medicine book “Lei Gong Concocting (Paozhi) Theory” also recorded during the Northern and Southern Dynasties in China that people with “asthenic yin causing excessive pyrexia” should avoid taking BGZ. Thus, the BGZ-induced DILI may be related to its inappropriate symptomatic treatment. However, whether it has a causal relationship with the imbalance of Yang syn and Yin syn in the body remains unclear. Continuously administered high doses of exogenous glucocorticoid (GC) (e.g., hydrocortisone) (Zhang et al., 2019 ) or thyroxine + reserpine (Wang, P. et al., 2010 ) in rats results in symptoms similar to Yang syn or Yin syn , respectively, and play important roles in the evaluation of protective or damaging effects and the TCM action mechanism against Yang syn or Yin syn . With a good description of the changes in the metabolic characteristics of endogenous metabolites and the differential expression changes of a series of functional genes in organisms, metabolomics and transcriptomics have been successfully applied to screen various diseases and their metabolic or gene profile changes in drug intervention (Lu et al., 2020 ; Ren et al., 2016 ). This study constructed Yang syn and Yin syn animal models to evaluate whether the liver damage induced by BGZ is related to its non-symptomatic use and the possible susceptible individual characteristics. 2. Experiment 2.1 Chemicals and reagents Hydrocortisone succinate sodium was provided by Tianjin Biochemical Pharmaceutical Co., Ltd. (Tianjin, China). Thyroxine and reserpine were provided by Shanghai MACKLIN Technology Co., Ltd. (Shanghai, China). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and serum creatinine (SCr) tests were purchased from Jiancheng Biological Technology, Co., Ltd. (Nanjing, China). Cyclic adenosine monophosphate (cAMP), and cyclic guanosine monophosphate (cGMP) were purchased from Mlbio Biotechnology Co., Ltd. (Shanghai, China). 2.2 BGZ Preparation BGZ was purchased from the Anhui Puren Herbal Pieces Co., Ltd. (Anhui, China) and authenticated by Professor Xiaohe Xiao of the institute of Hepatology, Fifth Medical Center, PLA General Hospital (Beijing, China). In addition, BGZ was crushed and filtered through 200 mesh sieves and then suspended in 0.5% sodium carboxymethyl cellulose (CMC-Na) for administration. 2.3 Animal maintenance and treatment Male Sprague–Dawley rats (180–200 g) were obtained from the SPF Biotechnology Co. Ltd. (License No. SCXK20190010, Beijing, China), and housed in the Laboratory Animal Center of the Fifth Medical Center, Chinese PLA General Hospital (animal ethics committee approval No. YFYDW2020017). All rats were raised under specific pathogen-free conditions under a 12 h light/dark cycle, with free access to adequate food and water. All animals were fed adaptively for 1 week before starting the experiments. The rats were randomly divided into six separate groups (N = 8) as follows: Control group (CON), BGZ group (BGZ), kidney-yang deficiency syndrome group (Yang syn ), kidney-yang deficiency syndrome-treated with BGZ group (Yang syn +BGZ), kidney-yin deficiency syndrome group (Yin syn ); kidney-yin deficiency syndrome-treated with BGZ group (Yin syn +BGZ). Rats in the treatment groups were consecutively administered intragastrically with BGZ suspension every afternoon for 21 consecutive days, while the control group received the same volume of 0.50% CMC-Na solution for the same amount of time. The changes in body weight were recorded before the end of the experiment. From the 8th day of administration, the Yang syn and Yang syn +BGZ group rats were given 25 mg/kg hydrocortisone subcutaneously once a day for 14 consecutive days to prepare the Yang syn model (Zhang et al., 2019 ). From the 15th day of administration, the Yin syn and Yin syn +BGZ group rats were given thyroxine (16 mg/ml) and reserpine (1 mg/ml) at the dosage of 0.5 ml/100 g via gastric perfusion once a day for 7 consecutive days to prepare the Yin syn model (Wang, P. et al., 2010 ). The CON and the BGZ groups were given the same volume of 0.50% CMC-Na solution. 2.4 Blood collection, organ index, and tissue preparation After the experiment, all animals were anesthetized with 2% pentobarbital sodium. Blood samples with and without anticoagulants were collected, and the liver and kidney were weighed immediately after sacrifice to calculate the organ index. Partial liver tissue was collected for histological examination and the remaining liver was quickly frozen with liquid nitrogen and stored at -80°C until needed. 2.5 Serum biochemistry and histopathological analysis After centrifugation (3500 rpm, 10 min, 4°C), serum biochemistry (ALT, AST, SCr) and neurotransmitters (cAMP, cGMP) were determined according to the microplate assay kit instructions, the left hepatic lobe was fixed with 4% paraformaldehyde for 48 h, embedded in wax, and sectioned at approximately 5 µm for Hematoxylin and Eosin (HE) pathological staining analysis. 2.6 RNA sequence analysis and data processing Total RNA was extracted from liver tissues using Trizol reagent (Invitrogen, USA) according to the manufacturer’s protocol, and genomic DNA was removed using DNase I (TaKara). RNA quality was assessed with a Bioanalyzer 2100 (Agilent) and measured with a NanoDrop 2000 spectrophotometer. Only a high-quality RNA sample (OD 260/280 = 1.8 ~ 2.2, OD 260/230 ≥ 2.0, RNA integrity number (RIN) ≥ 6.5, 28S:18S ≥ 1.0, > 1 µg) was used to construct the sequencing library. RNA-seq transcriptome libraries were prepared using a TruSeqTMRNA sample preparation kit from Illumina (San Diego, CA) and sequenced with the Illumina HiSeq xten/NovaSeq 6000 sequencer under standard protocols. All samples had a Q30 (bases of Q ≥ 30 /all bases of sequencing) of > 91%. Sequence readers were trimmed and quality controlled using SeqPrep ( https://github.com/jstjohn/SeqPrep ) and Sickle ( https://github.com/najoshi/sickle ) with default parameters and aligned to the reference genome through the orientation mode using hierarchical indexing for spliced alignment of transcripts 2 (HISAT2) (Kim et al., 2015 ). Messenger RNA levels were quantified using RSEM ( http://deweylab.biostat.wisc.edu/rsem/ ) (Li and Dewey, 2011 ). Differentially expressed genes (DEGs) were identified as those with | log2 (fold change) | >1 and Padjust < = 0.05 (DESeq2) (Love et al., 2014 ). 2.7 Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Liver tissue RNA was extracted by the Tissue RNA Purification Kit Plus (RN002plus, ES Science, China) and reverse-transcribed into cDNA using the Fast All-in-One RT Kit (RT001, ES Science, China) according to the manufacturer's instructions. The qPCR of aldehyde dehydrogenase 1 family member B1 (Aldh1b1), galectin 5 (Lgals5), solute carrier family 25 member 25 (Slc25a25), Pim-3 proto-oncogene, serine/threonine kinase (Pim3), and out at first (Oaf) were quantified by the SYBR Green PCR master mix (RN002plus, ES Science, China) with the QuantStudio 6 Flex PCR System (Applied Biosystems, USA). The amplification parameters were set according to the standard protocol. Primer sequences used in this study are shown in Supplemental Table 1. Relative gene expression was calculated using the 2 −△△Ct method 2 −∆∆Ct (Ish-Shalom and Lichter, 2010 ). 2.8 Liver sample processing The liver was homogenized with normal saline at 1:1 (1g:1mL) using a homogenizer. The 300 uL homogenized sample and 900 uL methanol were mixed and vortexed for 30 s. The supernatant was centrifuged at 12000 r/min for 10 min and concentrated to dry using a vacuum centrifugal concentrator. Then 100 uL methanol was added for redissolving and centrifuged at 13000 r/min for 10 min. Finally, a 4 uL supernatant was taken out for UPLC-QTOF/MS (Waters, Manchester, UK) detection. 2.9 UPLC-QTOF/MS analysis and data processing Metabolic profiling analysis of the biofluids was performed using the Waters Xevo G2-XS QTOF/MS (Waters, Manchester, UK). An analytical Acquity UPLC HSS T3 C18 column (temperature 30°C) was injected with 4 µL aliquots of each sample. For positive electrospray ionization source (ESI+) analysis, samples were isolated using a 30 min linear gradient of solvent A (water spiked with 0.1% formic acid) and solvent B (acetonitrile spiked with 0.1% formic acid) as mobile phases. The flow rate was fixed at 0.30 mL/min. For each sample, 10 µL was drawn as a quality control sample to ensure that the system was stable and the analyses were repeatable. Every 20th sample was injected with the control sample and subsequently analyzed. Masslynx software (v4.1, Waters Corp.) and Progenesis QI (v. 2.4, Waters Technologies, UK) were used for identifying the original mass spectral data and normalizing the total ion intensity of each chromatogram to acquire a data matrix containing the m/z value, retention time (RT), and normalized peak area. SIMCA-P 14.1 software (Umetrics, Umea, Sweden) was used for principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA). The PCA score chart was used to show the natural interrelation of observation results. Variable importance in the projection (VIP) ≥ 1 and with significant differences between groups ( P < 0.05 ) were selected as potential metabolites. Online metabolic databases including the Human Metabolome Database (HMDB) ( http://www.hmdb.ca/ ) and Kyoto Encyclopedia of Genes and Genomes (KEGG) ( https://www.genome.jp/kegg/ ) were combined with exact masses and secondary ion mass spectrometry of the metabolites acquired through Progenesis QI to identify the differential metabolites. 2.10 Statistical analysis Statistical analysis was performed with Prism 8.0 (GraphPad Software, San Diego, CA). All results are expressed as mean ± SD. A one-way analysis of variance (ANOVA) was used to statistically analyze the multiple-group analysis. The student’s t-test was used to statistically analyze the receiver operating characteristic (ROC) curve analysis. The significance threshold was set at P < 0.05 . 3 Results 3.1 Effects of BGZ treatment on weight, anal temperature, and organ indexes of liver and kidney in Yin syn and Yang syn rats As shown in Fig. 1 A, compared with the CON group, the body weight of the BGZ group, Yang syn group, Yang syn +BGZ group, and Yin syn group had no obvious changes, while the Yin syn +BGZ group decreased significantly compared with the Yin syn group ( P < 0.01 ). The anal temperature of rats in the Yang syn group and Yin syn group were significantly decreased and increased, respectively, compared with the CON group (both P < 0.01 ), while the anal temperature in the Yang syn +BGZ group and Yin syn +BGZ group were all significantly increased compared with the corresponding model groups (both P < 0.05 ) (Fig. 1 B). As shown in Fig. 1 C, the organ indexes of the liver in the Yang syn group and Yin syn group were all markedly increased compared with the CON group ( P < 0.001, P < 0.001 ), and the liver organ indexes in the Yin syn +BGZ group was further increased compared with the Yin syn group ( P < 0.001 ), while there was also no changed in the Yang syn +BGZ group compared with the Yang syn group. As for the kidney, only the Yin syn group had a significant increase in organ indexes compared with the CON group ( P < 0.001 ) (Fig. 1 D). 3.2 Effect of BGZ treatment on the serum levels of ALT, AST, SCr, and liver histopathological changes in Yin syn and Yang syn rats Serum ALT, AST, and SCr, which are well-recognized markers of various types of liver and kidney damage, were used for the analysis. Regarding liver function (Fig. 2 A-B), there were no obvious changes in the levels of ALT and AST between the BGZ group and the CON group. In contrast, the levels of ALT and AST in the Yang syn group increased prominently, while BGZ significantly reversed the phenomena (all P < 0.05 ). Though the AST level in the Yin syn group was significantly increased ( P < 0.01 ), there was no obvious change in the levels of AST between the Yin syn +BGZ group and the Yin syn group, while the ALT level was significantly increased after administration of BGZ in the Yin syn +BGZ group ( P < 0.001 ), illustrating that there was a certain risk of liver injury in rats with Yin syn after administration of BGZ. As for kidney function (Fig. 2 C), no significant SCr changes were found in almost all groups except for the Yin syn group ( P < 0.001 ). Combined with the changes in the kidney organ index, it revealed that BGZ may have no obvious renal toxicity in rats with Yang syn or Yin syn . As shown in Fig. 2 D, the liver sections of the CON group showed normal hepatocyte structures. The liver samples from BGZ-treated rats were almost indistinguishable from normal rats. The liver samples from the Yang syn group exhibited hepatocyte focal necrosis, loss of central vein intima, and inflammatory cell infiltration in portal vein areas, while the above symptoms were alleviated in the Yang syn +BGZ group. The liver samples from the Yin syn group exhibited slight inflammatory infiltration in the portal area but no evident hepatocyte injury, while the hepatocyte focal necrosis and inflammatory cell infiltration were aggravated in the Yin syn +BGZ group. Combined with the levels of ALT, AST, liver index, and liver pathological examination, it was suggested that BGZ has a preferable liver protection effect on rats with Yang syn and a certain risk of liver injury effect on rats with Yin syn . 3.3 DEG alterations of BGZ treatment in Yin syn and Yang syn rats To reveal the mechanism of different therapeutic effects of BGZ on Yin syn and Yang syn rats, liver gene expression profiles were obtained from the CON, BGZ, Yang syn , Yang syn +BGZ, Yin syn , and Yin syn +BGZ groups using RNA-Seq analysis. As shown in Fig. 3 A, compared with the CON group, BGZ barely influenced the gene expression with only 4 DEGs increased and 5 DEGs decreased, while 439 up-regulated DEGs and 652 down-regulated DEGs were found in the Yang syn group, and 508 up-regulated DEGs along with 847 down-regulated DEGs were found in the Yin syn group. Compared to the Yang syn group, the number of up-regulated and down-regulated DEGs decreased to 59 and 43 by BGZ treatment, respectively, while for the Yin syn group, the number of up-regulated and down-regulated DEGs decreased to 11 and 17 by BGZ treatment, respectively. To further display the above differences in DEGs more intuitively, heatmaps were constructed based on relative abundance (Fig. 3 B and 3 C). 3.4 Correlation analysis between DEGs and serum biochemistry By analyzing the correlations between serum biochemistry (ALT and AST) and DEGs changed using BGZ in Yin syn and Yang syn rats (Fig. 4 ), the results demonstrated that 36 DEGs, including 34 positive DEGs and 2 negative DEGs, were significantly correlated with ALT, and 12 DEGs, including 9 positive DEGs and 3 negative DEGs, were significantly correlated with AST. Among them, hyperpolarization-activated cyclic nucleotide-gated potassium channel 3 (Hcn3), aldehyde dehydrogenase 1 family member B1 (Aldh1b1), galectin 5 (Lgals5), solute carrier family 25 member 25 (Slc25a25), Pim-3 proto-oncogene, serine/threonine kinase (Pim3), and out at first (Oaf) were all significantly positively correlated with ALT and AST, and LOC100364769 was significantly negatively correlated with ALT and AST. The detailed relationship between DEGs and serum biochemistry (ALT and AST) was shown in Supplemental Table 2. 3.5 ROC curve analysis of DEGs To further explore the diagnostic efficacy of DEGs, a ROC curve analysis was performed using GraphPad Prism software (version 8.01). The results revealed that 22 DEGs could be better discriminated between the Yang syn +BGZ group and Yang syn group, and 10 DEGs had better discrimination between the Yin syn +BGZ group and Yin syn group (all the areas under the curve (AUC) of the ROC curves ≥ 0.9 and P < 0.05 ) (Supplemental Fig. 1). Venn analysis showed the ROC results of DEGs and their correlation with the levels of ALT and AST between the Yang syn +BGZ group and Yang syn group (Fig. 5 A), revealing that Aldh1b1, Slc25a25, Pim3, and Oaf can be used as potential biomarkers for the treatment of Yang syn (Fig. 5 B-E). Venn analysis also showed the ROC results of DEGs and their correlation with ALT and AST between the Yin syn +BGZ group and Yin syn group (Fig. 5 J), revealing that Lgals5 may be used as a potential biomarker for the treatment of Yin syn (Fig. 5 K). As shown in Fig. 5 F-I and 5 L, the expression of Slec25a25, Pim3, and Oaf in rats with Yang syn and Yin syn were all significantly increased compared with the CON group (all P < 0.05 ), while the level of Aldh1b1 in the Yang syn and the level of Lgals5 in the Yin syn were all significantly decreased compared with the normal group (both P < 0.05 ). After BGZ intervention, the levels of Aldh1b1, Lgals5, Slec25a25, Pim3, and Oaf were all significantly decreased in the Yang syn +BGZ group (all P < 0.05 ), while only Lgals5 was significantly increased in rats with Yin syn +BGZ compared the Yin syn group ( P < 0.05 ). 3.6 GO and KEGG functional enrichment analysis of DEGs Gene Ontology (GO) functional enrichment analysis (Fig. 6 A) found that 95 GOs were significantly changed in the Yang syn +BGZ group compared with the Yang syn group, such as GO:0009991 (response to extracellular stimulus), GO:0032922 (circadian regulation of gene expression), GO:0031667 (response to nutrient levels), and GO:0009892 (negative regulation of metabolic process), while 6 GOs including GO:0019825 (oxygen binding), GO:0020037 (heme-binding), GO:0046906 (tetrapyrrole binding), GO:0005833 (hemoglobin complex), GO:0005344 (oxygen carrier activity), and GO:0042743 (hydrogen peroxide metabolic process) were significantly changed in the Yin syn +BGZ group compared with the Yin syn group. Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis (Fig. 6 B) found that 4 KEGG orthologs (Kos) including ko00360 (phenylalanine metabolism), ko00350 (tyrosine metabolism), ko00380 (tryptophan metabolism), and ko00130 (ubiquinone and another terpenoid-quinone biosynthesis) were significantly changed in the Yang syn +BGZ group compared with the Yang syn group, while 2 Kos including ko00260 (glycine, serine, and threonine metabolism) and ko00591 (linoleic acid metabolism) were significantly changed in the Yin syn +BGZ group compared with the Yin syn group. 3.7 Metabolomic analysis of BGZ treatment in Yin syn and Yang syn rats As shown in Fig. 7 A and 7 B, the QC samples gathered closely in both PCA score plots, indicating the stability of the UPLC-QTOF/MS system throughout the analysis. The CON, Yang syn , and Yin syn groups can be well distinguished, suggesting that the metabolic information between the Yang syn and Yin syn groups has been changed. OPLS-DA analysis found that the comparison between the Yang syn group and the Yang syn +BGZ groups (Fig. 7 C and 7 D) could both be significantly separated under ESI + and ESI- modes, and the comparison between the Yin syn group and Yin syn +BGZ group (Fig. 7 E and 7 F) could also both be significantly separated under ESI + and ESI- modes, and the permutation tests for OPLS-DA analysis revealed the models were not overfitted (Supplemental Fig. 2), indicating that significant metabolic disturbance occurred in the Yang syn group and Yin syn group after BGZ treatment. A total of 40 metabolites were identified based on m/z and corresponding secondary fragment ion characteristic maps (Table 1 , Supplemental Fig. 3). Among the metabolites, 27 metabolites were markedly changed in the Yang syn +GBZ group compared with the Yang syn group, and 31 metabolites were significantly changed in the Yin syn +GBZ group compared with the Yin syn group. The heatmap for the above differential metabolites is presented in Fig. 7 G. Table 1 Identified differential metabolites. ESI m/z RT (min) KEGG Metabolite (Yang syn +BGZ) vs (Yang syn ) (Yin syn +BGZ) vs (Yin syn ) - 145.0132 0.9378 C00026 α-Ketoglutarate ↑* ↓ - 455.0965 2.9226 C00061 Riboflavin-5-phosphate ↑* ↓* - 104.0348 0.8028 C00065 L-Serine ↑ ↓* + 113.0352 0.9136 C00106 Uracil ↓* ↑* + 778.5407 13.2262 C00157 Phosphatidylcholine ↑ ↓ - 582.5100 13.2602 C00195 N-Acylsphingosine ↑** ↓* - 148.0378 1.3859 C00250 Pyridoxal ↑* ↓* - 243.0615 1.3509 C00299 Uridine ↓ ↑* - 102.0556 0.8313 C00334 gamma-Aminobutyric acid ↑* ↓* - 766.6037 9.3765 C00350 Phosphatidylethanolamine ↓** ↑* - 240.0224 1.5073 C00352 Glucosamine 6-phosphate ↑* ↓** - 151.0569 0.9235 C00379 D-Xylitol ↑ ↓* - 671.4653 13.3460 C00416 Phosphatidate ↓*** ↑* + 173.0922 0.8994 C00430 5-Aminolevulinate ↓** ↑ + 160.0756 1.9497 C00483 Tyramine ↓* ↑* - 583.2469 8.6720 C00486 Bilirubin ↑ ↓* + 583.2554 8.0917 C00500 Biliverdin ↑** ↓* + 90.0555 0.8565 C00546 Methylglyoxal ↑ ↓* - 397.2291 11.6884 C00584 Prostaglandin E2 ↓** ↑* - 229.0667 1.6209 C00588 Phosphorylcholine ↑* ↓* + 426.3162 15.2409 C00695 Cholic acid ↑ ↓** + 252.0724 2.1330 C00818 Glucaric acid ↑ ↓* + 465.3556 11.5639 C01724 Lanosterin ↑ ↓* + 799.6627 15.2528 C01829 Thyroxine ↑** ↓ + 196.0943 4.0050 C03264 D-Leucate ↑** ↓ - 187.1076 1.3080 C02727 N6-Acetyl-L-lysine ↑* ↓* + 560.3333 8.4013 C03033 beta-D-Glucuronoside ↑* ↓ - 205.0645 1.7274 C03227 3-Hydroxy-L-kynurenine ↓* ↑* + 522.3564 9.5634 C04230 1-Acyl-sn-glycero-3-phosphocholine ↑** ↓ - 401.0785 6.4160 C04352 (R)-4'-Phosphopantothenoyl-L-cysteine ↑** ↓*** + 381.0794 0.9636 C04778 N1-(5-Phospho-a-D-ribosyl)-5,6-dimethylbenzimidazole ↓* ↑** - 347.1835 8.9063 C05302 2-Methoxyestradiol ↑ ↓** - 526.0585 1.3859 C05381 3-Carboxy-1-hydroxypropylthiamine diphosphate ↓ ↑* + 389.2142 4.7813 C05476 Tetrahydrocorticosterone ↑** ↓ - 183.0346 0.8099 C05580 3,4-Dihydroxymandelate ↓ ↑* - 297.0703 0.9593 C05648 5-Hydroxy-N-formylkynurenine ↑ ↓* - 577.3444 13.7089 C05789 L-Urobilinogen ↓* ↑ - 365.2324 6.4089 C14777 12(S)-HETE ↓* ↑* + 353.2472 6.4221 C16513 Docosapentaenoic acid (22n-3) ↑* ↓* + 136.0396 1.6256 C22040 Methylcysteine ↑* ↓** *P < 0.05 , **P < 0.01 , ***P < 0.001 . 3.8 Correlation analysis between metabolites and serum biochemistry By analyzing the correlations between serum biochemistry (ALT and AST) and differential metabolites (Fig. 8 ), 20 metabolites, including 3 positive and 17 negative correlations, were significantly correlated with ALT, and 12 metabolites, including 3 positive and 9 negative correlations, were significantly correlated with AST. The correlation coefficient r and P-value are shown in Supplemental Table 3. Among them, Phosphatidate, Uracil, Prostaglandin E2, and N1-(5-Phospho-a-D-ribosyl)-5,6-dimethylbenzimidazole were all significantly positively correlated with ALT and AST, and Phosphatidylcholine, Lanosterin, N-Acylsphingosine, D-Leucate, Gamma-aminobutyric acid, Biliverdin, beta-D-Glucuronoside, and Docosapentaenoic acid (22n-3) were all significantly negatively correlated with ALT and AST. 3.9 ROC curve analysis of metabolites As shown in Supplemental Fig. 4, 9 metabolites had better discrimination between the Yang syn +BGZ group and Yang syn group, and 5 metabolites had better discrimination between the Yin syn +BGZ group and Yin syn group (all the areas under the curve (AUC) of the ROC curves ≥ 0.9 and P < 0.05 ). Venn analysis showed the ROC results of metabolites and their correlation with the levels of ALT and AST among the comparison of the Yang syn +BGZ group and Yang syn group (Fig. 9 A) and the comparison of the Yin syn +BGZ group and the Yin syn group (Fig. 9 F). Among them, Phosphatidate, Phosphatidylcholine, D-Leucate, and Biliverdin may be used as potential hepatoprotective biomarkers for the treatment of Yang syn (Fig. 9 B-E), while N1-(5-Phospho-a-D-ribosyl)-5,6-dimethylbenzimidazole may be used as a potential hepatotoxicity biomarker for the treatment of Yin syn (Fig. 9 G). 3.10 Comparison of overall metabolic profiles To further understand the metabolic disorder of susceptible syndromes, the KEGG identification code of differential metabolites was used for pathway analysis via MetaboAnalyst 5.0 ( https://www.metaboanalyst.ca/ ). The schematic diagram of the disturbed metabolic pathways changed by the treatment of BGZ for Yang syn and Yin syn rats is presented in Fig. 10 A and 10 B. The redder the bubble color, the smaller the P value of the metabolic pathway. The details for the value of P are shown in Supplemental Tables 4 and 5. The results indicated that there are significant differences in glycerophospholipid metabolism, arachidonic acid metabolism, pantothenate, and CoA biosynthesis pathways in the Yang syn +BGZ group compared with the Yang syn group, while pantothenate and the CoA biosynthesis pathway were significantly changed in the Yin syn +BGZ group compared with the Yin syn group. To compare the differences in metabolic profiles for BGZ treatment on Yin syn and Yang syn rats, a network map was constructed based on the identified metabolites and enriched metabolic pathways. As shown in Fig. 10 C, metabolic pathways and metabolites interacted with each other to form a complex network. 3.11 Integration analysis of metabolite and DEGs enrichment pathway. The expression changes of metabolites and DEGs in vivo can affect each other and cause a series of changes in body function. It is worth noting that, the pathways of BGZ regulating in rats with Yin syn and Yang syn are almost all involved in energy metabolism, amino acid metabolism, lipid metabolism, and metabolism of cofactors and vitamins (Fig. 11 A), and the above pathways were also cross-regulated by the “second messenger” of cAMP and cGMP (Cheng et al., 2019 ; Godfrey et al., 1987 ; Shen et al., 2011 ) (Fig. 11 B). As shown in Fig. 11 C-E, the study further found the protein expression cAMP was significantly decreased in the Yang syn group ( P < 0.01 ) and increased in the Yin syn group ( P < 0.05 ) while compared with the CON group. Meanwhile, the protein levels of cGMP were significantly increased in both model groups (both P < 0.05 ), accompanied by a significantly decreased ratio of cAMP/cGMP in the Yang syn group ( P < 0.001 ). The changes in levels of cAMP, cGMP, and cAMP/cGMP ratio are consistent with the changes in Yin syn and Yang syn models and are often used as indicators to evaluate the above syndrome models (Averaimo and Nicol, 2014 ). After BGZ intervention, there was no significant change in cGMP in the two model groups, while the levels of cAMP and cAMP/cGMP in the Yang syn +BGZ group and Yin syn +BGZ group were all significantly increased or decreased compared with corresponding models, respectively (all P < 0.05 ). 4 Discussion Individual variability in liver injury following drug ingestion is a major challenge for DILI research and clinical prevention (Andrade et al., 2019 ). In the study, it was found that Yin syn and Yang syn are two different predisposed individual states of hepatotoxicity or hepatoprotection caused by BGZ. From the perspective of the gene expression profile, BGZ mostly does not affect normal rats, but it has a mild or greater impact on the change numbers of DEGs in Yin syn (28 DEGs) and Yang syn (102 DEGs) rats, respectively. From the perspective of endogenous metabolites, Yin syn and Yang syn rats can be well distinguished on the PCA scatter plot, and the OPLS-DA plot can further better distinguish Yin syn , Yang syn , and their corresponding BGZ-treated groups, suggesting that BGZ exerts different metabolic perturbation patterns for different models. Among the DEGs with ROC ≥ 0.9 and significant correlation with ALT and AST, Slc25a25 may function as an ATP-Mg/Pi carrier to mediate the transport of Mg-ATP in exchange for phosphate. It is also likely responsible for the net uptake or efflux of adenine nucleotides into or from the mitochondria and is highly expressive in acute liver failure or induced by exogenous thyroxine (Fujimoto et al., 2018 ; Lee et al., 2022 ). This study confirmed the high expression of Slc25a25 in the liver of Yin syn and Yang syn rats, which is consistent with the phenomenon of abnormal liver function (Lee et al., 2022 ). However, only the expression of Slc25a25 in Yang syn rats was inhibited by BGZ, indicating that Slc25a25 may be a hepatoprotective gene for BGZ in Yang syn rats, and the way to aggravate liver injury in rats with Yin syn may not be through regulating Slc25a25. Pim3 is a liver growth-stimulating factor with serine/threonine kinase activity and is involved in gp130-mediated induction of cell proliferation and protection of apoptosis downstream of signal transducer and activator of transcription 3 (STAT3) (Liu et al., 2018 ; Wang et al., 2019 ). Pim3 is barely expressed in normal tissues but highly expressed in the prostate, large intestine, liver, and other cancer tissues (Brault et al., 2010 ). Therefore, its expression l evel is mainly used to evaluate tumor expression and metastasis (Qu et al., 2016 ; Zhou et al., 2016 ). Epidemiological studies have found and confirmed the correlations between tumors and the constitution of Yin syn and Yang syn (Chen and Wang, 2012 ; Ji et al., 2016 ). This study also found that Pim3 was abnormally highly expressed in the liver of Yin syn and Yang syn rats, suggesting that Yin syn and Yang syn may be susceptible to constitutions that induce tumors. Surprisingly, BGZ only decreased the expression of Pim3 in Yang syn rats with almost no change in Yin syn rats. The above results indicated that Pim3 may be a marker gene for BGZ to exert a hepatoprotective effect on Yang syn rats. In addition, BGZ may also have a potential therapeutic effect on tumor patients with Yang syn , and the anti-tumor effect of BGZ has been confirmed (Wu et al., 2013 ; Yu et al., 2019 ). Among the metabolites with ROC > 0.90 and significant correlation with ALT and AST, glycerophospholipids (including phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine) are the most abundant phospholipids in the body. In addition to constituting biofilm, they are also one of the components of bile and membrane surface-active substances and participate in protein recognition and signal transduction through the cell membrane (Hishikawa et al., 2014 ). Hepatocytes can express various glycerophospholipids activities, making the liver an important organ for glycerophospholipid metabolism. Dysregulation of glycerophospholipids is related to the development and progression of liver diseases including hepatitis, liver cancer, fatty liver, and liver fibrosis (van der Veen et al., 2017 ). For example, the abnormal increase or decrease of the ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) will affect energy metabolism and are closely related to disease progression (Li et al., 2006 ). The changes in hepatic phospholipid composition are also associated with fatty liver disease and impaired postoperative liver regeneration (Clemens et al., 2021 ; Ling et al., 2012 ; Sasabe et al., 2014 ). In the glycerophospholipid metabolic pathway, phosphatidate can activate hepatic interleukin 6 (IL-6) signaling through inter-organ crosstalk and alleviating acetaminophen-induced liver injury in mice (Clemens et al., 2021 ). Phosphatidate also acts as an ionophore in the brain between depolarization and the release of neurotransmitters such as gamma-Aminobutyric Acid (GABA) (Harris et al., 1981 ). The increase in the level of GABA can enhance the body’s immunity under stress conditions (Abdou et al., 2006 ) and has a good protective effect on acute liver injury or liver failure induced by ethanol, fluoride, and d-galactosamine (Hata et al., 2019 ; Wang et al., 2018 ; Yang et al., 2017 ). In this study, under the intervention of BGZ, the expression of PE and GABA in the liver of rats with Yin syn and Yang syn both showed opposite trends, suggesting that regulating glycerophospholipid metabolism may be another way in which BGZ exerts liver protection and liver injury in Yang syn and Yin syn rats, respectively. The metabolites of lipid metabolism participate in the biosynthesis of glycerophospholipid metabolism together with serine, providing raw materials for its metabolism (Cook et al., 1991 ). The metabolism of arachidonic acid, sphingolipid, glycine, serine, threonine, porphyrin, and chlorophyll can also interact directly or indirectly through serine. In the arachidonic acid metabolic pathway, 12(S)-HETE, as an important inflammatory marker, can affect the inflammatory process by stimulating the release of cytokines such as tumor necrosis factor alpha-like (TNF-α) and IL-6. Blocking the production of 12(S)-HETE can inhibit ischemia-reperfusion-induced liver dysfunction, inflammation, and cell death (Zhang et al., 2018 ). As a regulatory enzyme of arachidonic acid metabolism, arachidonate 15-lipoxygenase (Alox15) can catalyze it to 12(S)-HPETE, which is further reduced by glutathione peroxidase into 12(S)-HETE (Spector et al., 1988 ). Prostaglandin E2 (PGE2), another metabolite of arachidonic acid, has also been shown to induce acute and chronic inflammation and various autoimmune diseases through Th1 differentiation, Th17 cell proliferation, and activation of mast cells (Tsuge et al., 2019 ). In this study, the expressions of 12(S)-HETE, PGE2, and Alox15 were significantly increased in the Yin syn group under the intervention of BGZ, while the expressions of 12(S)-HETE, PGE2, and Alox15 were significantly decreased or did not change significantly in the Yang syn group, suggesting that the regulation of arachidonic acid metabolism may be another way in which BGZ regulates the effects of liver protection or liver damage in rats with Yang syn and Yin syn , respectively. In the metabolic pathway of porphyrin and chlorophyll, 5-aminolevulinate, the direct metabolite of glycine, can be used as a precursor of heme and participate in the regulation of the production of heme and its metabolite biliverdin (Sansaloni-Pastor et al., 2022 ). Biliverdin has a good anti-inflammatory effect, can inhibit the expression of toll-like receptor 4 and nitric oxide, and reduces the inflammatory induction of lipopolysaccharide on macrophages (Wegiel et al., 2011 ). It is used in the protection against various diseases (vascular injury, organ transplantation, etc.). As such, biliverdin can better reduce the ischemia-reperfusion injury in pig liver and have a liver protective effect (Andria et al., 2013 ). Biliverdin reductase B (Blvrb) is a non-redundant nicotinamide adenine dinucleotide (phosphate)-dependent biliverdin reductase that regulates the cellular redox state by converting biliverdin to bilirubin. Tts redox function also reduces intracellular reactive oxygen species accumulation (Wu et al., 2016 ) and maintains essential cytoprotective functions in recovery from hematopoietic stress (Nesbitt et al., 2021 ). Bilirubin is the main pigment in human bile and the main metabolite of iron porphyrin compounds in the body with certain damage to the brain and nerves (Watchko and Tiribelli, 2013 ). The increase in its level can also lead to impaired liver function, so it is also used as a test for one of the common indicators of jaundice (Fawaz et al., 2017 ). In the study, BGZ significantly increased the expressions of biliverdin in Yang syn rats accompanied by the inhibition expressions of bilirubin in Yin syn rats, suggesting that the regulation of porphyrin metabolism, especially Blvrb, may be a mechanism by which BGZ exerts hepatoprotective or liver damage effects on Yang syn and Yin syn rats, respectively. However, this is not related to bilirubin. Tryptophan is involved in immune regulation, neural function, and intestinal homeostasis through the kynurenine pathway for metabolism (Cervenka et al., 2017 ). For example, kynurenine is an endothelium-derived relaxation factor in the inflammatory process (Wang, Y. et al., 2010 ) and is metabolized to neurotoxic 3-Hydroxy-L-kynurenine under the action of kynureninase (Kynu), which is involved in the inflammatory process in psoriasis and other inflammatory diseases (Harden et al., 2016 ). Therefore, the imbalance of tryptophan metabolism in diseases ranging from cancer to neurodegenerative diseases has become a research hotspot for the therapeutic targeting of the kynurenine pathway (Platten et al., 2019 ). In this study, compared with the Yang syn group and the Yin syn group, the levels of 3-Hydroxy-L-kynurenine were significantly decreased and increased under the intervention of BGZ, respectively, suggesting the kynurenine metabolism pathway in regulating tryptophan metabolism may be another mechanism by which BGZ exerts hepatoprotective or liver damage effects on Yang syn and Yin syn rats. The above-discussed pathways changed by BGZ in Yin syn and (or) Yang syn , were almost all related to energy metabolism, amino acid metabolism, and lipid metabolism, and the disorder of the above pathways has also been reported in Yin syn and Yang syn (Chen et al., 2019 ; Jiang et al., 2015 ). The effects of BGZ on the metabolism of the above pathways have also been reported by Xu et al. (Xu et al., 2017 ). cAMP and cGMP, the potential biomarkers for Yin/Yang disharmony in TCM (Wang et al., 2021 ), can cross-activate protein kinases related to tissue biosynthesis and metabolism (including carbohydrate, lipid, amino acid, cofactors, and vitamins) as cyclic nucleotide effectors (Coelho Horta et al., 2005 ; Francis and Corbin, 1999 ; Jiang et al., 1992 ). Considering that BGZ can enhance the expression of cAMP-responsive element modulator-τ (CREMτ) (Wei et al., 2011 ), and the ability of BGZ to down-regulate and up-regulate the expression of cAMP and the ratio of cAMP/cGMP in rats with Yin syn and Yang syn , respectively. It is suggested that the cAMP signaling pathway may be one of the mechanisms by which BGZ regulates energy metabolism to affect rats with Yin syn or Yang syn . As for the different intervention effects of BGZ on the different syndromes, the interaction of endogenous metabolites and genes may be the main reason, which is worthy of further study in the future. In conclusion, this study found that BGZ has a double-edged sword-like effect that not only exerts a good hepatoprotective effect on Yang syn rats but also has a potential risk of inducing liver injury in Yin syn rats. The mechanism is mainly reflected in BGZ having different regulatory effects on amino acid metabolism, energy metabolism, lipid metabolism, and metabolism of cofactors and vitamins in the above two different syndromes. In response to the seemingly contradictory results that BGZ has both liver-damaging and liver-protective effects in previous reports (Wang et al., 2020 ; Zhang et al., 2016 ), this study found that BGZ may have both liver-damaging and liver-protecting effects on predisposed individuals from the perspective of TCM syndrome theory. The study screened out the endogenous markers that can characterize the corresponding predisposed individuals, which will provide a certain reference for the safe and rational application of TCM. Abbreviations Psoralea corylifolia Linn. BGZ Yang syn kidney-yang deficiency syndrome Yin syn kidney-yin deficiency syndrome TCM Traditional Chinese Medicine HE Hematoxylin and eosin HMDB Human Metabolome Database KEGG Kyoto Encyclopedia of Genes and Genomes PCA Principal components analysis OPLS-DA Orthogonal partial least-squares discriminate VIP variable importance in the projection UPLC Ultra-performance liquid chromatography LC-MS Liquid chromatography-tandem mass spectrometry DILI Drug-induced liver injury GC glucocorticoid ALT Alanine aminotransferase AST Aspartate aminotransferase Scr Serum creatinine cAMP cyclic adenosine monophosphate cGMP cyclic guanosine monophosphate DEGs Differentially expressed genes ESI+ Positive electrospray ionization source ROC Receiver operating characteristic GO Gene Ontology Kos KEGG orthology AUC Areas under the curve PGE2 Prostaglandin E2 Declarations Ethics approval and consent to participate Experimental animal protocols were approved by the Animal Ethics Committee of the First Affiliated Hospital of Henan University of Traditional Chinese Medicine and the procedures were conformed to the Guide for the Care and Use of Laboratory Animals. Consent for publication The authors declare that they have no competing interests. Competing interests The authors declare no competing financial interest. Funding This work was supported by the National Natural Science Foundation of China (No. 82173993, No. U1904129, No. 82204638, No. 82230118), the Key Project of Henan Province for Scientific Research of Traditional Chinese Medicine, China (No. 2019ZYBJ08), and the Key Scientific Research Projects of Colleges and Universities in Henan Province, China (No. 19A360007). Authors' contributions Jin-Fa Tang, Xiao-He Xiao, and Yan-Ling Zhao conceived and designed the experiments; Ming-Liang Zhang performed the experiments, analyzed the data, and wrote the manuscript; De-Xin Kong, Yuan Gao was responsible for helping the collection of experimental samples; Yu-Long Chen, Hui Zhang, Xiao-Yan Wang help to detect the metabonomic changes of liver samples, Xu Zhao and Ming Niu helped with data analysis, Zhao-Fang Bai, Wei-Xia Li, and Yu-Ming Guo reviewed the paper. All authors have reviewed the manuscript and approved the final version of the manuscript. All authors have read, revised, and approved the final manuscript. Acknowledgments We thank James thompson, PhD, from BEIJING LANGYI TRANSLATION CO., LTD., China, for mother tongue polishing the English text of this manuscript. Availability of data and material The research data generated from this study are included in the article and additional files. References Abdou AM, Higashiguchi S, Horie K, Kim M, Hatta H, Yokogoshi H. 2006. Relaxation and immunity enhancement effects of gamma-aminobutyric acid (GABA) administration in humans. BioFactors 26(3), 201–208. Andrade RJ, Chalasani N, Björnsson ES, Suzuki A, Kullak-Ublick GA, Watkins PB, Devarbhavi H, Merz M, Lucena MI, Kaplowitz N, Aithal GP. Drug-induced liver injury. Nat Rev Dis Primers. 2019;5(1):58. Andria B, Bracco A, Attanasio C, Castaldo S, Cerrito MG, Cozzolino S, Di Napoli D, Giovannoni R, Mancini A, Musumeci A, Mezza E, Nasti M, Scuderi V, Staibano S, Lavitrano M, Otterbein LE, Calise F. Biliverdin protects against liver ischemia reperfusion injury in swine. PLoS ONE. 2013;8(7):e69972. Averaimo S, Nicol X. Intermingled cAMP, cGMP and calcium spatiotemporal dynamics in developing neuronal circuits. Front Cell Neurosci. 2014;8:376. Brault L, Gasser C, Bracher F, Huber K, Knapp S, Schwaller J. PIM serine/threonine kinases in the pathogenesis and therapy of hematologic malignancies and solid cancers. Haematologica. 2010;95(6):1004–15. Cervenka I, Agudelo LZ, Ruas JL. Kynurenines: Tryptophan's metabolites in exercise, inflammation, and mental health. Science. 2017;357(6349):eaaf9794. Chen M, Borlak J, Tong W. High lipophilicity and high daily dose of oral medications are associated with significant risk for drug-induced liver injury. Hepatology. 2013;58(1):388–96. Chen M, Borlak J, Tong W. A Model to predict severity of drug-induced liver injury in humans. Hepatology. 2016;64(3):931–40. Chen M, Suzuki A, Borlak J, Andrade RJ, Lucena MI. Drug-induced liver injury: Interactions between drug properties and host factors. J Hepatol. 2015;63(2):503–14. Chen R, Wang J, Zhan R, Zhang L, Wang X. Integrated Systems Pharmacology, Urinary Metabonomics, and Quantitative Real-Time PCR Analysis to Uncover Targets and Metabolic Pathways of the You-Gui Pill in Treating Kidney-Yang Deficiency Syndrome. Int J Mol Sci. 2019;20(15):3655. Chen Z, Wang P. 2012. Clinical Distribution and Molecular Basis of Traditional Chinese Medicine ZHENG in Cancer. Evid Based Complement Alternat Med 2012, 783923. Cheng ZX, Guo C, Chen ZG, Yang TC, Zhang JY, Wang J, Zhu JX, Li D, Zhang TT, Li H, Peng B, Peng XX. Glycine, serine and threonine metabolism confounds efficacy of complement-mediated killing. Nat Commun. 2019;10(1):3325. Chinese Pharmacopoeia, Commission. 2020. Pharmacopoeia of the People's Republic of China. Beijing: Chinese Medical Science and Technology Press, 2020;1:195. Clemens MM, Kennon-McGill S, Vazquez JH, Stephens OW, Peterson EA, Johann DJ, Allard FD, Yee EU, McCullough SS, James LP, Finck BN, McGill MR. Exogenous phosphatidic acid reduces acetaminophen-induced liver injury in mice by activating hepatic interleukin-6 signaling through inter-organ crosstalk. Acta Pharm Sinica B. 2021;11(12):3836–46. Coelho Horta B, Perilo S, Caldeira Costa C, Nogueira-Machado D, Martins Chaves JA, M. Aging: functional metabolic balance among cAMP, cGMP and reactive oxygen intermediate generation by human granulocytes. Gerontology. 2005;51(6):363–8. Cook HW, Thomas SE, Xu Z. Essential fatty acids and serine as plasmalogen precursors in relation to competing metabolic pathways. Biochem Cell Biol. 1991;69(7):475–84. Deng PY, Cai HD, Cheng JH, Liu SA, Dao WB. 1996. Liver Damage Caused by Zhuanggu Joint Pill: A Report of 30 Cases. Chin J of New Drugs (03), 212–214. Dermatology Branch of China Association of Chinese Medicine. Expert Consensus on Chinese Medicine Treatment of Vitiligo. Chin J Dermato Veneral Integ Trad W Med. 2017;16(02):191–2. Dermatology Branch of China Association of Chinese Medicine. Expert consensus on traditional Chinese medicine treatment of psoriasis in Dermatology Branch. Chin J Dermato Venemol Integ Trad W Med. 2018;17(03):273–7. Fawaz R, Baumann U, Ekong U, Fischler B, Hadzic N, Mack CL, McLin VA, Molleston JP, Neimark E, Ng VL, Karpen SJ. Guideline for the Evaluation of Cholestatic Jaundice in Infants: Joint Recommendations of the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition and the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition. J Pediatr Gastroenterol Nutr. 2017;64(1):154–68. Francis SH, Corbin JD. Cyclic nucleotide-dependent protein kinases: intracellular receptors for cAMP and cGMP action. Crit Rev Clin Lab Sci. 1999;36(4):275–328. Fujimoto N, Kitamura S, Uramaru N, Miyagawa S, Iguchi T. Identification of hepatic thyroid hormone-responsive genes in neonatal rats: Potential targets for thyroid hormone-disrupting chemicals. Toxicol Lett. 2018;286:48–53. Ge FL, Niu M, Han ZX, Cao JL, Wang JB, Bai ZF, Song HB, Guo YM, Xiao XH. Landscape of Hepatobiliary Adverse Drug Reactions Related to Preparations Containing Psoraleae Fructus and Its Application in Pharmacovigilance. Chin J Integr Med. 2021;27(11):832–7. Ge JR, Wang HM, Zheng HX, Luo YW, Wang JB, Zhao YF, Wan XM, Lu M, He CJ, Dong Z, Zhou HJ, Li G, Sun SB, Lin XS. Traditional Chinese Medicine Expert Consensus on the prevention and treatment of primary osteoporosis. Chin J Osteoporos. 2020;26(12):1717–25. Godfrey RW, Manzi RM, Gennaro DE, Hoffstein ST. Phospholipid and arachidonic acid metabolism in zymosan-stimulated human monocytes: modulation by cAMP. J Cell Physiol. 1987;131(3):384–92. Gupta AK, Anderson TF. Psoralen photochemotherapy. J Am Acad Dermatol. 1987;17(5 Pt 1):703–34. Harden JL, Lewis SM, Lish SR, Suárez-Fariñas M, Gareau D, Lentini T, Johnson-Huang LM, Krueger JG, Lowes MA. The tryptophan metabolism enzyme L-kynureninase is a novel inflammatory factor in psoriasis and other inflammatory diseases. J Allergy Clin Immunol. 2016;137(6):1830–40. Harris RA, Schmidt J, Hitzemann BA, Hitzemann RJ. Phosphatidate as a molecular link between depolarization and neurotransmitter release in the brain. Science. 1981;212(4500):1290–1. Hata T, Rehman F, Hori T, Nguyen JH. GABA, γ-Aminobutyric Acid, Protects Against Severe Liver Injury. J Surg Res. 2019;236:172–83. Hishikawa D, Hashidate T, Shimizu T, Shindou H. Diversity and function of membrane glycerophospholipids generated by the remodeling pathway in mammalian cells. J Lipid Res. 2014;55(5):799–807. Hoofnagle JH, Björnsson ES. Drug-Induced Liver Injury - Types and Phenotypes. N Engl J Med. 2019;381(3):264–73. Ish-Shalom S, Lichter A. Analysis of fungal gene expression by Real Time quantitative PCR. Methods Mol Biol. 2010;638:103–14. Ji Q, Luo YQ, Wang WH, Liu X, Li Q, Su SB. Research advances in traditional Chinese medicine syndromes in cancer patients. J Integr Med. 2016;14(1):12–21. Jiang H, Shabb JB, Corbin JD. Cross-activation: overriding cAMP/cGMP selectivities of protein kinases in tissues. Biochem Cell Biol. 1992;70(12):1283–9. Jiang N, Liu HF, Li SD, Zhou WX, Zhang YX, Zhang Q, Yan XZ. An integrated metabonomic and proteomic study on Kidney-Yin Deficiency Syndrome patients with diabetes mellitus in China. Acta Pharmacol Sin. 2015;36(6):689–98. Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015;12(4):357–60. Lee YM, Choi DH, Cheon MW, Kim JG, Kim JS, Shin MG, Kim HR, Youn D. 2022. Changes in Mitochondria-Related Gene Expression upon Acupuncture at LR3 in the D-Galactosamine-Induced Liver Damage Rat Model. Evid Based Complement Alternat Med 2022, 3294273. Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011;12:323. Li CY, Niu M, Liu YL, Tang JF, Chen W, Qian G, Zhang MY, Shi YF, Lin JZ, Li XJ, Li RS, Xiao XH, Li GH, Wang JB. Screening for Susceptibility-Related Factors and Biomarkers of Xianling Gubao Capsule-Induced Liver Injury. Front Pharmacol. 2020;11:810. Li Z, Agellon LB, Allen TM, Umeda M, Jewell L, Mason A, Vance DE. The ratio of phosphatidylcholine to phosphatidylethanolamine influences membrane integrity and steatohepatitis. Cell Metab. 2006;3(5):321–31. Li ZJ, Abulizi A, Zhao GL, Wang T, Zhou F, Jiang ZZ, Aibai S, Zhang LY. Bakuchiol Contributes to the Hepatotoxicity of Psoralea corylifolia in Rats. Phytother Res. 2017;31(8):1265–72. Ling J, Chaba T, Zhu LF, Jacobs RL, Vance DE. Hepatic ratio of phosphatidylcholine to phosphatidylethanolamine predicts survival after partial hepatectomy in mice. Hepatology. 2012;55(4):1094–102. Liu J, Qu X, Shao L, Hu Y, Yu X, Lan P, Guo Q, Han Q, Zhang J, Zhang C. Pim-3 enhances melanoma cell migration and invasion by promoting STAT3 phosphorylation. Cancer Biol Ther. 2018;19(3):160–8. Liu YL, Ge FL, Zhu JX, Jing J, Wang JB, Zhang YM, Guo YM, Xiao XH. Re-evaluation of liver injury associated with Buguzhi Preparations based on passive monitoring data and hospital cases. Zhongguo Zhong Yao Za Zhi. 2019;44(19):4272–6. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550. Lu Y, Shao M, Xiang H, Zheng P, Wu T, Ji G. Integrative transcriptomics and metabolomics explore the mechanism of kaempferol on improving nonalcoholic steatohepatitis. Food Funct. 2020;11(11):10058–69. McNeely W, Goa KL. 5-Methoxypsoralen. A review of its effects in psoriasis and vitiligo. Drugs. 1998;56(4):667–90. Nam SW, Baek JT, Lee DS, Kang SB, Ahn BM, Chung KW. A case of acute cholestatic hepatitis associated with the seeds of Psoralea corylifolia (Boh-Gol-Zhee). Clin Toxicol (Phila). 2005;43(6):589–91. Nesbitt NM, Malone LE, Liu Z, Jares A, Gnatenko DV, Ma Y, Zhu W, Bahou WF. Divergent erythroid megakaryocyte fates in Blvrb-deficient mice establish non-overlapping cytoprotective functions during stress hematopoiesis. Free Radic Biol Med. 2021;164:164–74. Platten M, Nollen EAA, Röhrig UF, Fallarino F, Opitz CA. Tryptophan metabolism as a common therapeutic target in cancer, neurodegeneration and beyond. Nat Rev Drug Discov. 2019;18(5):379–401. Qu Y, Zhang C, Du E, Wang A, Yang Y, Guo J, Wang A, Zhang Z, Xu Y. Pim-3 is a Critical Risk Factor in Development and Prognosis of Prostate Cancer. Med Sci Monit. 2016;22:4254–60. Ren S, Shao Y, Zhao X, Hong CS, Wang F, Lu X, Li J, Ye G, Yan M, Zhuang Z, Xu C, Xu G, Sun Y. Integration of Metabolomics and Transcriptomics Reveals Major Metabolic Pathways and Potential Biomarker Involved in Prostate Cancer. Mol Cell Proteomics. 2016;15(1):154–63. Sansaloni-Pastor S, Varesio E, Lange N. Modulation and proteomic changes on the heme pathway following treatment with 5-aminolevulinic acid. J Photochem Photobiol B. 2022;233:112484. Sasabe N, Keyamura Y, Obama T, Inoue N, Masuko Y, Igarashi Y, Aiuchi T, Kato R, Yamaguchi T, Kuwata H, Iwamoto S, Miyazaki A, Hara S, Yoshikawa T, Itabe H. Time course-changes in phosphatidylcholine profile during oxidative modification of low-density lipoprotein. Lipids Health Dis. 2014;13:48. Shen B, Kwan HY, Ma X, Wong CO, Du J, Huang Y, Yao X. cAMP activates TRPC6 channels via the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB)-mitogen-activated protein kinase kinase (MEK)-ERK1/2 signaling pathway. J Biol Chem. 2011;286(22):19439–45. Shen T, Liu Y, Shang J, Xie Q, Li J, Yan M, Xu J, Niu J, Liu J, Watkins PB, Aithal GP, Andrade RJ, Dou X, Yao L, Lv F, Wang Q, Li Y, Zhou X, Zhang Y, Zong P, Wan B, Zou Z, Yang D, Nie Y, Li D, Wang Y, Han X, Zhuang H, Mao Y, Chen C. Incidence and Etiology of Drug-Induced Liver Injury in Mainland China. Gastroenterology. 2019;156(8):2230–41.e2211. Spector AA, Gordon JA, Moore SA. Hydroxyeicosatetraenoic acids (HETEs). Prog Lipid Res. 1988;27(4):271–323. Tsuge K, Inazumi T, Shimamoto A, Sugimoto Y. Molecular mechanisms underlying prostaglandin E2-exacerbated inflammation and immune diseases. Int Immunol. 2019;31(9):597–606. van der Veen JN, Kennelly JP, Wan S, Vance JE, Vance DE, Jacobs RL. The critical role of phosphatidylcholine and phosphatidylethanolamine metabolism in health and disease. Biochim Biophys Acta Biomembr Biomembranes. 2017;1859(9 Pt B):1558–72. Wang L, Wang Y, Wee A, Soon G, Gouw ASH, Yang R, Tian Q, Liu L, Ma H, Zhao X. Clinicopathological features of Bu Gu Zhi-induced liver injury, a long-term follow-up cohort study. Liver Int. 2020;40(3):571–80. Wang P, Sun H, Lv H, Sun W, Yuan Y, Han Y, Wang D, Zhang A, Wang X. Thyroxine and reserpine-induced changes in metabolic profiles of rat urine and the therapeutic effect of Liu Wei Di Huang Wan detected by UPLC-HDMS. J Pharm Biomed Anal. 2010;53(3):631–45. Wang S, Sui S, Liu Z, Peng C, Liu J, Luo D, Fan X, Liu C, Lu WY. Protective roles of hepatic gamma-aminobutyric acid signaling in acute ethanol exposure-induced liver injury. J Appl Toxicol. 2018;38(3):341–50. Wang X, Du Y, Wu C, Xu M, Liu Y, Di X. UHPLC-MS/MS analysis of cAMP and cGMP in rat plasma as potential biomarkers of Yin-Yang disharmony in traditional Chinese medicine. J Pharm Anal. 2021;11(4):458–64. Wang Y, Liu C, Hu L. Cholesterol regulates cell proliferation and apoptosis of colorectal cancer by modulating miR-33a-PIM3 pathway. Biochem Biophys Res Commun. 2019;511(3):685–92. Wang Y, Liu H, McKenzie G, Witting PK, Stasch JP, Hahn M, Changsirivathanathamrong D, Wu BJ, Ball HJ, Thomas SR, Kapoor V, Celermajer DS, Mellor AL, Keaney JF Jr, Hunt NH, Stocker R. Kynurenine is an endothelium-derived relaxing factor produced during inflammation. Nat Med. 2010;16(3):279–85. Watchko JF, Tiribelli C. Bilirubin-induced neurologic damage–mechanisms and management approaches. N Engl J Med. 2013;369(21):2021–30. Wegiel B, Gallo D, Csizmadia E, Roger T, Kaczmarek E, Harris C, Zuckerbraun BS, Otterbein LE. Biliverdin inhibits Toll-like receptor-4 (TLR4) expression through nitric oxide-dependent nuclear translocation of biliverdin reductase. Proc Natl Acad Sci U S A. 2011;108(46):18849–54. Wei SM, Yan ZZ, Zhou J. Psoralea corylifolia protects against testicular torsion/detorsion-induced ischemia/reperfusion injury. J Ethnopharmacol. 2011;137(1):568–74. Wu C, Sun Z, Ye Y, Han X, Song X, Liu S. Psoralen inhibits bone metastasis of breast cancer in mice. Fitoterapia. 2013;91:205–10. Wu S, Li Z, Gnatenko DV, Zhang B, Zhao L, Malone LE, Markova N, Mantle TJ, Nesbitt NM, Bahou WF. BLVRB redox mutation defines heme degradation in a metabolic pathway of enhanced thrombopoiesis in humans. Blood. 2016;128(5):699–709. Xu Y, Zhao Y, Xie J, Sheng X, Li Y, Zhang Y. 2017. The Evaluation of Toxicity Induced by Psoraleae Fructus in Rats Using Untargeted Metabonomic Method Based on UPLC-Q-TOF/MS. Evid Based Complement Alternat Med 2017, 6207183. Yang H, Xing R, Liu S, Yu H, Li P. Rescuing fluoride-induced damages in liver with gamma aminobutyric acid. Biochem Biophys Res Commun. 2017;491(1):19–24. Yu B, Wang AH, Zhou K, Chai LJ, Liu L. Molecular Pathway of Psoralidin-Induced Apoptosis in HepG2 Cell Line. Chin J Integr Med. 2019;25(10):757–62. Zhang C, Zhao JQ, Sun JX, Li HJ. Psoralen and isopsoralen from Psoraleae Fructus aroused hepatotoxicity via induction of aryl hydrocarbon receptor-mediated CYP1A2 expression. J Ethnopharmacol. 2022;297:115577. Zhang L, Niu M, Wei AW, Tang JF, Tu C, Bai ZF, Zou ZS, Xiao XH, Liu YP, Wang JB. Risk profiling using metabolomic characteristics for susceptible individuals of drug-induced liver injury caused by Polygonum multiflorum. Arch Toxicol. 2020;94(1):245–56. Zhang X, Zhao W, Wang Y, Lu J, Chen X. The Chemical Constituents and Bioactivities of Psoralea corylifolia Linn.: A Review. Am J Chin Med. 2016;44(1):35–60. Zhang XJ, Cheng X, Yan ZZ, Fang J, Wang X, Wang W, Liu ZY, Shen LJ, Zhang P, Wang PX, Liao R, Ji YX, Wang JY, Tian S, Zhu XY, Zhang Y, Tian RF, Wang L, Ma XL, Huang Z, She ZG, Li H. An ALOX12-12-HETE-GPR31 signaling axis is a key mediator of hepatic ischemia-reperfusion injury. Nat Med. 2018;24(1):73–83. Zhang Y, Xu SY, Liu MN, Jia TY, Qu WJ, Han T, Jia Z, Xu XF, Li XR. 2019. Comparative Studies on Chemical Contents and Effect in Kidney-Yang Deficiency Rats of Salt-Processed Product and Wine-Processed Product of Cuscutae Semen. Evid Based Complement Alternat Med 2019, 2049497. Zhou Z, Zhang R, Wang R, Zhang Y, Xu L, Chen J, Zhang J, Huang Z, Chen M, Pan Z. Expression of Pim-3 in colorectal cancer and its relationship with prognosis. Tumour Biol. 2016;37(7):9151–6. Supplementary Files SupplementaryTablesandFigures.docx Cite Share Download PDF Status: Published Journal Publication published 17 Aug, 2023 Read the published version in Chinese Medicine → Version 1 posted Editorial decision: Major revision 15 Feb, 2023 Reviewers agreed at journal 11 Jan, 2023 Reviewers invited by journal 03 Dec, 2022 Editor assigned by journal 25 Nov, 2022 First submitted to journal 24 Nov, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2301287","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":157200867,"identity":"856de113-9361-4447-9171-909e68c52578","order_by":0,"name":"Ming-Liang Zhang","email":"","orcid":"","institution":"The First Affiliated Hospital of Henan University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ming-Liang","middleName":"","lastName":"Zhang","suffix":""},{"id":157200868,"identity":"ac3fc15d-871c-407f-904d-6cd51eba7e35","order_by":1,"name":"Xu Zhao","email":"","orcid":"","institution":"5th Medical Center of Chinese PLA General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Zhao","suffix":""},{"id":157200869,"identity":"9b67112f-b642-4592-b081-aa7ca31e485b","order_by":2,"name":"Wei-Xia Li","email":"","orcid":"","institution":"The First Affiliated Hospital of Henan University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wei-Xia","middleName":"","lastName":"Li","suffix":""},{"id":157200870,"identity":"03e4f7b7-6a73-4185-bb18-157f73f6157f","order_by":3,"name":"Xiao-Yan Wang","email":"","orcid":"","institution":"The First Affiliated Hospital of Henan University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiao-Yan","middleName":"","lastName":"Wang","suffix":""},{"id":157200871,"identity":"03bf8679-2b6c-4401-8e04-3f1a7e0ea343","order_by":4,"name":"Ming Niu","email":"","orcid":"","institution":"5th Medical Center of Chinese PLA General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ming","middleName":"","lastName":"Niu","suffix":""},{"id":157200872,"identity":"9d46c3f2-ce22-4925-aa7f-46c360149878","order_by":5,"name":"Hui Zhang","email":"","orcid":"","institution":"The First Affiliated Hospital of Henan University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Zhang","suffix":""},{"id":157200873,"identity":"e8722e31-0cb7-418f-82d4-5b0dab3bb4b7","order_by":6,"name":"Yu-Long Chen","email":"","orcid":"","institution":"Henan University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu-Long","middleName":"","lastName":"Chen","suffix":""},{"id":157200874,"identity":"59dc6178-df98-481a-8518-c36e70dba7bf","order_by":7,"name":"De-Xin Kong","email":"","orcid":"","institution":"Henan University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"De-Xin","middleName":"","lastName":"Kong","suffix":""},{"id":157200875,"identity":"dab8434b-43d0-4bb1-937b-effcf2f88e85","order_by":8,"name":"Yuan Gao","email":"","orcid":"","institution":"Capital Medical University School of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Gao","suffix":""},{"id":157200876,"identity":"8205d2ae-5340-4f8b-8c95-6c940dea119a","order_by":9,"name":"Yu-Ming Guo","email":"","orcid":"","institution":"5th Medical Center of Chinese PLA General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu-Ming","middleName":"","lastName":"Guo","suffix":""},{"id":157200877,"identity":"7539aed4-deea-4752-a2d7-c0697b5dc530","order_by":10,"name":"Zhao-Fang Bai","email":"","orcid":"","institution":"5th Medical Center of Chinese PLA General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhao-Fang","middleName":"","lastName":"Bai","suffix":""},{"id":157200878,"identity":"10e01230-946f-4367-841a-b7540a58a040","order_by":11,"name":"Xiao-he Xiao","email":"","orcid":"","institution":"5th Medical Center of Chinese PLA General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiao-he","middleName":"","lastName":"Xiao","suffix":""},{"id":157200879,"identity":"06bbdf9e-a3d0-4035-bdd3-e38d023e38be","order_by":12,"name":"Yan-Ling Zhao","email":"","orcid":"","institution":"5th Medical Center of Chinese PLA General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yan-Ling","middleName":"","lastName":"Zhao","suffix":""},{"id":157200880,"identity":"1819ef63-4662-4133-9cc0-77f23fd7cb15","order_by":13,"name":"Jinfa Tang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArElEQVRIiWNgGAWjYDACZv4HBz5USMjJE6+FvYfx4IwzFsaGDURr4TnDfJi3rSKR4QCxOvhn5B44zDtPIoGxgfnhoxvEaJG4kZdwcO42iTx2BjZj4xyirLmRYHDg7TaJYsYGHjZporTIg7TwzpFIbDhArBaDM2cMDvI2kKLF8HhbwsEZxySMDZuJ9YvcYebDHz7U1MnJszc/fEyc9+GAmTTlo2AUjIJRMArwAQBEmTS4Cz36aQAAAABJRU5ErkJggg==","orcid":"","institution":"The First Affiliated Hospital of Henan University of CM","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jinfa","middleName":"","lastName":"Tang","suffix":""}],"badges":[],"createdAt":"2022-11-22 13:02:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2301287/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2301287/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13020-023-00793-x","type":"published","date":"2023-08-17T22:02:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":29985542,"identity":"3f2fadd0-41f9-4157-a119-66b079132a3e","added_by":"auto","created_at":"2022-12-06 21:04:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":93785,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of BGZ treatment on changes in body weight, anal temperature, and organ indexes (N=8). Weight (A), anal temperature (B), liver index (C), and kidney index (D). \u003cem\u003e*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001\u003c/em\u003e, compared with CON group; \u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u0026lt; 0.05,\u003c/em\u003e compared with Yang\u003csub\u003esyn\u003c/sub\u003e group; \u003csup\u003e\u003cem\u003e\u0026amp;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e, \u003csup\u003e\u003cem\u003e\u0026amp;\u0026amp;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e, \u003csup\u003e\u003cem\u003e\u0026amp;\u0026amp;\u0026amp;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u0026lt; 0.001\u003c/em\u003e, compared with Yin\u003csub\u003esyn\u003c/sub\u003e group.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2301287/v1/1d0be95a7be711032a8850a2.png"},{"id":29985932,"identity":"b0827d68-1df0-4d26-91dd-a1c6a2b7206d","added_by":"auto","created_at":"2022-12-06 21:28:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":613601,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of BGZ treatment on serum biochemistry and histopathological examination (N=6~8). Serum ALT (A), AST (B), and SCr (C) activity. \u003cem\u003e**P \u0026lt; 0.01\u003c/em\u003e, \u003cem\u003e***P \u0026lt; 0.001\u003c/em\u003e, compared with CON group; \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u0026lt; 0.01, \u003c/em\u003e\u003csup\u003e\u003cem\u003e###\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u0026lt; 0.001\u003c/em\u003e, compared with Yang\u003csub\u003esyn\u003c/sub\u003e group; \u003csup\u003e\u003cem\u003e\u0026amp;\u0026amp;\u0026amp;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u0026lt; 0.001\u003c/em\u003e, compared with Yin\u003csub\u003esyn\u003c/sub\u003e group. (D) Typical histopathological section photographs of rat liver specimens for HE analysis (Scale bar, 100 μm).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2301287/v1/bcf6bf8b47333040a0404db9.png"},{"id":29985781,"identity":"b8f69300-30bb-4a8e-baf6-04e250558f18","added_by":"auto","created_at":"2022-12-06 21:20:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":494672,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptomic alterations of BGZ treatment on Yin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e rats (N=5). (A) The number of DEGs compared with each group. The red color represents up-regulated genes and the green color represents down-regulated genes. (B) Heatmap showing the intersection of DEGs between the Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ group and the Yin\u003csub\u003esyn\u003c/sub\u003e group. (C) Heatmap showing the intersection of DEGs between the Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ group and the Yang\u003csub\u003esyn\u003c/sub\u003e group. DEGs: differentially expressed genes.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2301287/v1/c17200fb9f7fd390410c9cc2.png"},{"id":29985543,"identity":"4385bbc6-1df7-40e0-9903-9e2b3e9ba956","added_by":"auto","created_at":"2022-12-06 21:04:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":224768,"visible":true,"origin":"","legend":"\u003cp\u003eThe relationship between DEGs and the levels of serum biochemistry (ALT and AST). (A) Heatmap of correlation between the levels of serum biochemistry (ALT and AST) and DEGs (N=5). Color depth represents correlation strength, the red color represents positive correlation, and the blue color represents negative correlation.\u003cem\u003e *P \u0026lt; 0.05\u003c/em\u003e, \u003cem\u003e**P \u0026lt; 0.01\u003c/em\u003e, \u003cem\u003e***P \u0026lt; 0.001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2301287/v1/def974d8986478e06a983451.png"},{"id":29985611,"identity":"c592172a-b935-4894-b854-e60e9d033c30","added_by":"auto","created_at":"2022-12-06 21:12:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":231464,"visible":true,"origin":"","legend":"\u003cp\u003eROC analysis of DEGs associated with ALT and AST expression and RT-qPCR verification.\u003cstrong\u003e \u003c/strong\u003e(A) Venn diagram showing the number of DEGs associated with the levels of ALT and AST under ROC \u0026gt; 0.90 between the Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ group and the Yang\u003csub\u003esyn\u003c/sub\u003e group. (B-E) ROC curves of DEGs associated with the levels of ALT and AST under ROC \u0026gt; 0.90 between the Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ group and Yang\u003csub\u003esyn\u003c/sub\u003e group, Aldh1b1 (B), Slc25a25 (C), Oaf (D), and Pim3 (E). (J) Venn diagram showing the number of DEGs associated with the levels of ALT and AST under ROC \u0026gt; 0.90 between the Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ group and the Yin\u003csub\u003esyn\u003c/sub\u003e group. (K) ROC curves of Lgals5 between the Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ group and the Yin\u003csub\u003esyn\u003c/sub\u003e group. (F-I and L) The mRNA expression of Aldh1b1 (F), Slc25a25 (G), Oaf (H), Pim3 (I), and Lgals5 (L). \u003cem\u003e*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001\u003c/em\u003e, compared with CON group; \u003csup\u003e\u003cem\u003e###\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u0026lt; 0.001\u003c/em\u003e, compared with Yang\u003csub\u003esyn\u003c/sub\u003e group; \u003csup\u003e\u003cem\u003e\u0026amp;\u0026amp;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e, compared with Yin\u003csub\u003esyn\u003c/sub\u003e group. DEGs: differentially expressed genes; ROC: receiver operating characteristic; AUC: area under the ROC curve; CI: confidence interval.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2301287/v1/38b1ae87973f2bd1b75d4eb5.png"},{"id":29985933,"identity":"80882e56-a2eb-4dcf-9952-bde010657647","added_by":"auto","created_at":"2022-12-06 21:28:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":268606,"visible":true,"origin":"","legend":"\u003cp\u003eGO and KEGG\u003cstrong\u003e \u003c/strong\u003efunctional enrichment pathways analysis of DEGs.\u003cstrong\u003e \u003c/strong\u003e(A) GO enrichment analysis. (B) KEGG enrichment analysis. DEGs: differentially expressed genes; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2301287/v1/23d282fab5106b1cb1732044.png"},{"id":29985616,"identity":"37f72207-508e-4df0-9ff3-cabd5f18525b","added_by":"auto","created_at":"2022-12-06 21:12:41","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":377258,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolomic alterations of BGZ treatment on Yin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e rats (N=8). (A-B) Scatter plot of liver metabolites in all groups determined by PCA in ESI- mode (A) and ESI+ mode (B). (C-D) Scatter plot of liver metabolites between the Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ and Yang\u003csub\u003esyn\u003c/sub\u003e groups determined by OPLS-DA in ESI- mode (C) and ESI+ mode (D). (E-F) Scatter plot of liver metabolites between the Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ and Yin\u003csub\u003esyn\u003c/sub\u003e groups determined by OPLS-DA in ESI- mode (E) and ESI+ mode (F). (G) Heatmap of the metabolites identified. Color depth represents the variation trend of the relative abundance of metabolites, the red color represents up-regulated metabolites, and the blue color represents down-regulated metabolites. ESI+: electrospray ionization source positive; ESI-: electrospray ionization source negative; PCA: Principal Component Analysis; OPLS-DA: Orthogonal Projections to Latent Structures Discriminant Analysis.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-2301287/v1/c28f9bac3e5e8b9dabb92057.png"},{"id":29985614,"identity":"a8b8d0a0-32d1-4467-8a60-7a135833e852","added_by":"auto","created_at":"2022-12-06 21:12:41","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":217801,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of correlation between serum biochemistry (ALT and AST) and metabolites (N=6~8). The color depth represents correlation strength, the red color represents positive correlation, and the blue color represents negative correlation. \u003cem\u003e*P \u0026lt; 0.05\u003c/em\u003e, \u003cem\u003e**P \u0026lt; 0.01\u003c/em\u003e, \u003cem\u003e***P \u0026lt; 0.001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-2301287/v1/cc35e437fe34b0c7e41e0fce.png"},{"id":29985537,"identity":"8815d0f9-9240-459e-bc4a-b7918469cbf0","added_by":"auto","created_at":"2022-12-06 21:04:41","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":201287,"visible":true,"origin":"","legend":"\u003cp\u003eROC analysis of metabolites associated with ALT and AST expression (N=8). (A) Venn diagram showing the number of metabolites associated with the levels of ALT and AST under ROC \u0026gt; 0.90 between the Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ group and the Yang\u003csub\u003esyn\u003c/sub\u003e group. (B-E) ROC curves of metabolites associated with the levels of ALT and AST under ROC \u0026gt; 0.90 between the Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ group and the Yang\u003csub\u003esyn\u003c/sub\u003e group. Biliverdin (B), D-Leucate (C), Phosphatidylcholine (D), Phosphatidate (E). (F) Venn diagram showing the number of metabolites associated with the levels of ALT and AST under ROC \u0026gt; 0.90 between the Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ group and the Yin\u003csub\u003esyn\u003c/sub\u003e group. (G) ROC curves of N1-(5-Phospho-a-D-ribosyl)-5,6-dimethylbenzimidazole between the Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ group and the Yin\u003csub\u003esyn\u003c/sub\u003e group. ROC: receiver operating characteristic, AUC: area under the ROC curve, CI: confidence interval.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-2301287/v1/eb64b9b7a5fa02c5d454a836.png"},{"id":29985545,"identity":"f7d34a57-0ea6-4696-adc8-6d2305cf1f71","added_by":"auto","created_at":"2022-12-06 21:04:41","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":331341,"visible":true,"origin":"","legend":"\u003cp\u003eOverall metabolic profile.\u003cstrong\u003e \u003c/strong\u003e(A and B) Schematic diagram of the disturbed metabolic pathways for the BGZ treatment on Yang\u003csub\u003esyn\u003c/sub\u003e (A) and Yin\u003csub\u003esyn\u003c/sub\u003e (B) rats. (C) Correlation network diagram of DEGs and metabolism of BGZ intervention in Yin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e rats.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-2301287/v1/7e0876c703d1eff8fa93302b.png"},{"id":29985547,"identity":"be4bcb22-b22c-412e-8fac-088a17866657","added_by":"auto","created_at":"2022-12-06 21:04:41","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":248161,"visible":true,"origin":"","legend":"\u003cp\u003eIntegration analysis of metabolite and DEGs enrichment pathway. (A) Pathways of co-regulation of metabolites and (or) DEGs by BGZ in Yin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e rats, (B) Schematic diagram of the relationship between cAMP and cGMP and the pathway of BGZ regulating Yin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e. pathways of co-regulation or specific regulation of metabolites and DEGs by BGZ in Yin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e rats. Effect of BGZ treatment on the changes in the expression of cAMP (C), cGMP (D), and the ratio of cAMP/cGMP (E) (N=6~8). \u003cem\u003e*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001\u003c/em\u003e, compared with CON group; \u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u0026lt; 0.05,\u003c/em\u003e compared with Yang\u003csub\u003esyn\u003c/sub\u003e group; \u003csup\u003e\u003cem\u003e\u0026amp;\u0026amp;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u0026lt; 0.01, \u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026amp;\u0026amp;\u0026amp;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u0026lt; 0.001\u003c/em\u003e, compared with Yin\u003csub\u003esyn\u003c/sub\u003e group. DEGs: differentially expressed genes; cAMP: cyclic adenosine monophosphate; cGMP: cyclic guanosine monophosphate; TCA: tricarboxylic acid cycle.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-2301287/v1/c0ca7253c85b30dbf9be0d6e.png"},{"id":44735658,"identity":"50ef3286-47d1-45ee-a21f-9602d98f5d17","added_by":"auto","created_at":"2023-10-16 22:26:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3651889,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2301287/v1/7f9710d0-6247-4295-a44b-565d84aaf2cc.pdf"},{"id":29985548,"identity":"06694f8c-bcac-4204-b0c1-c9ba03d8aa86","added_by":"auto","created_at":"2022-12-06 21:04:41","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1542268,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTablesandFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-2301287/v1/acee6694d5c0e4fbc8751484.docx"}],"financialInterests":"","formattedTitle":"Yin/Yang associated differential responses to Psoralea corylifolia Linn. in rat models: an integrated metabolomics and transcriptomics study","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn recent years, the proportion of drug-induced liver injury (DILI) caused by traditional Chinese herbs and dietary supplements has increased year by year (Hoofnagle and Bj\u0026ouml;rnsson, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Shen et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This is particularly noticeable for non-toxic traditional Chinese medicine (TCM) such as \u003cem\u003ePolygonum multiflorum\u003c/em\u003e Thunb. (Zhang et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and \u003cem\u003ePsoralea corylifolia\u003c/em\u003e Linn. (BGZ) (Wang et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), which have been used for thousands of years in China. This increase is causing great confusion for doctors and patients.\u003c/p\u003e \u003cp\u003eDILI usually involves two factors, the \u0026ldquo;drug\u0026rdquo; and the \u0026ldquo;host\u0026rdquo; (Chen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). For drug factors, Chen et al. constructed a \u0026ldquo;role-of-two\u0026rdquo; model (Chen et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and modified the version used (Chen et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The model enhanced the ability to predict whether a drug will cause DILI, but it was unable to fully explain individual differences in drug usage. Therefore, it cannot be used to predict individuals who are predisposed to DILI. Host factors are also important in understanding the susceptibility to DILI. Host factors that are generally recognized by modern research include hosts who carry specific HLA genes or the immune homeostasis of the host (Chen et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This is consistent with the concept of TCM syndrome, which summarizes the pathological changes of the body at a certain stage of disease development based on TCM theory. TCM treatment requires \u0026ldquo;therapy based on syndrome differentiation.\u0026rdquo; Symptomatic treatment can result in a better therapeutic effect, otherwise, it can aggravate the disease process and induce new diseases. Therefore, it may be feasible to explore the predisposition of individuals to TCM liver injury based on TCM syndrome theory.\u003c/p\u003e \u003cp\u003eBGZ has been used for many years in China to treat symptoms such as impotence, nocturnal emission, enuresis, frequent urination, cold aching in the lower back and knees, kidney deficiency, and premature ejaculation (Chinese Pharmacopoeia Commission (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)). BGZ has definite pharmacological effects, such as anti-tumor, anti-oxidation, antibacterial, anti-inflammatory, anti-depression, estrogen level regulation, bone growth promotion, nerve protection, and influence on the liver (Zhang et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Its active ingredients, psoralen and 5-methoxypsoralen, have become commonly used in the clinical treatment of vitiligo and psoriasis (Gupta and Anderson, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; McNeely and Goa, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). In addition, BGZ and its related compounds are also commonly used in health food and dietary supplements. However, in recent years, BGZ and its related compound preparations Zhuanggu Guanjie Pill and Xianling Gubao Capsules were reported to induce DILI in China (Deng et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, Li et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The BGZ-induced liver injury also occurred in South Korea (Nam et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough some studies have confirmed that BGZ and some of its components have hepatotoxicity (Li et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), these studies only focused on the drug itself and did not consider the host factors that induced DILI. Accordingly, by integrating the basic disease characteristics of the population treated with BGZ, our previous study found that most BGZ-induced DILI patients had osteoporosis, psoriasis, osteoarthritis, and other basic diseases related to immune activation (Ge et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and such diseases were found to be closely related to the imbalance of kidney-yang deficiency syndrome (Yang\u003csub\u003esyn\u003c/sub\u003e) and kidney-yin deficiency syndrome (Yin\u003csub\u003esyn\u003c/sub\u003e) according to TCM diagnosis (Dermatology Branch of China Association of Chinese Medicine. 2017, Dermatology Branch of China Association of Chinese Medicine. 2018, Ge et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The ancient TCM medicine book \u0026ldquo;Lei Gong Concocting (Paozhi) Theory\u0026rdquo; also recorded during the Northern and Southern Dynasties in China that people with \u0026ldquo;asthenic yin causing excessive pyrexia\u0026rdquo; should avoid taking BGZ. Thus, the BGZ-induced DILI may be related to its inappropriate symptomatic treatment. However, whether it has a causal relationship with the imbalance of Yang\u003csub\u003esyn\u003c/sub\u003e and Yin\u003csub\u003esyn\u003c/sub\u003e in the body remains unclear.\u003c/p\u003e \u003cp\u003eContinuously administered high doses of exogenous glucocorticoid (GC) (e.g., hydrocortisone) (Zhang et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) or thyroxine\u0026thinsp;+\u0026thinsp;reserpine (Wang, P. et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) in rats results in symptoms similar to Yang\u003csub\u003esyn\u003c/sub\u003e or Yin\u003csub\u003esyn\u003c/sub\u003e, respectively, and play important roles in the evaluation of protective or damaging effects and the TCM action mechanism against Yang\u003csub\u003esyn\u003c/sub\u003e or Yin\u003csub\u003esyn\u003c/sub\u003e. With a good description of the changes in the metabolic characteristics of endogenous metabolites and the differential expression changes of a series of functional genes in organisms, metabolomics and transcriptomics have been successfully applied to screen various diseases and their metabolic or gene profile changes in drug intervention (Lu et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ren et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This study constructed Yang\u003csub\u003esyn\u003c/sub\u003e and Yin\u003csub\u003esyn\u003c/sub\u003e animal models to evaluate whether the liver damage induced by BGZ is related to its non-symptomatic use and the possible susceptible individual characteristics.\u003c/p\u003e"},{"header":"2. Experiment","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemicals and reagents\u003c/h2\u003e \u003cp\u003eHydrocortisone succinate sodium was provided by Tianjin Biochemical Pharmaceutical Co., Ltd. (Tianjin, China). Thyroxine and reserpine were provided by Shanghai MACKLIN Technology Co., Ltd. (Shanghai, China). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and serum creatinine (SCr) tests were purchased from Jiancheng Biological Technology, Co., Ltd. (Nanjing, China). Cyclic adenosine monophosphate (cAMP), and cyclic guanosine monophosphate (cGMP) were purchased from Mlbio Biotechnology Co., Ltd. (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 BGZ Preparation\u003c/h2\u003e \u003cp\u003eBGZ was purchased from the Anhui Puren Herbal Pieces Co., Ltd. (Anhui, China) and authenticated by Professor Xiaohe Xiao of the institute of Hepatology, Fifth Medical Center, PLA General Hospital (Beijing, China). In addition, BGZ was crushed and filtered through 200 mesh sieves and then suspended in 0.5% sodium carboxymethyl cellulose (CMC-Na) for administration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Animal maintenance and treatment\u003c/h2\u003e \u003cp\u003eMale Sprague\u0026ndash;Dawley rats (180\u0026ndash;200 g) were obtained from the SPF Biotechnology Co. Ltd. (License No. SCXK20190010, Beijing, China), and housed in the Laboratory Animal Center of the Fifth Medical Center, Chinese PLA General Hospital (animal ethics committee approval No. YFYDW2020017). All rats were raised under specific pathogen-free conditions under a 12 h light/dark cycle, with free access to adequate food and water. All animals were fed adaptively for 1 week before starting the experiments.\u003c/p\u003e \u003cp\u003eThe rats were randomly divided into six separate groups (N\u0026thinsp;=\u0026thinsp;8) as follows: Control group (CON), BGZ group (BGZ), kidney-yang deficiency syndrome group (Yang\u003csub\u003esyn\u003c/sub\u003e), kidney-yang deficiency syndrome-treated with BGZ group (Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ), kidney-yin deficiency syndrome group (Yin\u003csub\u003esyn\u003c/sub\u003e); kidney-yin deficiency syndrome-treated with BGZ group (Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ). Rats in the treatment groups were consecutively administered intragastrically with BGZ suspension every afternoon for 21 consecutive days, while the control group received the same volume of 0.50% CMC-Na solution for the same amount of time. The changes in body weight were recorded before the end of the experiment. From the 8th day of administration, the Yang\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ group rats were given 25 mg/kg hydrocortisone subcutaneously once a day for 14 consecutive days to prepare the Yang\u003csub\u003esyn\u003c/sub\u003e model (Zhang et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). From the 15th day of administration, the Yin\u003csub\u003esyn\u003c/sub\u003e and Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ group rats were given thyroxine (16 mg/ml) and reserpine (1 mg/ml) at the dosage of 0.5 ml/100 g via gastric perfusion once a day for 7 consecutive days to prepare the Yin\u003csub\u003esyn\u003c/sub\u003e model (Wang, P. et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The CON and the BGZ groups were given the same volume of 0.50% CMC-Na solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Blood collection, organ index, and tissue preparation\u003c/h2\u003e \u003cp\u003eAfter the experiment, all animals were anesthetized with 2% pentobarbital sodium. Blood samples with and without anticoagulants were collected, and the liver and kidney were weighed immediately after sacrifice to calculate the organ index. Partial liver tissue was collected for histological examination and the remaining liver was quickly frozen with liquid nitrogen and stored at -80\u0026deg;C until needed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Serum biochemistry and histopathological analysis\u003c/h2\u003e \u003cp\u003eAfter centrifugation (3500 rpm, 10 min, 4\u0026deg;C), serum biochemistry (ALT, AST, SCr) and neurotransmitters (cAMP, cGMP) were determined according to the microplate assay kit instructions, the left hepatic lobe was fixed with 4% paraformaldehyde for 48 h, embedded in wax, and sectioned at approximately 5 \u0026micro;m for Hematoxylin and Eosin (HE) pathological staining analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 RNA sequence analysis and data processing\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from liver tissues using Trizol reagent (Invitrogen, USA) according to the manufacturer\u0026rsquo;s protocol, and genomic DNA was removed using DNase I (TaKara). RNA quality was assessed with a Bioanalyzer 2100 (Agilent) and measured with a NanoDrop 2000 spectrophotometer. Only a high-quality RNA sample (OD\u003csub\u003e260/280\u003c/sub\u003e = 1.8\u0026thinsp;~\u0026thinsp;2.2, OD\u003csub\u003e260/230\u003c/sub\u003e \u0026ge; 2.0, RNA integrity number (RIN)\u0026thinsp;\u0026ge;\u0026thinsp;6.5, 28S:18S\u0026thinsp;\u0026ge;\u0026thinsp;1.0, \u0026gt; 1 \u0026micro;g) was used to construct the sequencing library. RNA-seq transcriptome libraries were prepared using a TruSeqTMRNA sample preparation kit from Illumina (San Diego, CA) and sequenced with the Illumina HiSeq xten/NovaSeq 6000 sequencer under standard protocols. All samples had a Q30 (bases of Q\u0026thinsp;\u0026ge;\u0026thinsp;30 /all bases of sequencing) of \u0026gt;\u0026thinsp;91%. Sequence readers were trimmed and quality controlled using SeqPrep (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/jstjohn/SeqPrep\u003c/span\u003e\u003cspan address=\"https://github.com/jstjohn/SeqPrep\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Sickle (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/najoshi/sickle\u003c/span\u003e\u003cspan address=\"https://github.com/najoshi/sickle\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with default parameters and aligned to the reference genome through the orientation mode using hierarchical indexing for spliced alignment of transcripts 2 (HISAT2) (Kim et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Messenger RNA levels were quantified using RSEM (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://deweylab.biostat.wisc.edu/rsem/\u003c/span\u003e\u003cspan address=\"http://deweylab.biostat.wisc.edu/rsem/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Li and Dewey, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Differentially expressed genes (DEGs) were identified as those with | log2 (fold change) | \u0026gt;1 and \u003cem\u003ePadjust\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;0.05\u003c/em\u003e (DESeq2) (Love et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)\u003c/h2\u003e \u003cp\u003eLiver tissue RNA was extracted by the Tissue RNA Purification Kit Plus (RN002plus, ES Science, China) and reverse-transcribed into cDNA using the Fast All-in-One RT Kit (RT001, ES Science, China) according to the manufacturer's instructions. The qPCR of aldehyde dehydrogenase 1 family member B1 (Aldh1b1), galectin 5 (Lgals5), solute carrier family 25 member 25 (Slc25a25), Pim-3 proto-oncogene, serine/threonine kinase (Pim3), and out at first (Oaf) were quantified by the SYBR Green PCR master mix (RN002plus, ES Science, China) with the QuantStudio 6 Flex PCR System (Applied Biosystems, USA). The amplification parameters were set according to the standard protocol. Primer sequences used in this study are shown in Supplemental Table\u0026nbsp;1. Relative gene expression was calculated using the 2\u003csup\u003e\u0026minus;△△Ct\u003c/sup\u003e method 2\u003csup\u003e\u0026minus;∆∆Ct\u003c/sup\u003e (Ish-Shalom and Lichter, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Liver sample processing\u003c/h2\u003e \u003cp\u003eThe liver was homogenized with normal saline at 1:1 (1g:1mL) using a homogenizer. The 300 uL homogenized sample and 900 uL methanol were mixed and vortexed for 30 s. The supernatant was centrifuged at 12000 r/min for 10 min and concentrated to dry using a vacuum centrifugal concentrator. Then 100 uL methanol was added for redissolving and centrifuged at 13000 r/min for 10 min. Finally, a 4 uL supernatant was taken out for UPLC-QTOF/MS (Waters, Manchester, UK) detection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 UPLC-QTOF/MS analysis and data processing\u003c/h2\u003e \u003cp\u003eMetabolic profiling analysis of the biofluids was performed using the Waters Xevo G2-XS QTOF/MS (Waters, Manchester, UK). An analytical Acquity UPLC HSS T3 C18 column (temperature 30\u0026deg;C) was injected with 4 \u0026micro;L aliquots of each sample. For positive electrospray ionization source (ESI+) analysis, samples were isolated using a 30 min linear gradient of solvent A (water spiked with 0.1% formic acid) and solvent B (acetonitrile spiked with 0.1% formic acid) as mobile phases. The flow rate was fixed at 0.30 mL/min. For each sample, 10 \u0026micro;L was drawn as a quality control sample to ensure that the system was stable and the analyses were repeatable. Every 20th sample was injected with the control sample and subsequently analyzed. Masslynx software (v4.1, Waters Corp.) and Progenesis QI (v. 2.4, Waters Technologies, UK) were used for identifying the original mass spectral data and normalizing the total ion intensity of each chromatogram to acquire a data matrix containing the m/z value, retention time (RT), and normalized peak area. SIMCA-P 14.1 software (Umetrics, Umea, Sweden) was used for principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA). The PCA score chart was used to show the natural interrelation of observation results. Variable importance in the projection (VIP)\u0026thinsp;\u0026ge;\u0026thinsp;1 and with significant differences between groups (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) were selected as potential metabolites. Online metabolic databases including the Human Metabolome Database (HMDB) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.hmdb.ca/\u003c/span\u003e\u003cspan address=\"http://www.hmdb.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genome.jp/kegg/\u003c/span\u003e\u003cspan address=\"https://www.genome.jp/kegg/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were combined with exact masses and secondary ion mass spectrometry of the metabolites acquired through Progenesis QI to identify the differential metabolites.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed with Prism 8.0 (GraphPad Software, San Diego, CA). All results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. A one-way analysis of variance (ANOVA) was used to statistically analyze the multiple-group analysis. The student\u0026rsquo;s t-test was used to statistically analyze the receiver operating characteristic (ROC) curve analysis. The significance threshold was set at \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cp\u003e \u003cem\u003e3.1 Effects of BGZ treatment on weight, anal temperature, and organ indexes of liver and kidney in Yin\u003c/em\u003e \u003csub\u003e \u003cem\u003esyn\u003c/em\u003e \u003c/sub\u003e \u003cem\u003eand Yang\u003c/em\u003e\u003csub\u003e\u003cem\u003esyn\u003c/em\u003e\u003c/sub\u003e \u003cem\u003erats\u003c/em\u003e\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, compared with the CON group, the body weight of the BGZ group, Yang\u003csub\u003esyn\u003c/sub\u003e group, Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ group, and Yin\u003csub\u003esyn\u003c/sub\u003e group had no obvious changes, while the Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ group decreased significantly compared with the Yin\u003csub\u003esyn\u003c/sub\u003e group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e). The anal temperature of rats in the Yang\u003csub\u003esyn\u003c/sub\u003e group and Yin\u003csub\u003esyn\u003c/sub\u003e group were significantly decreased and increased, respectively, compared with the CON group (both \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e), while the anal temperature in the Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ group and Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ group were all significantly increased compared with the corresponding model groups (both \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, the organ indexes of the liver in the Yang\u003csub\u003esyn\u003c/sub\u003e group and Yin\u003csub\u003esyn\u003c/sub\u003e group were all markedly increased compared with the CON group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), and the liver organ indexes in the Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ group was further increased compared with the Yin\u003csub\u003esyn\u003c/sub\u003e group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), while there was also no changed in the Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ group compared with the Yang\u003csub\u003esyn\u003c/sub\u003e group. As for the kidney, only the Yin\u003csub\u003esyn\u003c/sub\u003e group had a significant increase in organ indexes compared with the CON group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003e3.2 Effect of BGZ treatment on the serum levels of ALT, AST, SCr, and liver histopathological changes in Yin\u003c/em\u003e \u003csub\u003e \u003cem\u003esyn\u003c/em\u003e \u003c/sub\u003e \u003cem\u003eand Yang\u003c/em\u003e\u003csub\u003e\u003cem\u003esyn\u003c/em\u003e\u003c/sub\u003e \u003cem\u003erats\u003c/em\u003e\u003c/p\u003e \u003cp\u003eSerum ALT, AST, and SCr, which are well-recognized markers of various types of liver and kidney damage, were used for the analysis. Regarding liver function (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B), there were no obvious changes in the levels of ALT and AST between the BGZ group and the CON group. In contrast, the levels of ALT and AST in the Yang\u003csub\u003esyn\u003c/sub\u003e group increased prominently, while BGZ significantly reversed the phenomena (all \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Though the AST level in the Yin\u003csub\u003esyn\u003c/sub\u003e group was significantly increased (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e), there was no obvious change in the levels of AST between the Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ group and the Yin\u003csub\u003esyn\u003c/sub\u003e group, while the ALT level was significantly increased after administration of BGZ in the Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e), illustrating that there was a certain risk of liver injury in rats with Yin\u003csub\u003esyn\u003c/sub\u003e after administration of BGZ. As for kidney function (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), no significant SCr changes were found in almost all groups except for the Yin\u003csub\u003esyn\u003c/sub\u003e group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). Combined with the changes in the kidney organ index, it revealed that BGZ may have no obvious renal toxicity in rats with Yang\u003csub\u003esyn\u003c/sub\u003e or Yin\u003csub\u003esyn\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, the liver sections of the CON group showed normal hepatocyte structures. The liver samples from BGZ-treated rats were almost indistinguishable from normal rats. The liver samples from the Yang\u003csub\u003esyn\u003c/sub\u003e group exhibited hepatocyte focal necrosis, loss of central vein intima, and inflammatory cell infiltration in portal vein areas, while the above symptoms were alleviated in the Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ group. The liver samples from the Yin\u003csub\u003esyn\u003c/sub\u003e group exhibited slight inflammatory infiltration in the portal area but no evident hepatocyte injury, while the hepatocyte focal necrosis and inflammatory cell infiltration were aggravated in the Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ group. Combined with the levels of ALT, AST, liver index, and liver pathological examination, it was suggested that BGZ has a preferable liver protection effect on rats with Yang\u003csub\u003esyn\u003c/sub\u003e and a certain risk of liver injury effect on rats with Yin\u003csub\u003esyn\u003c/sub\u003e.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 DEG alterations of BGZ treatment in Yin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e rats\u003c/h2\u003e \u003cp\u003eTo reveal the mechanism of different therapeutic effects of BGZ on Yin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e rats, liver gene expression profiles were obtained from the CON, BGZ, Yang\u003csub\u003esyn\u003c/sub\u003e, Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ, Yin\u003csub\u003esyn\u003c/sub\u003e, and Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ groups using RNA-Seq analysis. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, compared with the CON group, BGZ barely influenced the gene expression with only 4 DEGs increased and 5 DEGs decreased, while 439 up-regulated DEGs and 652 down-regulated DEGs were found in the Yang\u003csub\u003esyn\u003c/sub\u003e group, and 508 up-regulated DEGs along with 847 down-regulated DEGs were found in the Yin\u003csub\u003esyn\u003c/sub\u003e group. Compared to the Yang\u003csub\u003esyn\u003c/sub\u003e group, the number of up-regulated and down-regulated DEGs decreased to 59 and 43 by BGZ treatment, respectively, while for the Yin\u003csub\u003esyn\u003c/sub\u003e group, the number of up-regulated and down-regulated DEGs decreased to 11 and 17 by BGZ treatment, respectively. To further display the above differences in DEGs more intuitively, heatmaps were constructed based on relative abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Correlation analysis between DEGs and serum biochemistry\u003c/h2\u003e \u003cp\u003eBy analyzing the correlations between serum biochemistry (ALT and AST) and DEGs changed using BGZ in Yin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), the results demonstrated that 36 DEGs, including 34 positive DEGs and 2 negative DEGs, were significantly correlated with ALT, and 12 DEGs, including 9 positive DEGs and 3 negative DEGs, were significantly correlated with AST. Among them, hyperpolarization-activated cyclic nucleotide-gated potassium channel 3 (Hcn3), aldehyde dehydrogenase 1 family member B1 (Aldh1b1), galectin 5 (Lgals5), solute carrier family 25 member 25 (Slc25a25), Pim-3 proto-oncogene, serine/threonine kinase (Pim3), and out at first (Oaf) were all significantly positively correlated with ALT and AST, and LOC100364769 was significantly negatively correlated with ALT and AST. The detailed relationship between DEGs and serum biochemistry (ALT and AST) was shown in Supplemental Table\u0026nbsp;2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5 ROC curve analysis of DEGs\u003c/h2\u003e \u003cp\u003eTo further explore the diagnostic efficacy of DEGs, a ROC curve analysis was performed using GraphPad Prism software (version 8.01). The results revealed that 22 DEGs could be better discriminated between the Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ group and Yang\u003csub\u003esyn\u003c/sub\u003e group, and 10 DEGs had better discrimination between the Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ group and Yin\u003csub\u003esyn\u003c/sub\u003e group (all the areas under the curve (AUC) of the ROC curves\u0026thinsp;\u0026ge;\u0026thinsp;0.9 and \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) (Supplemental Fig.\u0026nbsp;1). Venn analysis showed the ROC results of DEGs and their correlation with the levels of ALT and AST between the Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ group and Yang\u003csub\u003esyn\u003c/sub\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), revealing that Aldh1b1, Slc25a25, Pim3, and Oaf can be used as potential biomarkers for the treatment of Yang\u003csub\u003esyn\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-E). Venn analysis also showed the ROC results of DEGs and their correlation with ALT and AST between the Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ group and Yin\u003csub\u003esyn\u003c/sub\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ), revealing that Lgals5 may be used as a potential biomarker for the treatment of Yin\u003csub\u003esyn\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-I and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL, the expression of Slec25a25, Pim3, and Oaf in rats with Yang\u003csub\u003esyn\u003c/sub\u003e and Yin\u003csub\u003esyn\u003c/sub\u003e were all significantly increased compared with the CON group (all \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), while the level of Aldh1b1 in the Yang\u003csub\u003esyn\u003c/sub\u003e and the level of Lgals5 in the Yin\u003csub\u003esyn\u003c/sub\u003e were all significantly decreased compared with the normal group (both \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). After BGZ intervention, the levels of Aldh1b1, Lgals5, Slec25a25, Pim3, and Oaf were all significantly decreased in the Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ group (all \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), while only Lgals5 was significantly increased in rats with Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ compared the Yin\u003csub\u003esyn\u003c/sub\u003e group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.6 GO and KEGG functional enrichment analysis of DEGs\u003c/h2\u003e \u003cp\u003eGene Ontology (GO) functional enrichment analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) found that 95 GOs were significantly changed in the Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ group compared with the Yang\u003csub\u003esyn\u003c/sub\u003e group, such as GO:0009991 (response to extracellular stimulus), GO:0032922 (circadian regulation of gene expression), GO:0031667 (response to nutrient levels), and GO:0009892 (negative regulation of metabolic process), while 6 GOs including GO:0019825 (oxygen binding), GO:0020037 (heme-binding), GO:0046906 (tetrapyrrole binding), GO:0005833 (hemoglobin complex), GO:0005344 (oxygen carrier activity), and GO:0042743 (hydrogen peroxide metabolic process) were significantly changed in the Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ group compared with the Yin\u003csub\u003esyn\u003c/sub\u003e group. Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) found that 4 KEGG orthologs (Kos) including ko00360 (phenylalanine metabolism), ko00350 (tyrosine metabolism), ko00380 (tryptophan metabolism), and ko00130 (ubiquinone and another terpenoid-quinone biosynthesis) were significantly changed in the Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ group compared with the Yang\u003csub\u003esyn\u003c/sub\u003e group, while 2 Kos including ko00260 (glycine, serine, and threonine metabolism) and ko00591 (linoleic acid metabolism) were significantly changed in the Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ group compared with the Yin\u003csub\u003esyn\u003c/sub\u003e group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Metabolomic analysis of BGZ treatment in Yin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e rats\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, the QC samples gathered closely in both PCA score plots, indicating the stability of the UPLC-QTOF/MS system throughout the analysis. The CON, Yang\u003csub\u003esyn\u003c/sub\u003e, and Yin\u003csub\u003esyn\u003c/sub\u003e groups can be well distinguished, suggesting that the metabolic information between the Yang\u003csub\u003esyn\u003c/sub\u003e and Yin\u003csub\u003esyn\u003c/sub\u003e groups has been changed. OPLS-DA analysis found that the comparison between the Yang\u003csub\u003esyn\u003c/sub\u003e group and the Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD) could both be significantly separated under ESI\u0026thinsp;+\u0026thinsp;and ESI- modes, and the comparison between the Yin\u003csub\u003esyn\u003c/sub\u003e group and Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF) could also both be significantly separated under ESI\u0026thinsp;+\u0026thinsp;and ESI- modes, and the permutation tests for OPLS-DA analysis revealed the models were not overfitted (Supplemental Fig.\u0026nbsp;2), indicating that significant metabolic disturbance occurred in the Yang\u003csub\u003esyn\u003c/sub\u003e group and Yin\u003csub\u003esyn\u003c/sub\u003e group after BGZ treatment. A total of 40 metabolites were identified based on \u003cem\u003em/z\u003c/em\u003e and corresponding secondary fragment ion characteristic maps (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Supplemental Fig.\u0026nbsp;3). Among the metabolites, 27 metabolites were markedly changed in the Yang\u003csub\u003esyn\u003c/sub\u003e+GBZ group compared with the Yang\u003csub\u003esyn\u003c/sub\u003e group, and 31 metabolites were significantly changed in the Yin\u003csub\u003esyn\u003c/sub\u003e+GBZ group compared with the Yin\u003csub\u003esyn\u003c/sub\u003e group. The heatmap for the above differential metabolites is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIdentified differential metabolites.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eESI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003em/z\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRT (min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKEGG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMetabolite\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e(Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ) vs (Yang\u003csub\u003esyn\u003c/sub\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e(Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ) vs (Yin\u003csub\u003esyn\u003c/sub\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e145.0132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9378\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC00026\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eα-Ketoglutarate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e455.0965\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.9226\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC00061\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRiboflavin-5-phosphate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e104.0348\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.8028\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC00065\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eL-Serine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e113.0352\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC00106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUracil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026uarr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e778.5407\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13.2262\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC00157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePhosphatidylcholine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e582.5100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13.2602\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC00195\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN-Acylsphingosine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e148.0378\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.3859\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC00250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePyridoxal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e243.0615\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.3509\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC00299\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUridine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026uarr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e102.0556\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.8313\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC00334\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003egamma-Aminobutyric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e766.6037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.3765\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC00350\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePhosphatidylethanolamine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026uarr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e240.0224\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.5073\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC00352\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGlucosamine 6-phosphate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e151.0569\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9235\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC00379\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eD-Xylitol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e671.4653\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13.3460\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC00416\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePhosphatidate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026uarr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e173.0922\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.8994\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC00430\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5-Aminolevulinate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e160.0756\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.9497\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC00483\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTyramine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026uarr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e583.2469\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.6720\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC00486\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBilirubin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e583.2554\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.0917\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC00500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBiliverdin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e90.0555\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.8565\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC00546\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMethylglyoxal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e397.2291\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11.6884\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC00584\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eProstaglandin E2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026uarr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e229.0667\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.6209\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC00588\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePhosphorylcholine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e426.3162\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15.2409\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC00695\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e252.0724\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.1330\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC00818\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGlucaric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e465.3556\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11.5639\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC01724\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLanosterin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e799.6627\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15.2528\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC01829\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThyroxine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e196.0943\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.0050\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC03264\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eD-Leucate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e187.1076\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.3080\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC02727\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN6-Acetyl-L-lysine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e560.3333\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.4013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC03033\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ebeta-D-Glucuronoside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e205.0645\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.7274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC03227\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3-Hydroxy-L-kynurenine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026uarr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e522.3564\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.5634\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC04230\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1-Acyl-sn-glycero-3-phosphocholine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e401.0785\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.4160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC04352\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(R)-4'-Phosphopantothenoyl-L-cysteine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e381.0794\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9636\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC04778\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN1-(5-Phospho-a-D-ribosyl)-5,6-dimethylbenzimidazole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026uarr;**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e347.1835\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.9063\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC05302\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2-Methoxyestradiol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e526.0585\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.3859\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC05381\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3-Carboxy-1-hydroxypropylthiamine diphosphate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026uarr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e389.2142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.7813\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC05476\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTetrahydrocorticosterone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e183.0346\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.8099\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC05580\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3,4-Dihydroxymandelate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026uarr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e297.0703\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9593\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC05648\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5-Hydroxy-N-formylkynurenine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e577.3444\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13.7089\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC05789\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eL-Urobilinogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e365.2324\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.4089\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC14777\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12(S)-HETE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026uarr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e353.2472\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.4221\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC16513\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDocosapentaenoic acid (22n-3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e136.0396\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.6256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC22040\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMethylcysteine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026darr;**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003cem\u003e*P\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e, \u003cem\u003e**P\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e, \u003cem\u003e***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Correlation analysis between metabolites and serum biochemistry\u003c/h2\u003e \u003cp\u003eBy analyzing the correlations between serum biochemistry (ALT and AST) and differential metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), 20 metabolites, including 3 positive and 17 negative correlations, were significantly correlated with ALT, and 12 metabolites, including 3 positive and 9 negative correlations, were significantly correlated with AST. The correlation coefficient r and \u003cem\u003eP-value\u003c/em\u003e are shown in Supplemental Table\u0026nbsp;3. Among them, Phosphatidate, Uracil, Prostaglandin E2, and N1-(5-Phospho-a-D-ribosyl)-5,6-dimethylbenzimidazole were all significantly positively correlated with ALT and AST, and Phosphatidylcholine, Lanosterin, N-Acylsphingosine, D-Leucate, Gamma-aminobutyric acid, Biliverdin, beta-D-Glucuronoside, and Docosapentaenoic acid (22n-3) were all significantly negatively correlated with ALT and AST.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.9 ROC curve analysis of metabolites\u003c/h2\u003e \u003cp\u003eAs shown in Supplemental Fig.\u0026nbsp;4, 9 metabolites had better discrimination between the Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ group and Yang\u003csub\u003esyn\u003c/sub\u003e group, and 5 metabolites had better discrimination between the Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ group and Yin\u003csub\u003esyn\u003c/sub\u003e group (all the areas under the curve (AUC) of the ROC curves\u0026thinsp;\u0026ge;\u0026thinsp;0.9 and \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Venn analysis showed the ROC results of metabolites and their correlation with the levels of ALT and AST among the comparison of the Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ group and Yang\u003csub\u003esyn\u003c/sub\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA) and the comparison of the Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ group and the Yin\u003csub\u003esyn\u003c/sub\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eF). Among them, Phosphatidate, Phosphatidylcholine, D-Leucate, and Biliverdin may be used as potential hepatoprotective biomarkers for the treatment of Yang\u003csub\u003esyn\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB-E), while N1-(5-Phospho-a-D-ribosyl)-5,6-dimethylbenzimidazole may be used as a potential hepatotoxicity biomarker for the treatment of Yin\u003csub\u003esyn\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.10 Comparison of overall metabolic profiles\u003c/h2\u003e \u003cp\u003eTo further understand the metabolic disorder of susceptible syndromes, the KEGG identification code of differential metabolites was used for pathway analysis via MetaboAnalyst 5.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.metaboanalyst.ca/\u003c/span\u003e\u003cspan address=\"https://www.metaboanalyst.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The schematic diagram of the disturbed metabolic pathways changed by the treatment of BGZ for Yang\u003csub\u003esyn\u003c/sub\u003e and Yin\u003csub\u003esyn\u003c/sub\u003e rats is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA and \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB. The redder the bubble color, the smaller the \u003cem\u003eP\u003c/em\u003e value of the metabolic pathway. The details for the value of \u003cem\u003eP\u003c/em\u003e are shown in Supplemental Tables\u0026nbsp;4 and 5. The results indicated that there are significant differences in glycerophospholipid metabolism, arachidonic acid metabolism, pantothenate, and CoA biosynthesis pathways in the Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ group compared with the Yang\u003csub\u003esyn\u003c/sub\u003e group, while pantothenate and the CoA biosynthesis pathway were significantly changed in the Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ group compared with the Yin\u003csub\u003esyn\u003c/sub\u003e group. To compare the differences in metabolic profiles for BGZ treatment on Yin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e rats, a network map was constructed based on the identified metabolites and enriched metabolic pathways. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC, metabolic pathways and metabolites interacted with each other to form a complex network.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003e3.11 Integration analysis of metabolite and DEGs enrichment pathway.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe expression changes of metabolites and DEGs in vivo can affect each other and cause a series of changes in body function. It is worth noting that, the pathways of BGZ regulating in rats with Yin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e are almost all involved in energy metabolism, amino acid metabolism, lipid metabolism, and metabolism of cofactors and vitamins (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eA), and the above pathways were also cross-regulated by the \u0026ldquo;second messenger\u0026rdquo; of cAMP and cGMP (Cheng et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Godfrey et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Shen et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eB). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eC-E, the study further found the protein expression cAMP was significantly decreased in the Yang\u003csub\u003esyn\u003c/sub\u003e group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) and increased in the Yin\u003csub\u003esyn\u003c/sub\u003e group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) while compared with the CON group. Meanwhile, the protein levels of cGMP were significantly increased in both model groups (both \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), accompanied by a significantly decreased ratio of cAMP/cGMP in the Yang\u003csub\u003esyn\u003c/sub\u003e group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e). The changes in levels of cAMP, cGMP, and cAMP/cGMP ratio are consistent with the changes in Yin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e models and are often used as indicators to evaluate the above syndrome models (Averaimo and Nicol, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). After BGZ intervention, there was no significant change in cGMP in the two model groups, while the levels of cAMP and cAMP/cGMP in the Yang\u003csub\u003esyn\u003c/sub\u003e+BGZ group and Yin\u003csub\u003esyn\u003c/sub\u003e+BGZ group were all significantly increased or decreased compared with corresponding models, respectively (all \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eIndividual variability in liver injury following drug ingestion is a major challenge for DILI research and clinical prevention (Andrade et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In the study, it was found that Yin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e are two different predisposed individual states of hepatotoxicity or hepatoprotection caused by BGZ. From the perspective of the gene expression profile, BGZ mostly does not affect normal rats, but it has a mild or greater impact on the change numbers of DEGs in Yin\u003csub\u003esyn\u003c/sub\u003e (28 DEGs) and Yang\u003csub\u003esyn\u003c/sub\u003e (102 DEGs) rats, respectively. From the perspective of endogenous metabolites, Yin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e rats can be well distinguished on the PCA scatter plot, and the OPLS-DA plot can further better distinguish Yin\u003csub\u003esyn\u003c/sub\u003e, Yang\u003csub\u003esyn\u003c/sub\u003e, and their corresponding BGZ-treated groups, suggesting that BGZ exerts different metabolic perturbation patterns for different models.\u003c/p\u003e \u003cp\u003eAmong the DEGs with ROC\u0026thinsp;\u0026ge;\u0026thinsp;0.9 and significant correlation with ALT and AST, Slc25a25 may function as an ATP-Mg/Pi carrier to mediate the transport of Mg-ATP in exchange for phosphate. It is also likely responsible for the net uptake or efflux of adenine nucleotides into or from the mitochondria and is highly expressive in acute liver failure or induced by exogenous thyroxine (Fujimoto et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This study confirmed the high expression of Slc25a25 in the liver of Yin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e rats, which is consistent with the phenomenon of abnormal liver function (Lee et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, only the expression of Slc25a25 in Yang\u003csub\u003esyn\u003c/sub\u003e rats was inhibited by BGZ, indicating that Slc25a25 may be a hepatoprotective gene for BGZ in Yang\u003csub\u003esyn\u003c/sub\u003e rats, and the way to aggravate liver injury in rats with Yin\u003csub\u003esyn\u003c/sub\u003e may not be through regulating Slc25a25.\u003c/p\u003e \u003cp\u003ePim3 is a liver growth-stimulating factor with serine/threonine kinase activity and is involved in gp130-mediated induction of cell proliferation and protection of apoptosis downstream of signal transducer and activator of transcription 3 (STAT3) (Liu et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Pim3 is barely expressed in normal tissues but highly expressed in the prostate, large intestine, liver, and other cancer tissues (Brault et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Therefore, its expression l evel is mainly used to evaluate tumor expression and metastasis (Qu et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Epidemiological studies have found and confirmed the correlations between tumors and the constitution of Yin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e (Chen and Wang, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Ji et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This study also found that Pim3 was abnormally highly expressed in the liver of Yin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e rats, suggesting that Yin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e may be susceptible to constitutions that induce tumors. Surprisingly, BGZ only decreased the expression of Pim3 in Yang\u003csub\u003esyn\u003c/sub\u003e rats with almost no change in Yin\u003csub\u003esyn\u003c/sub\u003e rats. The above results indicated that Pim3 may be a marker gene for BGZ to exert a hepatoprotective effect on Yang\u003csub\u003esyn\u003c/sub\u003e rats. In addition, BGZ may also have a potential therapeutic effect on tumor patients with Yang\u003csub\u003esyn\u003c/sub\u003e, and the anti-tumor effect of BGZ has been confirmed (Wu et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the metabolites with ROC\u0026thinsp;\u0026gt;\u0026thinsp;0.90 and significant correlation with ALT and AST, glycerophospholipids (including phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine) are the most abundant phospholipids in the body. In addition to constituting biofilm, they are also one of the components of bile and membrane surface-active substances and participate in protein recognition and signal transduction through the cell membrane (Hishikawa et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Hepatocytes can express various glycerophospholipids activities, making the liver an important organ for glycerophospholipid metabolism. Dysregulation of glycerophospholipids is related to the development and progression of liver diseases including hepatitis, liver cancer, fatty liver, and liver fibrosis (van der Veen et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). For example, the abnormal increase or decrease of the ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) will affect energy metabolism and are closely related to disease progression (Li et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The changes in hepatic phospholipid composition are also associated with fatty liver disease and impaired postoperative liver regeneration (Clemens et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ling et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sasabe et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In the glycerophospholipid metabolic pathway, phosphatidate can activate hepatic interleukin 6 (IL-6) signaling through inter-organ crosstalk and alleviating acetaminophen-induced liver injury in mice (Clemens et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Phosphatidate also acts as an ionophore in the brain between depolarization and the release of neurotransmitters such as gamma-Aminobutyric Acid (GABA) (Harris et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). The increase in the level of GABA can enhance the body\u0026rsquo;s immunity under stress conditions (Abdou et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and has a good protective effect on acute liver injury or liver failure induced by ethanol, fluoride, and d-galactosamine (Hata et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In this study, under the intervention of BGZ, the expression of PE and GABA in the liver of rats with Yin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e both showed opposite trends, suggesting that regulating glycerophospholipid metabolism may be another way in which BGZ exerts liver protection and liver injury in Yang\u003csub\u003esyn\u003c/sub\u003e and Yin\u003csub\u003esyn\u003c/sub\u003e rats, respectively.\u003c/p\u003e \u003cp\u003eThe metabolites of lipid metabolism participate in the biosynthesis of glycerophospholipid metabolism together with serine, providing raw materials for its metabolism (Cook et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). The metabolism of arachidonic acid, sphingolipid, glycine, serine, threonine, porphyrin, and chlorophyll can also interact directly or indirectly through serine. In the arachidonic acid metabolic pathway, 12(S)-HETE, as an important inflammatory marker, can affect the inflammatory process by stimulating the release of cytokines such as tumor necrosis factor alpha-like (TNF-α) and IL-6. Blocking the production of 12(S)-HETE can inhibit ischemia-reperfusion-induced liver dysfunction, inflammation, and cell death (Zhang et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). As a regulatory enzyme of arachidonic acid metabolism, arachidonate 15-lipoxygenase (Alox15) can catalyze it to 12(S)-HPETE, which is further reduced by glutathione peroxidase into 12(S)-HETE (Spector et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1988\u003c/span\u003e). Prostaglandin E2 (PGE2), another metabolite of arachidonic acid, has also been shown to induce acute and chronic inflammation and various autoimmune diseases through Th1 differentiation, Th17 cell proliferation, and activation of mast cells (Tsuge et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this study, the expressions of 12(S)-HETE, PGE2, and Alox15 were significantly increased in the Yin\u003csub\u003esyn\u003c/sub\u003e group under the intervention of BGZ, while the expressions of 12(S)-HETE, PGE2, and Alox15 were significantly decreased or did not change significantly in the Yang\u003csub\u003esyn\u003c/sub\u003e group, suggesting that the regulation of arachidonic acid metabolism may be another way in which BGZ regulates the effects of liver protection or liver damage in rats with Yang\u003csub\u003esyn\u003c/sub\u003e and Yin\u003csub\u003esyn\u003c/sub\u003e, respectively.\u003c/p\u003e \u003cp\u003eIn the metabolic pathway of porphyrin and chlorophyll, 5-aminolevulinate, the direct metabolite of glycine, can be used as a precursor of heme and participate in the regulation of the production of heme and its metabolite biliverdin (Sansaloni-Pastor et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Biliverdin has a good anti-inflammatory effect, can inhibit the expression of toll-like receptor 4 and nitric oxide, and reduces the inflammatory induction of lipopolysaccharide on macrophages (Wegiel et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). It is used in the protection against various diseases (vascular injury, organ transplantation, etc.). As such, biliverdin can better reduce the ischemia-reperfusion injury in pig liver and have a liver protective effect (Andria et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Biliverdin reductase B (Blvrb) is a non-redundant nicotinamide adenine dinucleotide (phosphate)-dependent biliverdin reductase that regulates the cellular redox state by converting biliverdin to bilirubin. Tts redox function also reduces intracellular reactive oxygen species accumulation (Wu et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and maintains essential cytoprotective functions in recovery from hematopoietic stress (Nesbitt et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Bilirubin is the main pigment in human bile and the main metabolite of iron porphyrin compounds in the body with certain damage to the brain and nerves (Watchko and Tiribelli, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The increase in its level can also lead to impaired liver function, so it is also used as a test for one of the common indicators of jaundice (Fawaz et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In the study, BGZ significantly increased the expressions of biliverdin in Yang\u003csub\u003esyn\u003c/sub\u003e rats accompanied by the inhibition expressions of bilirubin in Yin\u003csub\u003esyn\u003c/sub\u003e rats, suggesting that the regulation of porphyrin metabolism, especially Blvrb, may be a mechanism by which BGZ exerts hepatoprotective or liver damage effects on Yang\u003csub\u003esyn\u003c/sub\u003e and Yin\u003csub\u003esyn\u003c/sub\u003e rats, respectively. However, this is not related to bilirubin.\u003c/p\u003e \u003cp\u003eTryptophan is involved in immune regulation, neural function, and intestinal homeostasis through the kynurenine pathway for metabolism (Cervenka et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). For example, kynurenine is an endothelium-derived relaxation factor in the inflammatory process (Wang, Y. et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and is metabolized to neurotoxic 3-Hydroxy-L-kynurenine under the action of kynureninase (Kynu), which is involved in the inflammatory process in psoriasis and other inflammatory diseases (Harden et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Therefore, the imbalance of tryptophan metabolism in diseases ranging from cancer to neurodegenerative diseases has become a research hotspot for the therapeutic targeting of the kynurenine pathway (Platten et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this study, compared with the Yang\u003csub\u003esyn\u003c/sub\u003e group and the Yin\u003csub\u003esyn\u003c/sub\u003e group, the levels of 3-Hydroxy-L-kynurenine were significantly decreased and increased under the intervention of BGZ, respectively, suggesting the kynurenine metabolism pathway in regulating tryptophan metabolism may be another mechanism by which BGZ exerts hepatoprotective or liver damage effects on Yang\u003csub\u003esyn\u003c/sub\u003e and Yin\u003csub\u003esyn\u003c/sub\u003e rats.\u003c/p\u003e \u003cp\u003eThe above-discussed pathways changed by BGZ in Yin\u003csub\u003esyn\u003c/sub\u003e and (or) Yang\u003csub\u003esyn\u003c/sub\u003e, were almost all related to energy metabolism, amino acid metabolism, and lipid metabolism, and the disorder of the above pathways has also been reported in Yin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e (Chen et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Jiang et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The effects of BGZ on the metabolism of the above pathways have also been reported by Xu et al. (Xu et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). cAMP and cGMP, the potential biomarkers for Yin/Yang disharmony in TCM (Wang et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), can cross-activate protein kinases related to tissue biosynthesis and metabolism (including carbohydrate, lipid, amino acid, cofactors, and vitamins) as cyclic nucleotide effectors (Coelho Horta et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Francis and Corbin, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Jiang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Considering that BGZ can enhance the expression of cAMP-responsive element modulator-τ (CREMτ) (Wei et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and the ability of BGZ to down-regulate and up-regulate the expression of cAMP and the ratio of cAMP/cGMP in rats with Yin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e, respectively. It is suggested that the cAMP signaling pathway may be one of the mechanisms by which BGZ regulates energy metabolism to affect rats with Yin\u003csub\u003esyn\u003c/sub\u003e or Yang\u003csub\u003esyn\u003c/sub\u003e. As for the different intervention effects of BGZ on the different syndromes, the interaction of endogenous metabolites and genes may be the main reason, which is worthy of further study in the future.\u003c/p\u003e \u003cp\u003eIn conclusion, this study found that BGZ has a double-edged sword-like effect that not only exerts a good hepatoprotective effect on Yang\u003csub\u003esyn\u003c/sub\u003e rats but also has a potential risk of inducing liver injury in Yin\u003csub\u003esyn\u003c/sub\u003e rats. The mechanism is mainly reflected in BGZ having different regulatory effects on amino acid metabolism, energy metabolism, lipid metabolism, and metabolism of cofactors and vitamins in the above two different syndromes. In response to the seemingly contradictory results that BGZ has both liver-damaging and liver-protective effects in previous reports (Wang et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), this study found that BGZ may have both liver-damaging and liver-protecting effects on predisposed individuals from the perspective of TCM syndrome theory. The study screened out the endogenous markers that can characterize the corresponding predisposed individuals, which will provide a certain reference for the safe and rational application of TCM.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003ePsoralea corylifolia\u003c/em\u003e Linn.\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBGZ\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eYang\u003csub\u003esyn\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ekidney-yang deficiency syndrome\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eYin\u003csub\u003esyn\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ekidney-yin deficiency syndrome\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTCM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTraditional Chinese Medicine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHematoxylin and eosin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHMDB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman Metabolome Database\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKEGG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKyoto Encyclopedia of Genes and Genomes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePrincipal components analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOPLS-DA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOrthogonal partial least-squares discriminate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVIP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003evariable importance in the projection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUPLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUltra-performance liquid chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLC-MS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLiquid chromatography-tandem mass spectrometry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDILI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDrug-induced liver injury\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglucocorticoid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eALT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlanine aminotransferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAspartate aminotransferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eScr\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSerum creatinine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ecAMP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecyclic adenosine monophosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ecGMP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecyclic guanosine monophosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDEGs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDifferentially expressed genes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eESI+\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePositive electrospray ionization source\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReceiver operating characteristic\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Ontology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKos\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKEGG orthology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAUC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAreas under the curve\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePGE2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProstaglandin E2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental animal protocols were approved by the Animal Ethics Committee of the First Affiliated Hospital of Henan University of Traditional Chinese Medicine and the procedures were conformed to the Guide for the Care and Use of Laboratory Animals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 82173993, No. U1904129, No. 82204638, No. 82230118), the Key Project of Henan Province for Scientific Research of Traditional Chinese Medicine, China (No. 2019ZYBJ08), and the Key Scientific Research Projects of Colleges and Universities in Henan Province, China (No. 19A360007).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJin-Fa Tang, Xiao-He Xiao, and Yan-Ling Zhao conceived and designed the experiments; Ming-Liang Zhang performed the experiments, analyzed the data, and wrote the manuscript; De-Xin Kong, Yuan Gao was responsible for helping the collection of experimental samples; Yu-Long Chen, Hui Zhang, Xiao-Yan Wang help to detect the metabonomic changes of liver samples, Xu Zhao and Ming Niu helped with data analysis, Zhao-Fang Bai, Wei-Xia Li, and Yu-Ming Guo reviewed the paper. All authors have reviewed the manuscript and approved the final version of the manuscript. All authors have read, revised, and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank James thompson, PhD, from BEIJING LANGYI TRANSLATION CO., LTD., China, for mother tongue polishing the English text of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research data generated from this study are included in the article and additional files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdou AM, Higashiguchi S, Horie K, Kim M, Hatta H, Yokogoshi H. 2006. Relaxation and immunity enhancement effects of gamma-aminobutyric acid (GABA) administration in humans. BioFactors 26(3), 201\u0026ndash;208.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndrade RJ, Chalasani N, Bj\u0026ouml;rnsson ES, Suzuki A, Kullak-Ublick GA, Watkins PB, Devarbhavi H, Merz M, Lucena MI, Kaplowitz N, Aithal GP. Drug-induced liver injury. Nat Rev Dis Primers. 2019;5(1):58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndria B, Bracco A, Attanasio C, Castaldo S, Cerrito MG, Cozzolino S, Di Napoli D, Giovannoni R, Mancini A, Musumeci A, Mezza E, Nasti M, Scuderi V, Staibano S, Lavitrano M, Otterbein LE, Calise F. Biliverdin protects against liver ischemia reperfusion injury in swine. PLoS ONE. 2013;8(7):e69972.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAveraimo S, Nicol X. Intermingled cAMP, cGMP and calcium spatiotemporal dynamics in developing neuronal circuits. Front Cell Neurosci. 2014;8:376.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrault L, Gasser C, Bracher F, Huber K, Knapp S, Schwaller J. PIM serine/threonine kinases in the pathogenesis and therapy of hematologic malignancies and solid cancers. Haematologica. 2010;95(6):1004\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCervenka I, Agudelo LZ, Ruas JL. Kynurenines: Tryptophan's metabolites in exercise, inflammation, and mental health. Science. 2017;357(6349):eaaf9794.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen M, Borlak J, Tong W. High lipophilicity and high daily dose of oral medications are associated with significant risk for drug-induced liver injury. Hepatology. 2013;58(1):388\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen M, Borlak J, Tong W. A Model to predict severity of drug-induced liver injury in humans. Hepatology. 2016;64(3):931\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen M, Suzuki A, Borlak J, Andrade RJ, Lucena MI. Drug-induced liver injury: Interactions between drug properties and host factors. J Hepatol. 2015;63(2):503\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen R, Wang J, Zhan R, Zhang L, Wang X. Integrated Systems Pharmacology, Urinary Metabonomics, and Quantitative Real-Time PCR Analysis to Uncover Targets and Metabolic Pathways of the You-Gui Pill in Treating Kidney-Yang Deficiency Syndrome. Int J Mol Sci. 2019;20(15):3655.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Z, Wang P. 2012. Clinical Distribution and Molecular Basis of Traditional Chinese Medicine ZHENG in Cancer. Evid Based Complement Alternat Med 2012, 783923.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng ZX, Guo C, Chen ZG, Yang TC, Zhang JY, Wang J, Zhu JX, Li D, Zhang TT, Li H, Peng B, Peng XX. Glycine, serine and threonine metabolism confounds efficacy of complement-mediated killing. Nat Commun. 2019;10(1):3325.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChinese Pharmacopoeia, Commission. 2020. Pharmacopoeia of the People's Republic of China. Beijing: Chinese Medical Science and Technology Press, 2020;1:195.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClemens MM, Kennon-McGill S, Vazquez JH, Stephens OW, Peterson EA, Johann DJ, Allard FD, Yee EU, McCullough SS, James LP, Finck BN, McGill MR. Exogenous phosphatidic acid reduces acetaminophen-induced liver injury in mice by activating hepatic interleukin-6 signaling through inter-organ crosstalk. Acta Pharm Sinica B. 2021;11(12):3836\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoelho Horta B, Perilo S, Caldeira Costa C, Nogueira-Machado D, Martins Chaves JA, M. Aging: functional metabolic balance among cAMP, cGMP and reactive oxygen intermediate generation by human granulocytes. Gerontology. 2005;51(6):363\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCook HW, Thomas SE, Xu Z. Essential fatty acids and serine as plasmalogen precursors in relation to competing metabolic pathways. Biochem Cell Biol. 1991;69(7):475\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeng PY, Cai HD, Cheng JH, Liu SA, Dao WB. 1996. Liver Damage Caused by Zhuanggu Joint Pill: A Report of 30 Cases. Chin J of New Drugs (03), 212\u0026ndash;214.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDermatology Branch of China Association of Chinese Medicine. Expert Consensus on Chinese Medicine Treatment of Vitiligo. Chin J Dermato Veneral Integ Trad W Med. 2017;16(02):191\u0026ndash;2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDermatology Branch of China Association of Chinese Medicine. Expert consensus on traditional Chinese medicine treatment of psoriasis in Dermatology Branch. Chin J Dermato Venemol Integ Trad W Med. 2018;17(03):273\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFawaz R, Baumann U, Ekong U, Fischler B, Hadzic N, Mack CL, McLin VA, Molleston JP, Neimark E, Ng VL, Karpen SJ. Guideline for the Evaluation of Cholestatic Jaundice in Infants: Joint Recommendations of the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition and the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition. J Pediatr Gastroenterol Nutr. 2017;64(1):154\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrancis SH, Corbin JD. Cyclic nucleotide-dependent protein kinases: intracellular receptors for cAMP and cGMP action. Crit Rev Clin Lab Sci. 1999;36(4):275\u0026ndash;328.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFujimoto N, Kitamura S, Uramaru N, Miyagawa S, Iguchi T. Identification of hepatic thyroid hormone-responsive genes in neonatal rats: Potential targets for thyroid hormone-disrupting chemicals. Toxicol Lett. 2018;286:48\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGe FL, Niu M, Han ZX, Cao JL, Wang JB, Bai ZF, Song HB, Guo YM, Xiao XH. Landscape of Hepatobiliary Adverse Drug Reactions Related to Preparations Containing Psoraleae Fructus and Its Application in Pharmacovigilance. Chin J Integr Med. 2021;27(11):832\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGe JR, Wang HM, Zheng HX, Luo YW, Wang JB, Zhao YF, Wan XM, Lu M, He CJ, Dong Z, Zhou HJ, Li G, Sun SB, Lin XS. Traditional Chinese Medicine Expert Consensus on the prevention and treatment of primary osteoporosis. Chin J Osteoporos. 2020;26(12):1717\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGodfrey RW, Manzi RM, Gennaro DE, Hoffstein ST. Phospholipid and arachidonic acid metabolism in zymosan-stimulated human monocytes: modulation by cAMP. J Cell Physiol. 1987;131(3):384\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupta AK, Anderson TF. Psoralen photochemotherapy. J Am Acad Dermatol. 1987;17(5 Pt 1):703\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarden JL, Lewis SM, Lish SR, Su\u0026aacute;rez-Fari\u0026ntilde;as M, Gareau D, Lentini T, Johnson-Huang LM, Krueger JG, Lowes MA. The tryptophan metabolism enzyme L-kynureninase is a novel inflammatory factor in psoriasis and other inflammatory diseases. J Allergy Clin Immunol. 2016;137(6):1830\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarris RA, Schmidt J, Hitzemann BA, Hitzemann RJ. Phosphatidate as a molecular link between depolarization and neurotransmitter release in the brain. Science. 1981;212(4500):1290\u0026ndash;1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHata T, Rehman F, Hori T, Nguyen JH. GABA, γ-Aminobutyric Acid, Protects Against Severe Liver Injury. J Surg Res. 2019;236:172\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHishikawa D, Hashidate T, Shimizu T, Shindou H. Diversity and function of membrane glycerophospholipids generated by the remodeling pathway in mammalian cells. J Lipid Res. 2014;55(5):799\u0026ndash;807.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoofnagle JH, Bj\u0026ouml;rnsson ES. Drug-Induced Liver Injury - Types and Phenotypes. N Engl J Med. 2019;381(3):264\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIsh-Shalom S, Lichter A. Analysis of fungal gene expression by Real Time quantitative PCR. Methods Mol Biol. 2010;638:103\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJi Q, Luo YQ, Wang WH, Liu X, Li Q, Su SB. Research advances in traditional Chinese medicine syndromes in cancer patients. J Integr Med. 2016;14(1):12\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang H, Shabb JB, Corbin JD. Cross-activation: overriding cAMP/cGMP selectivities of protein kinases in tissues. Biochem Cell Biol. 1992;70(12):1283\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang N, Liu HF, Li SD, Zhou WX, Zhang YX, Zhang Q, Yan XZ. An integrated metabonomic and proteomic study on Kidney-Yin Deficiency Syndrome patients with diabetes mellitus in China. Acta Pharmacol Sin. 2015;36(6):689\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015;12(4):357\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee YM, Choi DH, Cheon MW, Kim JG, Kim JS, Shin MG, Kim HR, Youn D. 2022. Changes in Mitochondria-Related Gene Expression upon Acupuncture at LR3 in the D-Galactosamine-Induced Liver Damage Rat Model. Evid Based Complement Alternat Med 2022, 3294273.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011;12:323.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi CY, Niu M, Liu YL, Tang JF, Chen W, Qian G, Zhang MY, Shi YF, Lin JZ, Li XJ, Li RS, Xiao XH, Li GH, Wang JB. Screening for Susceptibility-Related Factors and Biomarkers of Xianling Gubao Capsule-Induced Liver Injury. Front Pharmacol. 2020;11:810.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Z, Agellon LB, Allen TM, Umeda M, Jewell L, Mason A, Vance DE. The ratio of phosphatidylcholine to phosphatidylethanolamine influences membrane integrity and steatohepatitis. Cell Metab. 2006;3(5):321\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi ZJ, Abulizi A, Zhao GL, Wang T, Zhou F, Jiang ZZ, Aibai S, Zhang LY. Bakuchiol Contributes to the Hepatotoxicity of Psoralea corylifolia in Rats. Phytother Res. 2017;31(8):1265\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLing J, Chaba T, Zhu LF, Jacobs RL, Vance DE. Hepatic ratio of phosphatidylcholine to phosphatidylethanolamine predicts survival after partial hepatectomy in mice. Hepatology. 2012;55(4):1094\u0026ndash;102.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J, Qu X, Shao L, Hu Y, Yu X, Lan P, Guo Q, Han Q, Zhang J, Zhang C. Pim-3 enhances melanoma cell migration and invasion by promoting STAT3 phosphorylation. Cancer Biol Ther. 2018;19(3):160\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu YL, Ge FL, Zhu JX, Jing J, Wang JB, Zhang YM, Guo YM, Xiao XH. Re-evaluation of liver injury associated with Buguzhi Preparations based on passive monitoring data and hospital cases. Zhongguo Zhong Yao Za Zhi. 2019;44(19):4272\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLove MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu Y, Shao M, Xiang H, Zheng P, Wu T, Ji G. Integrative transcriptomics and metabolomics explore the mechanism of kaempferol on improving nonalcoholic steatohepatitis. Food Funct. 2020;11(11):10058\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcNeely W, Goa KL. 5-Methoxypsoralen. A review of its effects in psoriasis and vitiligo. Drugs. 1998;56(4):667\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNam SW, Baek JT, Lee DS, Kang SB, Ahn BM, Chung KW. A case of acute cholestatic hepatitis associated with the seeds of Psoralea corylifolia (Boh-Gol-Zhee). Clin Toxicol (Phila). 2005;43(6):589\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNesbitt NM, Malone LE, Liu Z, Jares A, Gnatenko DV, Ma Y, Zhu W, Bahou WF. Divergent erythroid megakaryocyte fates in Blvrb-deficient mice establish non-overlapping cytoprotective functions during stress hematopoiesis. Free Radic Biol Med. 2021;164:164\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePlatten M, Nollen EAA, R\u0026ouml;hrig UF, Fallarino F, Opitz CA. Tryptophan metabolism as a common therapeutic target in cancer, neurodegeneration and beyond. Nat Rev Drug Discov. 2019;18(5):379\u0026ndash;401.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQu Y, Zhang C, Du E, Wang A, Yang Y, Guo J, Wang A, Zhang Z, Xu Y. Pim-3 is a Critical Risk Factor in Development and Prognosis of Prostate Cancer. Med Sci Monit. 2016;22:4254\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen S, Shao Y, Zhao X, Hong CS, Wang F, Lu X, Li J, Ye G, Yan M, Zhuang Z, Xu C, Xu G, Sun Y. Integration of Metabolomics and Transcriptomics Reveals Major Metabolic Pathways and Potential Biomarker Involved in Prostate Cancer. Mol Cell Proteomics. 2016;15(1):154\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSansaloni-Pastor S, Varesio E, Lange N. Modulation and proteomic changes on the heme pathway following treatment with 5-aminolevulinic acid. J Photochem Photobiol B. 2022;233:112484.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSasabe N, Keyamura Y, Obama T, Inoue N, Masuko Y, Igarashi Y, Aiuchi T, Kato R, Yamaguchi T, Kuwata H, Iwamoto S, Miyazaki A, Hara S, Yoshikawa T, Itabe H. Time course-changes in phosphatidylcholine profile during oxidative modification of low-density lipoprotein. Lipids Health Dis. 2014;13:48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen B, Kwan HY, Ma X, Wong CO, Du J, Huang Y, Yao X. cAMP activates TRPC6 channels via the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB)-mitogen-activated protein kinase kinase (MEK)-ERK1/2 signaling pathway. J Biol Chem. 2011;286(22):19439\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen T, Liu Y, Shang J, Xie Q, Li J, Yan M, Xu J, Niu J, Liu J, Watkins PB, Aithal GP, Andrade RJ, Dou X, Yao L, Lv F, Wang Q, Li Y, Zhou X, Zhang Y, Zong P, Wan B, Zou Z, Yang D, Nie Y, Li D, Wang Y, Han X, Zhuang H, Mao Y, Chen C. Incidence and Etiology of Drug-Induced Liver Injury in Mainland China. Gastroenterology. 2019;156(8):2230\u0026ndash;41.e2211.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpector AA, Gordon JA, Moore SA. Hydroxyeicosatetraenoic acids (HETEs). Prog Lipid Res. 1988;27(4):271\u0026ndash;323.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsuge K, Inazumi T, Shimamoto A, Sugimoto Y. Molecular mechanisms underlying prostaglandin E2-exacerbated inflammation and immune diseases. Int Immunol. 2019;31(9):597\u0026ndash;606.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan der Veen JN, Kennelly JP, Wan S, Vance JE, Vance DE, Jacobs RL. The critical role of phosphatidylcholine and phosphatidylethanolamine metabolism in health and disease. Biochim Biophys Acta Biomembr Biomembranes. 2017;1859(9 Pt B):1558\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Wang Y, Wee A, Soon G, Gouw ASH, Yang R, Tian Q, Liu L, Ma H, Zhao X. Clinicopathological features of Bu Gu Zhi-induced liver injury, a long-term follow-up cohort study. Liver Int. 2020;40(3):571\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang P, Sun H, Lv H, Sun W, Yuan Y, Han Y, Wang D, Zhang A, Wang X. Thyroxine and reserpine-induced changes in metabolic profiles of rat urine and the therapeutic effect of Liu Wei Di Huang Wan detected by UPLC-HDMS. J Pharm Biomed Anal. 2010;53(3):631\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang S, Sui S, Liu Z, Peng C, Liu J, Luo D, Fan X, Liu C, Lu WY. Protective roles of hepatic gamma-aminobutyric acid signaling in acute ethanol exposure-induced liver injury. J Appl Toxicol. 2018;38(3):341\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Du Y, Wu C, Xu M, Liu Y, Di X. UHPLC-MS/MS analysis of cAMP and cGMP in rat plasma as potential biomarkers of Yin-Yang disharmony in traditional Chinese medicine. J Pharm Anal. 2021;11(4):458\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Liu C, Hu L. Cholesterol regulates cell proliferation and apoptosis of colorectal cancer by modulating miR-33a-PIM3 pathway. Biochem Biophys Res Commun. 2019;511(3):685\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Liu H, McKenzie G, Witting PK, Stasch JP, Hahn M, Changsirivathanathamrong D, Wu BJ, Ball HJ, Thomas SR, Kapoor V, Celermajer DS, Mellor AL, Keaney JF Jr, Hunt NH, Stocker R. Kynurenine is an endothelium-derived relaxing factor produced during inflammation. Nat Med. 2010;16(3):279\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWatchko JF, Tiribelli C. Bilirubin-induced neurologic damage\u0026ndash;mechanisms and management approaches. N Engl J Med. 2013;369(21):2021\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWegiel B, Gallo D, Csizmadia E, Roger T, Kaczmarek E, Harris C, Zuckerbraun BS, Otterbein LE. Biliverdin inhibits Toll-like receptor-4 (TLR4) expression through nitric oxide-dependent nuclear translocation of biliverdin reductase. Proc Natl Acad Sci U S A. 2011;108(46):18849\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei SM, Yan ZZ, Zhou J. Psoralea corylifolia protects against testicular torsion/detorsion-induced ischemia/reperfusion injury. J Ethnopharmacol. 2011;137(1):568\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu C, Sun Z, Ye Y, Han X, Song X, Liu S. Psoralen inhibits bone metastasis of breast cancer in mice. Fitoterapia. 2013;91:205\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu S, Li Z, Gnatenko DV, Zhang B, Zhao L, Malone LE, Markova N, Mantle TJ, Nesbitt NM, Bahou WF. BLVRB redox mutation defines heme degradation in a metabolic pathway of enhanced thrombopoiesis in humans. Blood. 2016;128(5):699\u0026ndash;709.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu Y, Zhao Y, Xie J, Sheng X, Li Y, Zhang Y. 2017. The Evaluation of Toxicity Induced by Psoraleae Fructus in Rats Using Untargeted Metabonomic Method Based on UPLC-Q-TOF/MS. Evid Based Complement Alternat Med 2017, 6207183.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang H, Xing R, Liu S, Yu H, Li P. Rescuing fluoride-induced damages in liver with gamma aminobutyric acid. Biochem Biophys Res Commun. 2017;491(1):19\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu B, Wang AH, Zhou K, Chai LJ, Liu L. Molecular Pathway of Psoralidin-Induced Apoptosis in HepG2 Cell Line. Chin J Integr Med. 2019;25(10):757\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang C, Zhao JQ, Sun JX, Li HJ. Psoralen and isopsoralen from Psoraleae Fructus aroused hepatotoxicity via induction of aryl hydrocarbon receptor-mediated CYP1A2 expression. J Ethnopharmacol. 2022;297:115577.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang L, Niu M, Wei AW, Tang JF, Tu C, Bai ZF, Zou ZS, Xiao XH, Liu YP, Wang JB. Risk profiling using metabolomic characteristics for susceptible individuals of drug-induced liver injury caused by Polygonum multiflorum. Arch Toxicol. 2020;94(1):245\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Zhao W, Wang Y, Lu J, Chen X. The Chemical Constituents and Bioactivities of Psoralea corylifolia Linn.: A Review. Am J Chin Med. 2016;44(1):35\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang XJ, Cheng X, Yan ZZ, Fang J, Wang X, Wang W, Liu ZY, Shen LJ, Zhang P, Wang PX, Liao R, Ji YX, Wang JY, Tian S, Zhu XY, Zhang Y, Tian RF, Wang L, Ma XL, Huang Z, She ZG, Li H. An ALOX12-12-HETE-GPR31 signaling axis is a key mediator of hepatic ischemia-reperfusion injury. Nat Med. 2018;24(1):73\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Xu SY, Liu MN, Jia TY, Qu WJ, Han T, Jia Z, Xu XF, Li XR. 2019. Comparative Studies on Chemical Contents and Effect in Kidney-Yang Deficiency Rats of Salt-Processed Product and Wine-Processed Product of Cuscutae Semen. Evid Based Complement Alternat Med 2019, 2049497.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou Z, Zhang R, Wang R, Zhang Y, Xu L, Chen J, Zhang J, Huang Z, Chen M, Pan Z. Expression of Pim-3 in colorectal cancer and its relationship with prognosis. Tumour Biol. 2016;37(7):9151\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Psoralea corylifolia Linn., liver injury, predisposed individual, metabolomics, transcriptomics","lastPublishedDoi":"10.21203/rs.3.rs-2301287/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2301287/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eAim\u003c/h2\u003e \u003cp\u003e \u003cem\u003ePsoralea corylifolia\u003c/em\u003e Linn. (BGZ) is a commonly used traditional Chinese medicine (TCM) for the treatment of kidney-yang deficiency syndrome (Yang\u003csub\u003esyn\u003c/sub\u003e) with good curative effect and security. However, BGZ was also reported to induce liver injury in recent years. According to TCM theory, taking BGZ may induce a series of adverse reactions in patients with kidney-yin deficiency syndrome (Yin\u003csub\u003esyn\u003c/sub\u003e), which suggests that BGZ-induced liver damage may be related to its unreasonable clinical use. The study aimed to investigate the differential responses to BGZ in Yang\u003csub\u003esyn\u003c/sub\u003e and Yin\u003csub\u003esyn\u003c/sub\u003e rat models and identify the corresponding characteristic biomarkers.\u003c/p\u003e\u003ch2\u003eMaterials and methods\u003c/h2\u003e \u003cp\u003eThe corresponding animal models of Yang\u003csub\u003esyn\u003c/sub\u003e and Yin\u003csub\u003esyn\u003c/sub\u003e were induced by hydrocortisone and thyroxine\u0026thinsp;+\u0026thinsp;reserpine respectively. Body weight, organ index, serum biochemistry, and Hematoxylin and Eosin (HE) staining were used to evaluate the liver toxicity effect of BGZ on rats with Yang\u003csub\u003esyn\u003c/sub\u003e and Yin\u003csub\u003esyn\u003c/sub\u003e. Transcriptomics and metabonomics were used to screen the representative biomarkers (including metabolites and differentially expressed genes (DEGs)) changed by BGZ in Yang\u003csub\u003esyn\u003c/sub\u003e and Yin\u003csub\u003esyn\u003c/sub\u003e rats, respectively.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe level changes of liver organ index, ALT, and AST suggested that BGZ has liver-protective and liver-damaging effects on Yang\u003csub\u003esyn\u003c/sub\u003e and Yin\u003csub\u003esyn\u003c/sub\u003e rats, respectively, and the results also were confirmed by the pathological changes of liver tissue. The results showed that 102 DEGs and 27 metabolites were significantly regulated related to BGZ\u0026rsquo;s protective effect on Yang\u003csub\u003esyn\u003c/sub\u003e, which is mainly associated with the glycerophospholipid metabolism, arachidonic acid metabolism, pantothenate, and CoA biosynthesis pathways. While 28 DEGs and 31 metabolites, related to the pathway of pantothenate and CoA biosynthesis, were significantly regulated for the BGZ-induced liver injury in Yin\u003csub\u003esyn\u003c/sub\u003e. Furthermore, 4 DEGs (Aldh1b1, Slc25a25, Pim3, Oaf) and 4 metabolites (phosphatidate, phosphatidylcholine, N-Acetylleucine, biliverdin) in the Yang\u003csub\u003esyn\u003c/sub\u003e group and 1 DEGs (Lgals5) and 1 metabolite (5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxylate) in Yin\u003csub\u003esyn\u003c/sub\u003e group were significantly correlated to the ALT and AST levels of BGZ treated and untreated groups (ROC\u0026thinsp;\u0026ge;\u0026thinsp;0.9).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eYin\u003csub\u003esyn\u003c/sub\u003e and Yang\u003csub\u003esyn\u003c/sub\u003e are the predisposed syndrome for BGZ to exert liver damage and liver protection respectively, which are mainly related to the regulation of amino acid metabolism, lipid metabolism, energy metabolism, and metabolism of cofactors and vitamins. The results further suggest that attention should be paid to the selection of predisposed populations when using drugs related to the regulation of energy metabolism, and the Yin\u003csub\u003esyn\u003c/sub\u003e/Yang\u003csub\u003esyn\u003c/sub\u003e animal models based on the theory of TCM syndromes may be a feasible method for identifying the susceptible population to receive TCM.\u003c/p\u003e","manuscriptTitle":"Yin/Yang associated differential responses to Psoralea corylifolia Linn. in rat models: an integrated metabolomics and transcriptomics study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-12-06 21:04:35","doi":"10.21203/rs.3.rs-2301287/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-02-15T21:04:18+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2023-01-11T16:05:20+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-12-04T01:13:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-11-25T17:01:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chinese Medicine","date":"2022-11-24T10:24:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1bbf8a2c-d9d3-4413-abb8-d1e77e23c87b","owner":[],"postedDate":"December 6th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T22:14:14+00:00","versionOfRecord":{"articleIdentity":"rs-2301287","link":"https://doi.org/10.1186/s13020-023-00793-x","journal":{"identity":"chinese-medicine","isVorOnly":false,"title":"Chinese Medicine"},"publishedOn":"2023-08-17 22:02:51","publishedOnDateReadable":"August 17th, 2023"},"versionCreatedAt":"2022-12-06 21:04:35","video":"","vorDoi":"10.1186/s13020-023-00793-x","vorDoiUrl":"https://doi.org/10.1186/s13020-023-00793-x","workflowStages":[]},"version":"v1","identity":"rs-2301287","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2301287","identity":"rs-2301287","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}