Eucommia ulmoides leaf extract attenuates cisplatin-induced kidney injury in mice through endoplasmic reticulum stress and biometabolic mechanism

Eucommia ulmoides leaf extract attenuates cisplatin-induced kidney injury in mice through endoplasmic reticulum stress and biometabolic mechanism | 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 Eucommia ulmoides leaf extract attenuates cisplatin-induced kidney injury in mice through endoplasmic reticulum stress and biometabolic mechanism Kexin Lin, Lijuan Xiong, Wen Zhang, xuan Chen, Xiaofei li, Jianyong Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3917893/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cisplatin (CP) is a widely utilized anticancer drug but is associated with significant side effects, notably acute kidney injury (AKI). Eucommia ulmoides leaf (EUL), a valuable Chinese herbal remedy, is known for its renoprotective properties. However, the function and underlying pathways of EUL in AKI therapy have remained largely unexplored. This research aimed to elucidate the protective roles of EUL in an AKI mouse model through biochemical assays, and histopathological andexaminations while also investigating the underlying mechanisms via endoplasmic reticulum (ER) stress-related protein expression analysis and metabolomics. The findings demonstrated that pretreatment with orally administered EUL significantly reduced blood urea nitrogen (BUN) and serum creatinine (SCr) levels, ameliorated CP-induced kidney histopathological injuries, and attenuated CP-induced ER stress by reducing the protein expressions of PERK, IRE 1α, GRP78, ATF6, ATF4, and CHOP. Additionally, metabolomics analysis identified 31 significant differential metabolites affected by EUL treatment in AKI mice, impacting pathways related to taurine and hypotaurine metabolism, lysine degradation, and steroid hormone biosynthesis. These findings suggested that EUL could offer valuable insights for potential CP-induced AKI treatment strategies. Cisplatin Eucommia ulmoides leaf Acute kidney injury Endoplasmic reticulum stress Metabolomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Cisplatin (CP), a platinum compound, has been used extensively as a chemotherapeutic drug in treating various cancers, including ovarian, testicular, lung, non-small cell, cervical, and bladder cancers(Holditch et al., 2019, Tchounwou et al., 2021). However, its clinical utility is hampered by severe nephrotoxicity(Almaghrabi, 2015), with 30% of patients suffering acute kidney injury (AKI)(Pabla and Dong, 2008 ). Current clinical approaches mainly involve the administration of fluids, diuretics, and dehydrating agents to mitigate CP-induced kidney injury(Chen et al., 2022). Nonetheless, these therapeutic strategies have drawbacks, including delayed onset of efficacy, disruption of water-electrolyte balance, acid-base equilibrium, and increased cardiac burden(Catalanotto et al., 2016, Lu and Rothenberg, 2018). Thus, there is an imperative need for research into new therapeutic interventions to prevent CP-induced AKI. Natural medicines have attracted increased interest in recent years for their potential role in the prevention and treatment of AKI(Chen et al., 2018). Traditional Chinese medicine (TCM), including angelica sinensis and astragaloside IV, has been extensively studied to treat and prevent CP-induced AKI (Bunel et al., 2015, Song et al., 2021). Eucommia ulmoides leaf (EUL), a by-product of Eucommia ulmoides, is considered both a food and a medicinal herb. It contains a variety of bioactive components, including chlorogenic acid and geniposidic acid (He et al., 2014, Ren et al., 2022). EUL is known for its health benefits in nourishing the liver and kidneys(Zhao et al., 2021), anti-endoplasmic reticulum (ER) stress(Lee et al., 2013), anti-inflammatory(Xu et al., 2010), and anti-hypertensive effects(Luo et al., 2010)according to TCM theory(Zhu and Sun, 2018 ). Moreover, previous studies have also shown the renoprotective role of EUL in a rat model of hyperuricemic kidney injury induced by intragastric adenine(Li et al., 2021). However, the mechanisms underlying the protective effects of EUL against CP-induced AKI warrant further investigation. The multi-component and multi-target characteristics of TCM pose challenges in elucidating their mechanisms using conventional pharmacological methods(Tao et al., 2020). Metabolomics, a system biology technology, offers comprehensive insights into global metabolic changes and has emerged as a powerful tool for understanding the metabolic alterations and pharmacological mechanisms of TCM in AKI treatment(Cui et al., 2018). Now, metabolomics has emerged as a potent tool for elucidating the intrinsic metabolic alterations and pharmacological mechanisms of TCM in treating AKI(Xiang et al., 2015, Zheng et al., 2021). Therefore, employing metabolomic analysis could be an effective approach to investigate the efficacy of pharmaceutical interventions against AKI. Our study examined the protective effects of EUL by inhibiting ER stress on a CP-induced AKI I mouse model. Additionally, Our study utilized UPLC/QTOF-MS to investigate how EUL affects metabolic changes associated with CP-induced AKI in mice. We identified key metabolites linked to the therapeutic effects of EUL using multivariate analysis. Furthermore, we enriched the metabolic pathways to elucidate the underlying biometabolic mechanisms. In conclusion, our investigation offers new insights into the mechanisms through which EUL acts on CP-induced AKI, potentially advancing its traditional application. 2. Material and methods 2. 1 Material CP was sourced from Qilu Pharmaceutical, China. Chlorogenic acid was obtained from Solaibao Life Science, China, and astragalin from Solaibao Life Science Co., Ltd., China. The BCA kit used for protein quantification was from Jian Cheng Bioengineering Institute, China. Primary antibodies used included protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1α (IRE1α), glucose-regulated protein-78 (GRP78), activating transcription factor 6 (ATF6), activating transcription factor 4 (ATF4), and C/EBP-homologous protein (CHOP) from CST, USA. GAPDH antibody was obtained from HuaAn, China. 2. 2. Preparation of EUL extract and quality analysis Fermented EUL was procured from Zhangjiajie Cha Kun Yuan Biotechnology Development Co., Ltd and identified by Prof. Jianyong Zhang from the College of Pharmacy of Zunyi Medical University. The EUL was extracted as follows: 100 g of EUL was dissolved in 1 L of 70% ethanol, refluxed at 90°C for 2 hours, and then filtered. The filter residue underwent two more identical extraction cycles. The filtrates from the two batches were mixed and concentrated into an extract, which was subsequently oven-dried at 80°C overnight and milled into powder. The chemical composition of the EUL extract, including chlorogenic acid and astragalin, was quantified using an LC-20A HPLC system (Shimazu, Kyoto, Japan). Separation was carried out on an Agilent Eclipse Plus C18 column (150 × 4.6 mm, 3.5 µm; Agilent, USA) at 25°C. The mobile phase consisted of solvent A (0.4% Formic acid in water) and solvent B (MeCN) with a linear gradient elution program (5%-13% B at 0–15 min; 13% – 25% B at 15–25 min; 25% – 50% B at 25–25.5 min; 50% B at 25.5–30 min). Detection was carried out at a wavelength of 327 nm, with a sample injection volume of 20 µL. The content of chlorogenic acid and astragalin (%) was calculated based on peak area. 2.3. Animal experiment, design, and drug administration Healthy male ICR mice (30–32 g) were obtained from Vital River Laboratories (Beijing, China) and kept under specific pathogen-free conditions in a controlled environment with regulated temperature and humidity. Forty-eight mice, after a week of acclimatization, were divided into four equal groups: normal Control group (NC), CP group (CP), and CP + EUL (100, 200 mg/kg) groups. NC group: Mice received normal saline for seven consecutive days, with a single intraperitoneal (i.p.) injection of saline on the 3rd day. CP group: Mice received CP (20 mg/kg) diluted with normal saline as a single i.p. injection. CP + EUL groups: Mice were given EUL (100, 200 mg/kg) orally daily for seven consecutive days, with a single i.p. injection of CP (20 mg/kg) on the 3rd day. The weight of the mice was recorded every two days. At the end of the experiment, blood samples were obtained by eye enucleation. The mice were sacrificed by decapitation, and the kidneys were immediately harvested. The left kidney of mice was removed and weighed to calculate the kidney index as follows: \(\text{K}\text{i}\text{d}\text{n}\text{e}\text{y} \text{i}\text{n}\text{d}\text{e}\text{x}=\frac{\text{k}\text{i}\text{d}\text{n}\text{e}\text{y} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t}}{\text{b}\text{o}\text{d}\text{y} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t}} \times 100\text{%}\) . The right kidney was placed in 4% paraformaldehyde through a coronal incision, and the rest of the kidney was stored in a refrigerator at − 80°C for subsequent metabolomics analysis. The animal experiment was conducted following approval by the animal ethics committee of Zunyi Medical University (Zunyi, China, ZMU21-2304-009) and adhered to the Helsinki Declaration. 2. 4. Serum detection in mice Mice serum samples were removed from the refrigerator testing. Blood urea nitrogen (BUN) and serum creatinine (SCr) of mice serum samples were measured using biochemical assay kits on an automatic biochemical analyzer (Beckman, USA) to evaluate kidney function. 2. 5. Kidney histological examination in mice Kidney tissue samples were collected and fixed in 4% neutral paraformaldehyde for 24 hours, embedded in paraffin, and cut into 4 µm thin slices. These slices were stained with hematoxylin and eosin (H&E) and examined using a light microscope (OLYMPUS, BX43F, Japan). 2. 6. Western blot analysis of ER stress-related proteins Total proteins were extracted from kidney tissues with RIPA lysis buffer and quantified using a BCA kit (Jian Cheng Bioengineering Institute, China). Proteins were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% nonfat milk for 1 hour, membranes were incubated overnight at 4°C with primary antibodies: PERK (1:1000), IRE 1α (1:1000), GRP78 (1:1000), ATF6 (1:1000), ATF4 (1:1000) and CHOP (1:1000). The relative expression of proteins was normalized to GAPDH (1:5000) (HuaAn, China), and Image lab software 6.1 was used for band analysis. 2. 7. Kidney metabolomics analysis in mice 2.7.1. Sample preparation Kidney samples were thawed slowly at 4°C before being weighed 20 mg was added to a prechilled methanol/acetonitrile/water solution (2:2:1, v/v), vortex mixed, and sonicated at low temperature for 30 min. The supernatant was dried under vacuum and reconstituted in 100 µL of aqueous acetonitrile (acetonitrile: water = 1:1, v/v) for mass spectrometry analysis. The supernatant was centrifuged at 14,000 × g for 15 minutes at 4°C before analysis. A quality control (QC) sample was prepared by mixing equal volumes (50 µL) of each test sample to estimate system reliability and repeatability during the analysis process. 2.7.2. LC-MS analytical conditions Supernatant separation was performed using an Agilent 1290 Infinity LC Ultra High-Performance Liquid Chromatography (UHPLC) system. The parameters were as described below: a HILIC column (ACQUITY UPLC BEH Amide column, 2.1 x 100 mm, 1.7 µm); column temperature at 25°C; flow rate at 0.5 mL/min; injection volume of 2 µL. The mobile phase comprised 25 mM ammonium acetate 25 m, M ammonia water (A) and acetonitrile (B). The gradient elution program was set as follows: 0-0.5 min, 95% B; 0.5-7 min, B linearly decreased from 95–65%; 7–8 min, B linearly decreased from 65–40%; 8–9 min, B maintained at 40%; 9-9.1 min, B linearly changes increased from 40–95%; 9.1-1min, B was maintained at 95%. Samples were maintained at 4°C throughout the analysis procedure. Mass spectrometry analysis was a Triple TOF 6600 mass spectrometer (AB SCIEX) with positive and negative ion modes of electrospray ionization (ESI) for detection. ESI source settings were: atomizing gas auxiliary heating gas 1 (Gas1) at 60, auxiliary heating gas 2 (Gas2) at 60, curtain gas (CUR) at 30 psi, ion source temperature at 600°C, and spray voltage (ISVF) at ± 5500 V (positive and negative modes). The primary mass-to-charge ratio detection range was 60-1000 Da, and the secondary sub-ion mass-to-charge ratio detection range was 25-1000 Da. Primary mass spectrometry scanning accumulated for 0.20 s/spectrum, and secondary mass spectrometry scanning accumulated for 0.05 s/spectrum. Data-dependent acquisition mode (IDA) and peak intensity value screening mode were employed for secondary mass spectrometry. De-clustering voltage (DP) was set at ± 60 V (positive and negative modes), and collision energy at 35 ± 15 eV (positive and negative modes). IDA settings included dynamic exclusion of isotope ions within a 4 Da range, with 10 fragment spectra collected per sweep. 2.7.3. Metabolic data processing Data preprocessing included filtering out data with less than 80% completeness, keeping RSD values below 30% within the same sample group, filling missing values in the same sample group with mean values (Zhang et al., 2022), and subsequent normalization. The processed three-dimensional data matrix included sample descriptions, normalized peak areas, and retention time-m/z pairs for multivariate analysis. This data was imported into SIMCA-P 14.0 (Umetrics AB, Umea, Sweden) for multivariate data analysis. Principal component analysis (PCA) and partial least-squared discrimination analysis (PLS-DA) were utilized to visualize overall differences. Orthogonal projection to latent structure-discriminant analysis (OPLS-DA) was employed to confirm the model and identify different metabolites between groups. Criteria for screening differential metabolites included VIP > 1, P 1.2 or FC < 0.833(Liu et al., 2023). Differential metabolite identification was initially performed using an in-house database (Shanghai Applied Protein Technology). Subsequently, differential metabolites were further identified based on UPLC-QTOF-MS/MS data by comparing with online databases, including HMDB ( http://www.hmdb.ca ), KEGG ( http://www.kegg.com ), and PubMed ( http://www.ncbi.nlm.nih.gov ). Pathway analysis of the differential metabolites was conducted using the online analytical tool MetaboAnalyst 14.0 ( http://www.metaboanalyst.ca ) to elucidate biological interpretation. 2. 8. Statistical analysis Data were presented as the mean ± SEM. Statistical analyses were conducted using SPSS 29.0 software. Multiple group data were statistically analyzed by one-way analysis of variance (ANOVA). P < 0.05 (marked as # or *) was considered statistically significant, while P < 0.01 (marked as ## or **) was considered highly statistically significant. 3. Results 3.1 Quality control analysis of EUL The chlorogenic acid and astragalin in the EUL extract were identified with HPLC (Fig. 1 ). The peak area and corresponding concentrations of the chlorogenic acid and astragalin standard were used to construct the standard curve chlorogenic acid was represented by y = 30.033x + 54.308, where R 2 = 0.9993, and for astragalin, it was y = 37.281x − 40.354, where R 2 = 0.9999 (Fig. S1 ). These results indicate a strong linear relationship between the peak area and chlorogenic acid and astragalin concentrations. The chlorogenic acid and astragalin content in EUL was determined to be 3.9% and 0.047%, respectively. 3. 2. EUL improved CP-induced AKI BUN and SCr levels are widely acknowledged markers of kidney dysfunction (Zhang et al., 2018) . To assess the impact of EUL on kidney function following CP intervention, in vivo experiments were conducted in mice. As depicted in Fig. 2 A and 2 B, CP injection at 72 hours led to a markedly decrease the body weight while the kidney index was attenuated compared to the Control group ( P < 0.01). However, this reduction was partially restored by treatment with 100 mg/kg or 200 mg/kg EUL (Fig. 2 C and 2 D), suggesting that EUL treatment significantly inhibited increased serum levels of BUN and SCr induced by CP ( P < 0.01). 3. 3. EUL ameliorated CP-induced histopathological injury in mice. To examine the protective impact of EUL in CP-treated mice, we evaluated alterations in kidney histology using H&E staining. In the Control group, kidney histology displayed a normal architecture with intact tubular and glomerular (Fig. 2 E). In the CP group, significant pathological changes were observed, including tubular disorganization, glomerular atrophy, and vascular degeneration. Compared with the CP group, the EUL 100 mg/kg group showed improvement in kidney damage, although few tubular exhibited proteinaceous material. In the 200 mg/kg EUL group, kidney damage was significantly alleviated. 3. 4. EUL inhibited CP-induced ER stress in mice To further examine the protective effects of EUL, the protein expression levels of PERK, IRE1α, GRP78, ATF6, ATF4, and CHOP, which are involved in ER stress, were examined. As shown in Fig. 2 F the expression levels of these ER stress-related proteins were significantly increased in the CP group compared to the Control group ( P < 0.05 or P < 0.01). However, the administration of EUL at both dosage levels significantly decreased these alterations in protein expression ( P < 0.05 or P < 0.01). 3. 5. Metabolomics analysis 3. 5. 1. Multivariate statistical analysis To investigate the potential effects of EUL on CP-induced AKI, differential metabolites in kidney samples from the CP and CP + EUL 200 mg/kg groups were identified and subjected to multivariate statistical analyses, including PCA and PLS-DA. PCA score plots demonstrated clear separation among kidney samples in all three groups, indicating significant metabolic changes (Fig. 3 A and 3 B). Subsequently, PLS-DA, a supervised statistical approach, exhibited a strong distinction between the CP and Control groups, with the CP + EUL 200 mg/kg group showing the closest similarity to the Control group compared to all the other groups (Fig. 3 C and 3 D). These results suggest that EUL can mitigate CP-induced alterations in kidney metabolism. 3. 5. 2. Metabolic pathway analysis OPLS-DA models were established to screen for differential metabolites. To evaluate the potential errors of the OPLS-DA models, 200 permutation tests were performed where the Q 2 values on the left were lower than the original points on the right, confirming the validity of the OPLS-DA models (Fig. 4A and 5A). A total of 87 differential metabolites were identified when comparing the CP group to the Control group (Fig. 4C) (VIP > 1.0, P 1.2, or FC < 0.833(He et al. , 2023). Among these, 31 differential metabolites were found to be regulated towards normal levels by EUL 200 mg/kg treatment, including 13 metabolites that showed upregulation and 18 metabolites that showed downregulation compared to the CP group (Fig.5C, Table 1). Additionally, a clustering heatmap (Fig. 4B and 5B) was utilized to visually represent changes in the differential metabolites among the three groups, further substantiating that EUL may ameliorate CP-induced AKI by modulating disruptions in these metabolic pathways. To obtain further insights into the therapeutic mechanisms of EUL at the metabolic level, the 31 differential metabolites from Table 1 were subjected to pathway topology analysis (Fig. 5D). This analysis revealed the involvement of pathways such as taurine and hypotaurine metabolism, lysine degradation, and steroid hormone biosynthesis (impact > 0.1). Our results showed that some biomarkers such as 2-Oxoadipic acid, L-2-Aminoadipate, Hypotaurine, and 17alpha-hydroxyprogesterone significantly decreased in CP-induced AKI (Figure. 6A). According to the correlative analyses (Figure. 6B and 6C), four biomarkers were significantly and negatively correlated With both BUN and SCr ( P < 0.05). Particularly, the EUL administration remarkably up-regulated the level of these biomarkers toward normal. 4. Discussion CP is a commonly applied chemotherapeutic agent for the treatment of malignant tumors (Hong et al., 2020). However, in clinical practice, nearly 30% of patients develop AKI due to CP administration (Jiang et al., 2018, Lameire et al., 2013). Therefore, finding ways to prevent CP-induced AKI has become an urgent priority. Traditional Chinese Medicine (TCM), known for its diverse biological activities, has been extensively employed in treating AKI and its associated complications (Li et al., 2019). Our study demonstrated that pretreatment with EUL significantly attenuated CP-induced AKI, employing a combination of traditional pharmacodynamic and metabolomics analyses. Figure 8 shows the full-text mechanism diagram of this study. 4.1 Traditional pharmacodynamic evaluation In our study, histopathological examination and the kidney index revealed characteristic features of CP-induced AKI, including inflammatory cell infiltration, tubular necrosis, and glomerular atrophy. Additionally, elevated levels of BUN and SCr, important indicators of kidney function evaluation (Terzi and Ciftci, 2022 ), were observed in the CP group. Importantly, pretreatment with EUL significantly ameliorated kidney histopathological damage and reduced the extent of kidney injury compared to the CP group. Emerging findings indicate that ER stress plays a crucial role in both the physiological and pathological processes of AKI(Tan et al., 2019). Prolonged ER stress can trigger apoptotic pathways, ultimately leading to renal cytotoxicity through three classical pathways: the PERK-eIF2α-ATF4-CHOP, IRE1α-XBP1, and ATF6 pathways (Ajoolabady et al., 2023, Walter and Ron, 2011 ). Our findings indicate that the administration of EUL resulted in a significant decrease in the expression of PERK, IRE1α, GRP78, ATF6, ATF4, and CHOP in kidney tissues from CP-induced AKI mice, suggesting that EUL may have a potential therapeutic effect in reducing the ER stress response in the context of AKI. 4.2 Biometabolic mechanisms analysis of EUL for treating AKI To unravel the underlying metabolic perturbations associated with CP-induced AKI and the potential kidney protective effect of EUL, we employed untargeted metabolomics to characterize differences among the Control, CP, and EUL groups. From a metabolic profile perspective, the samples from the Control, CP, and EUL groups exhibited distinct separation, with the EUL group displaying a clustering pattern closer to the Control group. Subsequently, we identified and analyzed 31 differential metabolites and found that three dysregulated metabolic pathways—taurine and hypotaurine metabolism, lysine degradation, and steroid hormone biosynthesis—were significantly affected by EUL treatment. These results imply that EUL may improve CP-induced AKI by regulating these metabolic disorders. 4.2.1 Taurine and hypotaurine metabolism In the present study, hypotaurine emerged as a critical metabolic biomarker in taurine and hypotaurine metabolism. Hypotaurine, a sulfur-containing organic acid abundant in tissues, plays a crucial role in kidney protection by regulating intracellular osmotic pressure and concentrations of reactive oxygen species (Jakaria et al., 2019, Sener et al., 2005). Increasing evidence suggests that ER stress, oxidative stress, and inflammation are central to the development of nephrotoxicity(Inagi et al., 2005, Lahmar et al., 2020). Importantly, taurine, a metabolite of hypotaurine, exerts significant anti-ER stress, antioxidant, and anti-inflammatory effects, thereby protecting against CP, 5-fluorouracil, and diabetes-induced kidney injury (Neog et al., 2019, Zhang et al., 2021). The level of hypotaurine decreased in the CP-induced AKI mice and was increased by pretreatment with EUL. These results suggest that EUL may decrease AKI-associated ER stress, oxidative stress, and inflammation, thereby preventing CP-induced AKI. 4.2.1 Lysine metabolism In our study, 2-oxoadipate and L-2-Aminoadipate were identified as important metabolic biomarkers associated with lysine degradation. Lysine is crucial in providing energy and essential substances for the body and maintaining homeostasis(Kawasaki et al., 2000, N. et al., 1979). Consistent with our research, other studies have demonstrated that lysine degradation is closely linked to diabetic nephropathy (Li et al., 2022). 2-Oxoadipate is catalyzed to acetyl-CoA, which enters the tricarboxylic acid cycle for energy production in mitochondria(Jordan et al., 2019). The kidneys rely on mitochondrial oxidative phosphorylation to generate significant amounts of ATP, facilitating blood filtration and toxin elimination (Tran et al., 2016). In our study, the levels of 2-Oxoadipate and L-2-Aminoadipate decreased in AKI mice and were downregulated by EUL pretreatment. These results suggest that EUL can correct energy metabolism disorders induced by CP in the kidneys, thereby playing a protective role in CP-induced AKI. 4.2.1 Steroid hormone biosynthesis In our study, 17 alpha-hydroxyprogesterone emerged as an important metabolic biomarker associated with steroid hormone biosynthesis. Steroid hormones regulate various physiological processes related to growth and development, and their reduced levels can disrupt the hypothalamus-pituitary-adrenal (HPA) axis, leading to kidney damage(Annane et al., 2017, Laulhé et al., 2021). Previous studies have demonstrated that 17alpha-hydroxyprogesterone possesses pro-inflammatory properties and can increase plasma TNF-α expression levels in non-pregnant women exposed to LPS(Amory et al., 2005). In this study, the level of 17alpha-hydroxyprogesterone was significantly decreased in AKI mice and increased following EUL pretreatment. These results suggest that the decrease in 17alpha-hydroxyprogesterone observed after AKI could be part of a self-protective mechanism, and EUL might help restore normal levels. 5. Conclusion In summary, based on traditional pharmacology and functional metabolomics strategy, we demonstrated that EUL exhibits a protective effect against AKI, as evidenced by improvements in BUN and SCr levels, amelioration of histopathological damage, and attenuation of ER stress. Furthermore, metabolomics analysis indicated that EUL treatment significantly reversed abnormal levels of 31 metabolites, particularly those involved in taurine and hypotaurine metabolism, lysine degradation, and steroid hormone biosynthesis. These findings can be concluded that EUL may mitigate the effects of CP-induced AKI, and provide a theoretical basis for its potential clinical use. Declarations Data availability The data and materials of the study can be obtained from the corresponding author upon request. Author information Authors and Affiliations School of Basic Medicine, Zunyi Medical University, Zunyi, 563000, China Kexin Lin , Xuan Chen, Xiaofei Li School of Pharmacy and Key Laboratory of Basic Pharmacology Ministry Education, Joint International Research Laboratory of Ethnomedicine Ministry of Education, Zunyi Medical University, Zunyi, 563000, China Lijuan Xiong, Xuan Chen, Jianyong Zhang Contributions KX Lin and LJ Xiong contributed equally to this work. KX Lin and JY Zhang wrote and modified the manuscript. LJ Xiong and XF Li designed the experiment. LJ Xiong, W Zhang, and X Chen completed the experiments. KX Lin constructed the illustration. KX Lin and JY Zhang revised and approved the final version of the manuscript. All authors confirmed the final manuscript. Corresponding authors Correspondence to Xiaofei Li ( [email protected] ) or Jianyong Zhang ( [email protected] ). Ethics declarations Ethical approval Animal experiments were conducted in accordance with the requirements of the Guizhou Provincial Experimental Animal Management Committee and were approved by the Ethical Committee of Zunyi Medical University. (Ethics approval number: ZMU21-2304-009). Declaration of Competing Interest The authors have no competing financial interests that could have appeared to influence this work. Funding This work was partially supported by the National Natural Science Foundation of China (82060754,81803838 and 82260812), Key project at central government level: The ability establishment of sustainable use for valuable Chinese medicine resources (2060302), Guizhou Provincial Science & Technology Program ([2020]1Y376, ZK[2021]532, ZK[2022]615,YQK[2023]038), Science and Technology Department of Zunyi city of Guizhou province of China (HZ（2022）420, HZ-[2020]-39,ZYK[2021]-3,[2020]-7), Science and technology project of Guizhou health and Health Committee (gzwkj2021-441), Guizhou Provincial Special research project on science and technology of traditional Chinese medicine and ethnic medicine (QZYY-2021-035), Science and Technology Department of Honghuagang District of Zunyi city of Guizhou province of China ([2020]-17), the Zunyi city of Guizhou Provincial Department of health outstanding young medical talents fund ([2021]-3), Science and Technology Department of Zunyi city of Guizhou province of China ([2020]-7). 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N., J., Weill-Thevnet, M., Hermann, J.-P., et al. Lysine Transport Systems in Pseudomonas in Relation to Their Physiological Function. Journal of General Microbiology. 1979. Neog MK, Chung H, Jang MJ, Kim DJ, Lee SH, Kim KS. Effect of Aging on Taurine Transporter (TauT) Expression in the Mouse Brain Cortex. Advances in experimental medicine and biology. 2019;1155:3-11. Pabla N, Dong Z. Cisplatin nephrotoxicity: mechanisms and renoprotective strategies. Kidney international. 2008;73:994-1007. Ren N, Gong W, Zhao Y, Zhao DG, Xu Y. Innovation in sweet rice wine with high antioxidant activity: Eucommia ulmoides leaf sweet rice wine. Frontiers in nutrition. 2022;9:1108843. Sener G, Sehirli O, Ipçi Y, Cetinel S, Cikler E, Gedik N, et al. Protective effects of taurine against nicotine-induced oxidative damage of rat urinary bladder and kidney. Pharmacology. 2005;74:37-44. Song Y, Hu T, Gao H, Zhai J, Gong J, Zhang Y, et al. Altered metabolic profiles and biomarkers associated with astragaloside IV-mediated protection against cisplatin-induced acute kidney injury in rats: An HPLC-TOF/MS-based untargeted metabolomics study. Biochem Pharmacol. 2021;183:114299. Tan Z, Guo F, Huang Z, Xia Z, Liu J, Tao S, et al. Pharmacological and genetic inhibition of fatty acid-binding protein 4 alleviated cisplatin-induced acute kidney injury. Journal of cellular and molecular medicine. 2019;23:6260-70. Tao YG, Huang XF, Wang JY, Kang MR, Wang LJ, Xian SX. Exploring Molecular Mechanism of Huangqi in Treating Heart Failure Using Network Pharmacology. Evidence-based complementary and alternative medicine : eCAM. 2020;2020:6473745. Tchounwou PB, Dasari S, Noubissi FK, Ray P, Kumar S. Advances in Our Understanding of the Molecular Mechanisms of Action of Cisplatin in Cancer Therapy. Journal of experimental pharmacology. 2021;13:303-28. Terzi F, Ciftci MK. Protective effect of silymarin on tacrolimus-induced kidney and liver toxicity. BMC complementary medicine and therapies. 2022;22:331. Tran MT, Zsengeller ZK, Berg AH, Khankin EV, Bhasin MK, Kim W, et al. PGC1α drives NAD biosynthesis linking oxidative metabolism to renal protection. Nature. 2016;531:528-32. Walter P, Ron D. The unfolded protein response: from stress pathway to homeostatic regulation. Science (New York, NY). 2011;334:1081-6. Xiang Z, Sun H, Cai X, Chen D, Zheng X. The study on the material basis and the mechanism for anti-renal interstitial fibrosis efficacy of rhubarb through integration of metabonomics and network pharmacology. Molecular bioSystems. 2015;11:1067-78. Xu Z, Tang M, Li Y, Liu F, Li X, Dai R. Antioxidant properties of Du-zhong (Eucommia ulmoides Oliv.) extracts and their effects on color stability and lipid oxidation of raw pork patties. Journal of agricultural and food chemistry. 2010;58:7289-96. Zhang J, Zhang Y, He Y, Du T, Shan D, Fan H, et al. Metabolome and transcriptome integration reveals insights into the process of delayed petal abscission in rose by STS. Frontiers in plant science. 2022;13:1045270. Zhang L, Gu Y, Li H, Cao H, Liu B, Zhang H, et al. Daphnetin protects against cisplatin-induced nephrotoxicity by inhibiting inflammatory and oxidative response. International immunopharmacology. 2018;65:402-7. Zhang Y, Wei Z, Yang M, Liu D, Pan M, Wu C, et al. Dietary taurine modulates hepatic oxidative status, ER stress and inflammation in juvenile turbot (Scophthalmus maximus L.) fed high carbohydrate diets. Fish & shellfish immunology. 2021;109:1-11. Zhao SB, Huang SY, Pan Z, Peng XM, Peng XJ, Wu SJ, et al. [The compatibility of coix leaves and epimedium against fatigue and hypoxia tolerance]. Zhongguo ying yong sheng li xue za zhi = Zhongguo yingyong shenglixue zazhi = Chinese journal of applied physiology. 2021;37:510-3. Zheng Y, Shi X, Hou J, Gao S, Chao Y, Ding J, et al. Integrating metabolomics and network pharmacology to explore Rhizoma Coptidis extracts against sepsis-associated acute kidney injury. Journal of chromatography B, Analytical technologies in the biomedical and life sciences. 2021;1164:122525. Zhu MQ, Sun RC. Eucommia ulmoides Oliver: A Potential Feedstock for Bioactive Products. Journal of agricultural and food chemistry. 2018;66:5433-8. Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Table1.docx Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-3917893","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":274396908,"identity":"467ce7e3-008a-4713-93cf-4bf0c86bc521","order_by":0,"name":"Kexin Lin","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Kexin","middleName":"","lastName":"Lin","suffix":""},{"id":274396909,"identity":"5dd43471-6e18-453a-82b0-a327ec2dc899","order_by":1,"name":"Lijuan Xiong","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lijuan","middleName":"","lastName":"Xiong","suffix":""},{"id":274396910,"identity":"9af57976-15b9-4053-94eb-ada0ec087f98","order_by":2,"name":"Wen Zhang","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Zhang","suffix":""},{"id":274396911,"identity":"7feae643-de02-4783-b414-9b203164ab56","order_by":3,"name":"xuan Chen","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"xuan","middleName":"","lastName":"Chen","suffix":""},{"id":274396912,"identity":"a6d05209-39dd-4b50-9ad2-ce3ee4bea20f","order_by":4,"name":"Xiaofei li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIie3QvQrCMBDA8ZPAucS6Vvx6hUhAF/FZIkKfoZsFIS7iLvgQBV/gSkGXPoDi4mJdXNwcFK26icS6OeS/HIT7wREAm+0PcxgAATQAi8HzoRB8I/giEpBTXvIaEsBVeUmRt+jsi4ZTOewXHLr1kFi6Mx+GKpokQmJVeVsOngwJO8JMGFFJX/q6qpYZifshcXTNpBBEVy2GuhLpjNzyEEZxSQuFLsOMUB6CKq4loqW5h5u5GMhZjG0jKZcTeTr6otkcr9L10e/Vp6tRaiRvPb6K/bBvs9lsts/dAWW3QXle2BFeAAAAAElFTkSuQmCC","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":true,"prefix":"","firstName":"Xiaofei","middleName":"","lastName":"li","suffix":""},{"id":274396913,"identity":"d8759a4b-b969-4de5-9657-1c3eb9c3133c","order_by":5,"name":"Jianyong Zhang","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jianyong","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-02-01 15:16:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3917893/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3917893/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51654882,"identity":"1654871f-805e-4d8c-943a-146c929b56b0","added_by":"auto","created_at":"2024-02-26 16:43:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":43320,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC chromatogram of standard (A) and EUL (B), 1: chlorogenic acid, 2: astragalinstandard.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3917893/v1/d4f4f87a5b9683e3fd5df677.png"},{"id":51654884,"identity":"cebd38ba-cf50-491c-aac5-95204e4fbc76","added_by":"auto","created_at":"2024-02-26 16:43:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2578878,"visible":true,"origin":"","legend":"\u003cp\u003eEUL therapy against CP-induced AKI. Body weight changes (A), Kindey index (B), BUN(C), SCr (D) (n=8), H\u0026amp;E staining in kidney sections with magnification × 200/400 (E) (n=3) (1: inflammatory\u0026nbsp;cell\u0026nbsp;infiltration, 2: \u0026nbsp;tubular\u0026nbsp;necrosis, 3: tubules containing proteinaceous casts, 4: vacuolar degeneration), Levels of ER stress maker proteins PERK, IRE1α, GRP78, ATF6, ATF4, and CHOP, were assessed using western blotting (F) (n=6). ( mean ± SEM, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs CP,\u0026nbsp; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs Control by One-way ANOVA).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3917893/v1/3da409ea800f91c8290b3d5a.png"},{"id":51654891,"identity":"3a421708-a977-4cc7-9008-71f1dfad7753","added_by":"auto","created_at":"2024-02-26 16:43:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":285248,"visible":true,"origin":"","legend":"\u003cp\u003eThe PCA scores in ESI+ modes (A) and in ESI- modes (B), and PLS-DA scores metabolic profiling of kidney samples in ESI+ modes (C) and in ESI- modes (D) among Control, CP, and CP + EUL 200 mg/kg groups (n=7).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3917893/v1/25539307c17c54d5fb5c3846.png"},{"id":51654887,"identity":"5891f180-165e-47fc-9f3f-f9a1288476cf","added_by":"auto","created_at":"2024-02-26 16:43:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":700663,"visible":true,"origin":"","legend":"\u003cp\u003eCP disturbed the metabolism in the kidney. OPLS-DA plot and 200 times permutation test in ESI+ modes (A) and in ESI- modes (B), heatmap of 87 differential metabolites (C), volcano plot (D) among Control, CP, and EUL 200 mg/kg groups, 10 disorder metabolic pathways in the kidney of AKI mice (E). (1: Riboflavin metabolism, 2: Lysine degradation, 3: Taurine and hypotaurine metabolism, 4: Nicotinate and nicotinamide metabolism, 5: Glycerophospholipid metabolism, 6: Tryptophan metabolism, 7:Arginine biosynthesis, 8: Pyrimidine metabolism, 9: Steroid hormone biosynthesis, 10: Selenocompound metabolism).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3917893/v1/81bd4c0ddbce1fa8abea7eba.png"},{"id":51654885,"identity":"a3449074-da4c-4d6a-b9d3-8d685e7194e2","added_by":"auto","created_at":"2024-02-26 16:43:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":493246,"visible":true,"origin":"","legend":"\u003cp\u003eKidney metabolism analysis of EUL in CP-induced AKI. OPLS-DA plot and 200 times permutation test in ESI+ modes (A) and in ESI- modes (B), heatmap of 31 differential metabolites (C), volcano plot (D) among Control, CP, and EUL 200 mg/kg groups, 3 metabolic pathways in the kidney regulated by EUL(E).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3917893/v1/f39a78eb64aaa8379036d602.png"},{"id":51654889,"identity":"f29ff63e-ffab-4a4b-942b-ca3e5c3a606c","added_by":"auto","created_at":"2024-02-26 16:43:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":699463,"visible":true,"origin":"","legend":"\u003cp\u003eKidney levels of identified biomarkers (A) among Control, CP, and EUL 200 mg/kg groups, and their correlations with kidney function indicators (B, BUN; C, SCr). Four metabolites were significantly negatively correlated with BUN (B)(\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) and SCr (C) (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). (n = 7, mean ± SEM, #\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05,##\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01 vs Control, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs CP by One-way ANOVA).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3917893/v1/51685374d002af66b0da242f.png"},{"id":51654888,"identity":"21c0604c-398f-4eb0-a32e-0a0665498346","added_by":"auto","created_at":"2024-02-26 16:43:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":192818,"visible":true,"origin":"","legend":"\u003cp\u003eDiagram of the possible mechanism by which EUL treatment improves CP-induced AKI. An increase in the EUL group compared with the CP group was marked in red. The decrease in the EUL group compared with the CP group was marked in blue.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3917893/v1/012e0fc1d8f2d786d1591466.png"},{"id":53158970,"identity":"fcb5bac9-c8a2-46d0-8d54-01b0e7c6c643","added_by":"auto","created_at":"2024-03-21 10:26:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2486312,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3917893/v1/49d7ef24-4b10-4b68-8f1f-d4f207f1574d.pdf"},{"id":51654883,"identity":"baae728e-0d45-4f72-89ce-9a626d9d2260","added_by":"auto","created_at":"2024-02-26 16:43:56","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":36453,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-3917893/v1/04cf763eff6c58f416146708.docx"},{"id":51654890,"identity":"4bfefe59-a899-480a-a481-04ae64cef720","added_by":"auto","created_at":"2024-02-26 16:43:58","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":97234,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3917893/v1/3a842f8aabcadc233cd05f30.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Eucommia ulmoides leaf extract attenuates cisplatin-induced kidney injury in mice through endoplasmic reticulum stress and biometabolic mechanism","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCisplatin (CP), a platinum compound, has been used extensively as a chemotherapeutic drug in treating various cancers, including ovarian, testicular, lung, non-small cell, cervical, and bladder cancers(Holditch et al., 2019, Tchounwou et al., 2021). However, its clinical utility is hampered by severe nephrotoxicity(Almaghrabi, 2015), with 30% of patients suffering acute kidney injury (AKI)(Pabla and Dong, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Current clinical approaches mainly involve the administration of fluids, diuretics, and dehydrating agents to mitigate CP-induced kidney injury(Chen et al., 2022). Nonetheless, these therapeutic strategies have drawbacks, including delayed onset of efficacy, disruption of water-electrolyte balance, acid-base equilibrium, and increased cardiac burden(Catalanotto et al., 2016, Lu and Rothenberg, 2018). Thus, there is an imperative need for research into new therapeutic interventions to prevent CP-induced AKI.\u003c/p\u003e \u003cp\u003eNatural medicines have attracted increased interest in recent years for their potential role in the prevention and treatment of AKI(Chen et al., 2018). Traditional Chinese medicine (TCM), including angelica sinensis and astragaloside IV, has been extensively studied to treat and prevent CP-induced AKI (Bunel et al., 2015, Song et al., 2021). \u003cem\u003eEucommia ulmoides\u003c/em\u003e leaf (EUL), a by-product of Eucommia ulmoides, is considered both a food and a medicinal herb. It contains a variety of bioactive components, including chlorogenic acid and geniposidic acid (He et al., 2014, Ren et al., 2022). EUL is known for its health benefits in nourishing the liver and kidneys(Zhao et al., 2021), anti-endoplasmic reticulum (ER) stress(Lee et al., 2013), anti-inflammatory(Xu et al., 2010), and anti-hypertensive effects(Luo et al., 2010)according to TCM theory(Zhu and Sun, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Moreover, previous studies have also shown the renoprotective role of EUL in a rat model of hyperuricemic kidney injury induced by intragastric adenine(Li et al., 2021). However, the mechanisms underlying the protective effects of EUL against CP-induced AKI warrant further investigation.\u003c/p\u003e \u003cp\u003eThe multi-component and multi-target characteristics of TCM pose challenges in elucidating their mechanisms using conventional pharmacological methods(Tao et al., 2020). Metabolomics, a system biology technology, offers comprehensive insights into global metabolic changes and has emerged as a powerful tool for understanding the metabolic alterations and pharmacological mechanisms of TCM in AKI treatment(Cui et al., 2018). Now, metabolomics has emerged as a potent tool for elucidating the intrinsic metabolic alterations and pharmacological mechanisms of TCM in treating AKI(Xiang et al., 2015, Zheng et al., 2021). Therefore, employing metabolomic analysis could be an effective approach to investigate the efficacy of pharmaceutical interventions against AKI.\u003c/p\u003e \u003cp\u003eOur study examined the protective effects of EUL by inhibiting ER stress on a CP-induced AKI I mouse model. Additionally, Our study utilized UPLC/QTOF-MS to investigate how EUL affects metabolic changes associated with CP-induced AKI in mice. We identified key metabolites linked to the therapeutic effects of EUL using multivariate analysis. Furthermore, we enriched the metabolic pathways to elucidate the underlying biometabolic mechanisms. In conclusion, our investigation offers new insights into the mechanisms through which EUL acts on CP-induced AKI, potentially advancing its traditional application.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cp\u003e2. 1 Material\u003c/p\u003e \u003cp\u003eCP was sourced from Qilu Pharmaceutical, China. Chlorogenic acid was obtained from Solaibao Life Science, China, and astragalin from Solaibao Life Science Co., Ltd., China. The BCA kit used for protein quantification was from Jian Cheng Bioengineering Institute, China. Primary antibodies used included protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1α (IRE1α), glucose-regulated protein-78 (GRP78), activating transcription factor 6 (ATF6), activating transcription factor 4 (ATF4), and C/EBP-homologous protein (CHOP) from CST, USA. GAPDH antibody was obtained from HuaAn, China.\u003c/p\u003e \u003cp\u003e2. 2. Preparation of EUL extract and quality analysis\u003c/p\u003e \u003cp\u003eFermented EUL was procured from Zhangjiajie Cha Kun Yuan Biotechnology Development Co., Ltd and identified by Prof. Jianyong Zhang from the College of Pharmacy of Zunyi Medical University.\u003c/p\u003e \u003cp\u003eThe EUL was extracted as follows: 100 g of EUL was dissolved in 1 L of 70% ethanol, refluxed at 90\u0026deg;C for 2 hours, and then filtered. The filter residue underwent two more identical extraction cycles. The filtrates from the two batches were mixed and concentrated into an extract, which was subsequently oven-dried at 80\u0026deg;C overnight and milled into powder. The chemical composition of the EUL extract, including chlorogenic acid and astragalin, was quantified using an LC-20A HPLC system (Shimazu, Kyoto, Japan). Separation was carried out on an Agilent Eclipse Plus C18 column (150 \u0026times; 4.6 mm, 3.5 \u0026micro;m; Agilent, USA) at 25\u0026deg;C. The mobile phase consisted of solvent A (0.4% Formic acid in water) and solvent B (MeCN) with a linear gradient elution program (5%-13% B at 0\u0026ndash;15 min; 13% \u0026ndash; 25% B at 15\u0026ndash;25 min; 25% \u0026ndash; 50% B at 25\u0026ndash;25.5 min; 50% B at 25.5\u0026ndash;30 min). Detection was carried out at a wavelength of 327 nm, with a sample injection volume of 20 \u0026micro;L. The content of chlorogenic acid and astragalin (%) was calculated based on peak area.\u003c/p\u003e \u003cp\u003e2.3. Animal experiment, design, and drug administration\u003c/p\u003e \u003cp\u003eHealthy male ICR mice (30\u0026ndash;32 g) were obtained from Vital River Laboratories (Beijing, China) and kept under specific pathogen-free conditions in a controlled environment with regulated temperature and humidity. Forty-eight mice, after a week of acclimatization, were divided into four equal groups: normal Control group (NC), CP group (CP), and CP\u0026thinsp;+\u0026thinsp;EUL (100, 200 mg/kg) groups. NC group: Mice received normal saline for seven consecutive days, with a single intraperitoneal (i.p.) injection of saline on the 3rd day. CP group: Mice received CP (20 mg/kg) diluted with normal saline as a single i.p. injection. CP\u0026thinsp;+\u0026thinsp;EUL groups: Mice were given EUL (100, 200 mg/kg) orally daily for seven consecutive days, with a single i.p. injection of CP (20 mg/kg) on the 3rd day. The weight of the mice was recorded every two days. At the end of the experiment, blood samples were obtained by eye enucleation. The mice were sacrificed by decapitation, and the kidneys were immediately harvested. The left kidney of mice was removed and weighed to calculate the kidney index as follows: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\text{K}\\text{i}\\text{d}\\text{n}\\text{e}\\text{y} \\text{i}\\text{n}\\text{d}\\text{e}\\text{x}=\\frac{\\text{k}\\text{i}\\text{d}\\text{n}\\text{e}\\text{y} \\text{w}\\text{e}\\text{i}\\text{g}\\text{h}\\text{t}}{\\text{b}\\text{o}\\text{d}\\text{y} \\text{w}\\text{e}\\text{i}\\text{g}\\text{h}\\text{t}} \\times 100\\text{%}\\)\u003c/span\u003e\u003c/span\u003e. The right kidney was placed in 4% paraformaldehyde through a coronal incision, and the rest of the kidney was stored in a refrigerator at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent metabolomics analysis. The animal experiment was conducted following approval by the animal ethics committee of Zunyi Medical University (Zunyi, China, ZMU21-2304-009) and adhered to the Helsinki Declaration.\u003c/p\u003e \u003cp\u003e2. 4. Serum detection in mice\u003c/p\u003e \u003cp\u003eMice serum samples were removed from the refrigerator testing. Blood urea nitrogen (BUN) and serum creatinine (SCr) of mice serum samples were measured using biochemical assay kits on an automatic biochemical analyzer (Beckman, USA) to evaluate kidney function.\u003c/p\u003e \u003cp\u003e2. 5. Kidney histological examination in mice\u003c/p\u003e \u003cp\u003eKidney tissue samples were collected and fixed in 4% neutral paraformaldehyde for 24 hours, embedded in paraffin, and cut into 4 \u0026micro;m thin slices. These slices were stained with hematoxylin and eosin (H\u0026amp;E) and examined using a light microscope (OLYMPUS, BX43F, Japan).\u003c/p\u003e \u003cp\u003e2. 6. Western blot analysis of ER stress-related proteins\u003c/p\u003e \u003cp\u003eTotal proteins were extracted from kidney tissues with RIPA lysis buffer and quantified using a BCA kit (Jian Cheng Bioengineering Institute, China). Proteins were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% nonfat milk for 1 hour, membranes were incubated overnight at 4\u0026deg;C with primary antibodies: PERK (1:1000), IRE 1α (1:1000), GRP78 (1:1000), ATF6 (1:1000), ATF4 (1:1000) and CHOP (1:1000). The relative expression of proteins was normalized to GAPDH (1:5000) (HuaAn, China), and Image lab software 6.1 was used for band analysis.\u003c/p\u003e \u003cp\u003e2. 7. \u003cb\u003eKidney metabolomics analysis in mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003e2.7.1. Sample preparation\u003c/p\u003e \u003cp\u003eKidney samples were thawed slowly at 4\u0026deg;C before being weighed 20 mg was added to a prechilled methanol/acetonitrile/water solution (2:2:1, v/v), vortex mixed, and sonicated at low temperature for 30 min. The supernatant was dried under vacuum and reconstituted in 100 \u0026micro;L of aqueous acetonitrile (acetonitrile: water\u0026thinsp;=\u0026thinsp;1:1, v/v) for mass spectrometry analysis. The supernatant was centrifuged at 14,000 \u0026times; g for 15 minutes at 4\u0026deg;C before analysis. A quality control (QC) sample was prepared by mixing equal volumes (50 \u0026micro;L) of each test sample to estimate system reliability and repeatability during the analysis process.\u003c/p\u003e \u003cp\u003e2.7.2. LC-MS analytical conditions\u003c/p\u003e \u003cp\u003eSupernatant separation was performed using an Agilent 1290 Infinity LC Ultra High-Performance Liquid Chromatography (UHPLC) system. The parameters were as described below: a HILIC column (ACQUITY UPLC BEH Amide column, 2.1 x 100 mm, 1.7 \u0026micro;m); column temperature at 25\u0026deg;C; flow rate at 0.5 mL/min; injection volume of 2 \u0026micro;L. The mobile phase comprised 25 mM ammonium acetate 25 m, M ammonia water (A) and acetonitrile (B). The gradient elution program was set as follows: 0-0.5 min, 95% B; 0.5-7 min, B linearly decreased from 95\u0026ndash;65%; 7\u0026ndash;8 min, B linearly decreased from 65\u0026ndash;40%; 8\u0026ndash;9 min, B maintained at 40%; 9-9.1 min, B linearly changes increased from 40\u0026ndash;95%; 9.1-1min, B was maintained at 95%. Samples were maintained at 4\u0026deg;C throughout the analysis procedure. Mass spectrometry analysis was a Triple TOF 6600 mass spectrometer (AB SCIEX) with positive and negative ion modes of electrospray ionization (ESI) for detection. ESI source settings were: atomizing gas auxiliary heating gas 1 (Gas1) at 60, auxiliary heating gas 2 (Gas2) at 60, curtain gas (CUR) at 30 psi, ion source temperature at 600\u0026deg;C, and spray voltage (ISVF) at \u0026plusmn;\u0026thinsp;5500 V (positive and negative modes). The primary mass-to-charge ratio detection range was 60-1000 Da, and the secondary sub-ion mass-to-charge ratio detection range was 25-1000 Da. Primary mass spectrometry scanning accumulated for 0.20 s/spectrum, and secondary mass spectrometry scanning accumulated for 0.05 s/spectrum. Data-dependent acquisition mode (IDA) and peak intensity value screening mode were employed for secondary mass spectrometry. De-clustering voltage (DP) was set at \u0026plusmn;\u0026thinsp;60 V (positive and negative modes), and collision energy at 35\u0026thinsp;\u0026plusmn;\u0026thinsp;15 eV (positive and negative modes). IDA settings included dynamic exclusion of isotope ions within a 4 Da range, with 10 fragment spectra collected per sweep.\u003c/p\u003e \u003cp\u003e2.7.3. Metabolic data processing\u003c/p\u003e \u003cp\u003eData preprocessing included filtering out data with less than 80% completeness, keeping RSD values below 30% within the same sample group, filling missing values in the same sample group with mean values (Zhang et al., 2022), and subsequent normalization. The processed three-dimensional data matrix included sample descriptions, normalized peak areas, and retention time-m/z pairs for multivariate analysis. This data was imported into SIMCA-P 14.0 (Umetrics AB, Umea, Sweden) for multivariate data analysis. Principal component analysis (PCA) and partial least-squared discrimination analysis (PLS-DA) were utilized to visualize overall differences.\u003c/p\u003e \u003cp\u003eOrthogonal projection to latent structure-discriminant analysis (OPLS-DA) was employed to confirm the model and identify different metabolites between groups. Criteria for screening differential metabolites included VIP\u0026thinsp;\u0026gt;\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and FC\u0026thinsp;\u0026gt;\u0026thinsp;1.2 or FC\u0026thinsp;\u0026lt;\u0026thinsp;0.833(Liu et al., 2023). Differential metabolite identification was initially performed using an in-house database (Shanghai Applied Protein Technology). Subsequently, differential metabolites were further identified based on UPLC-QTOF-MS/MS data by comparing with online databases, including 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), KEGG (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.kegg.com\u003c/span\u003e\u003cspan address=\"http://www.kegg.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and PubMed (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Pathway analysis of the differential metabolites was conducted using the online analytical tool MetaboAnalyst 14.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.metaboanalyst.ca\u003c/span\u003e\u003cspan address=\"http://www.metaboanalyst.ca\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to elucidate biological interpretation.\u003c/p\u003e \u003cp\u003e2. 8. Statistical analysis\u003c/p\u003e \u003cp\u003eData were presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical analyses were conducted using SPSS 29.0 software. Multiple group data were statistically analyzed by one-way analysis of variance (ANOVA). \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (marked as \u003csup\u003e#\u003c/sup\u003e or *) was considered statistically significant, while \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (marked as \u003csup\u003e##\u003c/sup\u003e or **) was considered highly statistically significant.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1 Quality control analysis of EUL\u003c/p\u003e \u003cp\u003eThe chlorogenic acid and astragalin in the EUL extract were identified with HPLC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The peak area and corresponding concentrations of the chlorogenic acid and astragalin standard were used to construct the standard curve chlorogenic acid was represented by y\u0026thinsp;=\u0026thinsp;30.033x\u0026thinsp;+\u0026thinsp;54.308, where R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9993, and for astragalin, it was y\u0026thinsp;=\u0026thinsp;37.281x \u0026minus;\u0026thinsp;40.354, where R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9999 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These results indicate a strong linear relationship between the peak area and chlorogenic acid and astragalin concentrations. The chlorogenic acid and astragalin content in EUL was determined to be 3.9% and 0.047%, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3. 2. EUL improved CP-induced AKI\u003c/p\u003e \u003cp\u003eBUN and SCr levels are widely acknowledged markers of kidney dysfunction\u003csup\u003e(Zhang et al., 2018)\u003c/sup\u003e. To assess the impact of EUL on kidney function following CP intervention, \u003cem\u003ein vivo\u003c/em\u003e experiments were conducted in mice. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, CP injection at 72 hours led to a markedly decrease the body weight while the kidney index was attenuated compared to the Control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, this reduction was partially restored by treatment with 100 mg/kg or 200 mg/kg EUL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), suggesting that EUL treatment significantly inhibited increased serum levels of BUN and SCr induced by CP (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3. 3. EUL ameliorated CP-induced histopathological injury in mice.\u003c/p\u003e \u003cp\u003eTo examine the protective impact of EUL in CP-treated mice, we evaluated alterations in kidney histology using H\u0026amp;E staining. In the Control group, kidney histology displayed a normal architecture with intact tubular and glomerular (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In the CP group, significant pathological changes were observed, including tubular disorganization, glomerular atrophy, and vascular degeneration. Compared with the CP group, the EUL 100 mg/kg group showed improvement in kidney damage, although few tubular exhibited proteinaceous material. In the 200 mg/kg EUL group, kidney damage was significantly alleviated.\u003c/p\u003e \u003cp\u003e3. 4. EUL inhibited CP-induced ER stress in mice\u003c/p\u003e \u003cp\u003eTo further examine the protective effects of EUL, the protein expression levels of PERK, IRE1α, GRP78, ATF6, ATF4, and CHOP, which are involved in ER stress, were examined. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF the expression levels of these ER stress-related proteins were significantly increased in the CP group compared to the Control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, the administration of EUL at both dosage levels significantly decreased these alterations in protein expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e3. 5. Metabolomics analysis\u003c/p\u003e \u003cp\u003e3. 5. 1. Multivariate statistical analysis\u003c/p\u003e \u003cp\u003eTo investigate the potential effects of EUL on CP-induced AKI, differential metabolites in kidney samples from the CP and CP\u0026thinsp;+\u0026thinsp;EUL 200 mg/kg groups were identified and subjected to multivariate statistical analyses, including PCA and PLS-DA. PCA score plots demonstrated clear separation among kidney samples in all three groups, indicating significant metabolic changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Subsequently, PLS-DA, a supervised statistical approach, exhibited a strong distinction between the CP and Control groups, with the CP\u0026thinsp;+\u0026thinsp;EUL 200 mg/kg group showing the closest similarity to the Control group compared to all the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These results suggest that EUL can mitigate CP-induced alterations in kidney metabolism.\u003c/p\u003e \u003cp\u003e3. 5. 2. Metabolic pathway analysis\u003c/p\u003e \u003cp\u003eOPLS-DA models were established to screen for differential metabolites. To evaluate the potential errors of the OPLS-DA models, 200 permutation tests were performed where the Q\u003csup\u003e2\u003c/sup\u003e values on the left were lower than the original points on the right, confirming the validity of the OPLS-DA models (Fig. 4A and 5A). A total of 87 differential metabolites were identified when comparing the CP group to the Control group (Fig. 4C) (VIP \u0026gt; 1.0, P \u0026lt; 0.05, FC \u0026gt; 1.2, or FC \u0026lt; 0.833(He et al. , 2023). Among these, 31 differential metabolites were found to be regulated towards normal levels by EUL 200 mg/kg treatment, including 13 metabolites that showed upregulation and 18 metabolites that showed downregulation compared to the CP group (Fig.5C, Table 1). Additionally, a clustering heatmap\u0026nbsp;(Fig. 4B and 5B) \u0026nbsp;was utilized to visually represent changes in the differential metabolites among the three groups, further substantiating that EUL may ameliorate CP-induced AKI by modulating disruptions in these metabolic pathways. To obtain further insights into the therapeutic mechanisms of EUL at the metabolic level, the 31 differential metabolites from\u0026nbsp;Table 1\u0026nbsp;were subjected to pathway topology analysis (Fig. 5D). This analysis revealed the involvement of pathways such as taurine and hypotaurine metabolism, lysine degradation, and steroid hormone biosynthesis (impact \u0026gt; 0.1). Our results showed that some biomarkers such as 2-Oxoadipic acid, L-2-Aminoadipate, Hypotaurine, and 17alpha-hydroxyprogesterone significantly decreased in CP-induced AKI (Figure. 6A). According to the correlative analyses (Figure. 6B and 6C), four biomarkers were significantly and negatively correlated With both BUN and SCr (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Particularly, the EUL administration remarkably up-regulated the level of these biomarkers toward normal.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eCP is a commonly applied chemotherapeutic agent for the treatment of malignant tumors (Hong et al., 2020). However, in clinical practice, nearly 30% of patients develop AKI due to CP administration (Jiang et al., 2018, Lameire et al., 2013). Therefore, finding ways to prevent CP-induced AKI has become an urgent priority. Traditional Chinese Medicine (TCM), known for its diverse biological activities, has been extensively employed in treating AKI and its associated complications (Li et al., 2019). Our study demonstrated that pretreatment with EUL significantly attenuated CP-induced AKI, employing a combination of traditional pharmacodynamic and metabolomics analyses. Figure\u0026nbsp;8 shows the full-text mechanism diagram of this study.\u003c/p\u003e \u003cp\u003e4.1 Traditional pharmacodynamic evaluation\u003c/p\u003e \u003cp\u003eIn our study, histopathological examination and the kidney index revealed characteristic features of CP-induced AKI, including inflammatory cell infiltration, tubular necrosis, and glomerular atrophy. Additionally, elevated levels of BUN and SCr, important indicators of kidney function evaluation (Terzi and Ciftci, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), were observed in the CP group. Importantly, pretreatment with EUL significantly ameliorated kidney histopathological damage and reduced the extent of kidney injury compared to the CP group.\u003c/p\u003e \u003cp\u003eEmerging findings indicate that ER stress plays a crucial role in both the physiological and pathological processes of AKI(Tan et al., 2019). Prolonged ER stress can trigger apoptotic pathways, ultimately leading to renal cytotoxicity through three classical pathways: the PERK-eIF2α-ATF4-CHOP, IRE1α-XBP1, and ATF6 pathways (Ajoolabady et al., 2023, Walter and Ron, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Our findings indicate that the administration of EUL resulted in a significant decrease in the expression of PERK, IRE1α, GRP78, ATF6, ATF4, and CHOP in kidney tissues from CP-induced AKI mice, suggesting that EUL may have a potential therapeutic effect in reducing the ER stress response in the context of AKI.\u003c/p\u003e \u003cp\u003e4.2 Biometabolic mechanisms analysis of EUL for treating AKI\u003c/p\u003e \u003cp\u003eTo unravel the underlying metabolic perturbations associated with CP-induced AKI and the potential kidney protective effect of EUL, we employed untargeted metabolomics to characterize differences among the Control, CP, and EUL groups. From a metabolic profile perspective, the samples from the Control, CP, and EUL groups exhibited distinct separation, with the EUL group displaying a clustering pattern closer to the Control group. Subsequently, we identified and analyzed 31 differential metabolites and found that three dysregulated metabolic pathways\u0026mdash;taurine and hypotaurine metabolism, lysine degradation, and steroid hormone biosynthesis\u0026mdash;were significantly affected by EUL treatment. These results imply that EUL may improve CP-induced AKI by regulating these metabolic disorders.\u003c/p\u003e \u003cp\u003e4.2.1 \u003cem\u003eTaurine and hypotaurine metabolism\u003c/em\u003e\u003c/p\u003e \u003cp\u003eIn the present study, hypotaurine emerged as a critical metabolic biomarker in taurine and hypotaurine metabolism. Hypotaurine, a sulfur-containing organic acid abundant in tissues, plays a crucial role in kidney protection by regulating intracellular osmotic pressure and concentrations of reactive oxygen species (Jakaria et al., 2019, Sener et al., 2005). Increasing evidence suggests that ER stress, oxidative stress, and inflammation are central to the development of nephrotoxicity(Inagi et al., 2005, Lahmar et al., 2020). Importantly, taurine, a metabolite of hypotaurine, exerts significant anti-ER stress, antioxidant, and anti-inflammatory effects, thereby protecting against CP, 5-fluorouracil, and diabetes-induced kidney injury (Neog et al., 2019, Zhang et al., 2021). The level of hypotaurine decreased in the CP-induced AKI mice and was increased by pretreatment with EUL. These results suggest that EUL may decrease AKI-associated ER stress, oxidative stress, and inflammation, thereby preventing CP-induced AKI.\u003c/p\u003e \u003cp\u003e4.2.1 \u003cem\u003eLysine metabolism\u003c/em\u003e\u003c/p\u003e \u003cp\u003eIn our study, 2-oxoadipate and L-2-Aminoadipate were identified as important metabolic biomarkers associated with lysine degradation. Lysine is crucial in providing energy and essential substances for the body and maintaining homeostasis(Kawasaki et al., 2000, N. et al., 1979). Consistent with our research, other studies have demonstrated that lysine degradation is closely linked to diabetic nephropathy (Li et al., 2022). 2-Oxoadipate is catalyzed to acetyl-CoA, which enters the tricarboxylic acid cycle for energy production in mitochondria(Jordan et al., 2019). The kidneys rely on mitochondrial oxidative phosphorylation to generate significant amounts of ATP, facilitating blood filtration and toxin elimination (Tran et al., 2016). In our study, the levels of 2-Oxoadipate and L-2-Aminoadipate decreased in AKI mice and were downregulated by EUL pretreatment. These results suggest that EUL can correct energy metabolism disorders induced by CP in the kidneys, thereby playing a protective role in CP-induced AKI.\u003c/p\u003e \u003cp\u003e4.2.1 \u003cem\u003eSteroid hormone biosynthesis\u003c/em\u003e\u003c/p\u003e \u003cp\u003eIn our study, 17 alpha-hydroxyprogesterone emerged as an important metabolic biomarker associated with steroid hormone biosynthesis. Steroid hormones regulate various physiological processes related to growth and development, and their reduced levels can disrupt the hypothalamus-pituitary-adrenal (HPA) axis, leading to kidney damage(Annane et al., 2017, Laulh\u0026eacute; et al., 2021). Previous studies have demonstrated that 17alpha-hydroxyprogesterone possesses pro-inflammatory properties and can increase plasma TNF-α expression levels in non-pregnant women exposed to LPS(Amory et al., 2005). In this study, the level of 17alpha-hydroxyprogesterone was significantly decreased in AKI mice and increased following EUL pretreatment. These results suggest that the decrease in 17alpha-hydroxyprogesterone observed after AKI could be part of a self-protective mechanism, and EUL might help restore normal levels.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, based on traditional pharmacology and functional metabolomics strategy, we demonstrated that EUL exhibits a protective effect against AKI, as evidenced by improvements in BUN and SCr levels, amelioration of histopathological damage, and attenuation of ER stress. Furthermore, metabolomics analysis indicated that EUL treatment significantly reversed abnormal levels of 31 metabolites, particularly those involved in taurine and hypotaurine metabolism, lysine degradation, and steroid hormone biosynthesis. These findings can be concluded that EUL may mitigate the effects of CP-induced AKI, and provide a theoretical basis for its potential clinical use.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data and materials of the study can be obtained from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSchool of Basic Medicine, Zunyi Medical University, Zunyi, 563000, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKexin Lin \u0026nbsp;, Xuan Chen, Xiaofei Li\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSchool of Pharmacy and Key Laboratory of Basic Pharmacology Ministry Education, Joint International Research Laboratory of Ethnomedicine Ministry of Education, Zunyi Medical University, Zunyi, 563000, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLijuan Xiong, Xuan Chen, Jianyong Zhang\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKX Lin\u0026nbsp;and LJ Xiong contributed equally to this work. KX Lin and JY Zhang wrote and modified the manuscript. LJ Xiong and XF Li designed the experiment. LJ Xiong, W Zhang, and X Chen completed the experiments. KX Lin constructed the illustration. KX Lin and JY Zhang revised and approved the final version of the manuscript. All authors confirmed the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Xiaofei Li ([email protected]) or Jianyong Zhang ([email protected]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal experiments were conducted in accordance with the requirements of the Guizhou Provincial Experimental Animal Management Committee and were approved by the Ethical Committee of Zunyi Medical University. (Ethics\u0026nbsp;approval\u0026nbsp;number: ZMU21-2304-009).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing financial interests that could have appeared to influence this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was partially supported by the National Natural Science Foundation of China (82060754,81803838 and 82260812), Key project at central government level: The ability establishment of sustainable use for valuable Chinese medicine resources (2060302), Guizhou Provincial Science \u0026amp; Technology Program ([2020]1Y376, ZK[2021]532, ZK[2022]615,YQK[2023]038), Science and Technology Department of Zunyi city of Guizhou province of China (HZ（2022）420, HZ-[2020]-39,ZYK[2021]-3,[2020]-7), Science and technology project of Guizhou health and Health Committee (gzwkj2021-441), Guizhou Provincial Special research project on science and technology of traditional Chinese medicine and ethnic medicine (QZYY-2021-035), Science and Technology Department of Honghuagang District of Zunyi city of Guizhou province of China ([2020]-17), the Zunyi city of Guizhou Provincial Department of health outstanding young medical talents fund ([2021]-3), Science and Technology Department of Zunyi city of Guizhou province of China ([2020]-7).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublisher\u0026apos;s Note\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAjoolabady A, Kaplowitz N, Lebeaupin C, Kroemer G, Kaufman RJ, Malhi H, et al. 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Journal of agricultural and food chemistry. 2018;66:5433-8.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cisplatin, Eucommia ulmoides leaf, Acute kidney injury, Endoplasmic reticulum stress, Metabolomics","lastPublishedDoi":"10.21203/rs.3.rs-3917893/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3917893/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCisplatin (CP) is a widely utilized anticancer drug but is associated with significant side effects, notably acute kidney injury (AKI). \u003cem\u003eEucommia ulmoides\u003c/em\u003e leaf (EUL), a valuable Chinese herbal remedy, is known for its renoprotective properties. However, the function and underlying pathways of EUL in AKI therapy have remained largely unexplored. This research aimed to elucidate the protective roles of EUL in an AKI mouse model through biochemical assays, and histopathological andexaminations while also investigating the underlying mechanisms via endoplasmic reticulum (ER) stress-related protein expression analysis and metabolomics. The findings demonstrated that pretreatment with orally administered EUL significantly reduced blood urea nitrogen (BUN) and serum creatinine (SCr) levels, ameliorated CP-induced kidney histopathological injuries, and attenuated CP-induced ER stress by reducing the protein expressions of PERK, IRE 1α, GRP78, ATF6, ATF4, and CHOP. Additionally, metabolomics analysis identified 31 significant differential metabolites affected by EUL treatment in AKI mice, impacting pathways related to taurine and hypotaurine metabolism, lysine degradation, and steroid hormone biosynthesis. These findings suggested that EUL could offer valuable insights for potential CP-induced AKI treatment strategies.\u003c/p\u003e","manuscriptTitle":"Eucommia ulmoides leaf extract attenuates cisplatin-induced kidney injury in mice through endoplasmic reticulum stress and biometabolic mechanism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-26 16:43:50","doi":"10.21203/rs.3.rs-3917893/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"109a55df-75f6-498d-9f72-095e5f144968","owner":[],"postedDate":"February 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-21T10:18:44+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-26 16:43:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3917893","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3917893","identity":"rs-3917893","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}