Gut Microbial Metabolism of Cinnabarinic Acid Promotes to the Role of Pseudolaric Acid B Against Metabolic Dysfunction-Associated Fatty Liver Disease

Gut Microbial Metabolism of Cinnabarinic Acid Promotes to the Role of Pseudolaric Acid B Against Metabolic Dysfunction-Associated Fatty Liver Disease | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 14 May 2025 V1 Latest version Share on Gut Microbial Metabolism of Cinnabarinic Acid Promotes to the Role of Pseudolaric Acid B Against Metabolic Dysfunction-Associated Fatty Liver Disease Authors : Tingyu Song , Xiufang Yang , Junyi Wang , Lianhong Yin , Xuerong Zhao , Ning Wang , Youwei Xu , Yan Qi , Cunquan Xiong , and L.N. Xu [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174721901.11057373/v1 205 views 141 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background and Purpose: Metabolic dysfunction-associated fatty liver disease (MAFLD) pathogenesis involves gut microbiota dysbiosis. This study investigated pseudolaric acid B (PAB), a diterpenoid from Pseudolarix kaempferi, for its therapeutic potential in high-fat diet-induced MAFLD mice. Experimental Approach: The effects of PAB on MAFLD were evaluated in a high-fat diet -induced C57BL/6J mouse model. The regulatory impact of PAB on the gut microbiota and metabolites was explored by 16s rRNA sequencing and metabolomics analysis. Fecal microbiota transplantation experiments further validated these results. Key Results: PAB treatment significantly improved body weight, liver index, serum biochemical indices, and histopathological damage in MAFLD mice. Gut microbiota analysis revealed PAB significantly reduced g_Faecalibaculum, g_Allobaculum, g_Ileibacterium, and g_Dubosiella abundance. Metabolomic profiling demonstrated PAB treatment increased the level of the microbiota-derived tryptophan metabolite, cinnabarinic acid (CA). The CA content was negatively correlated with the abundance of the four bacteria identified above. Fecal microbiota transplantation validated gut microbiota’s causal role in PAB’s therapeutic effects. Mechanistically, PAB activated the aryl hydrocarbon receptor (AhR)— CA’s target receptor— subsequently upregulating IL-22 expression and triggering JAK1/STAT3 signaling. This cascade suppressed key fatty acid synthesis regulators: SREBP-1c, ELOVL6, Acc, Fasn, and Scd1 at both mRNA and proten levels. Conclusion and Implications: PAB as a promising prebiotic agent against MAFLD through gut microbiota modulation, CA/AhR/IL-22 axis activation, and inhibition of hepatic lipogenesis pathways. The study elucidates a novel mechanism where microbial metabolite CA mediates PAB’s hepatoprotective effects via AhR — dependent signaling. Gut Microbial Metabolism of Cinnabarinic Acid Promotes to the Role of Pseudolaric Acid B Against Metabolic Dysfunction-Associated Fatty Liver Disease Ting-yu Song 1 , Xiu-fang Yang 1 , Jun-yi Wang 1 , Lian-hong Yin 1 , Xue-rong Zhao 1 , Ning Wang 1 , You-wei Xu 1 , Yan Qi 1 , Cun-quan Xiong 2, * , Li-na Xu 1, * 1 College of Pharmacy, Dalian Medical University, Western 9 Lvshunnan Road, Dalian 116044, China 2 Jiangsu Medical College, Jiefang South Road, Yancheng 224005, China * Correspondence: Lina Xu, College of Pharmacy , Dalian Medical University , Dalian, China Email: [email protected] Cunquan Xiong, Jiangsu Medical College , Yancheng, China Email: [email protected] ABSTRACT Background and Purpose: Metabolic dysfunction-associated fatty liver disease (MAFLD) pathogenesis involves gut microbiota dysbiosis. This study investigated pseudolaric acid B (PAB), a diterpenoid from Pseudolarix kaempferi, for its therapeutic potential in high-fat diet-induced MAFLD mice. Experimental Approach: The effects of PAB on MAFLD were evaluated in a high-fat diet -induced C57BL/6J mouse model. The regulatory impact of PAB on the gut microbiota and metabolites was explored by 16s rRNA sequencing and metabolomics analysis. Fecal microbiota transplantation experiments further validated these results. Key Results: PAB treatment significantly improved body weight, liver index, serum biochemical indices, and histopathological damage in MAFLD mice. Gut microbiota analysis revealed PAB significantly reduced g _Faecalibaculum , g _Allobaculum , g _Ileibacterium , and g _Dubosiella abundance. Metabolomic profiling demonstrated PAB treatment increased the level of the microbiota-derived tryptophan metabolite, cinnabarinic acid (CA). The CA content was negatively correlated with the abundance of the four bacteria identified above. Fecal microbiota transplantation validated gut microbiota’s causal role in PAB’s therapeutic effects. Mechanistically, PAB activated the aryl hydrocarbon receptor (AhR)— CA’s target receptor— subsequently upregulating IL-22 expression and triggering JAK1/STAT3 signaling. This cascade suppressed key fatty acid synthesis regulators: SREBP-1c, ELOVL6, Acc, Fasn, and Scd1 at both mRNA and proten levels. Conclusion and Implications: PAB as a promising prebiotic agent against MAFLD through gut microbiota modulation, CA/AhR/IL-22 axis activation, and inhibition of hepatic lipogenesis pathways. The study elucidates a novel mechanism where microbial metabolite CA mediates PAB’s hepatoprotective effects via AhR — dependent signaling. Keywords : Gut microbiota, Metabolic dysfunction-associated fatty liver disease, Pseudolaric acid B, Tryptophan metabolism, Aryl hydrocarbon receptor not-yet-known not-yet-known not-yet-known unknown Abbreviations Meaning 16S rRNA 16S ribosomal RNA AhR aryl hydrocarbon receptor ALT alanine aminotransferase AST aspartate aminotransferase BCA bicinchoninic acid assay CA cinnabarinic acid CKD chronic kidney disease CVD cardiovascular disease DAPI 4’, 6-diaminidine 2-phenylindole ECL enhanced chemiluminescence reagents FMT fecal microbiota transplantation GC-MS gas chromatography-mass spectrometry HDL-C high-density lippoprotein cholesterol H&E Hematoxylin-Eosin HFD high-fat diet IDO indoleamine-2,3-dioxygenase JAK Janus kinase KEGG kyoto encyclopedia of genes and genomes LC-MS liquid chromatograph-mass spectrometer LDL-C low-density lipoprotein cholesterol LEfSe linear discriminant analysis effect size MAFLD Metabolic dysfunction-associated fatty liver disease MRM multi-reaction detection NFFA nonesterified free fatty acids PAB pseudolaric acid B PCA principal component analysis SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis STAT signal transduction and transcriptional activator T2DM type 2 diabetes mellitus TC total cholesterol TG triglyceride Trp tryptophan UHPLC-MS ultra-high performance liquid chromatography-mass spectrometry 1. INTRODUCTION Metabolic dysfunction-associated fatty liver disease (MAFLD) is one of the most common liver diseases, previously konwn as non-alcoholic fatty liver disease (NAFLD), which was officially renamed in 2020 by various international academic organizations, including the International Liver Disease Association (Eslam et al., 2020). The incidence and prevalence of MAFLD is rapidly increasing worldwide, with a global prevalence of approximately 30%, growing in parallel with obesity and diabetes (Byrne et al., 2015). Recent studies have shown that pioglitazone and other thiazolidinediones may be promising drugs for the treatment of MAFLD, but these medications were associated with a series of adverse effects, such as weight gain, fluid retention, and consequently aggravation of heart failure (Ji et al., 2019). Therefore, it is urgent to find safe, effective and low toxic drugs to treat MAFLD. It has been reported that there are more microorganisms in the gut than in human cells, and these microorganisms are involved in metabolism, immunity and disease processes in the host body, and are important regulatory factors of health (Hui et al., 2024). Increasing evidence has defined a potential causal relationship between dysregulation of the gut flora and the development of MAFLD. Due to the presence of the gut-liver axis, the imbalance of intestinal bacteria has become a risk factor for the development of MAFLD (Albillos et al., 2020). In-depth investigations of the mechanism have revealed that changes in gut microorganisms that cause abnormalities in their metabolites are participate in the pathogenesis of MAFLD. For example, a high-fat diets (HFD) diet feeding exhausts Desulfovibrio vulgaris in mice (Hong et al., 2021). The production of its metabolite, short-chain fatty acids (SCFAs), inhibits fatty acid synthesis and stimulates the β-oxidation of fatty acids, thereby promoting MAFLD (Chen et al., 2021). Therefore, regulating the composition and function of the microbiome is essential for the improvement of disease. Tryptophan (Trp) is an essential amino acid that is vital for human health and plays a key role in a variety of physiological processes (Roager et al., 2018). The gut microbiota plays an important role in the metabolism of Trp and is able to convert it into a variety of molecules, such as indoles and their derivatives (Fiore et al., 2021). These metabolites contribute to the maintenance of intestinal homeostasis by regulating the balance of pro- and anti-inflammatory cytokines (Wojciech et al., 2023). An increasing number of researches have revealed the potential role of dysregulated Trp metabolism in liver-related diseases (Yang et al., 2023), suggesting that gut microbes homeostasis is critical for healthy liver. It has been indicated that Trp metabolites reverse intestinal microbiota disorders and alleviate liver damage caused by HFD. Indole-3-propionic acid (IPA) has been shown to inhibit liver inflammation and attenuate diet-induced NASH phenotype by inhibiting the production of endotoxins in the gut (Agus et al., 2018), and indole-3-acetic acid (IAA) can alleviate the development of MAFLD by attenuating liver lipogenesis, inflammatory response and oxidative stress (Basran et al., 2011). Trp could be used as a therapeutic target for many diseases, such as digestive system tumors, acute liver injury, cirrhosis and hepatic encephalopathy (Tsuji er al., 2023; Platten et al., 2019). Due to the lack of approved therapeutics for MAFLD, exploration of potentially active precursors from natural products has emerged as an effective way for therapy of MAFLD (Harvey et al., 2015). Pseudolaric acid B (PAB), a diterpenoid compound found mainly in the bark of Pseudolarix amabilis . PAB has a wide range of biological activities and has been verified to have anti-tumor, anti-fertility, anti-angiogenesis and anti-fungal effects (Liu et al., 2012; Wang et al., 2015; Tan et al., 2004; Liu et al., 2017). It can also improve the deterioration of fungal keratitis by inhibiting inflammation and reducing fungal load (Liu et al., 2023). In liver-related diseases, PAB triggers apoptosis through activation of the AMPK/JNK/DRP1 mitochondrial fission pathway in hepatocellular carcinoma (Yin et al., 2023). PAB also inhibits hepatitis B virus secretion through apoptosis and cell cycle arrest (Liu et al., 2024). However, the effect of PAB on MAFLD is still unknown, as well as whether it can ameliorate abnormality in hepatic fatty acid metabolism in MAFLD by modulating gut microbiota has not been systematically studied. In this study, we established a mouse MAFLD model by feeding HFD to evaluate the effect of PAB against MAFLD. Then, 16S rRNA sequencing, untargeted metabolomics, targeted metabolomics of tyrosine, and fecal microbiota transplantation (FMT) were employed to reveal the possible mechanism of PAB. This study could provide a theoretical basis for PAB as a potential agent for the treatment of MAFLD in the future. 2. METHODS not-yet-known not-yet-known not-yet-known unknown 2.1 Animals and treatment Male C57BL/6J mice (18-22 g) were purchased from Liaoning Changsheng Biotechnology Co., LTD. (SCXK: 2020-0001). All procedures followed the National Research Council Guidelines and were approved by the Animal Care and Use Committee of Dalian Medical University (AEE21009). Refer to the Supplementary data for more details. 2.2 Ultrasound detection One week before execution, anesthetized mice were fixed supine, and liver ultrasound images were captured using a high-resolution imaging system (Vinno, Suzhou, China). 2.3 Histopathological examination Liver tissues were collected, washed with cold saline, and fixed in 4% paraformaldehyde. Paraffin-embedded sections were stained with H&E, while OCT-embedded frozen sections were stained with Oil Red O. Images were acquired using fluorescence microscopy (Zeiss, Germany). 2.4 Serum biochemical analysis Serum levels of total cholesterol (TC), triglyceride (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), high-density lippoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and nonesterified free fatty acids (NFFA) were measured using commercial kits (Jiancheng Biotechnology, Nanjing, China). 2.5 Immunofluorescence assay Dewaxed liver sections were incubated overnight at 4°C with primary antibodies against AhR and IL-22 (1:500, Affinity Biosciences, China). After washing, sections were incubated with HRP-conjugated secondary antibodies at 4°C for 2 h, followed by DAPI staining (1:100) for 30 min. Fluorescence images were captured using an inverted microscope. not-yet-known not-yet-known not-yet-known unknown 2.6 Real-time PCR analysis Total RNA was extracted (RNAiso Plus, TransGen Biotech, Beijing, China) and analyzed using qRT-PCR (TransScript® Green One-Step qRT-PCR SuperMix, TransGen Biotech). Gene expression was normalized to GAPDH using the 2-ΔΔCt method. Primer sequences are listed in Supplementary Table S1. not-yet-known not-yet-known not-yet-known unknown 2.7 Western blotting assay Total liver protein was extracted, quantified (BCA Kit, SEVEN Innovation Biotechnology, Beijing, China), and separated by SDS-PAGE. After transfer to PVDF membranes (Millipore, USA), proteins were blocked, incubated with primary and secondary antibodies, and visualized via ECL using the Bio-Spectrum Gel Imaging System (UVP, California, USA). Tubulin was used as an internal control. Tubulin was used as an internal control. 2.8 16S ribosomal RNA (16S rRNA) sequencing Fecal samples from the control, model, and PAB (20 mg/kg) groups were collected, snap-frozen in liquid nitrogen, and stored at -80°C. Refer to the Supplementary data for more details. 2.9 Fecal microbiota transplantation (FMT) The procedure of FMT is based on previous research with some modifications (Zhao et al., 2021). Refer to the Supplementary data for more details. 2.10 Untargeted metabolomics analysis Metabolic extracts from fecal samples were prepared as previously described (Hui et al., 2024). Refer to the Supplementary data for more details. not-yet-known not-yet-known not-yet-known unknown 2.11 Targeted metabolomics analysis of Trp pathways Targeted metabolomics of tryptophan (Trp) in fecal samples was conducted using an ExionLC™ AD UPLC-QTRAP 6500+ MS (AB SCIEX, USA) with an HSS T3 column (2.1 × 150 mm, 1.7 μm, Waters, USA). Refer to the Supplementary data for more details. 2.12 Statistical analysis Data are presented as mean ± SD. Statistical analysis was performed using GraphPad Prism 10.0. One-way ANOVA was used for multiple group comparisons, with significance set at **p < 0.01 or *p < 0.05. 3. RESULTS 3.1 PAB attenuates HFD-induced MAFLD progress in mice The liver tissue of a randomly selected mouse from each group was photographed. Compared with the control group, the mouse in model group were significantly obese with whitish and mottled livers, which were significantly improved by the administration of PAB (Fig. 1A). Furthermore, mice in the model group showed a significant increase in body weight and liver index compared with control group, which were improved and decreased markedly by the intervention of PAB (10 mg/kg, 15mg/kg and 20mg/kg) (Fig. 1B-C). Compared with the model group, PAB significantly reduced the levels of AST and ALT (Fig. 1D), which are used to assess liver function and injury, and significantly reduced serum levels of TC, TG, TC/HDL, LDL and FFA (Fig. 1E). The changes in the liver of mice were observed by small animal ultrasonography (Fig. 1F). Under two-dimensional ultrasound, compared with the control group, mice in the model group had enlarged liver, diffuse and dense hepatic parenchyma, unclear intrahepatic ductal structures, fine dot-like echoes with high brightness, diffusely enhanced near-field echoes, and gradually attenuated far-field echoes. In PAB groups, the intrahepatic ductal structures were clearly defined and the far-field echoes were gradually enhanced. H&E staining observed changes in the structure of diseased tissues and cell morphology. Compared with the control group, the hepatocytes in the model group were obviously swollen and loosely arranged, the nuclei were shifted by the fusion of lipid droplets and the hepatocytes appeared to be degenerated (Fig. 1G). In PAB groups, the degree of liver steatosis was significantly improved and the histopathological score was decreased (Fig. 1G). Oil red O staining indicates steatosis and abnormal lipid deposits in tissues and organs. Mice in the model group showed obvious lipid droplet aggregation and deposition in hepatocytes, and those phenomena were significantly improved and the histopathological score was decreased in PAB groups (Fig. 1H). 3.2 PAB regulates the composition of intestinal flora in HFD-induced MAFLD mice We analyzed total DNA in mouse fecal samples by gut microbiota 16S rRNA sequencing to assess the effect of PAB on gut flora composition in MAFLD mice. According to the principal component analysis (PCA), samples of control, model and PAB group showed good separation in general, indicating that the microbiota composition differed within the three groups (Fig. 2A). The composition of the intestinal flora was revealed by Alpha diversity analysis. Based on the ACE, Sobs and Shannon indices, the results indicated a high community richness and the PAB treatment did not significantly alter the richness and diversity in the fecal samples (Supplementary Fig. S1). The results of the abundance analysis at the genus level show that unclassified_f__ Lachnospiraceae was mainly enriched in the fecal samples of mice in Model group and PAB group, while norank_f__ Muribaculaceae dominated in the Control group (Fig. 2B). The cluster analysis results at the genus level showed that compared with the Model group, PAB reverses the genera g_ Dubosiell , g_ Romboutsia and g_ Ileibacterium (Fig. 2C). The histogram analysis of differential bacterial genera (Fig. 2D-E) showed that among the top 10 differential genera in terms of abundance, PAB significantly down-regulated the abundance of the genera g _Allobaculum, g _Dubosiella, g _Faecalibaculum and g_Ileibacterium , and significantly up-regulated the abundance of g _Helicobacter and g _Odoribacter , as compared with those in Model group. Linear discriminant analysis Effect Size (LEfSe) analysis (Fig. 2F) allows for simultaneous statistical tests and difference analysis at different classification levels, analyzing the differences in community composition of different groups, and screening out the marker microbiomes that show the same performance in different groups. The increase in the abundance of Firmicutes and the decrease in the abundance of Bacteroides are indicators of obesity (Supplementary Fig. S2). Fig. 2F shows that g _Alloprevotella and g _Helicobacter were the dominant bacteria genera in the Control group, while g _Allobaculum, g _Faecalibaculum, g _Dubosiella and g _Ileibacterium were the dominant bacteria genera in the Model group. According to the results of the evolutionary branching diagram in Fig. 2G, g _Allobaculum , g _Faecalibaculum and g _Ileibacterium belong to o _Erysipelotrichales at the order level. Meanwhile, the abundance of Odoribacter at the generic level, which is thought to be critical for intestinal homeostasis, decreased in the Model group and recovered after PAB was administered (Supplementary Fig. S3). The dominant species in the Control group mainly contain genera such as g _Prevotellaceae_UCG-001, g _Alloprevotella, g _Rikenella and other genera. After the administration of PAB, the abundance of genera such as g _Colidextribacter, g _Acetatifactor, g _GCA900066575 and g _Anaerovorx increased significantly (Supplementary Fig. S4). Our findings of 16S rRNA sequencing confirmed that PAB remodeled the compositin of the gut microbiota and mitigated the dysbiosis caused by HFD feeding. not-yet-known not-yet-known not-yet-known unknown 3.3 FMT with PAB treated HFD mice improves MAFLD in HFD-induced mice A FMT experiment was conducted to investigate whether PAB improves MAFLD by regulating intestinal flora. The HFD-induced mice transplanted with fecal microbiota from model mice (Model-FMT group) appeared to be significantly obese with whitish and mottled livers compared with the Control-FMT group, which were significantly improved by the transplantation of fecal microbiota from HFD-induced mice with PAB treatment (PAB-FMT group). A randomly selected mouse and its liver tissue are shown in Fig. 3A. HFD-induced mice treated with PAB-FMT showed a marked decline in body weight and liver index compared with mice in Model-FMT group (Fig. 3B-C). Meanwhile, PAB-FMT significantly reduced the serum levels of AST and ALT in HFD-induced mice (Fig. 3D), and markedly decreased the serum levels of TC, TG, TC/HDL, LDL and FFA in HFD-induced mice compared with the Model-FMT group (Fig. 3E). In addition, under two-dimensional ultrasound, compared with Control-FMT group, the liver of mice in the Model-FMT group showd a diffuse and dense parenchyma as well as unclear intrahepatic ductal structures, which were notably improved in PAB-FMT group (Fig. 3F). In Fig. 3G, the liver was severely damaged in the Model-FMT group, while the pathologic changes and histopathological score were significantly improved in the PAB-FMT group. Although the PAB-FMT group showed improvement, steatosis was still present in Fig. 3H, compared with the Control group, the Model-FMT group mice showed significant lipid droplet aggregation and deposition in hepatocytes, whereas mice in the PAB-FMT group showed significantly improved lipid droplet aggregation and deposition in hepatocytes and Oil red O staining showed a significant reduction in fat deposition. Collectively, these results suggested that FMT could ameliorates high-fat diet-induced MAFLD and alleviate liver injury and hepatic steatosis in mice. not-yet-known not-yet-known not-yet-known unknown 3.4 PAB regulates metabolic levels in MAFLD mice To further investigate, we determined non-targeted metabolomics analysis of fecal samples. The PCA analysis showed (Fig. 4A) that the high-fat diet-fed Model and PAB groups had most of the overlap and showed an overall trend of aggregation, indicating that the types and levels of metabolites were similar in the three samples. The Venn diagram visualizes the similarities and differences of metabolites between the different samples. We identified a total of 1394 metabolites, of which 1102 metabolites were common to the three groups and 107 metabolites were common to the Model and PAB groups (Supplementary Fig. S5). As shown in Fig. 4B, there were 206 significantly up-regulated (dots in red) and 298 significantly down-regulated metabolites (dots in blue) in the Model group, compared with Control group (Supplementary Table S2). In PAB group, 43 significantly up-regulated (dots in red) and 47 significantly down-regulated metabolites (dots in blue) were found in PAB group, compared with Model group (Supplementary Table S3). According to the results of OPLS-DA analysis of the three groups, the top 10 differential metabolites contributing to pairwise comparisons were ranked according to VIP scores. Five of these metabolites were overlapping (Supplementary Fig. S6). The results of the differential metabolite clustering analysis (Fig. 4C) show the trends of differential metabolites in fecal samples from different groups. The analysis showed that among the top 100 metabolites, 38 metabolites were down-regulated in the PAB group compared to the model group. In Fig. 4D, KEGG pathway enrichment analysis revealed that the top 5 pathways altered by PAB-egulated metabolites were biosynthesis of terpenoids and steroids, tryptophan metabolism, biosynthesis of phenylpropanoids, pyruvate metabolism and glycolysis/gluconeogenesis. Among them, tryptophan metabolism pathway was most affected by PAB regulation. Fig. 4E showes that PAB significantly increased the levels of metabolites such as L-formyl kynurenine, and decreased the level of L-tryptophan in mice compared to the Model group. KEGG enrichment analysis identified all biological pathways involved in differential expression of metabolites (detailed in Supplementary Fig. S7). Combined with cluster analysis and pathway enrichment analysis, tryptophan metabolic pathway was selected as the target metabolic object for subsequent biological experiment verification or mechanism study. 3.5 PAB modulates cinnabarinic acid to improve MAFLD and is correlated to gut microbes The function of Trp metabolism was further investigated to explore the effects of PAB. According to the cluster analysis and histogram statistical results of the absolute content of differential metabolites (Fig. 5A-B), the levels of cinnabarinic acid (CA), kynurenic acid, 3-indolepropionic acid and indole-3-Iactic acid in Model group decreased significantly compared with the Control group. Among these metabolites, only the level of CA was markedly reversed after PAB treatment (Fig. 5A-B). Fig. 5C shows the metabolic process of Trp. Trp is first metabolized to L-formylkynurenine, which is then converted to L-Kynurenine by formamidases, followed by the conversion of L-Kynurenine to 3-hydroxyanthranilic acid catalyzed by Kynurenine-3-monooxygenase, and finally generates CA, the end product of Trp metabolic pathway. The results of Trp-targeted metabolomics showed that the metabolic catabolism of Trp was significantly enhanced by PAB treatment, noting a significant increase in the amount of CA in mouse feces. This finding suggests that CA may be critical for the function of PAB. We performed a joint analysis of differential abundance bacteria and metabolites, and the results revealed a correlation between bacteria and metabolite abundance in the Trp pathway (Fig. 5D). We found that there was a significant negative correlation between the level of CA and the abundance of g _Allobaculum, g _Faecalibaculum, g _Dubosiella and g _Ileibacterium . These results suggest that the effect of PAB on MAFLD may be related to the abundance of g _Allobaculum, g _Faecalibaculum, g _Dubosiella and g _Ileibacterium , which can affect the Trp metabolic pathway and increases the correlation of CA concentration. Therefore, our study not only revealed the effect of PAB on Trp metabolism, but also pointed out the possibility of CA as a potential biomarker and therapeutic target in MAFLD. 3.6 PAB elevates the expressions of AhR and IL-22 in MAFLD mice As an AhR agonist, CA can activate the AhR pathway to regulate immune responses. AhR regulates IL-22, while plays an important role in intestinal barrier, tissue repair and immunomodulation (Keir et al., 2020). Therefore, detecting the levels of AhR and IL-22 can reveal the mechanisms of CA in ameliorating metabolic diseases, such as MAFLD. We examined the expression levels of AhR and IL-22 in the liver tissues of mice using immunofluorescence and Western blot. Fig. 6A shows that the fluorescence intensity of AhR and IL-22 was significantly reduced in the liver tissues of the model mice compared with the control mice, indicating that the expressions of the two proteins were suppressed in MAFLD. In contrast, the fluorescence intensity of AhR and IL-22 was significantly elevated by PAB treatment (Fig. 6A). This result was also confirmed by further Western blot assay (Fig. 6A). Notably, after PAB treatment, AhR and IL-22 protein expression were increased significantly in the liver of mice with MAFLD. These results suggest that PAB may improve MAFLD by up-regulating the expression of AhR and IL-22. 3.7 PAB regulates the expression of AhR and IL-22 by modulating gut microbiota The levels of AhR and IL-22 in the liver tissue of mice with FMT were detected by immunofluorescence and Western blotting (Fig. 7A-B). Immunofluorescence staining revealed that the fluorescence intensity of AhR and IL-22 was significantly lower in the Model-FMT group compared to the Control-FMT group. In contrast, PAB-FMT intervention significantly increased the fluorescence intensity of AhR and IL-22 in the Model-FMT group (Fig. 7A). In parallel, Western blotting analysis revealed a decrease in the expression of both AhR and IL-22 in the Model-FMT group compared to the Control-FMT. However, their expression levels were increased markedly after PAB-FMT intervention, further supporting the notion that PAB treatment positively modulates the levels of AhR and IL-22 in the liver. These findings suggest that PAB affects CA by modulating the intestinal microbiota, thereby regulating the expressions of AhR and IL-22. not-yet-known not-yet-known not-yet-known unknown 3.8 PAB restores CA metabolism to promote AhR/IL-22-mediated fatty acid synthesis in MAFLD In vivo validation of the levels of IL-22 downstream proteins in the JAK1/STAT3 pathway showed that the levels of p-JAK1, JAK1, p-STAT3, and STAT3 were significantly down-regulated in the Model group compared to the Control group (Fig. 8A). This indicates that activation of the JAK1/STAT3 signaling pathway was impaired in the MAFLD model. However, in the PAB-treated group, the expressions of these proteins were significantly up-regulated, suggesting that PAB intervention effectively restored the metabolic levels of CA and activated the JAK1/STAT3 pathway. Additionally, PAB treatment significantly inhibited the expression of lipid synthesis-related proteins and genes that are downstream of the JAK1/STAT3 pathway. In the Model group, the expression levels of SREBP-1c and ELOVL6 were notably increased, while PAB intervention dramatically reduced the expressions of SREBP-1c and ELOVL6 (Fig. 8A). Correspondingly, compared to the Model group, the mRNA levels of genes related to de novo lipogenesis were significantly decreased in the PAB-treated group, including Fasn, Acc, and Scd1 (Fig. 8B). 3.9 PAB regulates gut microbiota to modulate AhR/IL-22-mediated lipid synthesis In Fig. 9A, the levels of p-JAK1, JAK1, p-STAT3 and STAT3 were significantly down-regulated in the Model-FMT group. However, after PAB-FMT intervention, the expressions of these proteins were markedly up-regulated, suggesting that PAB-FMT effectively restored the activation of the JAK1/STAT3 signaling pathway, potentially improving liver function. In addition, PAB-FMT also reduced the levels of SREBP-1c and ELOVL6 compared with Model-FMT group (Fig. 9A). Compared with the Model-FMT group, the mRNA levels of Fasn, Acc and Scd1 were significantly decreased in the PAB-FMT group (Fig. 9B). These results confirmed that PAB promotes CA production by modulating gut microbiota, and activating AhR/IL-22 to inhibit fatty acid synthesis, which subsequently attenuates MAFLD effectively. 4. DISCUSSION MAFLD is a metabolic stress liver injury, and its pathogenesis has gradually evolved from the ”two-strike theory” to the current ”multi-strike theory”, which believes that insulin resistance, mitochondrial dysfunction, gut microbiota and other factors are involved in the progression of MAFLD. Many studies have found that MAFLD increases the risk of developing type 2 diabetes mellitus (T2DM), cardiovascular disease (CVD) and chronic kidney disease (CKD) (Byrne et al., 2020). Therefore, seeking effective therapeutic agents for MAFLD in natural products and elucidating their mechanism play an integral role in the prevention and treatment of MAFLD. PAB showed antitumor activity in hepatocellular carcinoma by triggering cell apoptosis (Liu et al., 2023). Besides, PAB has been reported to exert a protective effect against atherosclerosis by inhibiting macrophage-mediated inflammatory response and cellular ox-LDL uptake, and promoting cholesterol efflux by suppressing NF-κB activation PPARγ-dependently (Li et al., 2018). This study aimed to explore the effect of PAB on MAFLD and investigate the underlying mechanism. The HFD-induced mice were utilized as the model of MAFLD with excessive hepatic lipid accumulation in this stuy. By orally administering of PAB to HFD-induced mice, the serum and hepatic lipids were dramatically declined, including the serum levels of TC, TG, TC/HDL, LDL and FFA, and the accumulation and deposition of fat droplets in liver evaluated by oil red O staining. It has been specifically emphasized that the most predictive lipid factor is the TC/HDL ratio, and changes in this ratio are an important reflection of risk degree of dyslipidemia (Criqui et al., 1998). Moreover, ultrasonography showed that the liver echoes were enhanced in the Model group, presenting a brighter image and suggesting excessive fat accumulation in the liver. In MAFLD, the overabundance of fatty acids in the liver can trigger liver injury by resulting in oxidative stress and inflammation (Pafili et al., 2021). Our study indicated that PAB markedly reduced AST and ALT levels, which are considered as the typical markers of liver damage. Additinally, H&E results showed that the degeneration of nuclei and inflammatory response due to lipid droplet fusion was significantly improved by PAB. Besides, the elevated lipogenesis is a critical process in the lipid metabolic dysfunction in MAFLD, which cause inhibiting de no synthesis of fatty acid become a central therapeutic target for MAFLD (Badmus et al., 2022). SREBP-1c is a transcription factor that primarily promotes factors associated with TG and fatty acid. ELOVL6 is a fatty acid elongase involved in the formation of long-chain fatty acids. SREBP-1c directly regulates the transcriptional activity of Elovl6 by binding to the SRE-1 and SRE-2 sites on its promoter (Han et al., 2015). In addition, SREBP-1c promotes fatty acid synthesis by activating downstream fatty acid synthase genes including FAS, ACC, and SCD1 (Matsuzaka et al., 2009), wihch are key enzymes in fatty acid synthesis by regulating fatty acid metabolism and influencing energy storage and fat accumulation (Ren et al., 2024). After administration of PAB, the expressions of SREBP-1c and ELOVL6 were reduced, which in turn inhibited the mRNA levles of FAS, ACC and SCD1, inhibited fatty acid synthesis, and thus reduced lipid accumulation in the liver. These results suggest that PAB could inhibit lipogenesis and alleviate hepatic impairment, and thus function as a therapeutic natural product for the treatment of MAFLD. More and more evidence shows that gut microbial dysbiosis alters host metabolism and has serious negative effects on various metabolic diseases, especially MAFLD (Aron-Wisnewsky et al., 2020). The gut microbiota lives mainly in the intestines, digesting and absorbing nutrients from food and producing metabolites (e.g., short-chain fatty acids and Trp, etc.). These metabolites enter the bloodstream through the liver-gut axis, then affects metabolic processes in the liver and is involved in the pathgenesis of MAFLD. In clinical trials, the abundance of beneficial bacteria, such as Faecalibacterium and Akkermansia , was significantly reduced in MAFLD patients, while the abundance of harmful bacteria was increased, such as Staphylococcaceae . This reveals a close association between MAFLD and the severity of gut microbial dysbiosis (Pan et al., 2021). Some natural products have been reported to alleviate MAFLD by modulating gut microbial diversity. Berberine, a natural alkaloid with anti-obesity and hepatoprotective properties, alleviates MAFLD by increasing the abundance of Clostridiales , Lactobacillaceae and Bacteroidales , modulating the intestinal flora and mediating intestinal FXR active tion (Shu et al.,2021). In our study, 16S rRNA sequencing showed that the abundance of the genera g_ Faecalibaculum,g_Allobaculum,g_Ileibacterium and g_ Dubosiella increased significantly and became the dominant species in the Model group as the course of MAFLD progressed. And all four genera belong to o_Erysipelotrichales at the level of order and c_Clostridia at the level of class. The related functions of these genera have been reported. g_ Faecalibaculum regulates intestinal integrity in diabetic mice by modulating tight junction protein expression (Min et al., 2014). g_ Allobaculum is thought to be associated with aging, high-fat diets, and fatty acid metabolism (van Muijlwijk et al., 2021). g_ Ileibacterium has been linked to obesity and intestinal inflammation through the production of IL-17, which may promote tumorigenesis (Fu et al., 2023). g_ Dubosiella produces butyrate which plays a protective role in sepsis-associated brain injury (Zhang et al., 2022). Meanwhile, several bacterial community sequencing studies on MAFLD have indicated that the abundance of g_ Faecalibaculum, g _Allobaculum, g _Ileibacterium and g_ Dubosiella were elevated in obese mice, and the inhibition of these characteristic bacteria would be extremelly effective in the tmanagement of MAFLD (Luo et al., 2022; Yu et al., 2022; Gu et al., 2022; Zhang et al., 2024). Administration of PAB reversed the abundance of these genera, suggesting an inhibitory effect of PAB on o_Erysipelotrichales/c_Clostridia -like bacteria is beneficial in treating MAFLD. FMT regulates the imbalance of intestinal flora to achieve therapeutic or adjunctive therapeutic effects on intestinal and extraintestinal diseases (Zhao et al., 2021), and a growing number of recent researches have reported that FMT could be useful in treating diseases such as metabolic diseases (Aron-Wisnewsky et al., 2019). FMT experiments confirmed that a citrus polymethoxyflavone-rich extract (PMFE) ameliorated MAFLD through significantly increasing the abundance of Bacteroides ovatus (Zeng et al., 2020). To confirm that the effect of PAB on gut microbial abundance is one of its key mechanisms for the therapy of MAFLD, the FMT assay was performed to confirm that asministration of PAB-treated gut microbiota to recipient mice was sufficient to protect the mice from HFD-induced MAFLD. Our results indicated that the gut microbiota influenced by PAB could improve lipid metabolism abnormalities, liver damage, and other MAFLD-related pathological and physiological indicators in mice. Collectively, we confirmed the ability of PAB to regulate the intestinal flora and to re-establish an intestinal micro-ecosystem with normal functioning, thereby improving MAFLD. Notably, one of the primary modalities by which the gut microbiota regulates health and disease is through metabolites produced by microbiota, which are small molecules that could diffuse from their initial intestinal location to affect local and systemic functions. The KEGG database presents metabolic pathways from multiple organisms. The biosynthesis of terpenoids and steroids pathway forms a series of terpenoid compounds and steroid hormones, which in turn regulate plant growth or animal metabolic processes (Chan et al., 2023). The biosynthesis of phenylpropanoids is a class of metabolites derived from phenylalanine, widely found in plants, helping them resist environmental stress (Zhang et al., 2015). Pyruvate metabolism is a core metabolic process in cells, and abnormalities in pyruvate metabolism can lead to the development of cancer and neurodegenerative diseases (Gray et al., 2014). The Trp metabolism pathway is involved in the synthesis of neurotransmitters and bioactive molecules, and it was found to play an important role in the improvement of MAFLD by PAB. In our untargeted metabolomics assay, L-formylkynurenine, kynurenic acid and 5-hydroxyindoleacetic acid metabolism levels were significantly down-regulated in the Model group, while L-tryptophan was significantly up-regulated. The L-formylkynurenine metabolism, which is the first product of Trp decomposition catalyzed by heme dioxygenases, wad significantly inhanced after administration of PAB (Basran et al., 2011). Trp metabolism has important functions and plays an important role in tissue metabolism, growth, maintenance and repair. It has been found that kynurenic acid attenuates MAFLD by increasing AMPK phosphorylation and expression of oxygen-regulated protein 150, which in turn reduces lipid accumulation and down-regulated the expression of adipogenic genes (Pyun et al., 2021). The major biosynthetic pathway for indole-3-acetic acid (IAA) is through Trp. IAA treatment down-regulated the expression of adipogenesis-related genes, including SREBP-1c, Scd1, and peroxisome proliferator-activated receptor gamma (PPARγ). In addition, IAA attenuated the inflammatory response in the liver and decreased the expression of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α), thereby alleviating MAFLD (Ji et al., 2019). Therefore, we performed a targeted metabolomics of Trp metabolic pathways to absolutely quantify all metabolites in the pathway, aiming to explore the potential role of Trp metabolites in alleviating MAFLD. The results displayed that the level of kynurenic acid was recovered after PAB treatment compared with the Model group, but levels of L-formylkynurenine, 5-hydroxyindoleacid and L-tryptophan were unexpectedly unchanged after PAB intervention. At the same time, we found that CA, one of the microbiota-derived Trp metabolites, was at low level in feces of MAFLD mice, which was significantly ameliorated by PAB. A recent study found that CA administration significantly reduced body weight, alleviated liver steatosis, lipid accumulation and liver damage in MAFLD mice (Patil et al., 2022). Additionally, CA treatment decreased levels of gene related to fatty acid synthesis, including ACC, FAS and SCD1. This suggests that CA improves MAFLD by reducing lipid uptake and synthesis, and inhibiting de novo lipogenesis. Then, a joint analysis of differential bacterial abundance and differential metabolite contents revealed that CA was significantly negatively correlated with g_ Faecalibaculum, g_ Allobaculum, g_ Ileibacterium and g_ Dubosiella at the genus level. When the abundance of these differential bacteria in PAB group was reduced compared with the Model grouop, while the content of CA increased. Collectively, PAB is most likely to modulate CA level by affecting the abundance of g_ Faecalibaculum, g_ Allobaculum, g_ Ileibacterium and g_ Dubosiella , thereby relieving MAFLD. Gut microbial regulation of Trp metabolism provides several ligands for AhR that can bind and activate the AhR, a series of processes known as the Trp-AhR pathway. The activated Trp-AhR pathway regulates the metabolism of indoleamine-2,3-dioxygenase (IDO) in dendritic cells and influences the differentiation and function of the IL-22-producing helper T lymphocyte populations (Yang et al., 2020). CA has been identified as a ligand for AhR that promotes IL-22 production by regulating Trp metabolism in the gut microbiota (Lowe et al., 2014; Gómez-Piñeiro et al., 2022). Numerous studies have shown that IL-22 can significantly induce the expression of phosphorylated Janus kinase (JAK) and key signaling molecules in the signal transduction and transcriptional activator (STAT) pathway, including JAK1, p-JAK1, STAT3 and p-STAT3. The activation of STAT3 signal transduction activates the downstream pathway genes such as Srebp-1c and ELOVL6. It provides new insights into the hepato-protective role of gut microbes in regulating AhR/IL-22 via CA in MAFLD (Joshi et al., 2022; Zhu et al., 2018). In this study, the expressions of AhR and IL-22 were significantly up-regulated after PAB administration, and the expression of JAK1, STAT3, and their phosphorylated proteins was found to be significantly activated. Consistent with the expected results, the expression of SREBP-1c and ELOVL6 was markedly suppressed by the administration of PAB. The results of the FMT experiments further validate these findings. In conclusion, PAB ameliorates MAFLD by modulating intestinal flora to up-regulate CA metabolism, activate the AhR/IL-22-mediated pathway, and thus reduce lipid synthesis. In summary, this study investigated the hepatoprotective effect of PAB on HFD-induced MAFLD in mice and revealed its mechanism based on 16S rRNA sequencing and metabolomics analysis. PAB treatment significantly improved body weight, liver index, serum biochemical indexes and histopathological injury in MAFLD mice. We identified g_ Faecalibaculum, g_ Allobaculum, g_ Ileibacterium and g_ Dubosiella as the core microorganisms regulated by PAB, that promote CA production and then activate AhR/IL-22 to inhibit fatty acid synthesis. Therefore, PAB could be used as a new natural prebiotic agent for MAFLD treatment. To more fully evaluate the long-term efficacy of PAB in MAFLD treatment and its clinical applicability, large-scale clinical trials are needed in the future. These trials will help to confirm the safety, efficacy, and optimal treatment regimen of PAB in different populations. 5. CONCLUSION In summary, this study investigated the hepatoprotective effect of PAB on HFD-induced MAFLD in mice and revealed its mechanism of action based on 16s rRNA sequencing and metabolomics analysis. .PAB treatment significantly improved body weight, liver index, serum biochemical indexes, and histopathological injury in MAFLD mice. We identified g_Faecalibaculum, g_Allobaculum, g_Ileibacterium and g_Dubosiella as the core microorganisms regulated by PAB, that promote CA production and then activate AhR/IL-22 to inhibit fatty acid synthesis. Therefore, PAB could be used as a new natural prebiotic agent for MAFLD treatment. To more fully evaluate the long-term efficacy of PAB in MAFLD treatment and its clinical applicability, large-scale clinical trials are needed in the future. These trials will help to confirm the safety, efficacy, and optimal treatment regimen of PAB in different populations. AUTHOR CONTRIBUTIONS Tingyu Song: Conceptualization, Formal analysis, Data curation, Writing - original draft. Xiufang Yang: Formal analysis, Investigation. Junyi Wang: Investigation, Methodology. Lianhong Yin: Resources, Software, Supervision. Xuerong Zhao: Software, Supervision. Ning Wang: Supervision, Validation. Youwei Xu: Supervision, Validation. Yan Qi: Validation, Supervision, Software, Data curation. Cunquan Xiong: Validation, Supervision, Software, Data curation. Lina Xu: Writing – review and editing, Visualization, Validation, Supervision, Software, Resources, Project administration, Funding acquisition, Conceptualization. not-yet-known not-yet-known not-yet-known unknown ACKNOWLEDGEMENTS The authors gratefully acknowledge the financial supports of the ”Qing Lan project” in Jiangsu Province (2022 and 2023). not-yet-known not-yet-known not-yet-known unknown CONFLICT OF INTEREST STATEMENT No potential conflict of interest was reported by the author(s) DATA AVAILABILITY STATEMENT All additional data supporting the results of this study are available in the article and in the Supplementary Information file or upon request from the corresponding authors. Source data are provided with this article. not-yet-known not-yet-known not-yet-known unknown DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design and Analysis and Animal Experimentation, and as recommended by funding agencies, publishers and other organizations engaged with supporting research. REFERENCES Albillos A, de Gottardi A, Rescigno M. The gut-liver axis in liver disease: Pathophysiological basis for therapy. J Hepatol. 2020 ;72(3):558-577. Agus A, Planchais J, Sokol H. Gut Microbiota Regulation of Tryptophan Metabolism in Health and Disease. Cell Host Microbe. 2018 ;23(6):716-724. Aron-Wisnewsky J, Vigliotti C, Witjes J, Le P, Holleboom AG, Verheij J, Nieuwdorp M, Clément K. Gut microbiota and human NAFLD: disentangling microbial signatures from metabolic disorders. 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(n=8). *p < 0.05 and **p < 0.01, compared with the Model group. Figure 2. PAB modulates the composition of gur flora in mice with MAFLD by 16S ribosomal RNA (rRNA) sequencing. (A) Score scatter plot of the principal component analysis (PCA) on the genus level. The plot indicates significant separation between the groups. (B) Community bar plot analysis at the genus level. It shows the relative abundance of different microbial communities in the three groups, with each color representing a different taxonomic group. (C) Heatmap of hierarchical clustering analysis. Cluster analysis highlighted differences in microbial composition between groups. (D) Bar graph of microbial abundance, focusing on genera of microorganisms that differ significantly under different experimental conditions. (E) Differential bacteria analysis based on the Kruskal-Wallis H-test showing the differences in the abundance of different microorganisms in each group at the genus level. (F-G) Linear discriminant analysis effect size. The relative abundance and compositional differences of microorganisms in the three groups are demonstrated. *p < 0.05 and **p < 0.01, compared with the Model group (n=8). PC: principal component; LDA: linear discriminant analysis. Figure 3. PAB improved MAFLD symptoms in mice after fecal microbiota transplantation (FMT). (A) Representative images of comparison of mice and liver. (B) Body weight change diagram in mice. (C) Liver index. (D) Serum biochemical analysis of AST and ALT Liver index. (E) Serum biochemical analysis of TC, TG, TC/HDL-C, LDL-C and FFA. (F) Representative ultrasound Imaging of mouse liver. (G) Hematoxylin-eosin (H&E) staining of liver tissue and histopathological score. (H) Oil red O staining of liver tissue and histopathological score. Data are presented as the mean ± SD. (n=8). *p < 0.05 and **p < 0.01, compared with the Model-FMT group. Figure 4. PAB regulates metabolite levels in MAFLD mice by untargeted metabolomics analysis. (A) Score scatter plot of the PCA. The differences in distribution in the graphs indicate that there are significant differences in metabolite composition between the different experimental groups. (B) Volcano plot shows metabolite differences in pairwise comparisons between the Control, Model, and PAB groups. Each point in the plot represents a metabolite. The significantly up-regulated, down-regulated and non-significant metabolites were shown in red, blue and gray, respectively. (C) Heatmap of hierarchical clustering analysis of metabolites in the three groups, using a color gradient (red for higher abundance and blue for lower abundance) to indicate the distribution of metabolites in each group. (D) KEGG metabolic pathway enrichment analysis. The size of the dots indicates the number of metabolites significantly enriched. The redder the color of the dots, the more statistically significant the KEGG pathway enrichment. (E) Box line plot of metabolite abundance of some important metabolites in the tryptophan metabolic pathway, including L-formyl kynurenine, kynurenic acid, L-tryptophan, and 5-hydroxyindoleacetic acid. *p < 0.05 and **p < 0.01, compared with the Model group (n=8). Figure 5. PAB modulates cinnabarinic acid to ameliorate MAFLD and correlates with gut microbes. (A) Cluster analysis of related metabolites in the tryptophan metabolic pathway. The heatmap demonstrates the differences in the abundance of different tryptophan metabolites in the three groups. Color changes (from red to blue) represent changes in metabolite abundance. (B) Metabolite content statistics of key metabolites in the tryptophan metabolic pathway. (C) Diagram of tryptophan metabolism process. This figure shows the regulatory role of PAB treatment in tryptophan metabolism and the key steps in the tryptophan metabolic pathway, including the pathway for the conversion of L-tryptophan to N-Formylkynurenine and 3-Hydroxyanthranilic acid, which ultimately generates the important metabolite cinnabarinic acid. (D) Conjoint analysis of gut microbiota and metabolites. *p < 0.05 and **p < 0.01, compared with the Model group (n=8). Figure 6. PAB significantly increases the expressions of aryl hydrocarbon receptor (AhR) and IL-22 in the liver of HFD-induced mice. (A) The expression of AhR and IL-22 in the liver was detected by immunofluorescence assay. (B) The expression levels of AhR and IL-22 detected by Western blotting. Data are presented as the mean ± SD (n=3). *p < 0.05 and **p < 0.01, compared with the Model group. Figure 7. PAB-FMT markedly increases the expressions of AhR and IL-22 in the liver of HFD-induced mice. (A) The expression of AhR and IL-22 in mouse liver was detected by immunofluorescence assay. (B) The expression levels of AhR and IL-22 were detected by Western blotting. Data are presented as the mean ± SD (n=3). *p < 0.05 and **p < 0.01, compared with the Model group. Figure 8. PAB inhibits the levels of lipid synthesis-related genes by promoting JAK/STAT3 pathway. (A) Effects of different doses of PAB treatment on the expression of JAK/STAT pathway proteins as well as SREBP-1c and ELOVL6 were examined by Western blotting. (B) Effect of PAB on mRNA expression of lipid metabolism-related genes (Acc, Fasn, and Scd1) in liver tissue by qRT-PCR analysis. Data are presented as the mean ± SD (n=3). *p < 0.05 and **p < 0.01, compared with the Model group. Figure 9. PAB-FMT promotes JAK/STAT3 pathway proteins and inhibits the expression of genes involved in lipid synthesis. (A) The expression levels of JAK/STAT pathway proteins as well as SREBP-1c and ELOVL6 detected by Western blotting. (B) The mRNA levels of Acc, Fasn, and Scd1 in liver tissue by qRT-PCR analysis in FMT experiments. Data are presented as the mean ± SD (n=3). *p < 0.05 and **p < 0.01, compared with the Model group. Information & Authors Information Version history V1 Version 1 14 May 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords hepatopharmacology metabolomics microbiome Authors Affiliations Tingyu Song Dalian Medical University View all articles by this author Xiufang Yang Dalian Medical University View all articles by this author Junyi Wang Dalian Medical University View all articles by this author Lianhong Yin Dalian Medical University View all articles by this author Xuerong Zhao Dalian Medical University View all articles by this author Ning Wang Dalian Medical University View all articles by this author Youwei Xu Dalian Medical University View all articles by this author Yan Qi Dalian Medical University View all articles by this author Cunquan Xiong Jiangsu Medical College View all articles by this author L.N. Xu [email protected] Dalian Medical University View all articles by this author Metrics & Citations Metrics Article Usage 205 views 141 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Tingyu Song, Xiufang Yang, Junyi Wang, et al. Gut Microbial Metabolism of Cinnabarinic Acid Promotes to the Role of Pseudolaric Acid B Against Metabolic Dysfunction-Associated Fatty Liver Disease. Authorea . 14 May 2025. DOI: https://doi.org/10.22541/au.174721901.11057373/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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