Background and Aim: Xuefu Zhuyu decoction (XZD), a traditional Chinese medicinal formula, was firstly recorded in the Qing dynasty of ancient China and previously demonstrated to ameliorate hepatic steatosis. In the present study, the effects of XZD on non-alcoholic fatty liver disease (NAFLD) induced by high-fat diet (HFD) were evaluated in mice and the hepatic transcriptome was detected to disclose the potential mechanisms of XZD. Experimental procedure: The effects of XZD (low- and high-dosage) on NAFLD induced by HFD for 16 weeks were evaluated. Obeticholic acid was used as control drug. Body weight, food intake and index of homeostatic model assessment for insulin resistance (HOMA-IR) were analyzed. Hepatic histology were observed in haematoxylin and eosin stained sections and quantified with NAFLD activity score (NAS). Lipid in hepatocytes was visualized by Oil red staining. Alanine aminotransferase (ALT) and hepatic triglyceride (TG) was measured. The hepatic transcriptom was detected with RNA-sequencing and validated with real-time polymerase chain reaction, western-blotting and hepatic quantitative metabolomics. Results: XZD ameliorated hepatic histology of NAFLD mice, accompanied with decreasing fasting insulin, HOMA-IR, NAS, ALT and hepatic TG. The hepatic transcriptom of NAFLD was significantly reversed by XZD treatment, especially the genes enriched in the pathways of arachidonic acid metabolism, fatty acid degradation, cytokine-cytokine receptor interaction and extracellular matrix (ECM) -receptor interaction. The hepatic quantitative metabolomics analysis confirmed fatty acid degradation as the key targeting pathway of XZD. Conclusions: XZD ameliorated NAFLD induced by HFD, which probably correlated closely to the pathways of fatty acid degradation.

Theaflavins are the characteristic polyphenols in black tea which can be enzymatically synthesized. In this review, the effects and molecular mechanisms of theaflavins on obesity and its comorbidities, including dyslipidemia, insulin resistance, hepatic steatosis, and atherosclerosis, were summarized. Theaflavins ameliorate obesity potentially via reducing food intake, inhibiting pancreatic lipase to reduce lipid absorption, activating the adenosine monophosphate-activated protein kinase (AMPK), and regulating the gut microbiota. As to the comorbidities, theaflavins ameliorate hypercholesterolemia by inhibiting micelle formation to reduce cholesterol absorption. Theaflavins improve insulin sensitivity by increasing the signaling of protein kinase B, eliminating glucose toxicity, and inhibiting inflammation. Theaflavins ameliorate hepatic steatosis via activating AMPK. Theaflavins reduce atherosclerosis by upregulating nuclear factor erythropoietin-2-related factor 2 signaling and inhibiting plasminogen activator inhibitor 1. In randomized controlled trails, black tea extracts containing theaflavins reduced body weight in overweight people and improved glucose tolerance in healthy adults. The amelioration on the hyperlipidemia and the prevention of coronary artery disease by black tea extracts were supported by meta-analysis.

OBJECTIVE:To evaluate the effects of Sanren Tang (SRT, ) on a high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) in mice and to investigate the hepatic transcriptome regulated by SRT.METHODS:The primary SRT components were identified using ultra-high-performance liquid chromatography-high-resolution accurate mass spectrometry. The SRT-induced pharmacological effects on HFD-induced NAFLD were evaluated in mice for 16 weeks. Obeticholic acid was used as a control drug. Body weight, food intake, and homeostatic model assessment for insulin resistance (HOMA-IR) index were analysed. Hepatic histological changes were observed in haematoxylin and eosin-stained sections and quantified using the NAFLD activity score (NAS). Serum alanine aminotransferase (ALT) and hepatic triglyceride (TG) levels were measured. Lipids in hepatocytes were visualised by Oil red staining. RNA-sequencing was performed to determine the transcriptome profile of the liver tissue. The differentially expressed genes were validated using real-time polymerase chain reaction and Western blotting.RESULTS:Four principal compounds were identified in the SRT: adenosine, amygdalin, luteoloside, and magnolol. SRT ameliorated hepatic histology and lipid deposition in the NAFLD mice, and decreased HOMA-IR, NAS and ALT, and hepatic TG levels. Hepatic transcriptome analysis revealed 232 HFD-regulated genes that were reversed by SRT simultaneously. Retinol metabolism, cytokine-cytokine receptor interaction, and peroxisome proliferator-activated receptor (PPAR) γ signalling were the top three SRT-regulated pathways in NAFLD.CONCLUSIONS:SRT significantly ameliorated HFD-induced NAFLD, which was correlated with the regulation of genes enriched in the retinol metabolism, cytokine-cytokine receptor interaction, and PPARγ signalling pathways.

Ethnopharmacological relevance: Qushi Huayu Decoction (QHD) is a traditional Chinese medicine formula con-sisting of five herbs, which has been used for non-alcoholic fatty liver disease (NAFLD) treatment in clinic for decades in China and validated in several NAFLD animal models. The hepatic de novo lipogenesis (DNL) is enhanced greatly to contribute to steatosis in NAFLD. The spliced form of X-box binding protein 1 (XBP1s) initiates DNL independently of sterol regulatory element-binding protein (SREBP) and carbohydrate-responsive element-binding protein (ChREBP). Aim of the study: To disclose the mechanism of inhibition on hepatic DNL by QHD and the responsible compounds. Methods: The effects of QHD on hepatic DNL were evaluated in mice induced by high-fructose diet (HFru). The effects of the serum-absorbed compounds of QHD on XBP1s were evaluated in HepG2 cells induced by tunica-mycin. Hepatic histology, triglyceride (TG) and nonesterified fatty acids were observed. Hepatic apolipoprotein B100 and very low-density lipoprotein were measured to reflect lipid out-transport. The mRNA expression of XBP1s and its target genes were detected by real-time polymerase chain reaction. The protein expression of TG synthetases and DNL enzymes, and inositol requirement enzyme 1 alpha (IRE1 alpha), phosphorylated IRE1 alpha and XBP1s were detected in liver tissue and HepG2 cells by western-blot. The binding activity of SREBP1, protein expression of ChREBP and XBP1s were detected in the nuclear extracts of liver tissue. Results: Dynamical observing suggested feeding with HFru for 2 weeks was sufficient to induce hepatic lipo-genesis and XBP1s. QHD ameliorated liver steatosis without enhancing out-transport of lipids, accompanied with more inhibitory effects on DNL enzymes than TG synthetases. QHD inhibits the nuclear XBP1s without affecting ChREBP and SREBP1. In QHD, chlorogenic acid, geniposide and polydatin inhibit lipogenesis initiated by XPB1s. Conclusion: QHD probably decreases hepatic DNL by inhibiting XBP1s independent of SREBP1 and ChREBP. Chlorogenic acid, geniposide and polydatin are the potential responsible compounds.

ObjectiveTo investigate the effect of Xuefu Zhuyu decoction on nonalcoholic fatty liver disease (NAFLD) and its material basis. MethodsIn experiment 1 for exploring the effect of Xuefu Zhuyu decoction on mice with NAFLD induced by high-fat diet, 50 healthy male C57BL/6J mice were randomly divided into normal group, model group, high- and low-dose Xuefu Zhuyu decoction groups, and obeticholic acid control group, with 10 mice in each group. The mice in the normal group were given control diet, and those in the other groups were given high-fat diet. Gastric administration was started at week 13, and related samples were collected at the end of week 16. Food intake and body weight were recorded, enzyme-linked immunosorbent assay was used to measure the serum level of fasting insulin, fasting blood glucose was measured, and insulin resistance index was calculated. HE staining and NAFLD activity score (NAS) were used to observe liver histopathology in mice, oil red O staining was used to observe lipid deposition, and triglyceride (TG) level in liver tissue and serum alanine aminotransferase (ALT) level were measured. In experiment 2 for exploring the effect of different extracts of Xuefu Zhuyu decoction on mice with NAFLD induced by high-fat diet, the methods of water decocting, water extraction and alcohol precipitation, and petroleum ether extraction were used to obtain the extracts 1, 2, and 3 of Xuefu Zhuyu decoction, and 54 healthy male C57BL/6J mice were randomly divided into normal group, model group, Xuefu Zhuyu decoction extract 1, 2, and 3 groups, and Xuefu Zhuyu decoction control group, with 9 mice in each group. The mice in the normal group were given control diet, and those in the other groups were given high-fat diet. Gastric administration was started at week 13, and related samples were collected at the end of week 16. Food intake and body weight were recorded, and enzyme-linked immunosorbent assay was used to measure the serum level of fasting insulin, fasting blood glucose was measured, and insulin resistance index was calculated. HE and NAS were used to observe liver histopathology in mice, oil red O staining was used to observe lipid deposition, and the levels of TG and gamma-glutamyl transpeptidase (GGT) in liver tissue and the serum level of ALT were measured. The t-test was used for comparison of normally distributed continuous data between two groups; a one-way analysis of variance was used for comparison between multiple groups, and the least significant difference t-test was used for further comparison between two groups. The Kruskal-Wallis H test was used for comparison of non-normally distributed continuous data between multiple groups and further comparison between two groups. ResultsIn experiment 1, compared with the model group, the high- and low-dose Xuefu Zhuyu decoction groups and the obeticholic acid control group had significant reductions in body weight, insulin resistance index, the distribution of vacuolar lipid droplets in liver tissue, intralobular inflammation, the ballooning degeneration of hepatocytes, NAS score, the level of TG in liver tissue, and the serum level of ALT (all P＜0.05). Compared with obeticholic acid, high- and low-dose Xuefu Zhuyu decoction had a significantly better effect in reducing body weight, insulin resistance index, and total NAS score (all P＜0.05), and low-dose Xuefu Zhuyu decoction had a significantly better effect in improving serum ALT (P＜0.05). In experiment 2, compared with the model group, the Xuefu Zhuyu decoction extract 1, 2, and 3 groups had significant reductions in fasting blood glucose, insulin resistance index, the distribution of lipid droplets in liver tissue, intralobular inflammation lesions, the ballooning degeneration of hepatocytes, total NAS score, and the level of TG in the liver (all P＜0.05). Compared with the model group, the extract 1 group had a significant reduction in body weight (P＜0.05); the extract 2 and 3 groups had a significant reduction in the serum level of ALT (P＜0.05); the extract 2 group had a significant reduction in the level of GGT in liver tissue (P＜0.05). The extract 2 of Xuefu Zhuyu decoction had the closest effect to compound Xuefu Zhuyu decoction. ConclusionXuefu Zhuyu decoction and its extracts can help to achieve varying degrees of improvement in NAFLD induced by high-fat diet in mice, and the extract 2 of Xuefu Zhuyu decoction might be the main material basis for Xuefu Zhuyu decoction.

Gut-derived lipopolysaccharide (LPS) leaking through the dysfunctional intestinal barrier contributes to the onset of non-alcoholic steatohepatitis (NASH) by triggering inflammation in the liver. In the present study, a combination consisting of Atractylodes macrocephala polysaccharide (A), chlorogenic acid (C), and geniposide (G) (together, ACG), was shown to ameliorate NASH in mice and reduce hepatic LPS signaling and endotoxemia without decreasing the abundance of identified Gram-negative bacteria through restoring the intestinal tight junctions. Our data indicated that inhibition of LPS gut leakage by the ACG combination contributed to its amelioration of NASH.

BACKGROUND:Gut microbiota is increasingly recognized as the key participant in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) by translocation of its products, such as lipopolysaccharide (LPS), via the dysfunctional intestinal barrier. Qushi Huayu decoction (QHD), a traditional Chinese medicine, is developed specially for NAFLD and used in clinic in China for more than a decade and previously found to ameliorate non-alcoholic steatohepatitis (NASH) induced by high-fat diet (HFD) in mice accompanied with inhibited metabolic endotoxemia and hepatic LPS signalling.PURPOSE:To investigate the mechanism of LPS gut-leakage inhibition by QHD in NASH.METHODS:Effects of QHD on gut microbioa and intestinal barrier were evaluated in NASH induced by HFD in mice. 16S rRNA sequencing is employed to analyse the gut microbiota composition. To identify the potential signalling pathway responsible for tight junction regulation, the colonic phosphoprotein profile is screened via the Phospho Explorer Antibody Array and verified in NASH, intestinal barrier dysfunctional mouse and Caco-2 cells.RESULTS:QHD ameliorates NASH accompanied with regulating the gut microbiota composition, protecting intestinal tight junctions and inhibiting LPS gut-leakage without decreasing the abundance of identified Gram-negative bacteria. The validated data of phosphorylated proteins suggested that mitogen-activated protein kinase (MAPK) pathway is predominantly responsible for the colonic tight junction regulation by QHD.CONCLUSION:QHD inhibits LPS gut-leakage in NASH, which is associated with downregulation of intestinal MAPK pathway.

Gut-liver axis is increasingly recognized to be involved in the pathogenesis and progression of non-alcoholic fatty liver disease (NAFLD). The gut microbiota and intestinal permeability have been demonstrated to be the key players in the gut-liver cross talk in NAFLD. Geniposide and chlorogenic acid (GC) combination is derived from a traditional Chinese medicine, Qushi Huayu Decoction (QHD), which has been used in clinic for NAFLD treatment for decades in China and validated in multiple animal models of NAFLD. GC combination previously has been demonstrated to treat NAFLD via modulation on the gut microbiota composition. In the present study, the effects of GC combination on gut barrier function in NAFLD were evaluated, and QHD and sodium butyrate (NaB), the intestinal mucosa protectant, were used as positive control. The therapeutic effect of GC combination on NAFLD were confirmed by amelioration on non-alcoholic steatohepatitis (NASH) induced by high-fat diet (HFD) in mouse, which was comparable to that of QHD. Simultaneously, GC combination was found to reduce the signaling of gut-derived lipopolysaccharide (LPS) including hepatic LPS binding protein, Toll like receptor 4, interleukin-1β, tumor necrosis factor -α, and Kupffer cells infiltration. Furthermore, GC combination reduced LPS and D-lactate in plasma, restoring the colonic tight junction (TJ) expression and inhibited colonic TJs disassembly by down-regulation on RhoA/ROCK signaling in NASH induced by HFD. On the other hand, NASH was also alleviated in NaB group. The results of the present study suggested the important role of protection on gut barrier function in NAFLD treatment, which contributed to the therapeutic effects of GC combination on NASH.