Protective Effect Of Water Extract Of Huangshan Mao Feng On Alcohol-associated Steatohepatitis

Backgrounds: Chronic liver disease has become the second most deadly disease worldwide,and alcohol-related liver disease(ALD)caused by alcohol consumption is becoming increasingly serious.As reported by the World Health Organization,alcoholism is the cause of 5.3% of deaths worldwide each year,or about three million people,with the growing trend of alcohol-related liver disease(ALD)in China being very striking.Due to the high prevalence of ALD and the heavy financial burden associated with ALD treatment,there has been a growing interest in ALD in the hepatic academic community.However,by far the most effective treatment to mitigate ALD is to stop drinking alcohol.Given that tea is a natural product and that it is native to China,it has been consumed in China for thousands of years.Green tea has been found to have a therapeutic effect on ALD,and Li Shizhen,a famous master of Chinese medicine,recorded as early as in the "Compendium of Materia Medica": "Tea is bitter and cold,dispels internal heat and strong......,and can also detoxify alcohol and detoxify.One of the proven health benefits of drinking green tea extract(GTWE)is anti-inflammatory.Therefore,it is of social and economic importance to study the hepatoprotective effect of tea drinking on alcoholic liver and its mechanism.Some data suggest that green tea drinkers have a lower incidence of alcohol-related liver disease,but the exact mechanism is unclear and needs to be studied in more depth.In this study,we combined an integrated approach of network pharmacology and experimental studies to elucidate the underlying mechanisms of green tea aqueous extract for the treatment of alcoholrelated hepatitis,using Huangshan Mao Feng as an example.Several components may be the main active compounds of green tea aqueous extract in the treatment of alcoholrelated hepatitis,as they affect most of the targets associated with alcohol-related hepatitis,and TLR4 is considered as the core target protein.A potential molecular mechanism for the affinity between the major compounds in green tea aqueous extracts and TLR4 was identified by molecular docking.The network pharmacological analysis of green tea aqueous extract(GTWE)on alcohol-associated steatohepatitis(ASH)predicted its potential targets and took ex vivo experiments to validate them,and the final experimental results obtained were consistent with the network pharmacological results.In addition,the present study revealed that green tea aqueous extract was effective in alleviating alcohol-induced intestinal inflammation and tending to improve the disrupted intestinal barrier through intestinal pathological analysis.This study provides evidence that green tea aqueous extract is a candidate source of drugs for further studies on alcohol-related hepatitis and that long-term consumption of green tea can be used as a preventive dietary therapy for alcohol-related hepatitis.Objective::(1)Predicting the potential targets of GTWE to alleviate ASH by network pharmacology method(2)To investigate the mechanism of action of green tea aqueous extract to alleviate alcohol-associated hepatic steatohepatitis by in vitro and in vivo experiments using Huangshan Mao Feng aqueous extract as an example;Methods: Network pharmacology was first used to predict the potential targets of green tea aqueous extract(GTWE)against ASH: the main active compound components in GTWE were determined by HPLC assay with gradient elution using acetonitrile,methanol and acetic acid as mobile phases.Seven active compounds were obtained using pubmed database analysis,followed by superpred analysis of the active targets of these compounds.Then,the disease targets of ASH were analyzed using three major databases,Drugbank,Disge NET,and Genecard,resulting in the same portion of active compound targets and disease targets as the initial targets of GTWE for ASH.Subsequently,mapping of PPI networks between targets,construction of GTWE-ASHtarget networks,enrichment analysis of pathways(GO and KECG),and use of molecular docking(Auto Dock)were used to obtain network pharmacology predicting the signaling pathways of GTWE for ASH mitigation.Then an in vivo model of alcohol-induced steatohepatitis was developed by liquid alcohol diet.The groups of this experiment were: normal control group,alcohol model group,and model GTWE group(200 mg/kg).At the end of modeling,mice were executed,and fresh liver and serum were taken.To observe the effect of GTWE in reducing ASH,pathological sections of mouse liver tissue were observed,and then the results of each group were compared.The effect of GTWE on the improvement of liver function in mice was analyzed by performing relevant biochemical indexes(such as ALT,etc.)on the serum of mice.To observe the protective effect of GTWE on the intestinal barrier and to analyze the effect of GTWE in reducing the release of LPS,the LPS content of mouse serum and tissues were measured by ELISA.The level of reactive oxygen species in mouse liver was analyzed and the effect of GTWE on oxidative stress was observed by in vivo tissue oxidative stress reactive oxygen species(ROS)kit.The effect of GTWE on reducing liver inflammation was observed by doing immunohistochemistry of sections and analyzing the inflammatory infiltration of mouse liver macrophages.Immunohistochemistry of sections was likewise done to observe the effect of GTWE on the expression level of TLR4 protein in ASH.To further analyze the specific mechanism,further in vitro experiments were performed: cell models were selected for lipopolysaccharide stimulation,cell types were selected for RAW264.7 cells,and experiments were grouped: normal group,model group(lipopolysaccharide group),and model + GTWE(10ug/ml).To observe the effect of GTWE on ASH inflammatory factors in vitro: RNA expression levels of cellular inflammatory factors,including IL-1β,TNF-α and IL-6,were analyzed by RT-PCR and immunoblotting,respectively,in order to observe the effect of GTWE on TLR4 protein expression in cells in vitro.In addition,the expression of related proteins in the cells,such as NF-κB and My D88,was also analyzed.In addition,pathological sections of mouse intestinal tissues were made to analyze the effect of GTWE on intestinal inflammation.The intestinal tissue sections were also subjected to immunohistochemistry to analyze the mouse intestinal barrier integrity marker proteins: ZO-1,Occludin and Claudin4 to determine whether GTWE affects intestinal integrity in mice.The effect of GTWE on intestinal microbial disorders was investigated by sequencing and analyzing mouse intestinal microbes after collecting fecal samples from mice in a clean environment.Results: GTWE reduced the inflammatory infiltration of liver macrophages and the aggregation of lipid droplets in the alcohol model group of mice,and decreased the levels of ALT and AST in the serum of mice.the results of ELISA experiments indicated that GTWE improved the levels of LPS in the serum and liver of mice.The fluorescence results of ROS in mouse liver showed that GTWE increased the antioxidant capacity of liver.q PCR results showed that GTWE reduced the level of inflammatory factors in LPS-stimulated cells.WB and immunohistochemistry results showed that GTWE reduced the expression of TLR4 and My D88 in liver and macrophages,and decreased the phosphorylation level of NF-κB P65.In addition,WB results and immunofluorescence results of ROS showed that GTWE reduced the recruitment of the cell membrane protein TRAF6 to mitochondria,attenuated oxidative stress in mitochondria,and reduced the release of ROS.Histopathological sections of mouse intestine as well as immunohistochemical results showed that GTWE improved macrophage inflammatory infiltration and increased the expression of mouse intestinal barrier integrity marker proteins: ZO-1,Occludin and Claudin4.Differences in Simpson’s index and Venn diagrams of species abundance tables both suggest that alcohol alters the composition of the mouse intestinal microbiotaConclusions: GTWE reduces alcohol-induced liver damage,reduces fat accumulation and inflammation in the liver,and increases antioxidant capacity.GTWE may alleviate alcohol-associated steatohepatitis through the TLR4/My D88/NF-κB signaling pathway.In addition,GTWE may affect intestinal inflammation,affect intestinal barrier integrity,and regulate intestinal microorganisms.Green tea water extract may protect intestinal barrier from damage by upregulating intestinal epithelial cell tight junction protein.This study is the first to analyze the potential active compounds,targets and pathways of GTWE in the treatment of ASH through a network pharmacological approach and experimental validation,which may support long-term consumption of green tea as a preventive dietary therapy for ASH.