Study On Mechanism Of Huang-Qi San Alleviating Non-alcoholic Fatty Liver Disease Via Regulating AMPK/mTOR Autophagy Signaling Pathway

BackgroundNon-alcoholic fatty liver disease（NAFLD）is a clinicopathologic syndrome mainly characterized by hepatocyte steatosis,in addition to alcohol and other factors that identify liver damage.Currently,there is no effective treatment for NALFD.The ancient prescription Huang-Qi San was first recorded in Sheng Ji Zong Lu of Song Dynasty.Modern studies have shown that Huang-Qi San can reduce insulin resistance by activating AMPK to play a hypoglycemic effect.Huang-Qi San can also regulate dyslipidemia and reduce hepatic steatosis,while improving type 2 diabetes mellitus（T2DM）,but its mechanism of action needs to be further explored.Therefore,the purpose of this study was to explore the effect and mechanism of Huang-Qi San on improving hepatic steatosis,and to provide experimental basis for drug treatment of NAFLD and the development of traditional Chinese medicine prescription.ObjectiveBased on adenosine 5’-monophosphate-activated protein kinase（AMPK）/mammalian target of rapamycin（mTOR）signaling pathway,high-fat diet（HFD）and palmitic acid（PA）induction were used to construct NAFLD models in vitro and in vivo,respectively,to explore the effect and potential mechanism of Huang-Qi San（HQS）on hepatic steatosis.Methods1.Discussion on the potential mechanism of Huang-Qi San improving NAFLD base on network pharmacologyThe main chemical compounds of Huangqi（HQ）,Gegen（GG）and Sangbaipi（SBP）were obtained through the Traditional Chinese Medicine Database and Analysis Platform（TCMSP）,and the main active compounds and its targets were screened according to oral bioavailability（OB）≥30%and drug-like drug（DL）≥0.18.The main targets of NAFLD was obtained from OMIM,TTD and Gene Card database,and the protein targets of all the selected compounds were standardized to the corresponding gene symbols with the help of Uniprot database.The Venn diagram was drawn of the intersection targets of HQS and NAFLD,and the protein-protein interaction（PPI）analysis and PPI network were conducted using Bisogenet plug-in.Cytoscape 3.8.2 software was used for network topology analysis,Cyto NCA plug-in was used to screen core targets,and finally Gene Ontology（GO）biological processes and Kyoto Encyclopedia of Genes and Genomes（KEGG）signaling pathway enrichment analysis and data visualization were carried out through the Matescape platform.2.Therapeutic effect of HQS on HFD-induced NAFLD miceSixty male C57BL/6J mice were randomly divided into five groups,normal control group（NCD）,model group（HFD）,Metformin group（MET）,Huang-Qi San with low dose（HQS-L）,and Huang-Qi San with high dose（HQS-H）,with 12 mice in each group.The mice in NCD group were given the basic maintenance diet,and the mice in other groups were given a high-fat diet,and intragastric administration were given at the same time as modelling establishment,lasting for 13 weeks.The mice body weight was recorded weekly and feeding blood glucose（GLU）was determined at the end of 12^th week.At the end of the13^th week,mice were dissected,and the blood samples were collected,and the effects of HQS on body weight,liver weight,liver index,blood lipid level,fasting blood glucose（FBG）,serum insulin level（FINS）,liver function,liver triglyceride（TG）content,liver pathological morphology and steatosis of mice were observed.to explore the therapeutic effect of HQS on HFD-induced NAFLD mice.3.Effects of HQS on PA-induced steatosis HepG2 cells（1）CCK-8 was used to detect the effects of HQS and PA on the proliferative activity of Hep G2 cells under different concentrations and treatment times.（2）PA-induced Hep G2cell adipohepatocytes model was established,the effects of HQS on cell TG content was determined,and oil red O staining was used to observe hepatocyte steatosis.4.Effects of HQS on AMPK/mTOR signalling pathway and autophagy in HFD-induced NAFLD miceTransmission electron microscopy（TEM）was used to observe the effect of Huang-Qi San on autophagosome and autolysosome in mice liver tissues,and real-time quantitative polymerase chain reaction（RT-PCR）and Western Blot were used to detect the changes of the expression of m RNA and protein levels of AMPK and mTOR,LC3 and p62 in mouse liver tissues,and to observe the effect of Huang-Qi San on AMPK/mTOR signalling pathway and autophagy in high-fat diet-induced NAFLD mice.Results1.The result of network pharmacology showed that 51 main active compounds and220 corresponding targets of HQS were obtained after preliminary screening,and 1536targets related to NAFLD were collected,and finally 95 intersecting targets of HQS and NAFLD were obtained.The biological processes involved in the improvement of NAFLD by HQS mainly including reactive oxygen metabolism process,response to lipopolysaccharide and nutrient levels,apoptotic signaling pathway etc.The signaling pathways involved mainly included AGE-RAGE,IL-17,PI3K-Akt,TNF,AMPK,and mTOR signaling pathway.Based on previous published studies on HQS,it was suggested that HQS could activate the AMPK signaling pathway to improve lipid accumulation in liver and insulin resistance.Combined with the result of KEGG analysis,mTOR as a key regulatory factor of autophagy,and AMPK/mTOR is one of the important signaling pathways of autophagy.Therefore,we reasonably put forward the hypothesis that HQS can alleviate hepatic steatosis and improve NAFLD by regulating autophagy via AMPK/mTOR signaling pathway.2.Compared with NCD mice,the mice’s body weight,liver weight,liver index,total cholesterol（TC）,low-density lipoprotein cholesterol（LDL-C）,non-esterified fatty acid（NEFA）,GLU,FBG and FINS levels,aspartate aminotransferase（AST）and alanine transaminase（ALT）in serum were significantly increased,high-density lipoprotein cholesterol（HDL-C）was also abnormally increased,and liver TG content was significantly increased.H&E staining of mouse liver showed extensive vacuolization of mouse liver lipid droplets（LDs）,and oil red staining showed lipid accumulation in hepatocytes.Compared with HFD group,the body weight,liver weight,serumTG and NEFA levels in HQS-L and HQS-H group were significantly deceased,and the HDL-C level was also significantly reduced,which was no significant difference with the NCD group.LDL-C and FBG levels showed a downward trend,but there was no significant difference.The liver index in HQS-L group was significantly decreased,the levels of TC,GLU,FINS,AST,ALT in serum and liver TG in HQS-H group were significantly decreased.H&E staining and oil red staining of mice in HQS-L and HQS-H group showed that the pathological changes in liver were obviously improved.The experimental results showed that Huang-Qi San could effectively improve the dyslipidemia in mice induced by high fat diet,and alleviate liver steatosis and protect the liver.3.Huang-Qi San could significantly improve PA-induced lipid accumulation in Hep G2 cells in vitro,reduce the TG content in hepatocytes,and reduce the intracellular lipid accumulation observed by oil red staining.4.At the transcriptional level,there were no significant differences in the m RNA expression of AMPK and mTOR in mice liver among all groups.In terms of protein level,compared with NCD group,protein expression of p-AMPK,ratio of p-AMPK/AMPK was significantly decreased,and p-mTOR was significantly increased in the liver of mice.After the intervention of Huang-Qi San,the protein expression of p-AMPK and the protein ratio p-AMPK/AMPK were significantly up-regulated,and the protein expression of p-mTOR was significantly inhibited.At the same time,a small amount of autophagosome and autolysosome were observed in mice administrated with HQS under TEM.The results of Western Blot and RT-PCR showed that HQS could significantly increase the m RNA expression levels of LC3A and LC3B and the protein ratio of LC3II/I expression in the liver of mice,and significantly decrease the m RNA and protein expression levels of p62.The results showed that the positive effect of Huang-Qi San on hepatic steatosis was related to promote autophagy and restore autophagy flux mediated by AMPK/mTOR signaling pathway.Conclusion1.Based on network pharmacology and combined with the previous studies basis on HQS,it is reasonably put forward the hypothesis that HQS may regulate autophagy via AMPK/mTOR signaling pathway to alleviate hepatic steatosis improving NAFLD.2.HQS could improve steatosis and lipid deposition in HFD-induced NAFLD mice in vivo and PA-induced Hep G2 cells in vitro.3.HQS may improve NAFLD by regulating AMPK/mTOR autophagy signaling pathway to promote autophagy and restore autophagy flux.