Study On The Effect And Mechanism Of Tangshen Formula And Its Active Compounds In Alleviating Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease(NAFLD)is one of the chronic liver diseases in the world currently.If there is no intervention at early stage,it can progress into liver fibrosis,liver cirrhosis,liver cancer or even lead to individual death.The most characterized pathological changes in NAFLD is excessive intracellular lipids accumulation in liver,which induces oxidative stress,endoplasmic reticulum stress,followed by hepatocyte damages and hepatocyte apoptosis,finally leading to liver inflammation and fibrosis,and loss of liver function.To date,there is little medication for NAFLD except lifestyle adjustment,energy intake restriction and weight loss surgery.Traditional Chinese medicine(TCM)is a treatment strategy based on the syndrome differentiation and is widely used to treat chronic liver diseases.Tangshen formula(TSF)is developed by Prof.Li based the clinical experience of domestic famous predecessors and hers own clinical practice and modern research results.Formononetin(FMN)is a isoflavone in TSF,which has been determined by liquid chromatograph-mass spectrometer(LC/MS)in serum of mice gavaed with TSF and can be absorbed into the blood circulation and subsequently entered liver through the intestinal tract.The previous application and research of TSF mainly focused on diabetic kidney diseases,but less on fatty liver diseases.Meanwhile,the specific effect and mechanism of formononetin on hepatic steatosis have been little studied in China and overseas.The effects and related mechanisms of TSF and formononetin on hepatic steatosis needs further theoretical support and exact experimental verification.Aims:1 To explore the specific effects and the underlying autophagy related mechanisms of TSF on non-alcoholic hepatic steatosis by using methionine choline deficient diet(MCDD)induced NAFLD in mouse,and sodium palmitate(PA)induced induced hepatic cells steatosis in HepG2 cell and primary mouse hepatocytes.2 To screen compounds with inhibing lipid accumulation activity from serum of mice gavaged with TSF by using free fatty acids(FFAs)induced hepatic cells steatosis in HepG2 cell and primary mouse hepatocytes.3 To explore the specific effects and the underlying lipophagy related mechanisms of FMN on non-alcoholic hepatic steatosis by using a high fat diet(HFD)induced NAFLD in mouse,and free fatty acids(FFAs)induced hepatic cells steatosis in HepG2 cell and primary mouse hepatocytes.Methods:1 Mice were fed on MCDD for 6 weeks.All mice were treated with TSF gavage(2.4 g/kg/day)from the 3rd week until dissection;in the week before dissection,food intake was assayed;after fasting,the serum and liver tissues were preserved;levels of serum triglyceride(TG),total cholesterol(TC),low density lipoprotein cholesterol(LDL-C),high density lipoprotein cholesterol(HDL-C),glutamic pyruvic transaminase(ALT)and glutamic oxaloacetic transaminase(AST)were detected by an automatic biochemical analyzer;liver triglycerides were detected with glycerophosphate oxidase peroxidase(GPO-PAP);liver H&E and oil red O staining were undertaken to observe the area of hepatic steatosis and the number of infiltrating inflammatory cell clusters,and to evaluate the NAFLD activity score.0.3 mM PA was used to stimulate HepG2 cells and primary mouse hepatocytes to induce hepatocyte steatosis,and then following TSF(100 pg/mL),chloroquine(CQ,20?M),bafilomycin A1(BafA1,10 nM),compound C(CC,10?M)and Sirtuin 1-small interfering RNA(SIRT1-siRNA)incubation respectively;cellular TG content were assayed with GPO-PAP;lipid droplets were determined by BODIPY493/503 staining and the integrated fluorescence intensity of lipid droplets was detected;the number of autophagy and autolysosome was counted in red fluorescent protein(RFP)-green fluorescent protein(GFP)-LC3 adenovirus infected HepG2 cells;the number of lipid droplets and autophagic vesicles in HepG2 cells were counted by transmission electron microscopy.Besides,the levels of adenosine monophosphate activated protein kinase(AMPK),phosphorylated AMPK(p-AMPK),SIRT1,light chain microtubule associated protein 3B-?(LC3B-?),and p62 in liver tissue,HepG2 cells and primary mouse hepatocytes,were assayed with western blot.21mM FFAs was used to stimulate HepG2 cells to induce hepatocyte steatosis,steatosis,and then following 40 ?M calycosin,40 ?M calycosin-7-O-beta-d-glucopyranoside,40?M hesperiden,40?M neohesperidin,320 ?M ginsenoside Rgl,320 ?M morroniside,80?M naringin,80 ?M astragaloside IV,20 ?M FMN,20?M formononetin glucoside,40?M ajugol and 320 ?M narirutin 24 h incubation respectively;intracellular lipids were determined by Nile red staining and the integrated fluorescence intensity of lipids was detected by widefield high content screening system.3 Mice were fed on high fat diet(HFD)for 16 weeks were treated with FMN gavage(100 mg/kg/day)from the 3rd week until dissection;in the week before dissection,food intake and intraperitoneal glucose tolerance test(IPGTT)were assayed;after fasting,the serum and liver tissues were preserved;levels of serum TG,TC,LDL-C,HDL-C,ALT and AST were detected by an automatic biochemical analyzer;liver TG content was detected with GPO-PAP;H&E and oil red O staining were undertaken to observe the area of hepatic steatosis and the number of infiltrating inflammatory cell clusters.1 mM FFAs was used to stimulate HepG2 cells and primary mouse hepatocytes to induce hepatocyte steatosis,and then following FMN(20 ?M),CQ(10 ?M),CC(10 ?M)and transcription factor EB(TFEB-siRNA)incubation respectively;cellular TG content were assayed with GPO-PAP;cellular lipid droplets were determined by BODIPY493/503 staining and autolysosomes in HepG2 cells were determined by Lysotracker staining,meanwile the number of colocalization of lipid dropltes and autolysosomes was counted;nuclear TFEB,autophagosomes and autolysosomes in HepG2 cells were detected by immunofluorescence of TFEB,LC3B and LAMP1,meanwile the number of colocalization of autolysosomes and lysosomes was counted;the number of autophagy and autolysosome was counted in RFP-GFP-LC3 adenovirus infected HepG2 cells.Besides,the levels of nuclear TFEB,lysosome-associated membrane protein 1(LAMP1),V-type proton ATPase catalytic subunit(ATP6V1A),peroxisome proliferator-activated receptor gamma coactivator 1(PGCla),AMPK,p-AMPK,phosphorylated ribosomal protein S6 kinase ?-1(p-S6K1),Beclinl,LC3B-II and p62 in liver tissue,HepG2 cells and primary mouse hepatocytes,were assayed with western blot.Results:1 TSF effectively alleviated hepatic steatosis in mice fed on MCDD,and ameliorated HepG2 and primary hepatic steatosis induced by PA;meanwhile,TSF enhanced the AMPK/SIRT1/autophagy pathway in PA-stimulated HepG2 cells and primary mouse hepatocytes,and liver of mice fed with MCDD through upregulating p-AMPK/AMPK ratio and SIRT1 expression,and increasing the LC3B-? expression and p62 degradation.2 Neohesperidin,FMN and ginsenoside Rgl reduced the integrated fluorescence intensity of lipids in FFAs-stimulated HepG2 cells and primary mouse hepatocytes,resulting in alleviating cellular steatosis.3 FMN significantly alleviated hepatic steatosis in mice fed on HFD,and ameliorated HepG2 and primary hepatic steatosis induced by FFAs;meanwhile,FMN promoted the TFEB mediated lysosomes biogenesis,enhanced lipophagy in FFAs-stimulated HepG2 cells and primary mouse hepatocytes,and liver of mice fed with HFD or MCDD,through upregulating the expression of LAMP1,ATP6V1A,PGCl?,p-S6K1 and Beclinl,along with p-AMPK/AMPK ratio,and increasing the LC3B-II expression and p62 degradation.Innovations:1 In this study,it was found for the first time that TSF could alleviate the hepatic lipid metabolism disorder induced by MCDD in mice,and the related autophagy pathway was further studied.It was found that TSF could enhance the hepatic autophagy function and reduce hepatic lipids accumulation by regulating AMPK/SIRT1/autophagy pathway.2 In this study,it was found for the first time formononetin extracted from TSF can enhance lysosome biogenesis and subsequent lipophagy by promoting TFEB nuclear translocation and alleviating non-alcoholic liver steatosis.Conclusions:TSF alleviates hepatic steatosis and NAFLD by enhancing AMPK/SIRT1/autophagy pathway.Its compound,FMN,alleviates hepatic steatosis and NAFLD via enhancing TFEB-mediated lipophagy pathway.Besides,Neohesperidin,FMN and ginsenoside Rgl owned activity of inhibing lipids accumulation in hepatic cells.The present study provided theoretical basis and exact experimental evidence for TSF's treatment against hepatic steatosis and NAFLD.