Research On The Mechanisms Of Poria Cocos Against Metabolic Dysfunction-Associated Fatty Liver Disease

Objective Metabolic dysfunction-associated fatty liver disease(MAFLD)is an umbrella name for the progression from simple steatosis and non-alcoholic steatohepatitis to steatohepatitis,fibrosis,and cirrhosis.It has become the No.1 chronic hematological disease worldwide,affecting 25 % of the global population.However,no specific drugs are approved for treating MAFLD,and the search for drugs from traditional Chinese medicine to improve the condition has become a promising direction in its therapeutic drug development.Bile acid(BAs)is a signaling molecule closely associated with the liver and extra-hepatic tissues.Its synthesis and metabolism in vivo can regulate hepatocyte cholesterol and lipid homeostasis,which is essential for MAFLD regulation.Poria cocos(Schw.)Wolf is the dry nucleus of the fungus Poria cocos(P.cocos)in the family Polyporaceae,strengthening the spleen and promoting dampness.It was used in the treatment of obesity,hyperlipidemia,and other disorders of lipid metabolism in both traditional and modern clinical practice.However,its efficacy in MAFLD has not been systematically demonstrated,and its possible mechanisms of action have not been studied.In this study,water-soluble polysaccharide(WPC),acid-soluble polysaccharide(also known as "water-insoluble polysaccharide," APC),and ethanolic extract of P.cocos(EPC)were isolated and purified from P.cocos.The effects of these extracts against MAFLD induced by a high-fat diet(HFD)were investigated.The pharmacological effectiveness and mechanism of action on MAFLD were also investigated by the activation of FXR-mediated metabolism of BAs by EPC monomeric active ingredient polyporenic acid C(PAC)and tumulosic acid(TA).This will provide a scientific basis for applying P.cocos in the prevention and treatment of MAFLD.MethodNetwork pharmacology: The association between P.cocos and MAFLD was established through the database.Secondly,the active ingredients and potential targets of P.cocos against MAFLD were screened by network pharmacology.Finally,functional prediction and active ingredient-pathway-target network diagram construction were performed.P.cocos extraction: Using 75 % ethanol as extraction solvent,heating reflux extraction,and freeze-drying to obtain EPC.Water was used as a solvent for P.cocos reflux extraction and separation to get and filtrate.The filter residue was dried and crushed to obtain APC.The filter residue was dried and crushed to obtain APC,the filtrate was added to ethanol to remove impurities and refined and dried at low temperature to obtain WPC.Evaluation of efficacy: 72 SPF-grade Sprague-Dawley male rats were randomly divided into randomly divided into nine groups(n = 8 per group)as follows: 1)normal diet control group(CON,containing 10 % kcal fat),2)high-fat diet group(HFD,containing 60 % kcal fat),3)20 mg/kg FC group,4)low dose(15 mg/kg)and 5)high dose(45 mg/kg)of WPC group(WPC-L and WPC-H),6)low dose(1.05 g/kg)and 7)high dose(3.15 g/kg)of APC group(APC-L and APC-H),8)low dose(56.3 mg/kg)and 9)high dose(168.9 mg/kg)of EPC group(EPC-L and EPC-H).Rats were treated daily from the first week to the end of the experiment(which lasted 12 weeks).The food intake of all the rats was recorded daily,and body weights were recorded weekly.At the end of this experiment,the rats were anesthetized using 1 % pentobarbital sodium.Blood,organ,and adipose tissue were collected,and each organ and adipose index were calculated.Hematoxylin&eosin(H&E)staining and Oil Red O staining observed pathological changes and lipid deposition.Total cholesterol(TC),triglycerides(TG),high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein d cholesterol(LDL-C),and total bile acids(TBA)were measured in serum and liver by the fully automated biochemical analyzer and biochemical kit,respectively.UHPLC Q-Trap/MS determined the concentrations of BAs in serum and ileal feces.Western blot and RT-qPCR measured the relative expression of BAs and lipid metabolism-related genes and proteins FXR,PPARα,FASN,and CYP7A1 in rat liver.Mechanisms explored: A total of 7 P.cocos triterpene active components,poricoic acid C,tumulosic acid(TA),polyporenic acid C(PAC),poricoic acid B,dehydrotumulosic acid,dehydroporicoic acid,and poricoic acid C,were investigated for molecular docking with FXR receptors using Auto Dock 1.5.6 with Obeticholic acid(FXR agonist),Ursodeoxycholic acid and Guggulsterone(FXR inhibitor)as controls.Eighty eight SPF-grade C57BL/6J male mice were randomly divided into a normal chow-fed group(CON,eight mice,containing 10 % kcal fat)and a high-fat chow-fed group(100 mice,containing 40 % kcal fat).After four weeks of feeding,the high-fat-fed group was randomly divided into nine groups(n = 8 per group)according to body weight by removing those that did not meet the requirements(below or above 20 % of the mean body weight).As follows: 1)high-fat diet group(HFD),2)100 mg/kg UDCA group,3)low dose(20 mg/kg)and 4)high dose(40 mg/kg)of PAC group(PAC.L and PAC.H),5)low dose(20 mg/kg)and 6)high dose(40 mg/kg)of TA group(TA.L and TA.H),7)10 mg/kg Guggulsterone Group(GS).The drug was administered intraperitoneally for four weeks.During the experiment,the daily food intake and weekly body weight changes of the mice were recorded.At the end of 8 weeks,blood was taken from the eyes and the organ indices(liver,heart,lung,spleen,and kidney)were measured.The serum and liver levels of TC,TG,HDL-C,LDL-C,and TBA were measured by an automatic biochemical analyzer and biochemical kits,respectively.H&E and Oil Red O staining to detect pathological changes and lipid deposition in the liver.UHPLC Q-Trap/MS determined BAs in the liver.The 16 S r DNA technique examined microbial diversity in the V3-V4 hypervariable region in fecal samples.Expression of BAs,lipid metabolism-related genes and proteins FXR,PPARα,FASN,and CYP7A1 in mouse liver was analyzed by Western blot and RT-qPCR.ResultNetwork pharmacological analysis: The results of the network pharmacological analysis showed that the total triterpenes of P.cocos represented by polyporenic acid C,poricoic acid B,and poricoic acid C could influence the development of MAFLD via the PPARα signaling pathway regulating lipid metabolism and BAs metabolism.Poria cocos extracts of different components: The extraction rates of WPC,APC,and EPC in P.cocos samples were determined to be 0.015 %,98.2 %,and 1.79 %,respectively,and their purity was80 %,80 %,and 57.43 %,respectively.Pharmacophore analysis: The MAFLD rat model was successfully induced after 12 weeks of HFD feeding.After administration of WPC,APC,and EPC treatment,the liver and white adipose tissue indexes decreased,while brown adipose tissue increased in rats.In addition,hepatocyte lipid droplet accumulation,swelling,degeneration,and necrosis were reduced,and the levels of TC,AST,and TBA in serum and liver were significantly decreased.EPC also considerably reduced the levels of TG and NEFA and increased the levels of HDL-C in the liver.WPC.L,APC.L/H,and EPC.H all markedly increased the serum concentrations of deoxycholic acid(DCA)and glycine deoxycholic acid(GDCA)and decreased the concentrations of taurine cholic acid(TCA)and taurine ursodeoxycholic acid(TUDCA).Furthermore,APC.L/H and EPC.H significantly increased serum bile acid(HCA)concentration.Moreover,APC.L/H and EPC.H also considerably increased the serum concentration of hyocholic acid(HCA).The further testing of BAs content in the feces of the EPC group showed that EPC dramatically decreased the attention of the primary BAs chenodeoxycholic acid3-Acyl-β-D-glucuronide and chenodeoxycholic acid 24-Acyl-β-D-glucuronide(p<0.001,p<0.01)and the secondary BAs DCA,HCA,UDCA,α-murichoclic acid(α-MCA),HDCA,THDCA,and lithocholic acid(LCA)in the feces concentrations(p<0.01,p<0.001,p<0.05).Additionally,EPC significantly down-regulated the relative expression of liver Srebp-1c,Fasn,Cyp7b1 mRNA(p<0.01,p<0.01,p=0.07)and up-regulated Fxr,Pparα,Cyp7a1,Cyp8b1,Ntcp,Bsep mRNA(p<0.01,p<0.05,p< 0.001).It also markedly decreased the protein expression of FASN,p-JNK,and CYP7A1(p<0.05,p<0.01,p<0.001)and increased the protein levels of PPARα and FXR(p<0.05).Mechanism of activation of FXR-mediated metabolism of BAs against MAFLD by PAC and TA explored: Molecular docking showed that PAC and TA with FXR had excellent binding activity.The MAFLD mouse model was successfully induced after 8 weeks of HFD feeding.After administration of PAC and TA treatment,the body weight and liver index of MAFLD mice were significantly reduced.Hepatocyte lipid droplet accumulation,swelling,and degeneration of hepatocytes were reduced.The liver function and kidney function impairment were improved considerably.PAC and TA also significantly reduced TC,TG,AST,and CREA levels in the serum and liver of MAFLD mice(p<0.05).PAC has significantly increased GCA,GCDCA,UDCA,β-MCA,MDCA,TLCA,GHCA,LCA-3-S(p<0.05,p<0.01,p<0.001).TA significantly increased β-MCA content in the liver of MAFLD mice(p<0.05)and dramatically decreased GLCA content(p<0.05).PAC and TA reversed the increase in abundance of the Firmicutes,Proteobacteria(p<0.001,p<0.05)and the decrease in the relative abundance of the Actinaobacteriota(p<0.001,p<0.05).PAC significantly up-regulated the relative expression of FXR,SHP(p<0.05)and downgraded the relative expression of Cyp7b1,Ntcp mRNA.TA was significantly up-regulated the relative expression of Pparα,Fxr,Cyp7b1,Shp mRNA(p<0.01,p<0.05,p<0.001)and BSEP(p<0.05).ConclusionWPC at 15/45 mg/kg dose and APC at 1.05/3.15 g/kg dose significantly improved hepatic steatosis and lipid metabolism disorders in HFD-induced MAFLD rats.In addition,EPC had regulatory effects on BAs disorders in serum,feces,and liver.P.cocos triterpenes and the monomeric components PAC acted as BAs acceptor activators to mediate FXR expression to interfere with BAs and lipid metabolism,thereby regulating the reversion of MAFLD.This study has implications for treating hepatic metabolic diseases with natural mushrooms and provides essential data to support P.cocos as a potential MAFLD treatment.