Baicalin Nanoliposomes Ameliorated Non-alcoholic Fatty Liver Disease By Suppression TLR4 Signaling Pathway In Mice

Background: Non-alcoholic fatty liver disease(NAFLD)is a common chronic metabolic disease in the liver,which is caused by factors except alcohol abuse.This disease is characterized by hepatic steatosis and fat accumulation.The disease spectrum of NAFLD includes simple steatosis,non-alcoholic steatohepatitis(NASH),liver fibrosis,liver cirrhosis and even hepatocellular carcinoma.The prevalence and mortality of NAFLD is increasing rapidly and assumes the younger age tendency.Up to now,the pathogenesis of NAFLD is poorly understood and effective therapies are lacking.Hence,looking for effective drug treatment is of great importance to the treatment of NAFLD.Baicalin(BA),a kind of flavonoid,has multiple pharmacological effects,such as anti-inflammation,anti-oxidant,regulating blood lipid,immunity adjustment and so on.A number of studies suggested that baicalin have effect on various liver injury.Objective: To explore the protective effects and the possible mechanism of baicalin and baicalin nanoliposomes on NAFLD in mice.Methods:1.Preparation and characterization of BA-NLBy the thin film dispersion method,used lecithin and cholesterol to prepare BA-NL.The morphology,particle size distribution,polydispersity index,Zeta potential,encapsulation efficiency and drug loading efficiency were characterized.2.The protect effect of BA-NL in NAFLD miceThe male C57BL/6J mice were randomly divided into control group,BA group,BA-NL group,MCD group,MCD-BA group,MCD-BA-NL group.Mice were fed with MCD diet 5 week to induce NAFLD,while BA and BA-NL(50mg/kg/d)were administered for last 4 weeks respectively.All mice general condition,body and liver weight were evaluated.The content of ALT,AST and TG in serum were measured by automatic biochemical analyzer.Liver histopathological changes,steatosis,lipid accumulation and fibrosis were observed by HE staining,oil red O staining and Masson staining.Hepatocyte apoptosis were determined by TUNEL.3.Effect of BA-NL on TLR4 signaling activated inflammatory in NAFLD miceMacrophages(F4/80)and neutrophils(Ly6G)in the liver tissues were detected by immunofluorescence.The hepatic mRNA expressions of IL-1?,IL-6,TNF-?,ICAM,ECAM,ELAM,CCL2 and CXCL2 were assayed by qRT-PCR.The hepatic protein expressions of TLR4,MyD88,P-P65,P-P38 were tested by WB.Results:1.The BA-NL were found to be spherical and small with smooth surface.The particle size distributions was 81.41 nm,polydispersity index was 0.31,Zeta potential was-13.9 ± 5.74 mV,The encapsulation efficiency was 81.10%,drug loading efficiency was 6.90%.2.Compare with MCD group,BA,BA-NL could notably ameliorate serum ALT and AST,liver injury,liver ballooning and hepatic fibrosis of NAFLD.However,mice treated with BA-NL is more effective.Besides BA-NL could notably increase liver index and decrease serum TG and hepatic steatosis.3.Relative to control group,the infiltration of Macrophages and neutrophils,mRNA expressions of inflammatory mediator,protein expressions of TLR4,MyD88,P-P65,P-P38 were up-regulated in MCD diet-fed mice,which was significantly declined by treatment with BA,BA-NL,and yet BA-NL is more effective.Conclusion: BA-NL ameliorated non-alcoholic fatty liver disease.The potential protective mechanism may be based on inhibiting the TLR4 signaling pathway,reduce the infiltration and activation of inflammatory cells.