Chrysin And Its Nanoliposomes Ameliorated Nonalcoholic Steatohepatitis Via Suppression Of TLR4 Signaling Pathway In Mice

Background: Nonalcoholic steatohepatitis(NASH)is a chronic liver disease histologically characterized by liver steatosis,inflammation,hepatocellular injury and different degrees of fibrosis.It's a critical stage in the progression of non-alcoholic fatty liver disease NAFLD from a reversible and benign non-alcoholic fatty liver(NAFL)to the irreversible stage of liver injury.Its progression leads to the development of cirrhosis,resulting in various liver?related adverse outcomes,and is now one of the leading causes for end-stage of liver disease.Early intervention is hopeful to halt or reverse its deterioration,and yet there continues to be a lack of fully proven pharmacological therapeutic options.Therefore,it is still necessary to search for new drugs with ideal therapeutic effects and find potential therapeutic targets on this basis.As a natural flavonoid compound,chrysin(CH)has been reported to exhibit hepatoprotective and anti-inflammatory properties,showing its good prospects in the treatment of NASH.Objective: To investigate the effects and the possible mechanism of CH and chrysin-loaded nanoliposomes(CH-NL)on NASH.Methods:1.Preparation and characterization of CH-NLThe CH was prepared into CH-NL with yolk lecithin and cholesterol using a film rehydration method.The particle size,zeta-potential,polydispersity index(PDI),morphology,encapsulation rate and drug loading rate of the CH-NL were characterized.Mice were intragastric administration with CH or CH-NL.The concentration of chrysin in plasma and liver were determined by UPLC-MS/MS,and the relative bioavailability of CH-NL was evaluated.2.The effect of CH and CH-NL in NASH miceAfter one week of diet adaptation,all mice were randomly divided into six groups(n=6 in each group)below: control group,CH group,CH-NL group,MCD group,M + CH group,M + CH-NL group.The last three groups of mice were fed with MCD for 4 weeks to induce NASH.And the other three groups of mice were fed a standard chow diet for the same period.The mice of CH group and M+CH group,CH-NL group and M+CH-NL group were intragastric administered with CH(100 mg/kg)or CH-NL(100 mg/kg)once every other day for the last 2 weeks,respectively.Mice in other groups received an equal volume of 0.5% CMC-Na.At the end of 4 weeks,all mice were anesthetized to collect blood and liver tissues.Liver injury were observed by hematoxylin and eosin(HE)staining and the levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in serum.Lipid accumulation in liver was evaluated by oil red O staining and determination of liver triglyceride content.The degree of liver fibrosis was assessed by Masson staining,Sirius red staining and ?-SMA immunohistochemical staining.DHE staining and the content of myeloperoxidase(MPO)in liver tissues were used to determine the degree of oxidative stress.3.Effect of CH and CH-NL on TLR4 signaling pathway in the liver induced by MCDImmunofluorescence staining(IF)was used to detect the recruitment and infiltration of inflammatory cells such as leukocytes,macrophages,and neutrophils in liver tissue.Immunohistochemical(IHC)was used to measure the levels of IL-6 and TNF-? in liver tissues.Western blot(WB)was used to detect the protein expression of TLR4 and its downstream signaling molecules in liver tissues.Results:1.The CH-NL were approximately spherical.The average particle sizes of CH-NL were 121±8 nm,zeta-potential were-37.9 ±3.4 mV,and the polydispersity index(PDI)was 0.22.The encapsulation rate of the prepared CH-NL was found to be 91.79%,and the drug loading rate was 10.49%.Compared with CH,CH-NL has increased the chrysin concentration in plasma and liver by 2.04 times and 3.32 times,respectively.2.Compare with MCD group,CH and CH-NL could significantly reduce serum ALT,AST activities and the level of oxidative stress in liver tissue,ameliorate the degree of hepatic steatosis,inflammation,hepatocyte ballooning and hepatic fibrosis.Besides CH-NL could notably decrease liver index and TG in serum.Interestingly,the CH-NL was more effective than the CH.3.Compared with control group,the infiltration of inflammatory cell in the liver was increased in MCD group.Besides,the levels of TLR4,MyD88,p-IRAK1,p-p38 and p-p65 were up-regulated in MCD group.However,after treatment with CH or CH-NL,the increased inflammatory cell infiltration in the liver was markedly inhibited.These upregulated and activated signaling molecules were inhibited by CH or CH-NL treatment.Conclusion: CH-NL have protective effects against MCD-induced nonalcoholic steatohepatitis,and the underlying mechanism may be associated with inhibition TLR4 signaling pathway,reducing hepatic pro-inflammatory mediator production and inflammatory cell infiltration.