| Chronic hepatitis B is highly infectious and the pathogenesis of steatohepatitis is complex.The clinical cases associated with the two diseases have increased recently,and the diagnosis interfere exist,which has become the difficulty in the treatment of chronic liver disease.However,the pathogenesis of chronic hepatitis B complicated with steatohepatitis has not been determined and there are no reports of effective therapeutic drugs.The study on the pathogenesis of complications depends on the models in vivo and in vitro,but there is no stable and reliable cell model at present.Therefore,it is of great significance to establish a stable model of chronic hepatitis B complicated with steatohepatitis in vivo and in vitro and to find natural and effective therapeutic drugs.In this study,HepG2.2.15,HepG2 and HL-7702 cells were induced by fat emulsion and oleic acid/ palmitic acid mixture in stages to establish a model of chronic hepatitis B complicated with steatohepatitis.And the cellular model was compared with the nutritional fatty liver model established by high fat diet mice.Then its mechanism of action was discussed in the paper.Furthermore,the paper researched the intervention effects of two natural products on the complication.1.Establishment of the model of chronic hepatitis B complicated with steatohepatitis in vitroThree different kinds of cells HepG2.2.15,HepG2 and HL-7702 were used to establish the model of fatty liver induced by FFA in stages.Fat emulsion was divided into three groups of different concentrations: 1%,0.5% and 0.25%,two different induction time: 12 h and 24 h.The mixture of OA/PA was set up with six concentrations: 5mM?2mM,1mM,0.5mM,0.25 mM and 0.125 mM,four induction time: 12 h,24h,48 h and 72 h.Cell viability,TG content and oil red O staining were employed to evaluate the formation of steatosis.The results showed that it is best induced 12 h by 1% fatty milk and 0.5mM OA/PA mixture for 24 h.Due to the steatosis was more and the cell viability was higher,the model was successfullyestablished.After induced steatosis of HepG2.2.15 cells,the content of HBV DNA in the model group was significantly lower than that in the normal group.2.Drug intervention of the model of chronic hepatitis B complicated with steatohepatitisTwo natural products A1:1806A and A2:1806C with different concentrations(100?g/mL,50?g/mL,10?g/mL)were used to treat HepG2.2.15,HepG2 and HL-7702 cells.The results indicated that both A1 and A2 could down-regulated HBV DNA level in HepG2.2.15 cells,inhibit the steatosis formation of HepG2 and protect HL-7702 from exogenous FFA-induced damage.3.Correlation research in mice with nutritional fatty liverThe mice were set up nine groups: normal,model,positive,high A1,middle A1,low A1,high A2,medium A2 and low A2.And the normal group mice were ordinary feed,the others were fed with high-fat diet.A1 and A2 are two natural phenolic acids A1:1806A and A2:1806C.Three concentrations of high,middle and low are50mg/kg,10mg/kg,2mg/kg respectively.Serological examination and TEM were carried out on liver tissues after three weeks of feeding,and the results showed that there were a lot of lipid drops in the hepatocytes of the model group.Therefore,the mice model of fatty liver was successfully established.It indicated that both A1 and A2 can significantly reduce TC,TG,AST,ALT,MDA and increase SOD,GSH-PX.In conclusion,this study used fat emulsion and OA/PA mixture to induce HepG2.2.15 cells transfected with HBV gene in stages,and successfully established a model of chronic hepatitis B complicated with fatty liver in vitro,which proved that cell steatosis could alleviate the replication of HBV virus.At the same time,the two kinds of cells HepG2 and HL-7702 were compared with HepG2.2.15.And we discussed the correlation with the nutritional fatty liver model established by high fat feeding mice.All these laid a foundation for the study of chronic hepatitis B complicated with steatohepatitis. |
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