Study On The Effect Of Qingzhuan Tea Extract On NAFLD Cell Lipid Deposition Via The MiR-33b-SREBP1c-FAS Pathway

Objective Non-alcoholic fatty liver(NAFLD)cell models were prepared using HepG2 cells induced by fetal calf serum(FBS)and LO2 cells induced by palmitic acid(PA).The effects of Qing Zhuan Tea(QZT)on the lipid deposition in non-alcoholic fatty liver cells and on the expression of mi R-33b-SREBP1 c / FAS in the adipogenesis signaling pathway were examined to investigate the molecular mechanism of QZT in preventing and treating non-alcoholic fatty liver cell steatosis.Methods(1)HepG2 cells were treated with different concentrations of FBS for 48 hours to establish the NAFLD cell models.The determination of cell viability in CCK-8 method combined with oil red O staining,intracellular triglyceride(TG)and total cholesterol(TC)measurement were used to determine the optimal FBS concentration for NAFLD cell model.(2)To observe the effect of QZT on HepG2 cell steatosis,cells were treated with QZT at different concentrations for 24 h.The CCK-8 method was used to detect the effect of different concentrations of QZT on cell proliferation.Then the cells were divided into 5 groups,namely normal control group,model group,QZT low and high dose group,positive drug group.The degree of cell steatosis was observed by oil red O staining.The TG and TC content changes were detected by enzyme method.The mi R-33 b and SREBP1 c m RNA expression were detected by q PCR.Western blot was used to detect the expression levels of lipid metabolism-related protein sterol regulatory element binding protein 1c(SREBP1c)and fatty acid synthase(FAS).(3)LO2 cells were used as the research object to give PA at different concentrations for 24 hours to establish the NAFLD cell models.The determination of cell viability in MTT assay combined with oil red O staining,and intracellular triglyceride(TG),alanine aminotransferase(ALT),and aspartate aminotransferase(AST)measurements to determine the optimal PA concentration for NAFLD cell models.(4)To observe the effect of QZT on LO2 cell steatosis,cells were pretreated with different concentrations of QZT for 24 h.MTT method was used to detect the effect of different concentrations of QZT on cell proliferation.Then the cells were divided into 5 groups,namely normal control group,model group,QZT low and high dose group,positive drug group.The degree of cell steatosis was observed by oil red O staining,and the changes of TG,ALT,and AST contents were detected by enzyme method.(5)q PCR was used to detect the expression of mi R-33 b,SREBP1c m RNA and FAS m RNA in cells.Cells were transfected with chemical fragments such as mi R-33 b mimicis and inhibitor.Western blot was used to detect the expression of downstream protein SREBP1 c to study on the molecular mechanism of QZT on LO2 cell steatosis.Results(1)HepG2 cells cultured with 50% FBS for 48 h can induce their steatosis to establish a NAFLD cell model.(2)When the concentration of QZT exceeds 240μg/m L,it has a significant inhibitory effect on the proliferation of HepG2 cells.Combined with the pre-experiment,60μg / m L was selected as the low dose group of QZT,and 240μg / m L was selected as the high dose group of QZT;After QZT treatment of the cell model,the results showed that the content of TG and TC in the model group were significantly increased,the expression of mir-33 b was decreased,and the expression of SREBP1 c and FAS were increased;QZT can significantly reduce the degree of cell steatosis(P<0.01),the content of TG and TC(P<0.01),increase mi R-33 b and reduce SREBP1 c m RNA expression level.At the same time,the expression of SREBP1 c and FAS protein could be down-regulated(P<0.01).(3)NAFLD cell model can be established by culturing LO2 cells with 0.4mmol/L PA for 24 h.(4)When the concentration of QZT exceeds 200μg/m L,it can significantly inhibit the proliferation of LO2 cells.Combined with the pre-experiment,50μg/m L was taken as the low dose group of QZT,200μg/m L was taken as the high dose group of QZT.The QZT pretreatment cell model results showed that the content of TG,ALT and AST in the model group were significantly increased.QZT pre-treated LO2 cell model can effectively reduce the degree of cell steatosis(P<0.01),and significantly reduce TG,ALT and AST content(P<0.01).(5)The expression of mi R-33 b in the model group cells was significantly reduced,and the expression of SREBP1 c and FAS was significantly increased.QZT pretreatment significantly up-regulated mi R-33 b and down-regulated the expression of SREBP1 c m RNA and FAS m RNA;PA or mi R-33 b inhibitor can increase the expression of SREBP1 c in LO2 cells,and application of mi R-33 b mimics can reverse the increase of SREBP1 c expression caused by PA.Conclusion Qingzhuan tea can affect the lipid production by up-regulating mi R-33 b,down-regulating the expression of SREBP1 c and FAS,thereby reducing the level of TG.The mi R-33b-SREBP1c-FAS pathway plays a protective role on NAFLD cells.