Protective Effect Of Ge Huang Granules Medicated Serum On The Injured L-02 Cells Induced By Ethanol In Vitro And The Study Of Its Mechanism

Objective: Through experiment in vitro,we set out to observe the efficacy of Ge Huang Granules Medicated Serum(GHGMS)to treat the injured L-02 Cells Induced By Ethanol,thus,discussing the protective effect and the possible molecular mechanism.Methods: 1.The preparation of medicated serum:60 SD rats were randomly divided into 3 groups: blank control group,Ge Huang Granules group(3.1g crude drug/100g/day),metadoxine group(0.1g/100g/day).20 rats were included in each group.To 2 times daily,morning and evening each once,for five consecutive days.The last delivery before 12 h of fasting,free drinking water,in the last delivery 1h after the treatment,2% pentobarbital sodium(0.3ml/100g)anesthesia animal,abdominal aortic blood serum was to be made.2.Model establishment and intervention : The concentrations of alcohol inducing hepatocyte damage,were detected by CCK8 assay.At the same time,cells supernatant AST,LDH was quantified by automatic biochemical analyzer.In combination with CCK8 consequences,to determine made mould time and ethanol concentration.Cell experiment divided into 6 groups:normal group,model group,high dosage GHGMS treatment group(high dosage group),middle dosage GHGMS treatment group(middle dosage group),low dosage GHGMS treatment group(low dosage group),metadoxine treatment group(positive control group).After cells inoculate 24 h,normal group rugular training,based on the above model group to join ethanol,the rest of group to join ethanol and different doses medicated serum,intervention 24 h,collected the cells supernatant and cells for subsequent testing.3.Cells supernatant AST,LDH was quantified by automatic biochemical analyzer.Flow cytometry instrument was employed to analyze the apoptosis.Real-time PCR was employed to analyze the RNA level of Fas,FasL,Caspase3,Caspase8.Immunohistochemical was used to determine the protein level of Fas,FasL.Results: 1.Model establishment:Using 3% ethanol intervention L-02 liver cells for 24 h,the content of AST and LDH increasing,the liver cell volume increases slightly,and the number of suspension cells increased slightly,the L-02 model of alcoholic liver disease were successfully established.2.Compared with the normal group,model group showed significantly increased apoptosis rate(P<0.05).However,these can be rescued markedly by middle dosage GHGMS treatment group(middle dosage group),high dosage GHGMS treatment group(high dosage group)and metadoxine treatment group(positive control group)(P<0.05).The significant difference between middle dosage group and positive control group(P<0.05),Still,no statistically significant difference was observed between high dosage group and positive control group(P>0.05).3.Compared with the normal group,model group L-02 cells showed significantly increased cells supernatant level of AST,LDH and elevated Fas,FasL,Caspase3,Caspase8 expression at mRNA(P<0.05),However,these can be rescued markedly by middle dosage GHGMS treatment group(middle dosage group),high dosage GHGMS treatment group(high dosage group)and metadoxine treatment group(positive control group)(P<0.05).The significant difference between middle dosage group and positive control group(P<0.05),Still,no statistically significant difference was observed between high dosage group and positive control group(P>0.05).4.Each group of the liver cells membrane has the express of Fas,FasL protein,which the dyeing lighter tan normal group,model group dyeing the most,dyeing high,middle and low dosage group were compared with model group becomes shallow.Staining positive control group compared with model group also becomes shallow.Conclusion: 1.Using 3% ethanol intervention L-02 liver cells for 24 h can sueeessful induction of liver cell injury.Using microscope to observe the liver cell volume increases slightly,and the number of suspension cells increased slightly.The model of alcoholic liver disease L-02 liver cells can be successfully established.2.GHGMS can improve the hepatic damage.Our results indicated efficient protective effect of GHGMS for ALD.3.GHGMS can lower the level of Fas,FasL,Caspase3,Caspase8 mRNA,decrease the express of Fas,FasL protein,reduce the rate of apoptosis,the regulation of apoptosis may be involved in the GHGMS mediated therapy for ALD.