The Study Of The Effects Of Liraglutide On The Proliferation Of Rat Hepatic Stellate Cells Under High Glucose Conditions And Its Relationship With ERK Signaling Pathway

Objective: Liver fibrosis is one of the many complications of diabetes.The hepatic stellate cell(HSC)in the core position in the formation of liver fibrosis is the main source of the cells which generate extracellular matrix during liver damage.Given the leading role HSC plays in the pathogenesis of hepatic fibrosis,it is seen as the target in anti-fibrosis research.Liraglutide is a new antidiabetic drugs which belongs to GLP-1 receptor agonists.Research shows that there is GLP-1 receptor expression on hepatic stellate cells.So we designed this experiment to explore the effects of liraglutide on the proliferation of rat hepatic stellate cells under high glucose conditions and its association with ERK signaling pathway.Methods:1 The cultivation of hepatic stellate cells : The rat hepatic stellate cells(HSC T6)were vaccinated in germ-free bottles at 37?,the volume fraction of 5% CO2 saturated humidity incubator with DMEM solution which includes 10% fetal bovine serum and 5.5mmol/L glucose.2 Use MTT assay to determine the most appropriate concentration of liraglutide.HSC was divided into 11 groups,which were given high glucose(25.0mmol/L)and different concentrations of liraglutide(0nmol/L?50nmol/L?100nmol/L?200nmol/L?300nmol/L?400nmol/L?500nmol/L?600nmol/L?700nmol/L?800nmol/L ?900nmol/L).MTT assay was used to detect the proliferation of cells of the 11 groups after the observation of 48 h.Select 700nmol/L the concentration of liraglutide whose inhibition of the proliferation of cells began to be the highest as optimum concentration.Use MTT assay to determine the most appropriate concentration of PD98059 the inhibitor of ERK signaling pathway.HSC was divided into 7 groups,whichwere given high glucose(25.0mmol/L)and different concentrations of PD98059(0umol/L ? 25umol/L ? 50umol/L ? 100umol/L ? 200umol/L ?300umol/L?400umol/L).MTT assay was used to detect the proliferation of cells of the 7 groups after the observation of 48 h.Select 200umol/L the concentration of PD98059 of the group whose inhibition of the proliferation of cells began to be the highest as optimum concentration.3 Divide into groups and intervene : Divide the rat hepatic stellate cells into 5 groups,low glucose control group(5.5 mmol/L glucose),high glucose control group(25.0 mmol/L glucose),hypertonic control group(5.5 mmol/L glucose+19.5 mmol/L mannitol),liraglutide group(high glucose+optimum concentration of liraglutide group),inhibitor group(25mmol/L glucose+ Inhibitor PD98059).Five groups of cells were cultured for 48 hours in DMEM solution with 10% fetal bovine serum.4 ELISA was used to detect the quantitative of type I collagen of supernatant of rat hepatic stellate cells.5 RT-PCR was used to detect the expression of ERK mRNA in rat hepatic stellate cells.6 Western blot method was used to detect the expression of ERK/p-ERK protein.7 Data analysis: Excell was used to make a data book.The data were presented as mean±standard deviation(x±s)and SPSS 17.0 software was used for statistical analys.One-Way ANOVA was used to make comparison between groups.P <0 05 represents there are significant differences.Results: 1 Determined by MTT method the most appropriate concentration of liraglutide is 700nmol/L and the most appropriate concentration of PD98059 is 200umol/L;2 Type I collagen content comparison:Compared with the control group,type I collagen in hypertonic group showed no significant changes(P>0.05),but in high glucose group it was significantly increased(P <0.05),however in ERK inhibitor group and liraglutide group it decreased significantly compared with that in high glucose group(P <0.05)and expression of Type I collagen content in ERK inhibitor group was lower than that in liraglutide group(P <0.05);3 ERK mRNA expression results : Compared with the control group,ERK mRNA in hypertonic group showed no significant changes(P>0.05),but in high glucose it was significantly increased(P <0.05),however in ERK inhibitor group and liraglutide group it decreased significantly compared with that in high glucose group(P <0.05)and expression of ERK mRNA in ERK inhibitor group was lower than that in liraglutide group(P <0.05);4 ERK expression results:Compared with the control group,p-ERK in hypertonic group showed no significant changes(P> 0.05),expression of p-ERK in high glucose was significantly increased(P <0.05),expression of p-ERK in ERK inhibitor group and liraglutide group decreased significantly compared with that in high glucose group(P <0.05)and expression of p-ERK in ERK inhibitor group was lower than that in liraglutide group(P <0.05).Conclusion:1 Hyperglycemia can promote the proliferation of rat hepatic stellate cells.2 Hyperglycemia inducing HSC proliferation is related to ERK signaling pathway.3 Liraglutide can inhibit the proliferation of HSC in high glucose condition by inhibiting ERK signaling pathway.