Effects Of SHP2 On The Apoptosis Of Activated Hepatic Stellate Cells In Vitro

Objectives To investigate the effects of src-homology domain 2-containing protein tyrosine phosphatase 2(SHP2)on the apoptosis in activated hepatic stellate cells(HSC)in vitro.Methods The recombinant adenovirus Ad-SHP2 harboring genes for both wild type SHP2(PTPN11)and green fluorescent protein(GFP),Ad-shRNA/SHP2 carrying RNA interference——short hairpin RNA(shRNA)targeting SHP2(PTPN11)and expressing GFP and control null adenovirus Ad-GFP expressing GFP alone used in the study were obtained by infecting the AD293 cells again and again and then the viral titer estimates were conducted.Human hepatic stellate cell line(LX-2)was cultured in vitro and transfected with the recombinant adenovirus Ad-SHP2,Ad-shRNA/SHP2 and the control null virus Ad-GFP.The expressions of SHP2 mRNA in HSC were measured by real-time fluorescent quantitation PCR and the protein expressions of SHP2,Bax and Bcl-2 in activated HSC were measured by Western blot.The apoptosis of activated HSC were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL),double labeling both of Annexin-V and Propidium iodide(PI)and flow cytometry.The SPSS 22.0 statistical software was used for data analysis,the measurement data was showed as mean±standard deviation(x±s),one-way analysis of variance was used for the comparison of mean among multiple groups,and LSD test was used for the comparison of mean between the two groups.P<0.05 was considered statistically significant.The experimental group as follows:(1)Control group,when adenovirus transfection,DMEM was used to replace virus venom.(2)Ad-GFP group,HSC was transfected with empty adenovirus Ad-GFP.(3)Ad-shRNA/SHP2 group,HSC was transfected with recombinant adenovirus Ad-shRNA/SHP2.(4)Ad-SHP2 group,HSC was transfected with recombinant adenovirus Ad-SHP2.Results 1 The adenovirus Ad-GFP,Ad-shRNA/SHP2,Ad-SHP2 were obtained by repeated infection of AD293 cells,the titers were 1.6 x 10~8 pfu/ml,1.4 x 10~8 pfu/ml and1.2 x 10~8 pfu/ml,respectively.2 At 48 hours after adenovirus infection in HSC at multiplicity of infection 100(Ad-GFP and Ad-shRNA/SHP2)or 300(Ad-SHP2),the transfection rate was measured by fluorescent microscopic counting.All of which were more than 80%.3 At 48 hours after adenovirus infection,SHP2 mRNA expressions in HSC were detected by real-time fluorescence quantitative PCR.The SHP2 mRNA expression of HSC in control group was 1,then the SHP2 mRNA expressions in Ad-GFP group,Ad-shRNA/SHP2 group,and Ad-SHP2 group were 1.109 times,0.256 times,and7.554 times,respectively,compared to the control group.The expression of SHP2 mRNA of HSC in Ad-SHP2 group was significantly higher than those in control group,Ad-GFP group and Ad-shRNA/SHP2 group(P<0.05).The expression level of SHP2 mRNA of HSC in Ad-shRNA/SHP2 group was significantly lower than those in control group,Ad-GFP group and Ad-shRNA/SHP2 group(P<0.05).However,there was no significant difference in the expression of SHP2 mRNA in HSC between the Ad-GFP group and the control group(P>0.05).Western blot was used to analyze the expression of SHP2 protein of HSC in each group.Ad-SHP2 group(1.852±0.138)was significantly higher than those in Ad-shRNA/SHP2 group(0.759±0.120),control group(1.271±0.206)and Ad-GFP(1.336±0.197),P<0.05.While the expression of SHP2 protein in Ad-shRNA/SHP2 group was significantly lower than those in Ad-SHP2 group,control group and Ad-GFP group(P<0.05),there was no statistically significant difference in the expression of SHP2 protein between the Ad-GFP group and the Control group(P>0.05).4.Apoptosis of activated HSC in vitro by TUNEL assay:the apoptosis rate of HSC in Ad-shRNA/SHP2 group(12.755±1.606)%was significantly higher than those in Ad-SHP2 group(1.520±0.259)%,control group(3.077±0.731)%and Ad-GFP group(3.250±0.851)%,P<0.05.The apoptosis rate of HSC in Ad-SHP2 group was significantly lower than those in control group,Ad-GFP group and Ad-shRNA/SHP2 group(P<0.05).There was no significant difference in the apoptosis rate of HSC between Ad-GFP group and control group(P>0.05).5.The double labeling of Annexin V/PI and flow cytometry were used to detect the apoptosis of HSC in each group.The apoptosis rate of HSC in Ad-shRNA/SHP2 group(19.340±2.505)%was significantly higher than those in Ad-SHP2 group(2.442±0.931)%,Ad-GFP group(8.893±1.982)%and control group(9.438±0.804)%,P<0.05.And the apoptosis rate of HSC in Ad-SHP2 group was significantly lower than those in control group,Ad-GFP group and Ad-shRNA/SHP2 group(P<0.05),but there was no significant difference in the apoptosis rate of HSC between Ad-GFP group and control group(P>0.05).6.Western blot was used to analyze the Bax protein expression of HSC at 48hours after adenovirus infection in each group.The Bax protein expression of HSC in Ad-shRNA/SHP2 group(2.493±0.203)was significantly higher than those in Ad-SHP2 group(1.545±0.208),control group(1.989±0.147)and Ad-GFP(1.999±0.162),P<0.05.The Bax protein expression of HSC in Ad-SHP2 group was significantly lower than those in control group,Ad-GFP group and Ad-shRNA/SHP2 group(P<0.05),but there was no significant difference between the Ad-GFP group and the control group(P<0.05).7.The Bcl-2 protein expression of HSC in each group was analyzed by Western blot,the Bcl-2 protein expression of HSC in Ad-shRNA/SHP2 group(1.042±0.148)was significantly lower than those in Ad-SHP2 group(2.134±0.247),control group(1.707±0.146)and Ad-GFP group(1.521±0.142),P<0.05.The Bcl-2 expression of HSC in Ad-SHP2 group was significantly higher than those in control group,Ad-GFP group and Ad-shRNA/SHP2 group(P<0.05),there was no significant difference between the Ad-GFP group and the control group(P>0.05).Conclusions 1 The changes of SHP2 expression have a significant effect on apoptosis in activated HSC in vitro,which is negatively correlated with apoptosis of activated HSC,that is,down-regulation of SHP2 expression can induce apoptosis of activated HSC,while up-regulation of SHP2 expression can inhibit apoptosis of activated HSC in vitro.2 The Bcl-2/Bax mechanism is involved in the regulation of SHP2 to the apoptosis of activated HSC in vitro,that is,the down-regulation of SHP2 expression leads to the increase of Bax expression and the decrease of Bcl-2 expression in activated HSC in vitro,while the up-regulation of SHP2 expression leads to the decrease of Bax expression and the increase of Bcl-2 expression in activated HSC in vitro.Figure6;Table6;Reference 75.