The Effects Of SHP2 Expression Changes On The Apoptosis Of Hepatic Stellate Cells In Vivo

Objectives To study the effects of SH2 domain-containing protein tyrosine phosphatase(SHP2)expression changes on apoptosis of in vivo hepatic stellate cells(HSC)and it’s mechanisms.Methods 160 healthy male SD rats were randomly divided into 5 groups according to the intervention method: control,model,Ad-GFP,Ad-SHP2 and Ad-sh RNA/SHP2 groups.Adenovirus control null viruses Ad-GFP showing only green fluorescent protein(GFP),and recombinant adenovirus Ad-SHP2 with wild-type SHP2 and GFP genes,and adenovirus Ad-sh RNA/SHP2 with RNA interference sequences targeting SHP2 short hairpin RNA(sh RNA)and GFP genes were amplified.The model group,Ad-GFP group,Ad-SHP2 group and Ad-sh RNA/SHP2 group were constructed by intraperitoneal injection of carbon tetrachloride(CCl4)into rats as a model of liver fibrosis.Ad-GFP group,AdSHP2 group and Ad-sh RNA/SHP2 group were injected with the above adenovirus via tail vein of rats.The control group was injected with saline only.Eight rats in each group were executed under anaesthesia at 2 weeks,4 weeks,6 weeks and 8 weeks of modeling time to obtain liver tissue specimens.Pathological changes in rats liver tissues were measured by HE staining and Masson trichromatic staining.SHP2 expressions in rats liver tissues were measured by Western blot technique.The apoptosis of HSC in vivo was measured by double-labelling with Tunel and α-SMA immunoimmunofluorescence.Changes in Bcl-2and Bax expressions in liver tissues were measured by Western blot and real-time fluorescence quantitative PCR.Results 1 HE and Masson trichromatic staining revealed that the liver tissue morphology of the control rats was normal,while the severity of liver fibrosis in the model group and each adenovirus-treated group(Ad-GFP,Ad-SHP2 and Ad-sh RNA/SHP2 groups)gradually increased as the modelling time progressed.When comparing the HE and Masson ttrichromatic staining of the liver tissues of the rats in each group at the same timepoint of modelling,the liver fibrosis of the rats in the Ad-SHP2 group was aggravated compared with the model and the Ad-GFP groups,and the difference between the Ad-GFP and model groups was not statistically significant(P>0.05),while the liver fibrosis of the rats in the Ad-sh RNA/SHP2 group was lessened compared with the model and the AdGFP groups.2 The Western blot technique revealed that the expression of SHP2 in the liver tissues of rats in the model and adenovirus-treated groups gradually increased as the modelling time progressed.Comparison of SHP2 expressions in the liver tissues of rats in each group at the time points of 2,4,6 and 8 weeks of modeling revealed that the AdSHP2 group was significantly higher than the model and Ad-GFP groups(P<0.05),the difference between the model and Ad-GFP groups was not statistically significant h(P>0.05),while the Ad-sh RNA/SHP2 group was significantly lower than the model and Ad-GFP groups(P<0.05).3 The results of Tunel and α-SMA immunofluorescence double staining revealed that the apoptotic index of activated HSC in liver tissues of the model group and each adenovirus-treated group decreased gradually with the extension of the modeling period.Comparison of apoptotic indices of activated HSC in liver tissue of rats in each group at time points of 2,4,6 and 8 weeks of modeling revealed that there was a marked decrease in the Ad-SHP2 group compared to the Ad-GFP and the model groups(P<0.05),and no statistically significant difference in the Ad-GFP group compared to the model group(P>0.05),while the Ad-sh RNA/SHP2 group was markedly elevated compared to the Ad-GFP group and the model group(P<0.05).4 The expressions of Bcl-2protein and m RNA in the liver tissues of rats in the model and adenovirus-treated groups increased gradually with the increase of modeling time.Comparison of Bcl-2 expression in the liver tissues of rats in each group at the time points of 2,4,6 and 8 weeks of modeling revealed that the Ad-SHP2 group was significantly higher compared to the Ad-GFP group and the model group(P<0.05),the difference between the Ad-GFP group and the model group was not statistically significant(P>0.05),while the Ad-sh RNA/SHP2 group was significantly lower compared to the Ad-GFP and the model groups(P<0.05).5 The expressions of Bax protein and m RNA in the liver tissues of rats in the model and adenovirus-treated groups increased gradually with the increase of modeling time.Comparison of Bax expressions in the liver tissue of rats in each group at the time points of2,4,6 and 8 weeks of modeling revealed that the Ad-SHP2 group was significantly lower compared to the Ad-GFP and the model groups(P<0.05),the difference between the AdGFP group and the model group was not statistically significant(P>0.05),while the Adsh RNA/SHP2 group was significantly higher compared to the Ad-GFP and the model groups(P<0.05).Conclusions 1 The apoptosis of activated hepatic stellate cells in vivo in rats is significantly influenced by changes of SHP2 expression,that is,down-regulation of SHP2 expression can promote apoptosis of activated hepatic stellate cells,whereas upregulation of SHP2 expression can suppress apoptosis of activated hepatic stellate cells in vivo in rats.2 The Bcl-2/Bax mechanism is involved in the regulation of apoptosis by SHP2 in activated hepatic stellate cells in vivo in rats.Down-regulation of SHP2 expressions increase Bax expressions and decrease Bcl-2 expressions in rats liver tissues,while up-regulation of SHP2 expressions decrease Bax expressions and increase Bcl-2expressions in rats liver tissues.3 Down-regulation of SHP2 expressions attenuates rats liver fibrosis by promoting apoptosis of activated hepatic stellate cells in vivo.Figure 11;Table 8;Reference 99...