| Objectives To investigate the effect of expression changes of SH2 domain-containing protein tyrosine phosphatase 2(SHP2)on the activation and proliferation of hepatic stellate cells(HSC)in vivo and its signal transduction mechanism.Methods The rats model of hepatic fibrosis was established by intraperitoneal injection of carbon tetrachloride(CCl4).The recombinant adenovirus Ad-SHP2 carrying wild-type SHP2(PTPN11)gene and expressing green fluorescent protein(GFP),the recombinant adenovirus Ad-sh RNA/SHP2 carrying RNA interference sequence targeting SHP2-short hairpin RNA(sh RNA)and expressing GFP,and empty virus Ad-GFP expressing only GFP were introduced into rats by tail vein injection.160 healthy male SD rats were randomly divided into 5 groups: control group(injection with saline only),model group(injection with CCl4),Ad-GFP group(injection with CCl4+Ad-GFP),Ad-SHP2 group(injection with CCl4+Ad-SHP2)and Ad-sh RNA/SHP2 group(injection with CCl4+Adsh RNA/SHP2).There were 32 rats in each group,at the 2nd,4th,6th and 8th week during modeling,8 rats in each group were taken for liver tissue samples.The histopathological changes of rats liver tissue were observed by HE and Masson trichromatic staining,the expression of SHP2 in rats liver tissue was detected by immunohistochemical staining,and the SHP2 expression of HSC in vivo was detected by SHP2 and α-smooth muscle action(α-SMA)immunofluorescence double-labeling.Western blot and real-time PCR techniques were used to detect the expression of protein and m RNA of SHP2,α-SMA,extracellular signal regulated kinase 1(ERK1),serinethreonine protein kinase B(Akt)and the protein expression of phosphorylated ERK(pERK)and phosphorylated Akt(p-Akt)in rats liver tissue.Results 1 HE and Masson trichrome staining showed that the rats model of hepatic fibrosis was successfully established,and the hepatic fibrosis gradually aggravated with the prolongation of modeling time;In Ad-SHP2 group,hepatic fibrosis was more severe;In Ad-sh RNA/SHP2 group,hepatic fibrosis was alleviated.2 Immunohistochemical staining,western blot and real-time PCR techniques to detect the expression of SHP2 protein and m RNA in rats liver tissue showed that,with the extension of modeling time,the expression of SHP2 in rats liver tissue increased gradually in model group and adenovirus treated groups(Ad-GFP group,Ad-SHP2 group and Ad-sh RNA/SHP2 group).The expression of SHP2 protein and m RNA in liver tissue of rats at the 2nd,4th,6th and 8th week during modeling were compared at the same time node,that in model group and adenovirus treated group was higher than that in control group(P>0.05),and compared with the model group and Ad-GFP group,SHP2 expressions in Ad-SHP2 group significantly increased(P<0.05),while that in Ad-sh RNA/SHP2 group significantly decreased(P<0.05),there was no significant difference between the Ad-GFP group and the model group(P>0.05).3 SHP2 and α-SMA immunofluorescence double labeling showed that with the extension of modeling time,the percentage of activated HSC expressing SHP2 to the total activated HSC increased gradually in the model group and each adenovirus treatment group;At the same time node,compared with the model group and Ad-GFP group,the percentage of activated HSC expressing SHP2 to the total activated HSC at the 2nd,4th,6th and 8th week after modeling was significantly increased in Ad-SHP2 group(P < 0.05),and significantly decreased in Ad-sh RNA/SHP2 group(P < 0.05),there was no significant difference between Ad-GFP group and model group(P > 0.05).4 α-SMA expression of protein and m RNA in rats liver tissue detected by western blot and real-time PCR showed that,with the extension of modeling time,the expression of α-SMA in liver tissue of rats in model group and adenovirus treatment group increased gradually.α-SMA expressions in liver tissues of rats at different modeling time points of the 2nd,4th,6th and 8th week were compared at the same time node.α-SMA expressions of protein and m RNA in liver tissue of rats in the model group and adenovirus treatment group were significantly higher than those in the control group(P<0.05).Compared with the model group and Ad-GFP group,α-SMA expression in AdSHP2 group increased significantly,while that in Ad-sh RNA/SHP2 group decreased significantly(P<0.05).There was no significant difference between the Ad-GFP group and the model group(P>0.05).5 The expression of ERK1 protein and m RNA and the expression of p-ERK1 protein in rats liver tissue were detected by western blot and realtime PCR,it showed that with the extension of modeling time,the above detection indexes increased gradually in the model group and each adenovirus treatment group.The expressions of ERK1 protein and m RNA and p-ERK1 protein in liver tissues of rats at different modeling time points of the 2nd,4th,6th and 8th week were compared at the same time node.The expression of ERK1 protein and m RNA and p-ERK1 protein in the liver tissue of the model group and each adenovirus treatment group was significantly higher than that in the control group,but there was no significant difference in the expression of ERK1 protein and m RNA between the model group and each adenovirus treatment group(P>0.05).Compared with the model group and the Ad-GFP group,the expression of p-ERK1 protein in Ad-SHP2 group was significantly increased,while that in Ad-sh RNA/SHP2 group was significantly decreased,there was no significant difference between the Ad-GFP group and the model group.6 Western blot and real-time PCR were used to detect the expression of Akt protein and m RNA and p-Akt protein in rat liver tissue.With the extension of modeling time,the above indexes in model group and adenovirus treated groups increased gradually.The expressions of Akt protein and m RNA and p-Akt protein in liver tissues of rats at different modeling time points of the 2nd,4th,6th and 8th week were compared at the same time node.The expressions of Akt protein and m RNA and p-Akt protein in the liver tissue of the model group and each adenovirus treatment group were significantly higher than that in the control group(P<0.05).There was no significant difference in the expression of Akt protein and m RNA between the model group and adenovirus treatment group.Compared with the model group and Ad-GFP group,the expression of p-Akt protein significantly increased in Ad-SHP2 group and decreased in Ad-sh RNA/SHP2 group(P<0.05),there was no significant difference between Ad-GFP group and model group(P>0.05).Conclusion 1 overexpression of SHP2 can promote the activation and proliferation of hepatic stellate cells in vivo,while down-regulation of SHP2 expression can inhibit the activation and proliferation of hepatic stellate cells in vivo.2 ERK and PI3K/Akt signal transduction pathways are involved in the regulation of SHP2 on the activation and proliferation of hepatic stellate cells in vivo.3 down-regulation of SHP2 expression can alleviate hepatic fibrosis by inhibiting the activation and proliferation of hepatic stellate cells in vivo.Figure 13;Table 15;Reference 70... |
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