The Dynamic Expression Of SHP2 In Fibrogenic Rats Liver Tissues And Its Relation With The Proliferation And Apoptosis Of Hepatic Stellate Cells In Vivo

Objectives To investigate the dynamic expression of SH2 domain containing protein tyrosine phosphatase 2?SHP2?in hepatic tissues of rats with hepatic fibrosis induced by intraperitoneal injection of carbon tetrachloride?CCI₄?and its relation with the proliferation and apoptosis of hepatic stellate cells?HSC?in vivo.Methods The rat model of liver fibrosis used in this study was induced by intraperitoneal injection of CCI₄.The histological changes of rats liver tissues were observed by Hematoxylin and eosin?HE?and Masson's trichrome staining.Immunohistochemical staining was used to detect the expression and localization of SHP2 and?-smooth muscle actin??-SMA?in hepatic tissues of rats.And SHP2 and?-SMA immunofluorescence double-labeling was used for detection of SHP2 expression in HSC in vivo.Dual staining both of the terminal deoxynucleotidy transferrase UTP-nick end laling?TUNEL?and?-SMA immunohistochemistry was used to detect apoptotic HSC in rat liver tissues.Western blot and real-time fluorescence quantitative PCR were used to detect the expressions of SHP2 proiten and mRNA in rat liver tissues.Excel 2003 was applied to establish experimental databases,and SPSS 17.0 statistical software was used for data analysis.Measured data were expressed as mean±standard deviation?x±s?.One-way analysis of variance was used to compare the mean among groups,the LSD test was used for comparison between the two groups.P<0.05 was considered with statistical significance.Results 1 The results of HE and Masson's trichrome staining showed that the rat model of hepatic fibrosis induced by CCl₄ was successfully established.With the progression of making model,liver fibrosis gradually aggravated,and the destruction of hepatic lobular was observed in the liver tissue sections at 6-8 weeks after modeling.At the same time,there were obvious proliferation of collagen fibersand and the formation of false leaflets,and the massive fibrous tissue in interlobular septum was observed.2 The immunohistochemical staining of?-SMA showed that the?-SMA was only weakly positively expressed in vascular smooth muscle cells of rat liver tissues in control group.With the development of hepatic fibrosis,the number of cell expressing?-SMA in liver tissues of rats gradually increased.The integral optical density?IOD?of?-SMA in fibrotic liver tissues of rats at 2 weeks,4 weeks,6 weeks and 8 weeks after modeling?0.13±0.01,0.18±0.01,0.24±0.02,0.28±0.02?were significantly?P<0.05?higher than that in the control group?0.09±0.01?,and the significant?P<0.05?differences in the IOD of?-SMA were observed in the liver tissues of rats among different time points after modeling.That was,with the progression of hepatic fibrosis,the number of cell expressing?-SMA gradually increased.3 The immunohistochemical staining of SHP2 showed that SHP2 was mainly expressed in the cytoplasm and some nuclei were also expressed.With the aggravation of hepatic fibrosis,the number of cell expressing SHP2 gradually increased.The IOD of SHP2 in the fibrotic liver tissues of rats at 2 weeks,4 weeks,6 weeks and 8weeks after modeling?0.23±0.01,0.27±0.01,0.30±0.01,0.33±0.01?were significantly?P<0.05?higher than that in the control group?0.19±0.01?,and there were significant differences in the IOD of SHP2 in fibrotic liver tissues of rats among different time points after modeling.In other words,the expressions of SHP2 in fibrotic rats liver tissues gradually increased with the progress of hepatic fibrosis.4.The observation of SHP2 and?-SMA immunofluorescence dual-labeled liver tissue sections in rats was conducted under laser scanning confocal microscopy?LSCM?.Single-channel scanning showed that the positive reaction products of SHP2 and?-SMA presented green and red fluorescent spots,respectively.When the scanning images were mixed,in addition to the green and red fluorescent spots,yellow spots formed under LSCM.Since only activated HSC and a few vascular smooth muscle cells express?-SMA in the fibrotic liver tissues,these yellow spots can be regarded as the co-expression product of SHP2 and?-SMA in HSC of fibrotic liver tissues.The co-expression products were mainly located in activated HSC cytoplasm.The results of image analysis showed that,at 2 weeks,4 weeks,6 weeks,and 8 weeks after modeling,SHP2-positive HSC accounted for 54%±3%,62%±2%,73%±4%,86%±3%of the cells expressing?-SMA?total activated HSC?,that was,with the progression of liver fibrosis,the proportion of activated HSC expressing SHP2 to total activated HSC gradually increased?P<0.05?.5.Dual staining both of TUNEL and of?-SMA immuno-histochemistry showed that apoptotic HSC were fewly observed in the liver tissue sections of the control group,whereas with the developing of liver fibrosis,the number of activated HSC increased,the number of apoptotic HSC also increased,thus the activated HSC and apoptotic HSC presented at the same time.Image analysis showed that,at 2weeks,4 weeks,6 weeks,and 8 weeks after modeling,the apoptotic index of HSC in rats liver tissues were 47%±1%,41%±2%,35%±1%and 29%±1%,respectively.Obviously,as the aggravation of liver fibrosis,the proportion of apoptotic HSC to total activated HSC gradually decreased?P<0.05?.6 Western blot showed that SHP2 protein expressions in the fibrotic hepatic tissues of rats at 2 weeks,4 weeks,6 weeks and 8 weeks after modeling?0.52±0.03,0.59±0.02,0.67±0.02,0.79±0.03?were significantly?P<0.05?higher than that of the control group?0.30±0.03?,and the differences of SHP2 protein expression in fibrotic liver tissues of rats at different time points had statistically significance?P<0.05?.That was to say,with the increase of hepatic fibrosis in rats,the expressions of SHP2 protein in liver tissues gradually increased.7 The expressions of SHP2?PTPN11?mRNA in rats liver tissues were detected by real-time fluorescence quantitative PCR.The expression level of SHP2?PTPN11?mRNA in the control group was assigned 1,the expressions of SHP2?PTPN11?mRNA in the fibrotic hepatic tissues of the rats at 2 weeks,4 weeks,6 weeks,and 8 weeks after modeling were 1.36-,1.60-,1.89-and 2.41-fold,respectively,all were significantly?P<0.05?higher than that in the control group,and gradually increased with the aggravation of liver fibrosis.8 Pearson's correlation analysis found that the immunohistochemistry results of SHP2 in fibrotic rat liver tissues and the proportion of SHP2-positive HSC to total activated HSC in rat liver tissues all had a significant positive correlation with the expressions of?-SMA,r values were 0.952 and 0.953,respectively?P<0.05?,but a significant negative correlation with the apoptotic index of activated HSC in fibrosis liver tissues in rats.r values were-0.958 and-0.963,respectively?P<0.05?.Conclusions 1 The protein and mRNA expressions of SHP2 in hepatic tissues of rats with CCl₄-induced hepatic fibrosis are up-regulated,and the ranges of up-regulationare are consistent with the degree of hepatic fibrosis.2 With the aggravation of hepatic fibrosis in rats,the expressions of SHP2 in activated HSC in vivo gradually increase.3 During the process of hepatic fibrosis in rats,the dynamic expressions of SHP2 in rat liver tissues and activated HSC in vivo all have a significant positive correlation with the activation and proliferation of HSC in vivo,but a significant negative correlation with the apoptosis of activated HSC in vivo.