The Dynamic Expression Of SHP1 In Fibrogenic Rats Liver Tissues And Its Relation With The Activation And Proliferation Of Hepatic Stellate Cells In Vivo

Objectives To investigate the dynamic expression of src-homology domain 2-containing protein tyrosine phosphatase 1?SHP1?in liver tissue of rats with hepatic fibrosis induced by intraperitoneal injection of carbon tetrachloride?CCI₄?and its relationship with activation and proliferation of hepatic stellate cells?HSC?in vivo.Methods The rat model of liver fibrosis used in this study was induced by intraperitoneal injection of carbon tetrachloride.The histological changes of rat liver tissues were observed by Masson's trichrome and Hematoxylin and eosin?HE?staining.Immunohistochemical staining was used for detecting the expressions and distributions of SHP1 and?-smooth muscle actin??-SMA?in rats liver tissues.At 4 time points,the localization and expression of SHP1 protein in hepatic tissues of rats were detected by western blot and real-time fluorescent quantitative PCR assay was used for detecting the expressions of SHP1 mRNA of fibrotic liver tissues in rats.Detection of SHP1 expression in HSC in vivo was conducted by using SHP1 and?-SMA immunofluorescence double labeling.Excel 2003 was used to establish an experimental database.Measured data were expressed as mean±standard deviation?x±s?.SPSS20.0 software was used for statistical analysis.One-way analysis of variance was used for comparison between groups and LSD test was used for comparison between groups.Test,P<0.05 was considered statistically significant.Results?1?HE and Masson's trichrome staining showed that CCI₄-induced rat liver fibrosis model was successfully constructed.With the prolongation of modeling,the degree of hepatic fibrosis gradually increased in rats.?2?The immunohistochemical staining of?-SMA showed that?-SMA was only weakly positively expressed in the smooth muscle cells of vessel wall in the liver tissues of the control group,and the number of cells expressing?-SMA in the liver tissues of the rats gradually increased with the development of liver fibrosis.The expressions of?-SMA were mainly distributed in the portal area,fibrous septum and hyperplastic bile duct of the rats liver tissues after modeling.The mean optical density?MOD?of?-SMA in the fibrotic liver tissues of rats at2 weeks,4 weeks,6 weeks and 8weeks after modeling?0.06±0.01,0.09±0.01,0.12±0.01,0.16±0.02?were significantly?P<0.05?higher than that in the control group?0.04±0.01?,and there were significant?P<0.05?differences in the MOD of?-SMA expressions among different time points of modeling.In brief,with the development of hepatic fibrosis,the cell number of positive?-SMA gradually increased,that was,the activation and proliferation of HSC in vivo in rats liver tissues gradually increased with the progression of hepatic fibrosis.?3?Immunohistochemical staining of SHP1 showed that SHP1 was mainly expressed in the cytoplasm and some nuclei were also expressed.The expression of SHP1 in hepatic tissues of normal rats was small,the cellular number of positive SHP1 in fibrotic rats liver tissues gradually increased with the aggravation of hepatic fibrosis,and the expressions of SHP1 were mainly distributed in fibrosis areas,portal areas,around the blood vessels,and hyperplastic bile ducts in fibrotic rats liver tissues.The MOD of SHP1 protein expressions in liver tissues of rats at 2 weeks,4 weeks,6 weeks,and 8 weeks after modeling?0.11±0.01,0.14±0.01,0.16±0.01,0.19±0.01?were significantly?P<0.05?higher than that of the control group?0.08±0.01?,and there were significant?P<0.05?differences in the expression of SHP1 in fibrotic rats liver tissues at the different time points after modeling.That was,the expression of SHP1 in fibrotic rats liver tissues gradually increased with the progression of hepatic fibrosis.?4?Western blot analysis showed that SHP1 protein expressions in the hepatic fibrotic tissues of rats at 2 weeks,4 weeks,6weeks,and 8 weeks after modeling?0.53±0.04,0.73±0.04,0.87±0.03,1.10±0.07?were significantly higher than that in the control group?0.35±0.07?,and at the different time after modeling,the differences of SHP1 protein expressions in the fibrotic liver tissues of rats were statistically significant?P<0.05?.That was to say,SHP1 protein expressions in rats liver tissues gradually increased with the progress of liver fibrosis.?5?Real-time fluorescence quantitative PCR was used to detect the expression of SHP1 mRNA in rats liver tissues.When the expression level of SHP1 mRNA in the control group was assigned1,the expression levels of SHP1 mRNA in the fibrotic liver tissues of the rats at 2 weeks,4weeks,6 weeks,and 8 weeks after modeling were 1.14-fold,1.36-fold,1.49-fold,1.61-fold,respectively,all were significantly?P<0.05?higher than that in the control group,and gradually increased with the progression of liver fibrosis?P<0.05?.?6?The observation of SHP1 and?-SMA immunofluorescence dual-labeled rats liver tissue sections were conducted under laser scanning confocal microscopy.There was SHP1 co-expressed with?-SMA,that was,SHP1 was positively expressed in activated HSC in liver tissues,and these co-expression products mainly located in the cytoplasm of activated HSC in vivo.The results of image analysis showed that,at weekly time points after modeling,SHP1-positive,activated HSC,accounted for 26.49%±3.44%,37.14%±4.57%,44.90%±2.94%and 58.09±5.33%?2,4,6 and 8 weeks,respectively?of the cells expressing?-SMA?total activated HSC?.That was,with the progression of liver fibrosis,the proportion of activated HSC expressing SHP1 to total activated HSC gradually increased?P<0.05?.?7?The pearson's correlation analysis between the results of immunohistochemical staining of SHP1 and the results of?-SMA immunohistochemical staining in rats liver tissues,or between the percentage of SHP1-positive activated HSC and the results of?-SMA immunohistochemical staining in rats liver tissues was conducted.And the results showed that the expressions of SHP1 and the percentage of SHP1-positive activated HSC all had a significant positive correlation with the expressions of?-SMA in fibrotic rats liver tissues?r values were 0.935,0.931,P<0.05,respectively?.Conclusions 1.The protein and mRNA expressions of SHP1 in hepatic tissues of rats with CCl4-induced hepatic fibrosis are up-regulated,and the ranges of up-regulation are consistent with the degree of hepatic fibrosis.2.The SHP1 expressions of activated HSC in fibrotic rats liver tissues gradually increase with the aggravation of hepatic fibrosis.3.During the pathological process of hepatic fibrosis in rats,the dynamic expressions of SHP1 in rats liver tissues and HSC in vivo are significantly positively correlated with the activation and proliferation of HSC in vivo.