急性溃疡性结肠炎小鼠结肠组织中SHP2的表达及其作用探讨

Expression and role of SHP2 in colon tissues of mice with acute ulcerative colitis

目的观察急性溃疡性结肠炎(UC)小鼠结肠组织中含C-Src同源序列的蛋白质酪氨酸磷酸酶2(SHP2)的表达,探讨SHP2在UC病程中的作用。方法将30只小鼠随机分成正常组、模型组、SHP2组各10只。模型组和SHP2组饮用4%DSS制作急性UC模型,正常组饮用高压过的饮用水。造模第1天,正常组、模型组、SHP2组分别注射腺病毒稀释液、腺病毒空载体、腺病毒SHP2突变载体。每天记录小鼠体质量、腹泻及便血情况,使用疾病活动指数(DAI)评分量表评价肠道临床炎症严重程度。饲养至20 d处死小鼠,取结肠组织,采用HE染色观察组织形态学变化,进行组织学评分,采用阿尔新蓝染色法检测杯状细胞数量。收集小鼠心脏血,ELISA法检测血清炎性因子COX-2、IL-6、TNF-α水平。取结肠组织,Western blotting法检测结肠组织中SHP2、p-P65、p-STAT3表达水平,免疫组化染色法检测Ki67表达水平。结果与正常组比较,模型组DAI评分、结肠组织病理学评分均升高,杯状细胞数量减少,血清COX-2、IL-6、TNF-α水平及结肠组织中p-P65、p-STAT3、Ki67表达水平均升高(P均<0. 05或<0. 01);与模型组比较,SHP2组DAI评分、结肠组织病理学评分均降低,杯状细胞数量增加,血清COX-2、IL-6、TNF-α水平及结肠组织中p-P65、p-STAT3、Ki67表达水平均降低(P <0. 05或<0. 01)。结论急性UC小鼠结肠组织中SHP2表达升高; SHP2可能是通过介导NF-κB/STAT3信号通路的激活,调节炎性因子COX-2、IL-6、TNF-α释放,导致黏膜损伤,促进UC的发生。

Objective To observe the expression of protein tyrosine phosphatase 2 containing C-Src homologous sequence(SHP2)in the colon tissues of mice with acute ulcerative colitis(UC),and to explore the role of SHP2 in the course of UC.Methods Thirty BALB/c mice were randomly divided into three groups:the normal control group,model group,and SHP2 group with 10 mice in each.The mice in the model and SHP2 groups drank 4%DSS solution to make acute UC models,while mice in the normal control group drank autoclaved drinking water.On the first day of modeling,the mice in the normal control group,model group,and SHP2 group were injected with adenovirus diluents,adenovirus vector,and adenovirus SHP2 mutant vectors,respectively.The changes in the body weight,diarrhea,and blood in the stool were recorded daily,and the disease activity index(DAI)scores were used to assess the severity of intestinal inflammation.After 20 days,the mice were sacrificed.Heart blood was collected from each group to detect the levels of serum inflammatory factors cyclooxygenase 2(COX-2),interleukin 6(IL-6)and tumor necrosis factor-α(TNF-α)by ELISA.Meanwhile,colonic tissues of mice were collected,HE staining was applied to detect the histomorphological changes and we performed histopathological scores,Alcian blue staining was used to detect the number of goblet cells,Western blotting was used to detect the expression of SHP2,phosphorylated P65(p-P65)and phosphorylated signal transducer and activator of transcription 3(p-STAT3),and the immunohistochemistry was used to detect the Ki67 expression.Results Compared with the normal control group,both the DAI scores and colon histopathological scores increased,the number of goblet cells decreased,the levels of serum COX-2,IL-6 and TNF-αand the expression levels of p-P65,p-STAT3 and Ki67 in the colon tissues increased in the model group(all P<0.05).Compared with the model group,the DAI scores and colon histopathological scores decreased,the number of goblet cells increased,the levels of serum COX-2,IL-6 and TNF-αand the expression levels of p-P65,p-STAT3 and Ki67 in the colon tissues decreased in the SHP2 group(P<0.01 or<0.05).Conclusion The expression of SHP2 increases in the colon tissues of acute UC mice;SHP2 may regulate the release of inflammatory factors COX-2,IL-6 and TNF-αby mediating the activation of NF-κB/STAT3 signaling pathway,leading to mucosal damage and thereby promoting the occurrence of UC.