CaSR表达在大鼠糖尿病性肝硬化损伤和纤维化发生中的作用

The role of CaSR expression in diabetic cirrhosis injury and fibrosis in rats

目的:观察钙敏感受体(CaSR)在糖尿病性肝损伤发生中的作用。方法:本实验分别制备糖尿病大鼠和高糖处理HSC系大鼠肝星形细胞模型。40只Wistar大鼠随机分为正常对照组(Control,n=10),糖尿病组(T1D,STZ 60 mg/kg一次性腹腔注射,n=30),造模成功后分别在2、4、8周检测大鼠的体重、血糖、血清中谷草转氨酶(AST)和谷丙转氨酶(ALT)活性,观察形态学和超微结构改变,以及Western blot检测CaSR和肝纤维化相关指标表达的变化。HSC系大鼠肝星形细胞随机分为正常对照组(Control,10%FBS-DMEM+5.6 mmol/L葡萄糖),高糖组(HG,10%FBS-DMEM+40 mmol/L葡萄糖下培养48 h)和CaSR抑制剂组(HG+Calhex 231,10%FBS-DMEM+40 mmol/L葡萄糖+2.5μmol/L CaSR抑制剂(Calhex 231)下培养48 h,每组n=5)。结果:动物模型中,与正常组相比,糖尿病大鼠体重减轻,血糖、AST和ALT显著升高,CaSR和胶原Ⅰ(COⅠ)、胶原Ⅲ(COⅢ)、基质金属蛋白酶1(MMP1)、基质金属蛋白酶2(MMP2)、基质金属蛋白酶9(MMP9)蛋白表达上调;细胞模型结果与大体基本一致,与正常组相比,高糖组细胞分化标志性蛋白α-平滑肌肌动蛋白(α-SMA)表达增加,表明HSC分化成肌成纤维细胞,细胞外间质(ECM)主要成分COⅠ和COⅢ表达增加,降解ECM的关键酶MMP9同样增加,Calhex 231可减轻上述变化。结论:CaSR表达上调参与大鼠糖尿病性肝损伤和纤维化的发生。

Objective:To observe the role of calcium sensitive receptor(CaSR)in the pathogenesis of diabetic liver injury.Methods:Forty Wistar rats were randomly divided into normal control group(control,n=10)and diabetes group(T1D,STZ 60 mg/kg intraperitoneal injection,n=30),and the samples were collected at the 2nd,4th and 8th week.Rats hepatic stellate cells(HSC)were randomly divided into normal control group(Control,10%FBS-DMEM culture,n=5),high glucose group(HG,10%FBS-DMEM+40 mmol/L glucose,treated for 48 h,n=5)and CaSR inhibitor group(HG+Calhex 231,10%FBS-DMEM+40 mmol/L glucose+2.5μmol/L Calhex 231 for 48h,n=5).The body weight,blood glucose,serum glutamic oxaloacetic transaminase(AST)and alanine aminotransferase(ALT)activities were measured dynamically.The changes of liver morphology and ultrastructure were observed by HE staining and Masson staining by transmission electron microscopy.The changes of CaSR and liver fibrosis related indexes were detected by Western blot.Results:Compared with the control group,diabetic rats lost weight,while blood glucose,AST and ALT increased significantly,and the expression of CaSR,collagenⅠ(COⅠ),collagenⅢ(COⅢ),matrix metalloproteinase(MMP)-1,-2 and-9 increased significantly.The results of the cell model were basically the same as those in vivo.Compared with the control group,the expression ofα-smooth muscle actin(α-SMA)was increased,indicating that HSC differentiated into myofibroblasts in HG group.The expression of the main components of ECM(COⅠand COⅢ),and the key enzyme of ECM degradation(MMP9)were also increased,while CaSR inhibitor,Calhex 231,could reduce the above changes.Conclusion:The up-regulation of CaSR expression is involved in the occurrence of diabetic liver injury and fibrosis.