肾缺血再灌注通过促凋亡途径加重糖尿病小鼠肾损伤

Renal ischemia reperfusion participates in renal injury in diabetic mice through proapoptotic pathway

目的通过检测凋亡相关蛋白在糖尿病小鼠肾缺血再灌注(I/R)损伤中的表达变化,探讨肾I/R加重糖尿病肾损伤的可能机制。方法 28只健康清洁级雄性C57BL/6J小鼠,随机分为4组:正常血糖假手术组(Sham组)(n=6),糖尿病假手术组(DM+Sham组)(n=6),正常血糖肾缺血组(I/R组)(n=8),糖尿病肾缺血组(DM+I/R组)(n=8)。通过连续5 d腹腔注射链脲佐菌素(55 mg/kg)的方法制备1型糖尿病模型,4周后,通过开腹用无创微动脉夹夹闭双侧肾动静脉30 min,然后松开动脉夹再灌注12 h建立肾缺血再灌注损伤小鼠模型。常规生化检测血肌酐、尿素氮反应肾功能情况。Western blot方法检测肾组织中Bcl-2、Bax、Caspase-3蛋白的表达变化。结果糖尿病小鼠空腹血糖明显高于正常血糖小鼠(P <0. 05),而糖尿病小鼠空腹体重明显低于正常血糖小鼠(P <0. 05)。I/R组和DM+Sham组小鼠血清肌酐、尿素氮高于Sham组(P <0. 05),DM+I/R组小鼠血清肌酐、尿素氮高于DM+Sham组和I/R组(P <0. 05)。与Sham组比较,Bax和Caspase-3蛋白表达在I/R组和DM+Sham组小鼠肾组织中升高(P <0. 05),Bcl-2蛋白表达降低(P <0. 05);与DM+Sham和I/R组比较,DM+I/R组小鼠肾组织中Bax和Caspase-3蛋白表达明显升高(P<0. 05),Bcl-2蛋白表达明显降低(P <0. 05)。结论肾缺血再灌注损伤导致糖尿病小鼠肾组织促凋亡蛋白Bax和Caspase-3表达升高,而抗凋亡蛋白Bcl-2降低,肾缺血再灌注通过促凋亡途径加重糖尿病肾损伤。

Objective To explore the possible mechanism of renal ischemia-reperfusion(I/R)aggravating diabetic renal injury by detecting the expression changes of apoptosis related proteins in diabetic mice with renal I/R.Methods Twenty eight healthy and clean male C57BL/6J mice were randomly divided into 4 groups:normal blood glucose sham operation group(Sham group)(n=6),diabetic sham operation group(DM+Sham group)(n=6),normal blood glucose operation group(I/R group)(n=8),Diabetic operation group(DM+I/R group)(n=8).The model of type 1 diabetes was established by intraperitoneal injection of streptozotocin(55 mg/kg)for 5 consecutive days.After 4 weeks,mouse model of renal ischemia reperfusion injury was established by clamping the bilateral renal artery and vein for 30 minutes by non-invasive micro-arterial clip in opened laparotomy,and then releasing the artery clamp and reperfusion for 12 h.The renal function was detected by serum creatinine and urea nitrogen.The expression of Caspase-3,Bcl-2 and Bax was assessed by Western blot methods.Results The fasting blood glucose of diabetic mice was significantly higher than that of normal mice(P<0.05),while the fasting body weight of diabetic mice was significantly lower than that of normoglycemic mice(P<0.05).The serum creatinine and urea nitrogen in I/R group and DM+Sham group were higher than those in Sham group(P<0.05).The serum creatinine and urea nitrogen in DM+I/R group were significantly higher than those in I/R group and DM+I/R group(P<0.05).Compared with Sham group,the expression of Bax and Caspase-3 protein increased in the I/R group and DM Sham group,while the expression of Bcl-2 protein decreased(P<0.05).Compared with DM+Sham group and I/R group,the expression of Bax and Caspase-3 protein in renal tissue of DM+I/R group was significantly increased(P<0.05),and the expression of Bcl-2 protein was significantly decreased(P<0.05).Conclusion Renal ischemia-reperfusion injury resultes in increased expression of pro-apoptotic protein bax and Caspase-3 in renal tissue of diabetic mice,while decreases expression of anti-apoptotic protein Bcl-2.Renal I/R may aggravate diabetic kidney injury through pro-apoptotic pathway.