抑制环氧化酶2表达对糖尿病肾病足细胞损伤的影响

Inhibition of cyclooxygenase-2 expression contributes to podocyte injury in streptozotocin-induced diabetic rats

目的 观察环氧化酶2(cyclooxygenase-2,COX-2)介导的低密度脂蛋白受体(low density lipoprotein receptor,LDLr)表达失调在糖尿病肾病(diabetic nephropathy,DN)早期足细胞损伤中的作用.方法 选取8周龄雄性Sprague-Dawley(SD)大鼠,采用链尿菌素(streptozotocin,STZ)诱导糖尿病大鼠模型,造模成功后按照简单随机抽样原则分为对照(Control)组、糖尿病(diabetic mellitus,DM)组、DM+阿司匹林(Aspirin)组,每组10只.于第12周收集24 h尿液、检测尿微量白蛋白/肌酐比值(microalbumin to creatinine ratio,ACR),并于第12周处死,收集血标本,检测血脂谱,留取肾组织.免疫组化及Western印迹法观察COX-2、LDLr、胆固醇调节元件结合蛋白2(sterol regulatory element-binding protein-2,SREBP-2)及其裂解激活蛋白(SREBP cleavage-activating protein,SCAP)在肾脏的表达情况,免疫荧光法观察COX-2与WT-1在肾脏的共表达情况.透射电镜观察足细胞超微结构的改变,免疫组化及Western印迹法观察足细胞特异性标志蛋白WT-1及nephrin在肾脏的表达情况.分析COX-2与LDLr表达的相关性.油红0染色及胞内游离胆固醇定量测定法观察肾小球脂质沉积情况.结果 与对照组相比,DM组大鼠尿ACR显著升高(P＜0.01),肾脏COX-2及LDLr、SCAP、SREBP-2蛋白的表达显著增加(P＜0.01),足细胞损伤加重(P＜0.01),肾小球内有显著的脂质沉积(P＜ 0.01),而DM+Aspirin组上述改变明显减轻(P＜0.05).并且COX-2的表达与LDLr的表达呈正相关(r=0.85,P＜0.01).激光共聚焦荧光显微镜观察证实,大鼠肾脏COX-2与足细胞特异性标志物WT-1存在共表达.结论 COX-2的过度表达加重了DN足细胞损伤,其机制可能与破坏LDLr负反馈调节、上调肾脏足细胞LDLr表达、增加细胞内胆固醇摄入,致使肾小球足细胞内脂质过度沉积有关.抑制COX-2表达能减轻糖尿病足细胞损伤.

Objective To investigate the role of cyclooxygenase-2 (COX-2) in podocyte injury in diabetic rats mediated by the disruption of low-density lipoprotein receptor (LDLr) pathway.Methods Eight-week old male Sprague-Dawley (SD) rats were treated for 12 weeks by dividing into three groups:control rats,streptozotocin (STZ) induced diabetic rats (DM),and diabetic rats treated with aspirin (DM+Aspirin).The plasma lipid profile was checked by clinical biochemistry assay.The ratio of urinary microalbumin to creatinine (ACR) was detected by enzyme-linked immunosorbent assay.Intracellular lipid accumulation was evaluated by Oil Red O staining and a free cholesterol quantitative assay.The glomerular podocyte injury and the expression of molecules related with LDLr pathway were evaluated by electron microscope,immunohistochemical staining,immunofluorescent staining,and Western blotting.Results There were increased levels of urinary ACR (P ＜ 0.01) and podocyte injury(P ＜ 0.01) in DM rats compared with the controls.Additionally,lipid accumulation in kidneys of DM rats were significantly increased (P ＜ 0.01),due to increased protein expressions of COX-2,LDLr,sterol regulatory element-binding protein (SREBP) cleavage activating protein (SCAP),and SREBP-2 (P ＜ 0.01).However,these changes were significantly inhibited by an inhibitor of COX-2,Aspirin (P ＜ 0.05).It's worth noting that,COX-2 protein expression was closely correlated with LDLr protein expression (r=0.85,P ＜ 0.01).Conclusion Dysregulation of LDLr pathway contributes to podocyte injury in diabetic nephropathy,which may be mediated through the increased COX-2 expression.