ARA290抑制糖尿病大鼠肾脏氧化应激反应

ARA290 inhibits oxidative stress in kidney of diabetic rats

目的:探讨促红细胞生成素(EPO)衍生肽ARA290能否抑制糖尿病大鼠肾脏氧化应激损伤,发挥肾脏保护作用。方法:链脲菌素腹腔注射制备糖尿病大鼠模型。将成模大鼠随机分为糖尿病模型组(DM组)、糖尿病模型+ARA290 50 U/kg治疗组(DA1组)和糖尿病模型+ARA290 100 U/kg治疗组(DA2组),同时设立正常对照组(NC组)。药物干预12周后检测大鼠尿白蛋白/尿肌酐(ACR)、尿β2微球蛋白(β2-MG)。肾组织切片PAS染色,观察肾脏病理变化。免疫荧光、免疫共沉淀检测大鼠肾脏EPO受体(EPOR)、CD131的表达,实时荧光定量PCR和Western印迹检测大鼠肾脏NOX4表达,ELISA法检测大鼠肾脏活性氧(ROS)水平,免疫组化检测大鼠肾脏丙二醛(MDA)及8-羟基脱氧鸟苷(8-OHdG)表达。结果:免疫荧光检测显示大鼠肾脏表达EPOR、CD131,且两者存在共定位表达;免疫共沉淀显示EPOR、CD131在肾脏内存在相互结合;ARA290可降低糖尿病大鼠尿ACR、β2-MG,减轻糖尿病大鼠肾脏病理改变;ARA290可抑制糖尿病大鼠肾脏NOX4表达,降低肾脏ROS水平,减少肾脏MDA及8-OHdG表达。结论:大鼠肾脏表达EPOR和CD131,且两者在肾脏内存在相互结合,ARA290可能通过EPOR-CD131蛋白复合物介导抑制糖尿病大鼠肾脏氧化应激损伤,发挥肾脏保护作用。

Objective:To investigate whether erythropoietin(EPO) derived peptide ARA290 can inhibit oxidative stress injury of kidney and play a renal protective role in diabetic rats. Methodology:Diabetic rats were induced by intraperitoneal injection of streptozotocin. Diabetic rats were randomly divided into diabetic model group,ARA290 50 U/kg treatment group,ARA290 100 U/kg treatment group,and normal control group was set up at the same time. After 12 weeks of drug intervention,urinary albumin/creatinine(ACR) and β2-microglobulin(β2-MG) were measured. The pathological changes of kidney were observed by PAS staining. The expressions of EPO receptor(EPOR) and CD131 in rat kidney were detected by immunofluorescence and co-immunoprecipitation. The expression of NOX4 in rat kidney was detected by quantitative real-time PCR and western bloting. The ROS level in rat kidney was detected by ELISA. The expressions of malondialdehyde(MDA) and 8-hydroxydeoxyguanosine(8-OHdG) in rat kidney were detected by immunohistochemistry. Results:ARA290 could reduce ACR and β2-MG in diabetic rat urine. Immunofluorescence assay showed that EPOR and CD131 were expressed in rat kidneys,and they were co-localized;co-immunoprecipitation showed that EPOR and CD131 were interlinked in the kidney;ARA290 could alleviate renal pathological damage,inhibit the expression of NOX4,reduce ROS level,and decrease expressions of MDA and 8-OHdG in the kidney of diabetic rats. Conclusion:EPOR and CD131 are expressed in rat kidneys,and they bind to each other in kidney. ARA290 inhibit oxidative stress injury,plays a renal protective role in diabetic rats,which might be mediated by EPOR-CD131 protein complex.