氧化应激和P53参与波动高糖诱导的肾小管上皮细胞凋亡

Oxidative stress and P53 involve in fluctuant high glucose-induced apoptosis of renal tubular epithelial cells

目的:探讨波动性高糖引起的肾小管上皮细胞凋亡的机制。方法:体外培养人肾小管上皮细胞(HK-2),给予稳定高糖或波动高糖干预,并使用抗氧化剂和P53特异性抑制剂验证氧化应激和P53在波动高糖诱导的肾小管上皮细胞凋亡中的作用;此外,SD大鼠随机分为正常对照组(A)、糖尿病稳定高血糖组(B)和糖尿病波动高血糖组(C)。采用链脲佐菌素(STZ)65 mg/kg腹腔注射诱发糖尿病,血糖波动组每天定时腹腔注射速效胰岛素,并错时给予葡萄糖,造成一天中血糖浓度大幅波动模型。采用比色法检测超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量,免疫组化法和Western blotting检测NADPH氧化酶4(Nox4)和P53蛋白表达,流式细胞术和原位缺口末端标记法(TUNEL)检测肾脏细胞凋亡。结果:(1)与稳定高糖比较,波动高糖引起更加明显的HK-2细胞凋亡,P53蛋白表达明显上调,同时培养上清液中MDA含量增加和SOD活性下降,抗氧化剂和P53抑制剂可明显抑制磷酸化P53蛋白表达和细胞凋亡。(2)在体动物实验制模12周后,与B组比较,C组肾组织Nox4表达明显增加,肾小管磷酸化P53蛋白表达上调,细胞凋亡数增加。结论:氧化应激和P53参与波动性高糖诱导的肾小管上皮细胞凋亡。

AIM： To explore the mechanism of fluctuant high blood glucose-induced apoptosis of renal tubular epithelial cells.METHODS： Cultured human renal tubular epithelial cells（HK-2） were treated with stable high glucose or fluctuant high glucose.Antioxidant and specific inhibitor of P53 were applied for identifying the role of oxidative stress and P53 in fluctuant high glucose-induced apoptosis of renal tubular epithelial cells.Additionally,SD rats were randomly divided into normal control group（A）,stable high blood glucose group（B） and fluctuant high blood glucose group（C）.Diabetic rats were induced by intraperitoneal injection of streptozocin（STZ,65 mg/kg）,and the fluctuant high blood glucose animal model was induced by intraperitoneal injection of ordinary insulin and glucose at different time points every day.The activity of superoxide dismutase（SOD） and the content of malonaldehyde（MDA） were detected by the method of colorimetry.The protein expression of NADPH oxidase 4（Nox4） and P53 were examined by immunohistochemistry and Western blotting.Apoptosis was assessed by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling（TUNEL）.RESULTS： The cultured HK-2 cells treated with fluctuant high glucose had significantly higher apoptotic rate and expression level of P53 protein than those in the cells treated with stable high glucose.Compared with the culture solution of the cels treated with stable high glucose,the SOD activity was decreased and the concentration of MDA was increased in the culture solution of the cells treated with fluctuant high glucose.The antioxidant and specific inhibitor of P53 significantly inhibited the p-P53 expression and decreased the apoptotic rate.After 12 experimental weeks,the cell apoptotic index and protein expression of Nox4 and p-P53 in the kidney tubular epithelial cells isolated from the diabetic rats were significantly increased in C group as compared with B group.CONCLUSION： Oxidative stress and P53 are involved in fluctuant high glucose-induced apoptosis of renal tubular epithelial cells.