探讨过度内质网应激在慢性环孢素A（ylsprineA，sA）肾毒性细胞凋亡中的作用机制.docVIP

目的：探讨过度内质网应激在慢性环孢素A (Cy-closporine A, CsA)肾毒性细 胞凋亡中的作用机制。 方法：将Sprague-Dawley (SD)人鼠随机分为正常对照组和慢性CsA肾毒性组， 分别给予皮下注射橄榄油(lmg . kg1 . d_1,对照组)和CsA (15mg . kg1 . d_1, 毒性组)1周及4周后处死大鼠。通过末端脱氧核苷酸转移酶介导的dUTP缺口 末端标记测定法(TUNEL染色法)和三色染色评估肾小管间质纤维化和细胞凋亡; 使用免疫组化学染色和免疫印迹依次检测分子免疫球蛋白结合蛋白(BIP)、磷酸 化的真核细胞翻译起始因子2 a (elF2 a )、生长抑制DNA损伤基因153(GADD153)、 细胞凋亡相关基因caspase-12及caspase-3蛋白表达。 结果：CsA注射1周观察不到肾小管间质纤维化和TUNEL阳性细胞，而给予CsA 注射4周大鼠表现为明显的肾小管间质纤维化和大量TUNEL阳性细胞蛋白的表达 1周达到高峰，4周后降至正常水平蛋白表达呈时间依赖性的增加。 结论：在慢性CsA肾毒性屮，过度的内质网应激耗尽分子伴侣、激活凋亡前途径 从而导致肾小管上皮细胞凋亡参与肾小管间质损伤。 关键词：慢性环孢素A肾毒性；内质网应激；细胞凋亡 Abstract Objective: To investigate the impact of excessive endoplasmic reticulum stress on apoptotic cell death in a rat model of chronic cyclosporine A (CsA) nephrotoxicity. Method: Male Sprague-Dawley rats maintained on a low salt diet were treated daily with vehicle (olive oil, ImL/kg/d, s.c.) and CsA (15mg/kg s.c.) for 1 and 4 week. Tubulointerstitial fibrosis and apoptotic cell death was estimated by trichrome staining and TUNEL staining. In addition, immunohistochemistry and immunoblotting were used to evaluated the expressions of BIP, elF2 a , GADD153, caspase-12, and caspase-3. Results: Rats treated with CsA for lweek did not develop tubulointerstitial fibrosis and TUNEL-positive cells, whereas 4weeks treatment of CsA induced typical tubulointerstitial fibrosis and increased TUNEL-positive cells. At a molecular level, CsA induced a significant increase in BIP and caspase-12 expression peaked at lweek, ant then return to normal levels at 4wccks In contrast, the expressions of eIF2 ex , GADD153， and caspase-3 in CsA-treated rat kidneys were significantly increased in a time-dependent manner. Conclusion: These findings suggest that excessive endoplasmic reticulum stress causes apoptotic cell death by depleting molecular chaperones and stimulating the proapoptotic pathway in chronic CsA nephrotoxicity. Key words: Chronic CsA nephrotoxicity; endoplasmic reticulum stress; apoptosis 刖目 环孢素A(CsA)是一种强力的免疫抑制药物，于1972年Borel首