肾康注射液对慢性肾衰竭大鼠肾间质纤维化、内质网应激状态和线粒体氧化损伤的影响

Effects of Shenkang Injection on renal interstitial fibrosis,endoplasmic reticulum stress and mitochondrial oxidative damage in chronic renal failure rats

目的探索肾康注射液对慢性肾衰竭大鼠肾损伤及肾纤维化的作用机制。方法采用腺嘌呤法建立大鼠慢性肾衰竭模型,采用叔丁基过氧化氢(TBHP)诱导NRK-52E细胞,建立慢性肾衰竭肾损伤细胞模型。采用苏木精–伊红(HE)染色评估肾康注射液对慢性肾衰竭大鼠的肾脏病理学损伤,免疫组化法检测模型大鼠肾损伤及纤维化的关键蛋白表达,免疫荧光法检测NRK-52E细胞葡萄糖调节蛋白78(GRP78)蛋白表达,Western blotting法进行肾组织和细胞中的关键蛋白定量,活性氧荧光探针检测NRK-52E细胞中的活性氧(ROS)水平,采用JC-1检测试剂盒检测NRK-52E细胞中线粒体膜电位水平。结果肾康注射液可以显著缓解慢性肾衰竭大鼠的肾间质纤维化状态,减轻病理损伤;显著抑制肾损伤分子1(KIM-1)、GRP78、内质网应激相关蛋白(CHOP)阳性表达表达,从而抑制内质网过度应激状态;可显著抑制I型胶原蛋白(Collagen I)沉积,抑制α-平滑肌肌动蛋白(α-SMA)表达,从而抑制肾间质纤维化形成(P<0.01);肾康注射液能够显著抑制NRK-52E细胞内ROS的水平,减轻NRK-52E线粒体氧化损伤,稳定线粒体膜电位水平(P<0.01)。结论肾康注射液能够减轻肾间质纤维化、缓解肾损伤状态,增强肾功能,其机制可能与抑制肾脏过度的内质网应激,抗氧化损伤以及减少肌成纤维细胞的增殖和减少细胞外基质的沉积实现有关。

Objective To explore the protective effect of Shenkang Injection on renal injury and renal fibrosis caused by chronic renal failure and its mechanism.Methods The rat model of chronic renal failure was established by adenine method,and NRK-52E cells were induced by TBHP.HE staining was used to evaluate the renal pathological injury of Shenkang Injection on rats with chronic renal failure.The expression of key proteins in renal injury and fibrosis was detected by immunohistochemical method.The expression of GRP78 in NRK-52E cells was detected by immunofluorescence method.Western Blotting method was used to quantify key proteins in tissues and cells,ROS levels in NRK-52E cells were detected by ROS fluorescent probes,and mitochondrial membrane potential levels in NRK-52E cells were detected by JC-1 detection kit.Results Shenkang Injection can significantly alleviate the renal interstitial fibrosis of chronic renal failure and alleviate pathological injury.It can significantly inhibit the positive expressions of KIM1,GRP78,and CHOP,so as to inhibit the overstress state of ER.Collagen I deposition andα-SMA were significantly inhibited,thus inhibiting the formation of renal interstitial fibrosis(P<0.01).Shenkang Injection could significantly inhibit the ROS level in NRK52E cells,alleviate the mitochondrial oxidative damage of NRK-52E,and stabilize the mitochondrial membrane potential(P<0.01).Conclusion Shenkang Injection can alleviate renal interstitial fibrosis,alleviate renal injury and enhance renal function,the mechanism of which may be related to inhibiting excessive endoplasmic reticulum stress,anti-oxidative injury,reducing proliferation of myofibroblasts and reducing ECM deposition.