基于Nrf2/HO-1信号通路探讨三黄益肾胶囊对糖尿病肾病大鼠氧化应激的影响

Effect of Sanhuang Yishen Capsule on oxidative stress in rats with diabetic nephropathy based on Nrf2/HO-1 signaling pathway

关键词:  三黄益肾胶囊  糖尿病肾病  氧化应激  Nrf2/HO-1信号通路

Key words:  Sanhuang Yishen Capsule  diabetic kidney disease  oxidative stress  Nrf2/HO-1 signaling pathway

摘要:

[目的] 研究三黄益肾胶囊（SHYS）对糖尿病肾病（DKD）大鼠的氧化应激水平和Nrf2/HO-1信号通路的影响。[方法] 选用SPF级雄性SD大鼠40只，选取8只为对照组，其余大鼠通过高脂饲料联合链脲佐菌素（STZ）注射造模为DKD大鼠，随机分为模型组，SHYS低（0.81 g/kg）、高（1.62 g/kg）剂量组，阳性药组给予厄贝沙坦（30 mg/kg）进行灌胃，正常组、模型组予以等体积蒸馏水灌胃，灌胃四周。首先，观察各组大鼠造模给药后血清及尿液中相关生化指标及肾组织病理学变化，评估SHYS对DKD模型大鼠的治疗作用；其次，检测各组大鼠肾组织中抗氧化相关指标超氧化物歧化酶（SOD）、丙二醛（MDA）及谷胱甘肽过氧化物酶（GSH-PX）水平，明确SHYS对DKD模型大鼠肾组织氧化应激的影响；同时，运用酶联免疫吸附实验（ELISA）法检测各组大鼠肾组织匀浆中白细胞介素（IL）-1β、IL-6、肿瘤坏死因子-α（TNF-α）水平，明确SHYS对DKD模型大鼠肾组织炎症反应的影响；最后，运用定量聚合酶链反应（qPCR）和蛋白免疫印迹（Western blot）法检测SHYS干预后，各组大鼠肾组织中核因子红系2相关因子2（Nrf2）/血红素氧合酶-1（HO-1）信号通路关键因子Nrf2、HO-1基因及蛋白水平，初步探究SHYS抑制DKD模型大鼠肾组织氧化应激的作用机制。[结果] SHYS可明显降低DKD模型大鼠血清中肌酐（Cr）、尿素氮（BUN）水平，降低24 h尿蛋白定量（UTP）水平，同时改善DKD模型大鼠肾组织中肾小管局灶变性、肾小球基底膜增厚等病理学变化。此外，SHYS增加DKD大鼠肾组织中SOD和GSH-PX活性、降低MDA水平，降低血清中炎症因子TNF-α、IL-1β及IL-6水平。qPCR及Western blot表明，SHYS可显著上调DKD模型大鼠肾组织中Nrf2和HO-1基因与蛋白水平。[结论] SHYS对DKD大鼠具有治疗作用，作用机制与激活肾组织中Nrf2/HO-1信号通路，改善其氧化应激有关。

Abstract:

[Objective] To study the therapeutic effect of Sanhuang Yishen Capsule on diabetic nephropathy（DKD） model rats and its effect on Nrf2/HO-1 signaling pathway. [Methods] Forty male SD rats of SPF grade were selected. Eight were randomly chosen as normal control group，and the rest of the rats were injected with high-fat chow and streptozotocin（STZ） to establish the DKD model rats，which were randomly divided into the model group，the Sanhuang Yishen Capsule low（0.81g/kg） and high（1.62 g/kg） dose group. The positive group was given Irbesartan（30 mg/kg） by gavage，while the normal group and the model group were given equal volume of distilled water by gavage for four weeks. Firstly，the therapeutic effect of Sanhuang Yishen Capsule on DKD model rats was evaluated by examining the relevant biochemical indexes and renal histopathological changes in the serum and urine of rats in each group after modeling. Secondly，the levels of antioxidant-related indexes of superoxide dismutase（SOD），malondialdehyde（MDA），and glutathione peroxidase（GSH-PX） were detected in the renal tissues of the rats in each group，so as to clarify the therapeutic effect of Sanhuang Yishen Capsule on DKD model rats. Meanwhile the levels of interleukin（IL）-1β，IL-6 and tumor necrosis factor α（TNF-α） in the homogenates of renal tissues of rats in each group were detected by ELISA to clarify the effect of Sanhuang Yishen Capsule on the inflammatory response of renal tissues of rats in the DKD model. Finally，qPCR and Western blot were used to detect the levels of Nrf2 and HO-1 genes and proteins，which are key factors of Nrf2/HO-1 signaling pathway，in the renal tissues of rats in each group，so as to preliminarily investigate the mechanism of inhibition of oxidative stress by Sanhuang Yishen Capsule in the renal tissues of rats in the DKD model. [Results] Sanhuang Yishen Capsule could significantly reduce the levels of Cr and BUN in the serum of DKD model rats，reduce the 24 h urinary protein（UTP） level，and improve the focal degeneration of renal tubules，glomerular basal pathological changes such as membrane thickening simultaneously. In addition，Sanhuang Yishen Capsule can significantly increase the activities of SOD and GSH-PX in the kidney tissue of DKD model rats，reduce the level of MDA，and reduce the levels of IL-6，IL-1β and TNF-α in the serum of DKD rats. The results of qPCR and Western blot showed that Sanhuang Yishen Capsule could significantly up-regulate the gene and protein levels of Nrf2 and HO-1 in the kidney tissue of DKD model rats. [Conclusion] Sanhuang Yishen Capsule has a therapeutic effect on DKD model rats，and its mechanism may be related to up-regulating Nrf2/HO-1 signaling pathway in renal tissue and improving its oxidative stress.