基于IRE1/GPR78信号通路研究三黄益肾胶囊抑制DKD大鼠肾组织细胞凋亡的作用机制

Study on the mechanism of Sanhuang Yishen Capsule in inhibiting apoptosis of renal tissue cells in DKD rats based on IRE1/ GPR78 signaling pathway

关键词:  三黄益肾胶囊  糖尿病肾病  内质网应激  细胞凋亡  IRE1/GPR78信号通路

Key words:  Sanhuang Yishen Capsule  diabetic kidney disease  endoplasmic reticulum stress  apoptosis  IRE1/GPR78 signaling pathway

摘要:

[目的] 探讨三黄益肾胶囊对糖尿病肾病（DKD）模型大鼠的治疗作用，同时研究三黄益肾胶囊对 DKD 模型大鼠肌醇酶 1（IRE1）/G 蛋白偶联受体 78（GPR78）信号通路的影响。[方法] 将 30 只大鼠平均分为正常组、模型组、阳性药组、三黄益肾低剂量及三黄益肾高剂量组，每组 6 只。除正常组外，其余各组运用高糖高脂饲料联合链脲佐菌素（STZ）注射建立 DKD 模型，造模后正常组与模型组每天灌胃生理盐水，阳性药组每天灌胃厄贝沙坦 11.51 mg/kg，三黄益肾低剂量及三黄益肾高剂量组每天分别灌胃三黄益肾胶囊 0.81、1.62 g/kg，分别干预 8 周。通过观察给药期间各组大鼠血糖、 体质量水平， 检测造模给药后生化指标水平及肾组织病理学染色评估三黄益肾胶囊对 DKD 的治疗作用；其次，运用试剂盒及酶联免疫吸附（ELISA）法研究三黄益肾胶囊对 DKD 大鼠肾组织中氧化应激及炎症因子水平的影响；最后运用免疫荧光、Tunel 染色、蛋白免疫印迹（Western blot）等方法研究三黄益肾胶囊对 DKD 大鼠肾组织细胞凋亡以及 IRE1/GPR78 信号通路的影响。[结果] 经三黄益肾胶囊干预后，DKD 大鼠体质量显著增加， 血糖水平降低，血清肌酐（Cr）、尿素氮（BUN）及 24 h 尿蛋白水平下降，同时也可改善 DKD 大鼠肾组织病理学变化，肾组织炎细胞浸润及纤维化减轻。此外，三黄益肾胶囊可显著升高 DKD 模型大鼠肾组织中超氧化物歧化酶（SOD）和谷胱甘肽过氧化酶（GSH-Px）活性、降低丙二醛（MDA）水平，并降低 DKD 大鼠血清中白细胞介素-6（IL-6）、白细胞介素-1β（IL-1β）及肿瘤坏死因子-α（TNF-α）水平。Western blot 结果显示，三黄益肾胶囊显著降低了 DKD 模型大鼠肾组织中内质网应激标志性因子 GRP78、IRE1 蛋白表达水平， 同时有效降低了模型大鼠肾组织中促凋亡蛋白 BcL2 关联 X 蛋白（Bax）、半胱氨酸蛋白酶-3（Caspase-3）、半胱氨酸蛋白酶-9（Caspase-9）的表达，提高了抑制凋亡蛋白 BcL2 的表达。[结论] 三黄益肾胶囊对 DKD 模型大鼠具有显著治疗作用， 其作用机制可能与抑制肾组织中 IRE1/GPR78 信号通路激活，改善内质网应激有关。

Abstract:

[Objective] To investigate the therapeutic effect of Sanhuang Yishen Capsule on diabetic kidney disease （DKD） model rats， and to study the effect of Sanhuang Yishen Capsule on IRE1/GPR78 signaling pathway in DKD model rats. [Methods] The 30 rats were divided into the normal group，model group，positive drug group，Sanhuang Yishen low dose group，and Sanhuang Yishen high dose group，6 rats in each group. Except for the normal group，the DKD model was established by a high-sugar and high-fat diet combined with streptozotocin （STZ） injection in the other groups. After modeling，the normal group and the model group were given normal saline every day，the positive drug group was given irbesartan 11.51 mg/kg every day，and Sanhuang Yishen low dose and Sanhuang Yishen high dose groups were given Sanhuang Yishen Capsule 0.81 g/kg and 1.62 g/kg respectively every day for 4 weeks. The therapeutic effect of Sanhuang Yishen Capsule on DKD was evaluated by observing the blood glucose and body weight levels of rats in each group during administration，detecting the levels of biochemical indicators，and renal histopathological staining after administration. Secondly，the effects of Sanhuang Yishen Capsule on oxidative stress and inflammatory factors in renal tissue of DKD rats were studied by kit and ELISA. Finally，the effects of Sanhuang Yishen Capsule on renal cell apoptosis and IRE1/GPR78 signaling pathway in DKD rats were studied by immunofluorescence，Tunel staining，and Western blot. [Results] After the intervention of Sanhuang Yishen Capsule，the body weight of DKD rats increased significantly，the blood glucose level decreased，the levels of serum creatinine （Cr），urea nitrogen（BUN） and 24-hour urine protein decreased，and the pathological changes of renal tissue in DKD rats were improved. The infiltration of inflammatory cells and fibrosis in renal tissue were reduced. In addition，Sanhuang Yishen Capsule can significantly increase the activity of SOD and GSH-Px in renal 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. Western blot results showed that Sanhuang Yishen Capsule significantly reduced the protein expression levels of endoplasmic reticulum stress marker factors GRP78 and IRE1 in renal tissue of DKD model rats，and effectively reduced the expression of pro-apoptotic proteins Bax，Caspase-3，and Caspase-9 in renal tissue of model rats，and increased the expression of anti-apoptotic protein BCL2. [Conclusion] Sanhuang Yishen Capsule has a significant therapeutic effect on DKD model rats，and its mechanism may be related to inhibiting the activation of the IRE1/GPR78 signaling pathway in renal tissue and improving endoplasmic reticulum stress.