槐杞黄颗粒对糖尿病肾病大鼠及高糖环境下肾细胞的保护机制

The Protection Mechanism of Huaiqihuang Granules from Diabetic Kidney Disease in Rats and for Renal Cells in High-Glucose Environment

【目的】观察槐杞黄颗粒对糖尿病肾病(DKD)大鼠及高糖环境下肾细胞的保护机制。【方法】(1)体内实验:采用高脂肪饲料喂养结合链脲佐菌素(STZ)腹腔注射法构建DKD大鼠模型。实验设正常组,模型组,槐杞黄颗粒低、中、高剂量组及氯沙坦组,每组10只大鼠。给予对应处理后,测定尿微量白蛋白、空腹血糖、血清肌酐、总胆固醇和低密度脂蛋白胆固醇等主要生化指标,分别采用苏木素-伊红(HE)染色、高碘酸希夫(PAS)染色和马松(Masson)染色观察肾组织病理改变。(2)体外实验:构建高糖诱导肾小管上皮细胞HK2模型。实验分为正常糖组、高糖组和槐杞黄颗粒组及甘露醇组。给予对应处理后,MTT法检测细胞增殖情况,采用2’,7’-二氢二氯荧光素二乙酸酯(DCFH-DA)法和MitoSOX/Hoechst 33342染色法分别检测细胞内、线粒体活性氧簇(ROS)的生成,蛋白免疫印迹(Western Blot)法检测炎症小体相关蛋白NOD样受体蛋白3(NLRP3)、裂解型半胱天冬酶3(cleaved-Caspase-3)和白细胞介素1β(IL-1β)及肾纤维化相关蛋白纤维连接蛋白(FN)和Ⅳ型胶原蛋白(ColⅣ)的表达。【结果】(1)在体内实验中,槐杞黄颗粒可显著改善DKD大鼠尿微量白蛋白排泄、血清肌酐异常,高血糖和脂质代谢紊乱,减轻肾脏组织病理损害;(2)在体外实验中,槐杞黄颗粒可显著抑制高糖诱导HK2细胞增殖和ROS的过量产生,降低高糖诱导HK2细胞NLRP3炎症小体的激活和肾纤维化相关蛋白FN和ColⅣ的表达。【结论】槐杞黄颗粒可有效改善DKD大鼠,其对高糖环境下肾细胞的保护机制可能与抑制ROS生成、NLRP3炎症小体活化及纤维化蛋白高表达有关。

To observe the protection mechanism of Huaiqihuang Granules from diabetic kidneydisease(DKD)in rats and for renal cells in high-glucose environment.Methods(1)In vivo experiment:the rat model was constructed by high-fat diet feeding combined with STZ intraperitoneal injection.The experiment was divided into normal group,model group,Huaiqihuang Granules low-,medium-and high-dose groups and Losartan group,with 10 rats in each group.After corresponding treatment,the main biochemical indexes such as urinary microalbumin,fasting blood glucose,serum creatinine,total cholesterol and low density lipoprotein cholesterol were measured,and the pathological changes of renal tissue were observed by hematoxylin-eosin staining,periodic acid Schiff’s staining and Masson staining,respectively.(2)In vitro experiments:a high glucoseinduced renal tubular epithelial cell HK2 model was constructed.The experiment was divided into normal glucose group,high-glucose group,Huaiqihuang Granules group and mannitol group.After corresponding treatment,the cell proliferation was detected by MTT assay,and the generation of reactive oxygen species(ROS)in cells and mitochondria was detected by 2’,7’-Dihydrodichlorofluorescein diacetate(DCFH-DA)method and MitoSOX/Hoechst 33342 staining method,respectively.Western Blot was used to detect the expressions of inflammasomerelated proteins NOD-like receptor protein 3(NLRP3),cleaved-caspase-3 and interleukin-1β(IL-1β)and renal fibrosis-related proteins fibronectin(FN)and typeⅣcollagen(ColⅣ).Results(1)In vivo experiments,Huaiqihuang Granules significantly improved the urinary microalbumin excretion and serum creatinine,hyperglycemia and lipid metabolism disorders in DKD rats,and reduced the pathological damage of renal tissue.(2)In vitro studies,Huaiqihuang Granules significantly inhibited proliferation and ROS overproduction of highglucose-induced HK2 cells,reduced the activation of NLRP3 inflammasome and the expressions of renal fibrosisrelated proteins FN and ColⅣin high-glucose-induced HK2 cells.Conclusion Huaiqihuang Granules has an impovement effect on DKD in rats,and its protection mechanism for renal cells in high-glucose environment is relavent to inhibiting ROS generation,activation of NLRP3 inflammasome and expression of fibrosis-related proteins.