基于NADPH/ROS/ERK信号通路探讨肾康降酸颗粒对高尿酸血症大鼠肾小管上皮细胞损伤的作用机制

Discussion on the mechanism of Shenkang Jiangsuan granule on the injury of renal tubular epithelial cells in rats with hyperuricemia based on the NADPH/ROS/ERK signal pathway

目的观察肾康降酸颗粒对高尿酸血症大鼠肾小管上皮细胞损伤及NADPH/ROS/ERK信号通路的影响。方法将40只雄性SD大鼠随机分成空白组、模型组、别嘌呤醇组及肾康降酸颗粒组,每组10只。除空白组外,其余组均给予腺嘌呤联合酵母粉连续灌胃14 d制备高尿酸血症模型。造模结束后第2天起,别嘌呤醇组给予27 mg/(kg·d)的别嘌呤醇水混液灌胃,肾康降酸颗粒组给予3.24 g/(kg·d)的肾康降酸颗粒溶液灌胃,空白组和模型组给予等量生理盐水灌胃,均持续灌胃6周。灌胃结束后采用ELISA法检测大鼠血尿酸、肌酐、尿素氮水平,计算肾小管上皮细胞损伤评分,采用Annexin-V-PI双染法流式细胞技术检测肾小管上皮细胞凋亡率,分别采用比色法和ELISA法检测肾小管上皮细胞中NADPH氧化酶活性和活性氧(ROS)水平,采用免疫组化和Western blot法检测组织中ERK1及其磷酸化水平。结果干预结束后,模型组大鼠血尿酸、肌酐、尿素氮水平,肾小管上皮细胞损伤评分,肾小管上皮细胞凋亡率,肾组织中NADPH氧化酶活性、ROS水平和ERK1磷酸化水平均显著高于空白组(P均<0.05),别嘌呤醇组和肾康降酸颗粒组上述指标均明显低于模型组(P均<0.05),且肾康降酸颗粒组上述指标均明显低于别嘌呤醇组(P均<0.05)。结论肾康降酸颗粒可通过抑制NADPH/ROS/ERK信号的活化,进而降低高尿酸血症大鼠尿酸,减轻肾组织损伤,起到保护肾小管上皮细胞的作用。

Objective It is to observe the effects of Shenkang Jiangsuan granules on renal tubular epithelial cell damage and NADPH/ROS/ERK signaling pathway in rats with hyperuricemia.Methods Forty male SD rats were randomly divided into blank group,model group,allopurinol group and Shenkang Jiangsuan granule group,with 10 rats in each group.Except for the blank group,the other groups were treated with adenine combined with yeast powder for 14 days to prepare hyperuricemia models.From the second day after the completion of the modeling,the allopurinol group was given allopurinol water mixture 27 mg/(kg·d)by gavage,and the Shenkang Jiangsuan granule group was given Shenkang Jiansuan granule solution 3.24 g/(kg·d)by gavage,and the blank group and the model group were given the same amount of normal saline by gavage,all the groups were treated for 6 weeks.After the gavage,the ELISA method was used to detect the levels of blood uric acid,creatinine,and urea nitrogen in rats,and the renal tubular epithelial cell injury score was calculated.The Annexin-V-PI double staining flow cytometry technique was used to detect the apoptosis rate of renal tubular epithelial cells.Colorimetric method and ELISA method were respectively used to detect NADPH oxidase activity and reactive oxygen species(ROS)levels in renal tubular epithelial cells.Immunohistochemistry and Western blot methods were used to detect the level of ERK1 and its phosphorylation in tissues.Results After the intervention,the levels of blood uric acid,creatinine and urea nitrogen,renal tubular epithelial cell injury score,renal tubular epithelial cell apoptosis rate,NADPH oxidase activity,ROS level and ERK1 phosphorylation level in renal tissue of the model group were significantly higher than those of the blank group(all P<0.05),the above indicators of the allopurinol group and the Shenkang jiangsuan granule group were significantly lower than the model group(all P<0.05),and the above indicators of the Shenkang Jiangsuan granule group were significantly lower than those in the allopurinol group(all P<0.05).Conclusion Shenkang Jiangsuan granules can inhibit the activation of NADPH/ROS/ERK signals,thereby reducing uric acid,reducing kidney tissue damage in rats with hyperuricemia,thus to protect renal tubular epithelial cells.