南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (10): 1519-1526.doi: 10.12122/j.issn.1673-4254.2021.10.10

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氧化苦参碱通过抑制CHK1/2磷酸化改善糖尿病大鼠肾组织的纤维化和炎症

李志阳，梁 丹，肖雅文，代云莉，艾福军，丁 菁，石明隽，肖 瑛，郭 兵   

  1. 贵州医科大学基础医学院病理生理学教研室//贵州省常见慢性疾病发病机制及药物研究重点实验室，贵州 贵阳 550025
  • 出版日期:2021-10-20 发布日期:2021-11-11

Oxymatrine improves renal fibrosis and inflammation in diabetic rats by modulating CHK1/2 phosphorylation

LI Zhiyang, LIANG Dan, XIAO Yawen, DAI Yunli, AI Fujun, DING Jing, SHI Mingjun, XIAO Ying, GUO Bing   

  1. Department of Pathophysiology, Basic Medical College, Guizhou Medical University/ Guizhou Provincial Key Laboratory of Pathogenesis and Drug Research of Common Chronic Diseases, Guiyang 550025, China
  • Online:2021-10-20 Published:2021-11-11

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摘要： 目的 探讨氧化苦参碱（OMT）抗炎抗纤维化是否是通过影响细胞周期检测点激酶1/2（CHK1/2）来发挥。方法 SD大鼠采用完全随机设计分为正常对照组（NC）、糖尿病模型组（DM）和氧化苦参碱治疗组（OMT），6只/组，尾静脉注射链脲佐菌素（STZ）（55 mg/kg）复制糖尿病模型。HE、Masson染色观察病理组织形态学变化；免疫组织化学染色观察CHK1、CHK2、p-CHK1、p-CHK2的表达部位；ELISA检测肾组织上清中IL-6和IL-1β的含量；Western blot检测CHK1、CHK2、p-CHK1、p-CHK2、 Ⅲ型胶原（Col-Ⅲ）、Ⅳ型胶原（Col-Ⅳ）、纤维连接蛋白（FN）蛋白的表达水平；NRK-52E细胞按处理方式分为正常糖对照组（NG组）、高糖模型组（HG组）、正常糖用药组（NG+OMT组）、高糖用药组（HG+OMT组）、正常糖空载组（NG+con组）、正常糖敲低组（NG+siCHK1/2）、高糖空载组（HG+con组）、高糖敲低组（HG+siCHK1/2），Western blot检测CHK1、CHK2、p-CHK1、p-CHK2、Col-Ⅲ、Col-Ⅳ、FN蛋白的表达水平。结果 OMT可降低大鼠血糖、血肌酐和24 h尿蛋白（P<0.05）；DM组大鼠肾脏已出现炎性细胞浸润和纤维化表型，OMT组有所改善；OMT有明显抗炎作用（P<0.05）；CHK1/2主要表达在肾小管胞浆及胞核，DM组CHK1/2磷酸化水平高于NC组、OMT组；大鼠肾组织和NRK-52E细胞中经OMT治疗后p-CHK1、p-CHK2、Col-Ⅲ、Col-Ⅳ、FN蛋白表达含量均明显下降（P<0.05）；敲低CHK1/2后p-CHK1/2、Col-Ⅲ、Col-Ⅳ、FN蛋白表达均明显下降（P<0.05）。结论 OMT在糖尿病大鼠肾脏中发挥抗炎、抗纤维化的作用机制可能是通过影响CHK1、CHK2的表达从而影响其磷酸化水平，减少下游炎症介质的释放，减轻细胞外基质的分泌和沉积。

关键词: 糖尿病肾病, 氧化苦参碱, 细胞周期检测点激酶1/2, 炎症, 纤维化

Abstract: Objective To explore the role of cell cycle checkpoint kinase 1/2 (CHK1/2) in mediating the inhibitory effect of oxymatrine (OMT) against renal inflammation and fibrosis in diabetic rats. Methods SD rats were randomly divided into normal control group, diabetes model group (DM) and OMT treatment group (n=6). HE and Masson staining were used to observe histopathological changes of the renal tissue, and the expressions of CHK1, CHK2, p-CHK1 and p-CHK2 were localized by immunohistochemical staining. The contents of interleukin-6 (IL-6) and IL-1β in the renal tissue were detected using ELISA, and the expression levels of CHK1, CHK2, p-CHK1, p-CHK2, type III collagen (Col-III), type IV collagen (Col-IV), and fibronectin (FN) were determined using Western blotting. The changes in the expressions of CHK1, CHK2, p-CHK1, p-CHK2, Col-III, Col-IV and FN proteins were also examined with Western blotting in NRK-52E cells in response to high glucose exposure, OMT treatment and siRNA-mediated CHK1/2 knockdown. Results In diabetic rats, OMT treatment significantly decreased the levels of blood glucose, serum creatinine and 24 h urinary protein (P<0.05) and obviously improved inflammatory cell infiltration and fibrosis phenotype in the renal tissue (P<0.05). CHK1 and CHK2 were mainly expressed in the cytoplasm and nuclei of renal tubule cells, and their phosphorylation levels were significantly higher in DM group than in the control group and OMT group. OMT treatment significantly decreased the protein expression levels of p-CHK1, p-CHK2, Col-Ⅲ, Col-Ⅳ and FN in the renal tissue of diabetic rats and in NRK-52E cells exposed to high glucose (P<0.05). In NRK-52E cells, CHK1/2 knockdown resulted in significant reduction of the protein expressions of p-CHK1/2, Col-Ⅲ, Col-Ⅳ and FN (P<0.05). Conclusion The inhibitory effects of OMT against renal inflammation and fibrosis in diabetic rats are mediated probably by lowered phosphorylation levels of CHK1 and CHK2, which result in reduced release of the downstream inflammatory mediators and decreased secretion and deposition of extracellular matrix.

Key words: diabetic kidney disease, oxymatrine, checkpoint kinase 1/2, inflammation, fibrosis