Z Li1, D Liang1, Y Xiao1, Y Dai1, F Ai1, J Ding1, M Shi1, Y Xiao1, B Guo1. 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.
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.
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.