Effect and mechanism of cysteine-rich protein 61 on transforming growth factor-β1-activated renal fibroblasts / 中华肾脏病杂志

ABSTRACT

Objective To observe the expression of cysteine-rich protein 61 (Cyr61) in transforming growth factor-β1 (TGF-β1)-activated renal fibroblasts (NRK-49F),and to explore its effect and mechanism.Methods (1) NRK-49F cells were activated by TGF-β1 with different concentrations (0.0,0.5,1.0,2.0,5.0 μg/L).Western blotting was used to detect the expression of Cyr61 protein,and CCK-8 assay was used to test the proliferative activity of NRK-49F cells.(2) NRK-49F cells with low expression and over expression of Cyr61 were established by plasmid transfection.The cells were divided into control group (null vector transfection),over-expression group and lowexpression group.The proliferation was discovered by CCK-8 assay after 24,48 and 72 h.Further,5.0 μg/L TGF-β1 activated these three groups.The proliferation was also discovered by CCK-8 assay and the cell cycle was analyzed by flow cytometry.The mRNA expressions of fibrosis markers (Collα1,Col3αl,MMP9,MMP13) and factors of cell senescence signal pathway (p53,p21,Rb,p16) were ascertained by real time PCR,and the protein expressions of Col3 and MMP9 were detected by Western blotting.Results (1) Compared with 0.0 μg/L TGF-β1 group,the proliferation of NRK-49F cells was enhanced in 0.5,1.0,2.0 and 5.0 μg/L TGF-β1 groups (all P ＜ 0.05),while the expression of Cyr61 protein was decreased in 1.0 μg/L group and increased in 5.0 μg/L group (all P ＜0.05).(2) The proliferation of over-expression group was lower than that of control group after 24,48and 72 h (all P＜ 0.05),which was in a time-dependent manner.(3) Compared with control group activated by TGF-β1,the over-expression group expressed less fibrosis factors (Col1α1 and Col3α1)and more anti-fibrosis factors (MMP9 and MMP13) with decreased proliferation (all P ＜ 0.05).Simultaneously,the proportion of cells bogged down in G1 phases,as well as the expressions of p53,p21 and Rb mRNA increased (all P ＜ 0.05).The above effects of low-expression group were just opposite to over-expression group.Moreover,there was no significant difference in the expression of p16 gene among the three groups (P ＞ 0.05).Conclusions Cyr61 can curb the proliferation and fibrotic phenotypes of fibroblasts,thereafter slowing down the process of renal fibrosis.The p53/p21/Rb interrelated cell senescence signal pathway may be involved in the anti-fibrosis process.