| Objective:To investigate the effect of intermedin(IMD) on angiotensin Ⅱ-induced rat proximal tubular cell line(NRK-52E) fibrosis and its possible mechanisms.Methods:NRK-52 E cells were cultured and randomly allotted to the following 4 groups: control group, Ang Ⅱ(10-6 mol/L) treated group, IMD+Ang Ⅱ treated group, and empty plasmid+Ang Ⅱ treated group. Cells in IMD+Ang Ⅱ treated group and empty plasmid +Ang Ⅱ treated group were transfected with p IRES2-IMD or p IRES2-empty vector by transfection complex comprising optimal proportion of plasmid and reagents, respectively. The transfer efficiency was detected by flow cytometry. The m RNA expression of Collagen I(Col I)were detected by real-time RT-PCR. Protein levels of Col I and E-cadherin were examined by Western blot analysis. The content of e NOS and c GMP in the supernatant were determined by ELISA.Results:The express of EGFP was significant under microscopy, and the transfection efficiency measured by flow cytometry was 35.73±7.28%. Compared with control group, the expression of Col significantly decreased while E-cadherin markedly increased in Ang Ⅱ treated cells(Col I m RNA level t=4.835, P=0.008; Col I protein level P<0.001; E-cadherin protein level t=4.284 P=0.013); e NOS and c GMP levels in the supernatant of Ang Ⅱ stimulated cells were significantly higher than that in the control cells(e NOS t=20.718,P<0.001;c GMP t=8.324, P=0.001). IMD gene transfer reversed the above changes(Col I m RNA level t=3.302,P=0.039; Col I protein level P<0.001; E-cadherin protein level t=6.045 P=0.004;e NOS t=10.682,P<0.001;c GMP t=18.974,P<0.001), while empty plasmid had no effects on them(Col I m RNA level t=0.951, P=0.395;Col I protein level t=1.208,P=0.294; E-cadherin protein level t=1.613, P=0.182;e NOS t=2.039,P=0.111;c GMP t=1.469,P=0.216).Conclusion:IMD inhibits Ang Ⅱ induced tubular cell fibrosis, and the effects might be achieved, at least partly, by inhibiting e NOS-c GMP pathway. |
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