Influence Of Angiotensin-(1-7) On Angiotensin â…¡ Induced Rat's Renal Interstitial Fibroblast Activation

bjective:To observe the influence and explore the mechanism of angiotensin-(1-7)[Ang-(1-7)] on angiotensionⅡ(AngⅡ) induced rat's renal interstitial fibroblast(NRK-49F) activation and the secretion of extracellular matrix. Methods:Study One:determine the best concentration and the best time of AngⅡand Ang-(1-7). (1) To determine the best concentration and the best time of AngⅡinduce rat's renal interstitial fibroblast activation,the NRK-49F were maintained and sub-cultured. Study had been performed after the cells were prepared 1×105/ml suspension with DMEM and cultured in 6-well plates.The cells were divided into four groups according to the different intervention factors:①control group;②AngⅡ10-7mol/L group;③AngⅡ10-6mol/L group;④AngⅡ10-5mol/L group;The cells were cultured for 24h,48h,72h, then we detected the expressions ofα-smooth muscle actin(α-SMA) of every group at different time by immunocytochemistry method.The result show that AngⅡ10-6mol/L stimulated 72h induce renal interstitial fibroblast activation is the best. (2) To determine the best concentration of Ang-(1-7) inhibit AngⅡ-induced NRK-49F activation, the cells were divided into three groups according to the different intervention factors:①AngⅡ+Ang-(1-7) 10-7mol/L group;②AngⅡ+Ang-(1-7) 10-6mol/L group;③AngⅡ+Ang-(1-7) 10-5mol/L group; Final concentration of AngⅡwas 10-6 mol/L. The cells were cultured for 72h,then we detected the expressions ofα-SMA of every group by immunocytochemistry method. The result show that Ang-(1-7)10-5mol/L inhibit AngⅡ-induced rat's renal interstitial fibroblast activation is the best. Study Two:influence of Ang-(1-7) on AngⅡinduced rat's renal interstitial fibroblast activation. According to preliminary study's results (final concentration of AngⅡwas 10-6 mol/L and Ang-(1-7) was 10-5 mol/L), the cells were divided into four groups according to the different intervention factors:①control group:the NRK-49F were cultured without any intervention factor;②AngⅡgroup:the NRK-49F were stimulated by AngⅡ;③Ang-(1-7) group:the NRK-49F were stimulated by Ang-(1-7);④AngⅡ+Ang-(1-7) group:the NRK-49F were co-stimulated by AngⅡand Ang-(1-7). The NRK-49F cells were cultured for 72h.①The cells expressions ofα-SMA, TGF-β1 and IGF-Ⅰby immunocytochemistry method.②The content of ColⅠ, TGF-β1 and IGF-Ⅰin the cultured supernatant were measured by ELISA. Results:Study One:(1) There were basic expressions ofα-SMA in control group when the cells were cultured for 24h,48h,72h. When the concentration of AngⅡincreased and intervention time was extended, the expressions ofα-SMA in other there groups were increased. The expressions ofα-SMA increased significantly of AngⅡ10-6mol/L and AngⅡ10-5mol/L group than that of control group (P<0.05), but the change of these two groups has not statistically significant (P>0.05). AngⅡ10-6mol/L stimulated 72h induce renal interstitial fibroblast activation is the best.(2)Cultured for 72h, the expressions ofα-SMA were decreased when the concentration of Ang-(1-7) increased (P<0.05). Ang-(1-7)10-5mol/L inhibit AngⅡ-induced rat's renal interstitial fibroblast activation is the best. Study Two:(1) immunocytochemistry:①the change of expressions of a-SMA of NRK-49F: In control group, there were basic expressions of a-SMA, Ang-(1-7) group was similar with it; AngⅡgroup increased significantly than that of control group (P<0.05); AngⅡ+Ang-(1-7) group decreased than that of AngⅡgroup (P<0.05);②the change of expressions of TGF-β1 and IGF-I of NRK-49F:In control group, there were no expressions of TGF-β1 and IGF-I, Ang-(1-7) group was similar with it; AngⅡgroup increased significantly than that of control group (P<0.05); AngⅡ+Ang-(1-7) group decreased than that of AngⅡgroup (P<0.05); (2) ELISA:cultured for 72h, content of ColⅠ, TGF-β1 and IGF-I in the Ang-(1-7) group were no significant change, AngⅡgroup increased significantly than that of control group (P> 0.05); AngⅡ+Ang-(1-7) group significantly decreased than that of AngⅡgroup (P<0.05). Conclusion:(1) Ang-(1-7) can inhibit renal interstitial fibroblast activation induced by AngⅡ; (2) Ang-(1-7) may play a role by inhibiting; the secretion of TGF-β1 and IGF-Ⅰ.