肾素通过非血管紧张素Ⅱ途径促进大鼠血管平滑肌细胞钙化

Renin Increases Calcification of Rat Vascular Smooth Muscle Cells Through Angiotesin Ⅱ

目的探讨肾素通过非血管紧张素Ⅱ(AngⅡ)途径对大鼠血管平滑肌细胞钙化的影响及其可能的分子机制。方法采用组织块贴壁法培养原代大鼠主动脉平滑肌细胞,利用β-甘油磷酸钠联合丙酮酸钠制备血管平滑肌细胞钙化模型。细胞随机分为6组:空白对照组(予常规培养基)、钙化组(予钙化培养基)、钙化+AngⅡ受体阻断剂组(予钙化培养基,再予氯沙坦10-6mol/L和PD123,31910-5mol/L分别阻断AngⅡ受体AT1、AT2)、钙化+肾素(10-10、10-9和10-8mmol/L)组(于钙化培养基中,先加入氯沙坦10-6mol/L和PD123,31910-5mol/L,再分别予10-10、10-9和10-8mmol/L肾素)。用Von Kossa染色鉴定钙化细胞,测定各组细胞中钙含量、碱性磷酸酶(ALP)活性判断钙化程度。用RT-PCR检测成骨细胞标志物核结合因子α1(Cbfα1)和转化生长因子β1(TGF-β1)mRNA表达,Western blot测定各组细胞Cbfα1蛋白含量。结果与空白对照组相比,钙化组钙含量、ALP活性显著增加,Cbfα1和TGF-β1 mRNA及Cbfα1蛋白表达明显增加(P<0.01)。与钙化组相比,钙化+AngⅡ受体阻断剂组对钙化的影响无统计学差异。与钙化+AngⅡ受体阻断剂组相比,钙化+肾素(10-10、10-9和10-8mmol/L)组呈剂量依赖地促进大鼠血管平滑肌细胞的钙含量、ALP活性,以及Cbfα1和TGF-β1 mRNA及Cbfα1蛋白表达(P<0.05)。结论肾素可通过非AngⅡ途径作用促进β-甘油磷酸钠诱导的血管平滑肌细胞钙化,其作用可能与TGF-β1、Cbfα1表达上调有关。

Aim To explore the impact of renin through angiotesin Ⅱ -independent mechanisms on calcification of rat vascular smooth muscle cells in vitro, and its molecular mechanisms. Methods Primary cultured cells were ob- tained by tissue-piece inoculation. Calcification of cultured rat vascular smooth muscle cells was produced by incubation with β-glycerophosphate and sodium pyruvate. The vascular smooth muscle cells were divided into 6 groups： the blank control group （cultured in normal medium）, the calcification group （incubated in calcified medium）, the calcification ＋ angiotesin Ⅱ receptor blocker group （ cultured in calcified medium, giving Losartan 10 -6 mol/L to block AT1 and PD12a,319 10^-5 mol/L to block AT2）, calcification ＋ renin group （incubated in calcified medium, giving Losartan 10-6 mol/L and 10 ^-5 moL/L PD123, 319, then added 10^ -10, 10-9, and 10 ^-5 mmol/L renin）. Calcification was confirmed by Von Kossa staining. Calcium content and alkaline phosphatase （ALP） activity were measured to estimate the extent of calcification. The mRNA expression of core binding faetorctl （ Cbfotl ） and transforming growth factor-β1 （ TGF-β1 ） was measured by competitive quantitative RT-PCR. The expression of Cbfctl protein content was measured by Western blot.Results Compared with the blank control group, the calcium content and ALP activity of the calcification group in creased significantly, as well as the mRNA expression of Cbfod, TGF-β1 and the expression of Cbfotl protein increased significantly （P 〈0. 01 ）. Compared with calcification group, calcification ＋ angiotensin Ⅱ receptor blocker group had no difference on calcification of vascular smooth muscle cells. Compared with calcification ＋ angiotensin Ⅱ receptor blocker group, interven- tion of rennin （ 10 -10, 10 -9, 10 -s mmol/L） in a dose-dependent manner further promoted the calcium content and the ALP activity of rat vascular smooth muscle cells, as well as the mRNA expression of Cbfod, TGF-β1 and Cbfotl protein expression （P 〈 0. 05）. Conclusions Renin can promote β-glycerophosphate-induced calcification of vascular smooth muscle cells through angiotesin Ⅱ-independent mechanisms, the mechanisms of renin promoted calcification of vascular smooth muscle cells through angiotesin Ⅱ-independent way may be through promoting the expression of TGF-β1, Cbfα1.