肾上腺髓质素对血管紧张素Ⅱ促血管外膜成纤维细胞胶原生成作用的影响

Effects of adrenomedullin on angiotensin Ⅱ-induced collagen synthesis in vascular adventitial fibroblasts

目的探讨肾上腺髓质素(ADM)对血管紧张素Ⅱ(AngⅡ)诱导的血管外膜成纤维细胞胶原生成的影响及机制。方法体外培养大鼠主动脉外膜成纤维细胞,通过放射免疫法测定培养上清中 ADM 含量,采用酶联免疫吸附法(ELISA)测定培养上清中Ⅰ、Ⅲ型胶原蛋白含量,用逆转录-聚合酶链反应(RT-PCR)及 Western 印迹法检测转化生长因子β1(TGFβ1)和基质金属蛋白酶-2(MMP-2)mRNA 及蛋白的表达。结果 AngⅡ呈剂量依赖性地刺激血管外膜成纤维细胞分泌 ADM,在 AngⅡ(10^(-6)mol/L)刺激前30 min 加入氯沙坦或(和)PD123319,氯沙坦(10^(-5)mol/L)可明显降低AngⅡ刺激的 ADM 分泌,其抑制率为45%(P<0.01),而 PD123319(10 mmol/L)作用后抑制率仅为3%(P>0.05),氯沙坦+PD123319组与单独氯沙坦组相比差异无统计学意义(P>0.05);AngⅡ显著增加培养上清中Ⅰ、Ⅲ型胶原蛋白含量,ADM 呈剂量依赖地抑制 AngⅡ上述作用,其中 ADM(10^(-8)mol/L)组中Ⅰ、Ⅲ型胶原合成分别抑制了30%和31%(P<0.01),ADM(10^(-7)mol/L)组则分别抑制了43%和42%(P<0.01)。ADM 受体拮抗剂 ADM_(22-52)可增强 AngⅡ上述作用,Ⅰ、Ⅲ型胶原合成分别增加了38%和43%(P<0.01);ADM 呈剂量依赖性抑制 AngⅡ刺激的 TGFβ1 mRNA 及蛋白表达,其中ADM(10^(-8)mol/L)组中 TGFβ1 mRNA 及蛋白表达分别抑制了55%和45%(P<0.01),ADM(10^(-7)mol/L)组则分别抑制了70%和59%(P<0.01);AngⅡ明显下调细胞内 MMP-2 mRNA 及蛋白表达,ADM 呈剂量依赖性抑制上述作用,其中10^(-8)mol/L ADM 组细胞内 MMP-2 mRNA 及蛋白表达分别增加了1.0和0.9倍。结论 AngⅡ可刺激血管外膜成纤维细胞释放 ADM,而自分泌旁分泌的 ADM 可能通过下调细胞内 TGFβ1表达和上调 MMP-2表达,抑制 AngⅡ刺激的Ⅰ、Ⅲ型胶原蛋白生成,从而发挥有效的抗血管重构作用。

Objective To explore the effects of adrenomedullin （ADM） on Angiotensin Ⅱ （Ang Ⅱ ）-induced collagen synthesis in cultured rat vascular adventitial fibroblasts. Methods Rat vascular adventitial fibroblasts were cultured in vitro. ADM produced and secreted from adventitia in the presence of Ang Ⅱ was detected by radioimmunoassay, type Ⅰ , Ⅲ collagen contents in adventitia fibroblasts were measured by ELISA and the expressions of TGFβ1 and MMP-2 were determined by RT-PCR and Western blotting. Results Ang Ⅱ significandy induced ADM secretion in adventitial fibroblasts in a dose-dependent manner. These effects could be reduced by 45%, 3% and 46%, through pre-treatment with Losartan, PD123319 or both, respectively for 30 min in culture medium. The Ang Ⅱ -induced type Ⅰ , Ⅲ collagen secretion in adventitial fibroblasts was significantly reduced by AMD in a dose-dependent manner, （ P 〈 0.01 ） while ADM agonist ADM22.52 significantly potentiated the effect of Ang Ⅱ ; ADM also reduced Ang Ⅱ - induced expression of TGFβ1 at mRNA and protein levels in a dose-dependent manner. Ang Ⅱ reduced the expression of MMP-2 at mRNA and protein levels in adventitial fibroblasts and these effects could be reversed by AMD（ 10 ^-8 mol/L）. Conclusion Ang Ⅱ stimulated ADM secretion in adventitia fibroblasts, ADM in turn can inhibit Ang Ⅱ-induced type Ⅰ , Ⅲ collagen synthesis in adventitial fibroblasts probably by downregulating the TGFβ1 expression and upregulating MMP-2 expression. ADM therefore served as an antifibrotic factor in vascular remodeling process.