ADM和ADT对肺动脉平滑肌细胞胶原合成及磷酸化ERK1/2表达的影响

Effects of adrenomedullin and adrenotensin on collagen synthesis and expression of phosphorylated ERK1/2 in pulmonary arterial smooth muscle cells

目的:检测不同浓度的肾上腺髓质素(ADM)和肾上腺加压素(ADT)对培养的Wistar大鼠肺动脉平滑肌细胞(PASMCs)Ⅰ、Ⅲ型胶原合成和磷酸化细胞外信号调节激酶(p-ERK1/2)表达的影响,探讨PASMCs增殖过程中ERK途径是否被激活。方法:取健康雄性Wistar大鼠,行大鼠远端PASMCs分离并进行原代培养,采用小鼠抗人平滑肌α-actin单克隆抗体对培养细胞进行鉴定;在培养基内分别加入10-7 mol/L ADM或ADT培养72h后加入兔抗大鼠Ⅰ型、Ⅲ型胶原抗体和兔抗大鼠p-ERK1/2抗体,0.01 mol/L PBS作阴性对照,FITC标记山羊抗兔IgG为Ⅱ抗,免疫荧光法观察PASMCs内Ⅰ、Ⅲ型胶原及p-ERK1/2表达。Western blotting检测10-7mol/L、10-8mol/L和10-9 mol/L ADM或ADT对p-ERK1/2蛋白表达的影响。结果:经平滑肌α-actin单克隆抗体对培养细胞进行鉴定,其纯度达97%。10-7 mol/L ADM可抑制PASMCsⅠ、Ⅲ型胶原及p-ERK1/2的表达(P<0.05,P<0.01);10-7 mol/L的ADT刺激后大鼠PASMCsⅠ、Ⅲ型胶原及p-ERK1/2的表达增强(P<0.05,P<0.01)。West-ern blotting结果显示ADM可呈剂量依赖性抑制p-ERK1/2蛋白表达(P<0.01,P<0.05);而ADT也可呈剂量依赖性促进p-ERK1/2蛋白表达(P<0.01,P<0.05)。结论:ADM可抑制大鼠PASMCsⅠ、Ⅲ型胶原及p-ERK1/2表达,而ADT可促进PASMCsⅠ、Ⅲ型胶原及p-ERK1/2表达,提示PASMCs增殖过程中ERK通路被激活,ADM和ADT可能通过ERK1/2信号通路来调节PASMCs增殖。

AIM： To investigate the activation of extracellular signal - regulated kinase （ERK.） during the proliferation of cultured rat pulmonary artery smooth muscle ceils （PASMCs）, and to study the regulatory effact of adrenomedullin （ADM） and adrenotensin （ADT） on the proliferation of PASMCs through ERK signaling pathway. METHODS： Healthy male Wistar rats weighing about 100 g were used in the study. The distal PASMCs were isolated and primarily cultured. The monoclonal antibody of mouse anti - human smooth muscle α- actin was adopted to identify the quality of the cultured PASMCs. ADM or ADT at 10 -7 mol/L was added to the culture medium, and cultured for 72 h. Immunofluorescence method was used to observe the expression levels of collagen Ⅰ, collagen In and p - ERK1/2 in cultured PASMCs. The effects of ADM or ADT at 10-7 mol/L, 10-8 mol/L and 10-9 mol/L on the protein expression of p - ERK1/2 in PASMCs were detected by Western blotting. RESULTS： The purity of cultured PASMCs was 97%, identified by adopting smooth muscleα -actin monoclonal antibody. Compared with control group, ADM at 10-7moL/L inhibited the productions of collagen Ⅰand Ⅲ, and the expression of p - ERK1/2 in cultured PASMCs. However, ADT showed promotive effect on the productions of collagen Ⅰand Ⅲ,and p, ERK1/2 expression in cultured PASMCs. ADM inhibited the protein expression of phosphorylated ERKI/2 in a dose - dependent manner, while ADT significantly promoted the phosphorylation of ERK1/2 protein in a dose -dependent manner. CONCLUSION： In cultured PASMCs, ADM inhibits, while ADT promotes the synthesis and expression of collagen Ⅰand Ⅲ, and phosphorylation of ERKi/2, indicating that ERK signaling pathway might be activated in the proliferation process of PASMCs, and ADM or ADT may regulate the proliferation of PASMCs through ERK signaling pathway.