Abstract:

Objective　To investigate the role of recombinant anoctamin 1 (ANO1) inhibitors in angiotensin Ⅱ (AngⅡ) -induced proliferation of vascular smooth muscle cells (VSMC). Methods　The study period was from July 2019 to June 2022. The rat thoracic aorta smooth muscle cell line A7r5 was cultured in vitro; the AngⅡ stimulated A7r5 cells were used to establish the cell proliferation models; 10, 100, and 500 nmol/L ANO1 inhibitors (A01) were used for the intervention; a control group was set up (intervention with the same amount of normal saline); Hoechst 33342 fluorescence staining was used to observe the morphological changes of VSMC induced by AngⅡ after the intervention of ANO1 inhibitors; the protein western blotting was used to detect the expression levels of apoptosis-related proteins in the VSMC cells induced by AngⅡ after the intervention of ANO1 inhibitors. One-way analysis of variance was used for multi-group comparison, and LSD-t test was used for pairwise comparison between groups check. Results　The Hoechst 33342 fluorescence staining results showed that the ANO1 inhibitor concentration increased, the bright blue cells (chromatin condensation and apoptotic bodies) were getting more, indicating that the number of apoptotic cells increased. The apoptosis rates of the low-, medium-, and high-dose groups were (17.60±1.36)%, (36.30±3.50)%, and (61.25±6.33)%, respectively, which were higher than that of the control group [(4.32±0.51)%], with statistical differences between the groups (all P<0.05), indicating a dose-dependent relationship between AngⅡ-induced post-proliferative apoptosis of VSMC and ANO1 inhibitor concentration. The results of protein western blotting in the detection of AngⅡ-induced apoptosis related proteins in VSMC after ANO1 inhibitor intervention showed that there was a statistical difference in the main effect of EPK, AKT, CDK4, Cyclin D1 protein relative expression levels between the groups (P<0.05); compared with those in the control group, the relative protein expressions of EPK, AKT, CDK4, and Cyclin D1 in the low-, medium-, and high-dose groups decreased in a dose-dependent manner, with statistical differences between the groups (all P<0.05). Conclusions　ANO1 inhibitors can significantly inhibit AngⅡ-induced VSMC proliferation, and the mechanism may be related to the inhibition of the EPK/AKT signaling pathway and cell cycle progression.

Key words:

Angiotensin Ⅱ, Vascular smooth muscle cells, Proliferation, ANO1 inhibitor

摘要：

目的　研究钙激活氯通道蛋白1（ANO1）抑制剂在血管紧张素Ⅱ（AngⅡ）诱导的血管平滑肌细胞（VSMC）增殖中的作用。方法　研究时间为2019年7月至2022年6月。体外培养大鼠胸主动脉平滑肌细胞A7r5细胞株，AngⅡ刺激A7r5细胞建立细胞增殖模型，分别采用10 nmol/L、100 nmol/L、500 nmol/L不同浓度ANO1抑制剂（A01）干预，同时设置对照组（以等量生理盐水干预），采用Hoechst 33342荧光染色法观察ANO1抑制剂干预后AngⅡ诱导的VSMC形态学变化，蛋白免疫印迹法检测ANO1抑制剂干预后AngⅡ诱导的VSMC细胞凋亡相关蛋白表达水平。多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。结果　Hoechst 33342荧光染色结果显示，随着ANO1抑制剂浓度增加，呈亮蓝色的细胞愈来愈多（染色质凝聚、出现凋亡小体），即细胞凋亡数量越多。低剂量组、中剂量组、高剂量组凋亡率分别为（17.60±1.36）%、（36.30±3.50）%、（61.25±6.33）%，均高于对照组凋亡率（4.32±0.51）%，组间差异均有统计学意义（均P<0.05），即AngⅡ诱导的VSMC增殖后凋亡与ANO1抑制剂浓度之间存在剂量依赖性。蛋白免疫印迹法检测ANO1抑制剂干预后AngⅡ诱导的VSMC凋亡相关蛋白结果显示，不同组间EPK、AKT、CDK4、Cyclin D1蛋白相对表达量主效应差异有统计学意义（P<0.05）；与对照组比较，低剂量组、中剂量组、高剂量组EPK、AKT、CDK4、Cyclin D1蛋白相对表达量均降低，且呈剂量依赖性，各组间差异均有统计学意义（均P<0.05）。结论　ANO1抑制剂可显著抑制AngⅡ诱导的VSMC增殖作用，其机制可能与抑制EPK/AKT信号通路及细胞周期进程有关。

关键词:

血管紧张素Ⅱ, 血管平滑肌细胞, 增殖, ANO1抑制剂