同型半胱氨酸通过Rho激酶信号通路促进大鼠脑基底动脉平滑肌细胞活力与迁移

目的:探讨同型半胱氨酸(homocysteine,Hcy)对大鼠脑基底动脉平滑肌细胞(basilar arterial smooth muscle cells,BASMCs)活力和迁移的作用及可能的分子机制。方法:分离培养大鼠BASMCs,给予不同浓度的Hcy刺激后,用CCK-8法初步测定细胞活力的变化,Western blot检测Rho激酶信号通路的活性。然后固定Hcy的浓度(1 mmol/L),并采用ROCK抑制剂Y-27632作为工具药对细胞进行处理,流式细胞术检测细胞的周期分布,划痕实验和Transwell实验检测细胞的迁移情况;检测细胞抗氧化酶超氧化物歧化酶(superoxide dismutase,SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)的活性以及丙二醛(malondialdehyde,MDA)的水平反映细胞的氧化应激状态。结果:Hcy可浓度依赖性地提高BASMCs的活力,并上调细胞中GTP-RhoA和ROCK2的蛋白表达(P<0.05);同浓度的Hcy处理细胞后,48 h处理组的细胞活力显著高于24 h处理组(P<0.05)。Hcy处理组BASMCs中S期细胞的比例明显升高,G0/G1期细胞的比例显著降低(P<0.05);加入Y-27632预处理可显著抑制Hcy处理组BASMCs的G1/S期转换。划痕实验和Transwell的实验结果显示,Hcy处理组BASMCs的迁移率显著高于对照组;加入Y-27632预处理可抑制Hcy处理组BASMCs的迁移(P<0.05)。此外,Hcy可降低BASMCs中SOD和GSH-Px的活性,同时升高MDA的含量;相较Hcy处理组,加入Y-27632预处理后,细胞内SOD和GSH-Px的活性升高,MDA的含量降低(P<0.05)。结论:同型半胱氨酸可促进大鼠脑基底动脉平滑肌细胞的活力及迁移,其作用机制可能与激活Rho激酶信号通路并提高细胞内氧化应激的水平有关。

Homocysteine activates Rho kinase signaling pathway to promote viability and migration of rat basilar arterial vascular smooth muscle cells

AIM: To investigate the regulatory effects of homocysteine(Hcy) on the viability and migration of rat basilar arterial smooth muscle cells(BASMCs) and its potential molecular mechanisms. METHODS: BASMCs were isolated, cultured in vitro and treated with Hcy at different concentrations. The cell viability was measured by CCK-8 assay, and the activation of Rho kinase pathway was measured by Western blot. The cells were treated with Hcy at fixed concentration(1 mmol/L), and ROCK inhibitor Y-27632 was also used. The cell cycle distribution was analyzed by flow cytometry. The cell migration ability was detected by wound healing assay and Transwell assay. The activation of antioxidant enzymes, including superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px), and the level of malondialdehyde(MDA) were measured for determining the status of oxidative stress.RESULTS: Hcy increased the viability of BASMCs and the protein expression of GTP-RhoA and ROCK2 in a dose-dependent manner(P<0.05). Compared with the cells treated with Hcy for 24 h, the cells treated with Hcy for 48 h had enhanced viability(P<0.05). Compared with control group, treatment with Hcy increased cell population in S phase and decreased cell population in G0/G1 phase, while pre-incubation with Y-27632 reversed Hcy-induced G1/S phase transition in BASMCs(P<0.05). The cell migration rate in Hcy treatment group was remarkably higher than that in control group(P<0.05), while pre-incubation with Y-27632 reversed Hcy-induced cell migration(P<0.05). Furthermore, Hcy inhibited the activation of SOD and GSH-Px, accompanied with increased MDA level(P<0.05). Compared with Hcy treatment group, pre-incubation with Y-27632 increased the activation of SOD and GSH-Px, but decreased MDA level(P<0.05). CONCLUSION: Homocysteine induces the viability and migration of rat BASMCs, and its mechanisms may be related to activation of Rho kinase pathway.