背景:黄芩苷是一种黄酮类化合物,在抑制增殖方面发挥特定的药理作用,迁移,低氧诱导的肺动脉平滑肌细胞(PASMCs)的凋亡抵抗。然而,潜在的机制尚未完全阐明。尽管我们以前的研究表明A2aR的激活减弱了CXCR的表达,对低氧PASMC中A2aR与SDF-1/CXCR4轴的关系知之甚少。在这项研究中,我们旨在研究A2aR对低氧PASMC中SDF-1/CXCR4轴的影响,这种效应背后的机制,以及黄芩苷是否通过A2aR发挥其保护功能。
方法:大鼠PASMCs在常氧/缺氧条件下培养,分为9组:常氧,缺氧,缺氧+AMD3100(一种CXCR4拮抗剂),缺氧+黄芩苷,缺氧+阴性病毒,常氧+A2aR敲低,缺氧+A2aR敲低,缺氧+CGS21680(A2aR激动剂),缺氧+A2aR敲除+黄芩苷。采用慢病毒转染方法建立PASMCs中A2aR敲低模型。将细胞在低氧条件下孵育24小时。A2aR的表达水平,使用RT-qPCR和蛋白质印迹检测SDF-1和CXCR4。通过CCK-8和Transwell方法观察增殖和迁移速率。通过流式细胞术(FCM)和原位细胞死亡检测试剂盒(荧光素)测量细胞周期分布和细胞凋亡。
结果:在低氧条件下,A2aR的水平,与常氧下相比,SDF-1和CXCR4显着增加。黄芩苷干预组A2aR上调时SDF-1和CXCR4被抑制的趋势更为明显。黄芩苷直接增强A2aR的表达,A2aR基因敲除削弱了黄芩苷的功能。缺氧+A2aR敲低组SDF-1和CXCR4表达水平升高,以及PASMC的增殖和迁移率,而凋亡率降低。黄芩苷和CGS21680显示相反的作用。
结论:我们的数据表明黄芩苷可有效减弱缺氧诱导的PASMC增殖,迁移,和凋亡抗性,以及SDF-1的分泌,通过上调A2aR和下调SDF-1/CXCR4轴。
BACKGROUND: Baicalin is a flavonoid compound that exerts specific pharmacological effect in attenuating the proliferation, migration, and apoptotic resistance of hypoxia-induced pulmonary artery smooth muscle cells (PASMCs). However, the underlying mechanism has not been fully elucidated yet. Although our previous studies had indicated that activation of A2aR attenuates CXCR expression, little is known about the relationship between A2aR and SDF-1/CXCR4 axis in hypoxic PASMCs. In this study, we aimed to investigate the effect of A2aR on the SDF-1/CXCR4 axis in hypoxic PASMCs, the mechanism underlying this effect, and whether baicalin exerts its protective functions though A2aR.
METHODS: Rat PASMCs were cultured under normoxia/hypoxia and divided into nine groups: normoxia, hypoxia, hypoxia + AMD3100 (a CXCR4 antagonist), hypoxia + baicalin, hypoxia + negative virus, normoxia + A2aR knockdown, hypoxia + A2aR knockdown, hypoxia + CGS21680 (an A2aR agonist), and hypoxia + A2aR knockdown + baicalin. Lentiviral transfection methods were used to establish the A2aR knockdown model in PASMCs. Cells were incubated under hypoxic conditions for 24 h. Expression levels of A2aR, SDF-1, and CXCR4 were detected using RT-qPCR and western blot. The proliferation and migration rate were observed via CCK-8 and Transwell methods. Cell cycle distribution and cell apoptosis were measured by flow cytometry (FCM) and the In-Situ Cell Death Detection kit (Fluorescein).
RESULTS: Under hypoxic conditions, levels of A2aR, SDF-1, and CXCR4 were significantly increased compared to those under normoxia. The trend of SDF-1 and CXCR4 being inhibited when A2aR is up-regulated was more obvious in the baicalin intervention group. Baicalin directly enhanced A2aR expression, and A2aR knockdown weakened the function of baicalin. SDF-1 and CXCR4 expression levels were increased in the hypoxia + A2aR knockdown group, as were the proliferation and migration rates of PASMCs, while the apoptotic rate was decreased. Baicalin and CGS21680 showed opposite effects.
CONCLUSIONS: Our data indicate that baicalin efficiently attenuates hypoxia-induced
PASMC proliferation, migration, and apoptotic resistance, as well as SDF-1 secretion, by up-regulating A2aR and down-regulating the SDF-1/CXCR4 axis.