Huang Yuqi, Tian Li, Liu Yan, Liu Jiangwei, Huang Jianzhao
Department of Cardiology, Shulan (Hangzhou) Hospital Affiliated to Zhejiang Shuren University Shulan International Medical College, Hangzhou, Zhejiang, China.
Department of Hepatobiliary Surgery, Guizhou Provincial People's Hospital, Guiyang, Guizhou, China.
Appl Bionics Biomech. 2022 Jul 31;2022:6148361. doi: 10.1155/2022/6148361. eCollection 2022.
CCK-8 (cell counting kit-8), Western blotting and Annexin V-FITC/PI staining were used to detect cell viability and apoptosis after hypoxia stimulation. The level of microRNA-92 a (miR-92 a) was detected by qRT-PCR.. Then, the assays of flow cytometry and the annexin V/PI staining kit were applied to value the impact of FA on hypoxia-induced cell proliferation, cell cycle distribution, and apoptosis. Furthermore, the inhibitor and mimic of miR-92a were also administrated to explore the role of miR-92a in this process. Student's -test was used to explore the differences between two groups, while one-way analysis of variance (ANOVA) was used to explore the differences between more than two groups.
The results showed that hypoxia stimulation significantly inhibited HUVEC viability and proliferation, such as remarkably decreasing the expression of CDK2, CDK4, and cyclin D1 in HUVECs. The results of annexin V-FITC/PI apoptosis detection showed that hypoxia culture significantly induced HUVEC apoptosis, which indicated that hypoxia stimulation significantly inhibited viability and proliferation of HUVECs but caused cell apoptosis and the expression of miR-92a. Meanwhile, FA remarkably protected HUVECs from hypoxia-induced inhibition of viability and proliferation, as well as the enhancement of apoptosis and miR-92a expression. Furthermore, suppression of miR-92a enhanced the protective effects of FA on hypoxia-induced HUVECs, while activation of miR-92a reversed those effects.
Our study reported that FA preserved HUVECs from hypoxia-induced injury via regulating miR-92a, which facilitated the understanding of the protective capacity of FA in hypoxia-caused HUVEC injury.
采用CCK-8（细胞计数试剂盒-8）、蛋白质印迹法及膜联蛋白V-异硫氰酸荧光素/碘化丙啶染色检测缺氧刺激后的细胞活力及凋亡情况。采用qRT-PCR检测微小RNA-92a（miR-92a）水平。然后，应用流式细胞术检测及膜联蛋白V/碘化丙啶染色试剂盒评估叶酸对缺氧诱导的细胞增殖、细胞周期分布及凋亡的影响。此外，还给予miR-92a抑制剂和模拟物以探讨miR-92a在此过程中的作用。采用学生t检验探讨两组间差异，采用单因素方差分析（ANOVA）探讨两组以上差异。
结果显示，缺氧刺激显著抑制人脐静脉内皮细胞（HUVEC）活力和增殖，如显著降低HUVEC中细胞周期蛋白依赖性激酶2（CDK2）、细胞周期蛋白依赖性激酶4（CDK4）和细胞周期蛋白D1的表达。膜联蛋白V-异硫氰酸荧光素/碘化丙啶凋亡检测结果显示，缺氧培养显著诱导HUVEC凋亡，这表明缺氧刺激显著抑制HUVEC的活力和增殖，但导致细胞凋亡及miR-92a表达。同时，叶酸显著保护HUVEC免受缺氧诱导的活力和增殖抑制，以及凋亡和miR-92a表达增强的影响。此外，抑制miR-92a增强了叶酸对缺氧诱导的HUVEC的保护作用，而激活miR-92a则逆转了这些作用。
我们的研究报告称，叶酸通过调节miR-92a保护HUVEC免受缺氧诱导的损伤，这有助于理解叶酸在缺氧导致的HUVEC损伤中的保护能力。
