冠心康调控FTO的m^(6)A甲基化抑制炎症和凋亡抗动脉粥样硬化的研究

Guanxinkang inhibits inflammation and apoptosis and prevents atherosclerosis by regulating m^(6)A methylation of FTO

目的观察冠心康对脂多糖诱导的人脐静脉内皮细胞(HUVEC)中去甲基化酶FTO甲基化水平、炎症和凋亡的影响。方法将体外培养的HUVEC细胞随机分为空白对照(NC)组、脂多糖(LPS)组和冠心康(GXK)组。采用CCK8法检测不同浓度LPS和冠心康对HUVEC细胞增殖活性的影响,筛选出造模和冠心康干预的最佳浓度;造模及冠心康治疗24h后,利用m^(6)A RNA定量试剂盒(比色法)检测m^(6)A RNA甲基化水平,实时荧光定量PCR(RT-qPCR)及蛋白质印迹法(Western blotting)法检测FTO mRNA及蛋白的表达量,ELISA法检测IL-1β、IL-6、TNF-α炎症因子变化,流式细胞术检测各种细胞凋亡水平。结果(1)分别选择1μg/mL和6μg/mL作为脂多糖和冠心康的最佳浓度;(2)与NC组比较,LPS组m^(6)A RNA甲基化水平升高(P<0.05),与LPS组比较,冠心康组m^(6)A RNA甲基化水平降低(P<0.05);(3)与NC组比较,LPS组FTO mRNA及蛋白表达量降低(P<0.05),与LPS组比较,冠心康干预后FTO mRNA及蛋白表达量升高(P<0.05);(4)与NC组比较,LPS组细胞上清液中IL-1β、IL-6、TNF-α炎症因子水平升高(P<0.05),与LPS组比较,冠心康组IL-1β、IL-6、TNF-α炎症因子水平下降(P<0.05);(5)与NC组比较,LPS组细胞凋亡率增加(P<0.05),与LPS组比较,冠心康组细胞凋亡率下降(P<0.05)。结论冠心康可能通过调控去甲基化酶FTO抑制LPS诱导的炎症和凋亡抗动脉粥样硬化。

Objective To observe the effects of Guanxinkang on lipopolysaccharide-induced inflammation and apoptosis of human umbilical vein endothelial cells(HUVEC)and the methylation level of demethylase FTO.Methods The cultured HUVEC cells were randomly divided into control group(NC),lipopolysaccharide(LPS)and Guanxinkang(GXK).The effects of LPS and Guanxinkang on the proliferation activity of HUVEC cells were detected by CCK8 method,and the optimal concentration of Guanxinkang and Guanxinkang were selected.After molding and Guanxinkang treatment for 24h,m^(6) A RNA methylation level was detected by m^(6) A RNA quantitative kit(colorimetric method),and FTO mRNA and protein expression levels were detected by real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting.The changes of IL-1β,IL-6 and TNF-αinflammatory factors were detected by ELISA,and apoptosis levels of various cells were detected by flow cytometry.Results(1)The optimal concentrations of LPS and GXK were determined to be 1μg/mL and 6μg/mL.(2)Compared with the NC group,the methylation level of m^(6) A RNA increased in the LPS group(P<0.05),while it decreased in the GXK group compared to the LPS group(P<0.05).(3)The expression levels of FTO mRNA and protein decreased in the LPS group compared with the NC group(P<0.05),whereas after GXK intervention,these levels increased compared to the LPS group(P<0.05).(4)The levels of inflammatory factors IL-1β,IL-6,and TNF-αin the cell supernatant were elevated in the LPS group compared to the NC group(P<0.05),while they decreased in the GXK group compared to the LPS group(P<0.05).(5)The apoptotic rate of cells increased in the LPS group compared to the NC group(P<0.05),whereas it decreased in the GXK group compared to the LPS group(P<0.05).Conclusion Guanxinkang may inhibit LPS-induced inflammation and apoptosis by regulating demethylase FTO to prevent atherosclerosis.