甘草酸抑制呼吸道合胞病毒感染支气管上皮细胞凋亡的机制研究

Mechanism of Inhibition of Apoptosis in Respiratory Syncytial Virus-Infected Bronchial Epithelial Cells by Glycyrrhizic Acid

目的:探讨甘草酸对呼吸道合胞病毒(RSV)感染的支气管上皮细胞凋亡的影响,分析其机制是否与内源性酪氨酸激酶2(JAK2)/信号传导和转录激活因子3(STAT3)信号通路有关。方法:将HBE细胞分为对照组、感染组(RSV组)、低剂量甘草酸组(RSV+L-甘草酸组)、中剂量甘草酸组(RSV+M-甘草酸组)、高剂量甘草酸组(RSV+H-甘草酸组)、RSV+Colivelin组、RSV+H-甘草酸+SD-1029组。对照组细胞不经任何处理;RSV组用含0.0001 PFU/mL的RSV病毒溶液培养细胞2h,再更换为正常培养液培养24h;RSV+L-甘草酸组、RSV+M-甘草酸组和RSV+H-甘草酸组在RSV感染HBE细胞模型的基础上,用30、60、120μg/mL甘草酸处理的培养基培养的细胞;RSV+Colivelin组在RSV感染HBE细胞模型的基础上,用含有浓度为0.5μmoL/L JAK2/STAT3信号通路激活剂Colivelin处理的培养基培养细胞;RSV+H-甘草酸+SD-1029组在RSV感染HBE细胞模型的基础上,用120μg/mL甘草酸和10μmoL/L JAK2/STAT3信号通路抑制剂SD-1029共同处理的培养基培养细胞。24h后收集细胞,CCK-8试剂盒检测细胞活性;流式细胞仪检测细胞凋亡;ELISA检测细胞上清液中IL-6、TNF-α水平;western blot检测细胞中Bax、cleaved-Caspase-3、Bcl-2、p-JAK2、JAK2、p-STAT3、STAT3蛋白表达情况。结果:与对照组比较,RSV组OD值、Bcl-2、cleaved-Caspase-3、p-JAK2/JAK2、p-STAT3/STAT3蛋白表达均显著降低(P<0.05),细胞凋亡率、IL-6、TNF-α水平、Bax蛋白表达量显著升高(P<0.05);与RSV组比较,RSV+L-甘草酸组、RSV+M-甘草酸组、RSV+H-甘草酸组、RSV+Colivelin组OD值、Bcl-2、cleaved-Caspase-3、p-JAK2/JAK2、p-STAT3/STAT3蛋白表达均显著升高(P<0.05),细胞凋亡率、IL-6、TNF-α水平、Bax蛋白表达量显著降低(P<0.05);与RSV+H-甘草酸组比较,RSV+H-甘草酸+SD-1029组OD值、Bcl-2、cleaved-Caspase-3、p-JAK2/JAK2、p-STAT3/STAT3蛋白表达均显著降低(P<0.05),细胞凋亡率、IL-6、TNF-α水平、Bax蛋白表达量显著升高(P<0.05)。结论:甘草酸可能通过激活JAK2/STAT3信号通路,促进RSV感染的支气管上皮细胞增殖,抑制RSV感染的支气管上皮细胞的凋亡。

Objective:To explore the effect of glycyrrhizic acid on the apoptosis of bronchial epithelial cells infected with respiratory syncytial virus(RSV)and to analyze whether the mechanism is related to the endogenous Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3)signaling pathway.Methods:Human bronchial epithelial(HBE)cells were divided into seven groups:control group,infection group(RSV group),low-dose glycyrrhizic acid group(RSV+L-glycyrrhizic acid group),medium-dose glycyrrhizic acid group(RSV+M-glycyrrhizic acid group),high-dose glycyrrhizic acid group(RSV+H-glycyrrhizic acid group),RSV+Colivelin group,and RSV+H-glycyrrhizic acid+SD-1029 group.The control group cells were untreated.The RSV group cells were cultured with RSV virus solution at 0.0001 PFU/mL for 2 hours and then switched to normal culture medium for 24 hours.The RSV+L-glycyrrhizic acid group,RSV+M-glycyrrhizic acid group,and RSV+H-glycyrrhizic acid group were cultured with media containing glycyrrhizic acid at concentrations of 30,60,and 120μg/mL respectively after RSV infection.The RSV+Colivelin group cells were cultured with media containing 0.5μmoL/L of JAK2/STAT3 signaling pathway activator Colivelin after RSV infection.The RSV+H-glycyrrhizic acid+SD-1029 group cells were cultured with media containing 120μg/mL glycyrrhizic acid and 10μmoL/L JAK2/STAT3 signaling pathway inhibitor SD-1029 after RSV infection.After 24 hours,cells were collected.Cell viability was detected using the CCK-8 kit.Apoptosis was detected by flow cytometry.Levels of IL-6 and TNF-αin cell supernatants were measured by ELISA.Protein expression of Bax,cleaved-Caspase-3,Bcl-2,p-JAK2,JAK2,p-STAT3,and STAT3 was analyzed by western blot.Results:Compared to the control group,the RSV group showed significantly lower OD value,Bcl-2,cleaved-Caspase-3,p-JAK2/JAK2,and p-STAT3/STAT3 protein expression(P<0.05),and significantly higher cell apoptosis rate,IL-6,TNF-αlevels,and Bax protein expression(P<0.05).Compared to the RSV group,the RSV+L-glycyrrhizic acid group,RSV+M-glycyrrhizic acid group,RSV+H-glycyrrhizic acid group,and RSV+Colivelin group showed significantly higher OD value,Bcl-2,cleaved-Caspase-3,p-JAK2/JAK2,and p-STAT3/STAT3 protein expression(P<0.05),and significantly lower cell apoptosis rate,IL-6,TNF-αlevels,and Bax protein expression(P<0.05).Compared to the RSV+H-glycyrrhizic acid group,the RSV+H-glycyrrhizic acid+SD-1029 group showed significantly lower OD value,Bcl-2,cleaved-Caspase-3,p-JAK2/JAK2,and p-STAT3/STAT3 protein expression(P<0.05),and significantly higher cell apoptosis rate,IL-6,TNF-αlevels,and Bax protein expression(P<0.05).Conclusion:Glycyrrhizic acid may promote the proliferation of bronchial epithelial cells infected with RSV and inhibit their apoptosis by activating the JAK2/STAT3 signaling pathway.