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甘草酸对感染人巨细胞病毒的人胚肺成纤维细胞凋亡和炎性反应的影响

Effects of glycyrrhizin on apoptosis and inflammatory response of human embryonic lung fibroblasts infected with human cytomegalovirus

    摘要
  • 摘要:
    目的 探讨甘草酸对感染人巨细胞病毒(HCMV)的人胚肺成纤维细胞MRC-5增殖、凋亡和炎性反应的影响。
    方法 体外培养MRC-5细胞，并将其分为对照组、病毒组、更昔洛韦组(50 μg/mL)、甘草酸低剂量组(0.25 mg/mL)、甘草酸中剂量组(0.50 mg/mL)、甘草酸高剂量组(1.00 mg/mL)。接种HCMV不同时间后，分别使用CCK8试剂盒检测细胞增殖活性，使用流式细胞仪检测细胞凋亡情况，采用蛋白质印迹法(Western blot)检测细胞中活化含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved-caspase3)等蛋白表达情况，采用酶联免疫吸附试验(ELISA)检测细胞培养上清液中肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)水平。
    结果 CCK8实验结果显示，经HCMV感染48、72 h时，病毒组增殖活性均低于对照组，更昔洛韦组、甘草酸中剂量组、甘草酸高剂量组增殖活性均高于病毒组，差异有统计学意义(P < 0.05)；甘草酸中剂量组与甘草酸高剂量组增殖活性比较，差异无统计学意义(P>0.05)，故选择甘草酸中剂量组(0.50 mg/mL)进行后续实验，并简称甘草酸组。流式细胞仪检测结果显示，经HCMV感染48、72 h时，病毒组细胞凋亡率均高于对照组，更昔洛韦组、甘草酸组则低于病毒组，差异有统计学意义(P < 0.05)。Western blot检测结果显示，经HCMV感染48 h后，病毒组细胞中Cleaved-caspase3、TNF-α、IL-6蛋白相对表达量均高于对照组，更昔洛韦组、甘草酸组则低于病毒组，差异有统计学意义(P < 0.05)。ELISA检测结果显示，经HCMV感染24、48、72 h时，病毒组MRC-5细胞TNF-α、IL-6水平均高于对照组，更昔洛韦组、甘草酸组则低于病毒组，差异有统计学意义(P < 0.05)。
    结论 甘草酸具有体外抗HCMV效应，可促进感染HCMV的人胚肺成纤维细胞MRC-5增殖，并抑制HCMV感染诱导的细胞凋亡和细胞炎性反应。

     

    Abstract:
    Objective To investigate the effects of glycyrrhizin on the proliferation, apoptosis and inflammatory response of human embryonic lung fibroblast MRC-5 infected with human cytomegalovirus (HCMV).
    Methods MRC-5 cells were cultured in vitro and were divided into control group, virus group, ganciclovir group(50 μg/mL), low dose glycyrrhizin group(0.25 mg/mL), medium dose glycyrrhizin group(0.50 mg/mL) and high dose glycyrrhizin group(1.00 mg/mL). After different periods of virus inoculation, cell proliferation activity was detected by CCK8 assay, cell apoptosis was detected by flow cytometry, cleaved-caspase3 caspase was detected by western blot, and tumor necrosis factor α(TNF-α) and interleukin-6 (IL-6) levels in cell culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA).
    Results At 24 and 48 h of HCMV infection, the results of CCK8 experiment showed that the proliferation activity of cells in virus group was significantly reduced compared with the control group, and was higher in the ganciclovir group, medium dose glycyrrhizin group and high dose glycyrrhizin group than that in the virus group(P < 0.05). There were no statistically significant difference in proliferative activity between medium dose glycyrrhizic acid group and high dose glycyrrhizic acid group (P>0.05), so the medium dose glycyrrhizic acid group (0.50 mg/mL) was selected for follow-up experiment and was called glycyrrhizic acid group for short. Flow cytometry showed that after 48 and 72 h of HCMV infection, the apoptosis rate of the virus group was higher than that of the control group, but the apoptosis rates in the ganciclovir group and glycyrrhizic acid group were lower than that of the virus group (P < 0.05). Western blot analysis showed that 48 h after HCMV infection, the relative expression levels of cleaved caspase3, TNF-α, and IL-6 in the virus group were higher than those in the control group, but were lower in the ganciclovir group and glycyrrhizic acid group than those in the virus group (P < 0.05). ELISA results showed that the levels of TNF-α and IL-6 in MRC-5 cells in the virus group were higher than those in the control group at 24, 48 and 72 h after HCMV infection, while those in ganciclovir group and glycyrrhizic acid group were lower than those in the virus group (P < 0.05).
    Conclusion Glycyrrhizic acid has anti-HCMV effect in vitro, which could promote the proliferation of infected MRC-5 cells, inhibit cell apoptosis and inflammatory response induced by HCMV infection.

     

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