毛蕊异黄酮对流感病毒感染HUVEC细胞通透性及其MLC磷酸化的影响
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摘要
目的:通过检测毛蕊异黄酮(Calycosin)对流感病毒A/PR/8/34(H1N1)感染的脐静脉内皮细胞(Human Umbilical Vein Endothelial Cells,HUVECs)通透性升高及其肌球蛋白轻链(MLC)磷酸化的影响来探讨其改善内皮细胞通透性的机制。方法:设置正常组、病毒组和毛蕊异黄酮组;将HUVEC接种于96孔板,MTT法检测不同浓度毛蕊异黄酮的细胞毒性;30 MOI PR8病毒感染病毒组与毛蕊异黄酮组HUVECs后,毛蕊异黄酮组加入20μg/mL毛蕊异黄酮溶液,采用内皮细胞跨膜电阻法检测各组在不同时间点的跨膜电阻值;激光共聚焦显微镜观察各组细胞内丝状肌动蛋白(Filamentous actin,F-actin)的表达和分布;Western Blot检测各组MLC、p-MLC蛋白表达水平;将MDCK接种于96孔板,用PR8病毒感染细胞,MTT法检测不同浓度毛蕊异黄酮的抗病毒活性。结果:毛蕊异黄酮对HUVEC的最大无毒浓度为40μg/mL,细胞存活率约为91.69%;毛蕊异黄酮组细胞通透性与正常组细胞相比无显著差异,但通透性显著低于病毒组(P<0.05);病毒组的F-actin发生变构、细胞中出现粗大的应力纤维,分布于胞浆中,而毛蕊异黄酮组的F-actin分布于细胞膜、应力纤维减少;与正常组相比,病毒组的p-MLC蛋白表达显著增加,差异有统计学意义(P<0.05)、与病毒组相比,毛蕊异黄酮组的p-MLC蛋白表达明显下降,差异有统计学意义(P<0.05);毛蕊异黄酮对流感病毒感染的MDCK细胞的抗病毒作用并不明显。结论:毛蕊异黄酮可通过抑制p-MLC蛋白的表达来抑制流感病毒引起的内皮细胞通透性升高及F-actin变构的作用,从而达到保护内皮细胞的功能。
Objective: To explore the mechanism of improving endothelial cell permeability by investigating the effects of calycosin on the increased permeability and the phosphorylation of myosin light chain(MLC) of human umbilical vein endothelial cells(HUVECs) infected with influenza virus A/PR/8/34(H1 N1).Methods:The normal group,virus group and calycosin group were set up. HUVECs were inoculated into 96-well plates and MTT assay was used to determine the cytotoxicity of different concentrations of calycosin.Virus group and calycosin group were infected with 30 MOI PR8 virus and 20μg/mL of calycosin was added to the calycosin group. The endothelial cell transmembrane resistance method was used to detect the transmembrane resistance of each group at different time points.The expression and distribution of filamentous actin in each group were detected by laser confocal microscopy.Western Blot was used to determine the protein expressions of MLC and p-MLC.MDCK was inoculated into 96-well plates and infected with PR8 virus. MTT assay was used to detect the antiviral activity of different concentrations of calycosin.Results: The maximum non-toxic concentration of calycosin to HUVECs was 40μg/mL, and the cell survival rate was about 91.69%. There was no significant difference in the cell permeability of the calycosin group compared with that of the normal group, but the permeability was significantly lower than that of the virus group(P<0.05).The F-actin of the virus group was allosteric, and coarse stress fibers appeared in the cells, distributed in the cytoplasm, whereas the F-actin in the calycosin group was distributed in the cell membrane and the stress fiber was reduced.Compared with the normal group, the expression of p-MLC protein in the virus group increased significantly, and the difference was significant statistically(P<0.05).Compared with the virus group, the expression of p-MLC protein in the calycosin group decreased significantly, and the difference was significant statistically(P<0.05).The antiviral effect of calycosin on MDCK cells infected by influenza virus is not obvious.Conclusion:By inhibiting the expression of p-MLC protein, calycosin can inhibit the increased permeability of the endothelial cells caused by influenza virus and the allosteric effect of F-actin, achieving the function of protecting endothelial cells.
Objective: To explore the mechanism of improving endothelial cell permeability by investigating the effects of calycosin on the increased permeability and the phosphorylation of myosin light chain(MLC) of human umbilical vein endothelial cells(HUVECs) infected with influenza virus A/PR/8/34(H1 N1).Methods:The normal group,virus group and calycosin group were set up. HUVECs were inoculated into 96-well plates and MTT assay was used to determine the cytotoxicity of different concentrations of calycosin.Virus group and calycosin group were infected with 30 MOI PR8 virus and 20μg/mL of calycosin was added to the calycosin group. The endothelial cell transmembrane resistance method was used to detect the transmembrane resistance of each group at different time points.The expression and distribution of filamentous actin in each group were detected by laser confocal microscopy.Western Blot was used to determine the protein expressions of MLC and p-MLC.MDCK was inoculated into 96-well plates and infected with PR8 virus. MTT assay was used to detect the antiviral activity of different concentrations of calycosin.Results: The maximum non-toxic concentration of calycosin to HUVECs was 40μg/mL, and the cell survival rate was about 91.69%. There was no significant difference in the cell permeability of the calycosin group compared with that of the normal group, but the permeability was significantly lower than that of the virus group(P<0.05).The F-actin of the virus group was allosteric, and coarse stress fibers appeared in the cells, distributed in the cytoplasm, whereas the F-actin in the calycosin group was distributed in the cell membrane and the stress fiber was reduced.Compared with the normal group, the expression of p-MLC protein in the virus group increased significantly, and the difference was significant statistically(P<0.05).Compared with the virus group, the expression of p-MLC protein in the calycosin group decreased significantly, and the difference was significant statistically(P<0.05).The antiviral effect of calycosin on MDCK cells infected by influenza virus is not obvious.Conclusion:By inhibiting the expression of p-MLC protein, calycosin can inhibit the increased permeability of the endothelial cells caused by influenza virus and the allosteric effect of F-actin, achieving the function of protecting endothelial cells.
引文
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[19] 齐晓宇,吴莹,张淑静,等.犀角地黄汤合银翘散对流感病毒感染小鼠肺组织MLC磷酸化信号通路的影响[J].世界中西医结合杂志,2016,11(11):1526-1531.
[20] Haidari M,ZHANG W,Ganjehei L,et al.Inhibition of MLC Phosphorylation Restricts Replication of Influenza Virus-A Mechanism of Action for Anti-Influenza Agents[J].PLOS ONE,2011,6(6):618-320.
[21] 王沙沙,王莹,郭泽,等.黄芪总黄酮对小鼠的急性毒性和致突变性研究[J].动物医学进展,2016,37(3):71-73.
[2] 卢娜娜,刘琪,顾立刚,等.疏风宣肺方和解表清里方干预流感病毒性肺炎小鼠细胞凋亡的研究[J].医学研究生学报,2013,26(11):1134-1137.
[3] 齐有胜,孙毅坤,刘为萍.单味中药抗流感病毒研究进展[J].中国实验方剂学杂志,2017,23(14):210-218.
[4] 高燕菁,王融冰.中医药治疗流感的研究进展[J].临床药物治疗杂志,2018,16(1):17-20.
[5] 蒋荣猛.流感不是“感冒”应重视高危人群早期抗病毒治疗[J].中国感染控制杂志,2018,17(2):93-96.
[6] 吴莹,李季倩,孟建,等.小檗碱改善流感病毒性肺炎小鼠肺血管通透性的作用及机制[J].现代生物医学进展,2012,12(17):3205-3208,3270.
[7] 李连达,张金艳,李贴奎,等.积极发挥中医药治疗流感的优势[J].中医杂志,2009,50(11):970-972.
[8] 孙平华.中药抗流感病毒的最新进展[J].中医药学刊,2006,24(4):733-735.
[9] 郭杰霞,王永胜.黄芪颗粒防治反复呼吸道感染56例[J].现代中西医结合杂志,2008,17(35):5420.
[10] 谢敏,吕旭艳.黄芪颗粒治疗小儿反复呼吸道感染(营卫失和型)65例临床疗效分析[J].中医儿科杂志,2015,11(4):20-21.
[11] 赵文,任永凤,樊建航.新疆黄芪抗病毒作用研究[J].中国药学杂志,2001,36(1):23-25.
[12] 张传杰,刘丽娟,张蓬华,等.黄芪和秦艽提取物抗甲型流感病毒研究[J].郧阳医学院学报,2010,29(2):138-140.
[13] 张小鸿,徐先祥,汪宁卿.黄芪保护血管内皮细胞作用机制研究进展[J].中国药学杂志,2013,48(18):1526-1530.
[14] 刘珂,邱炳勋,邹利,等.黄芪及其有效成分对内皮细胞及其连接的保护作用及机制研究进展[J].中草药,2016,47(21):3912-3917.
[15] ZHANG SJ,WU Y,XUAN Z,et al.Screening differential miRNAs responsible for permeability increase in HUVECs infected with influenza A virus[J].PLOS ONE,2017,12(10):1002-1004.
[16] 陈晓鸣,张淑静,齐晓宇,等.小檗碱对流感病毒感染HUVEC细胞骨架及通透性的影响[J].辽宁中医药大学学报,2017,19(12):52-55.
[17] 王伟.细胞骨架重构在VEGF与TNF-α促进汉坦病毒感染致血管内皮细胞通透性增高中的作用及其机制[D].西安:第四军医大学,2012.
[18] Kolavennu V,Zeng L,Peng H,et al.Targeting of RhoA/ROCK signaling ameliorates progression of diabetic nephropathy independent of glucose control[J].Diabetes,2008,57:714-723.
[19] 齐晓宇,吴莹,张淑静,等.犀角地黄汤合银翘散对流感病毒感染小鼠肺组织MLC磷酸化信号通路的影响[J].世界中西医结合杂志,2016,11(11):1526-1531.
[20] Haidari M,ZHANG W,Ganjehei L,et al.Inhibition of MLC Phosphorylation Restricts Replication of Influenza Virus-A Mechanism of Action for Anti-Influenza Agents[J].PLOS ONE,2011,6(6):618-320.
[21] 王沙沙,王莹,郭泽,等.黄芪总黄酮对小鼠的急性毒性和致突变性研究[J].动物医学进展,2016,37(3):71-73.