摘要
病毒性心肌炎严重影响患者身体健康,中药材中黄酮类物质被证实对病毒性心肌炎有治疗作用,但其中三七总黄酮对柯萨奇B3病毒导致的心肌炎发挥治疗作用的分子机制尚不明确。以探讨三七总黄酮缓解病毒性心肌炎炎症反应及细胞损伤的作用机制。采用RT-qPCR检测心肌细胞中miR-223-3p的表达水平;Western blotting检测心肌细胞中转录因子叉头框蛋白O1(Forkhead box O1,FOXO1)蛋白表达水平;MTT实验检测心肌细胞存活率;流式细胞术检测心肌细胞凋亡率;ELISA检测炎症因子肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)和白细胞介素1β(Interleukin-1β,IL-1β)、心肌酶谱磷酸肌酸激酶(Creative kinase,CK)和乳酸脱氢酶(Lactic dehydrogenase,LDH)、心肌损伤标志物心肌肌钙蛋白T(Cardiac troponin T,cTnT)和B型尿钠肽(Brain natriuretic peptide,BNP)的水平;双荧光素酶报告基因检验miR-223-3p和FOXO1之间的靶向关系。实验结果显示,三七总黄酮能够缓解病毒性心肌炎模型细胞的炎症反应及细胞损伤,并显著上调模型细胞中miR-223-3p的水平。敲除模型细胞中的miR-223-3p能够逆转三七总黄酮对病毒性心肌炎的治疗作用。通过双荧光素酶报告基因实验验证miR-223-3p靶向负调控FOXO1蛋白的表达。进一步研究发现,过表达FOXO1可抑制三七总黄酮对病毒性心肌炎的治疗作用;但同时过表达miR-223-3p后,过表达FOXO1对三七总黄酮疗效的抑制作用被逆转。由此得出结论,三七总黄酮可缓解病毒性心肌炎模型细胞的炎症反应及细胞损伤,其作用机制是通过调控miR-223-3p/FOXO1分子轴实现的。
Viral myocarditis seriously affects the health of patients,chinese herbal medicinal ingredients have been proved to be effective in treating viral myocarditis.However,the molecular mechanism of radix notoginseng flavone(RNF)in treating myocarditis caused by coxsackievirus B3 is not clear.In order to investigate the mechanism of RNF in alleviating inflammatory response and cell damage in viral myocarditis model cells.In this study,RT⁃qPCR was used to detect the expression of miR⁃223⁃3p in cardiomyocytes.Western blotting was used to detect the protein levels of FOXO1.MTT assay were used to detect cell survival rate of cardiomyocytes.Flow cytometry were used to detect cell apoptosis rate of cardiomyocytes.ELISA was used to measure the level of tumor necrosis factor⁃α(TNF⁃α),interleukin⁃1β(interleukin⁃1β),creatine kinase(CK),lactate dehydrogenase(LDH),cardiac troponin T(cTnT)and brain natriuretic peptide(BNP).Dual⁃luciferase reporter gene assay was used to verify the targeted relationship between miR⁃223⁃3p and FOXO1.The results showed that RNF alleviated the inflammatory response and cell damage of viral myocarditis cell model,and significantly up⁃regulated the level of miR⁃223⁃3p in the viral myocarditis model cell.Besides,knockdown of miR⁃223⁃3p reversed the therapeutic effect of RNF on viral myocarditis.Dual⁃luciferase reporter gene assay confirmed that FOXO1 is one of the target genes of miR⁃223⁃3p,and miR⁃223⁃3p inhibited the expression of FOXO1 by bound it.Furthermore,overexpression of FOXO1 significantly inhibited therapeutic effect of RNF on viral myocarditis,however,overexpression of miR⁃223⁃3p at the same time reversed the inhibitory effect of FOXO1.In conclusion,RNF alleviated the inflammatory response and cell damage of viral myocarditis model cells,and one of its mechanisms was achieved by regulating mir⁃223⁃3p/FOXO1 axis.
作者
许德星
万发银
张静怡
戴若竹
XU Dexing;WAN Fayin;ZHANG Jingyi;DAI Ruozhu(Quanzhou No.1 Hospital Affiliated to Fujian Medical University,Quanzhou 362000,China)
出处
《病毒学报》
CAS
CSCD
北大核心
2021年第4期781-789,共9页
Chinese Journal of Virology