IL-33通过miR-19a-3p/MAPK6通路影响心肌缺氧/复氧损伤的机制研究

Mechanism of IL-33 affecting myocardial hypoxia/reoxygenation injury through miR-19a-3p/MAPK6 pathway

目的 探讨白细胞介素-33(IL-33)通过miR-19a-3p/MAPK6通路对心肌缺氧/复氧(H/R)损伤的影响及可能的分子机制。方法 建立H/R诱导的新生小鼠心肌细胞体外模型,使用IL-33进行预处理,向心肌细胞中转入miR-19a-3p mimic,以上调miR-19a-3p表达,转入miR-19a-3p inhibitor,以抑制miR-19a-3p表达,转入pc DNA-MAPK6以上调MAPK6表达。通过Caspase-3活性、细胞凋亡ELISA法和原位细胞凋亡染色检测心肌细胞凋亡。Target Scan数据库预测miR-19a-3p的潜在靶点,双荧光素酶检测报告显示miR-19a-3p与MAPK6之间关系;RT-qPCR法检测心肌细胞miR-19a-3p和MAPK6 mRNA表达,Western blot法检测MAPK6蛋白表达。结果 IL-33预处理可以抑制H/R诱导的miR-19a-3p表达下调和细胞凋亡。双荧光素酶测定报告显示,miR-19a-3p靶向调控MAPK6。IL-33抑制了H/R诱导的MAPK6表达上调,过表达MAPK6减弱了IL-33对H/R的抗凋亡作用。过表达miR-19a-3p抑制了H/R诱导的MAPK6表达上调,而敲低miR-19a-3p可以消除IL-33对H/R诱导的MAPK6表达下调。结论 IL-33通过调控miR-19a-3p/MAPK6信号通路,减少H/R诱导的心肌细胞凋亡。

Objective To investigate the effect of interleukin-33(IL-33)on myocardial hypoxia/reoxygenation(H/R)injury via miR-19a-3p/MAPK6 pathway and its possible molecular mechanism.Methods The in vitro model of H/R-induced cardiomyocytes neonatal mouse was established and pre-treated with IL-33.The expression of miR-19a-3p was up-regulated through transferring miR-19a-3p mimic into the cells,the inhibition of miR-19a-3p was down-regulated through transferring miR-19a-3p inhibitor into the cells,and MAPK6 expression was up-regulated through transferring pc DNA-MAPK6 into the cells,myocardial cell apoptosis was detected by Caspase-3 activity,apoptosis ELISA and in situ apoptosis staining.Target Scan database was used to predict the potential targets of miR-19a-3p.Double luciferase report was used to study the relationship between miR-19a-3p and MAPK6;qRT-PCR was used to detect the expression of miR-19a-3p and MAPK6 mRNA in myocardial cells,and Western blot was used to detect the expression of MAPK6 protein.Results IL-33 pretreatment inhibited the down-regulation of miR-19a-3p expression and apoptosis induced by H/R.The dual-luciferase assay revealed that miR-19a-3p targeted and regulated MAPK6.IL-33 suppressed the up-regulation of MAPK6 expression induced by H/R,while overexpression of MAPK6 attenuated the anti-apoptotic effect of IL-33 against H/R.Overexpression of miR-19a-3p inhibited the up-regulation of MAPK6 expression induced by H/R,while knockdown of miR-19a-3p abolished the down-regulation of MAPK6 expression of IL-33 against H/R.Conclusion IL-33 can reduce H/R-induced myocardial cell apoptosis by regulating the signal pathway of miR-19a-3p/MAPK6.