miR-199a在LPS诱导的大鼠原代心肌细胞中通过激活NF-κB信号通路加重心肌细胞损伤

miR-199a increases myocardial injury in a mouse model with myocarditis by activating the NF-κB signaling pathway

目的探究miR-199a在LPS诱导的大鼠原代心肌细胞中的作用。方法将大鼠原代心肌细胞分为对照组(NC)、LPS组、LPS+miR-199a mimic组、LPS+miR-199a inhibitor组。本实验采取CCK8方法探究miR-199a对于大鼠原代心肌的细胞活力影响。随后运用ELISA法检测miR-199a对于LPS诱导大鼠原代心肌细胞的炎症因子如TNF-α及IL-1β的释放情况。通过流式细胞仪检测miR-199a对于LPS诱导大鼠原代心肌细胞的凋亡蛋白变化。运用Western blot实验研究miR-199a模拟物或抑制剂的miR-199a表达情况以及研究miR-199a对于LPS诱导大鼠原代心肌细胞的p-p65、p65和p-iκBa、iκBa及凋亡蛋白表达情况。结果CCK8法发现与NC组相比,LPS抑制了大鼠原代心肌细胞的活力(P<0.01),然而经模拟物处理后,经LPS诱导的大鼠原代心肌活力受抑制更加严重(P<0.05)。同时,与模拟物组相比,miR-199a抑制剂组大鼠原代心肌细胞活力受抑制降低(P<0.01)。ELISA结果表明,与NC组相比,LPS诱导了TNF-α及IL-1β的释放(P<0.05)。与LPS组相比,miR-199a模拟物组加重了TNF-α(P<0.01)及IL-1β(P<0.05)的释放,而miR-199a抑制剂组TNF-α(P<0.01)及IL-1β(P<0.05)的释放减少。流式凋亡、Western blot结果表明,50 nmol/L的miR-199a模拟物可通过上调caspase3、bax表达和下调bcl2表达而加剧LPS引起的大鼠原代心肌细胞的凋亡(P<0.05)。进一步检测NF-κΒ通路发现LPS可通过p-iκBa与p-p65表达的上调诱激活大鼠原代心肌细胞NF-κΒ通路,但与LPS组相比,在模拟物处理后,p-p65和p-iκBa上调更加明显。与此同时,阻断miR-199a后,与模拟物组相比,p-p65和p-iκBa表达下调。这些发现表明用模拟物处理可以加重体内LPS诱导的心肌炎中NF-κB信号传导途径的激活。进一步使用NF-κB通路抑制剂发现,miR-199a模拟物并不能加剧LPS对于大鼠原代心肌细胞的活力抑制,这进一步提示miR-199a可能通过NF-κB通路加剧心肌细胞的损伤。结论miR-199a可加重心肌细胞的损伤,这种损伤主要通过NF-κB信号通路来实现的。

Objective To explore the role of miR-199a in LPS-induced rat primary cardiomyocytes.Methods The primary rat cardiomyocytes were divided into control group(NC),LPS group,LPS+miR-199a mimic group and LPS+miR-199a inhibitor group.CCK8 assays were used to explore the effect of miR-199a on cell viability of rat primary cardiomyocytes.ELISAs were used to detect the release of inflammatory factors such as TNF-αand IL-1βby HRNA-199a induced by LPS in rat primary cardiomyocytes.Expression of apoptotic proteins in rat primary cardiomyocyte induced by LPS by miR-199a was detected by flow cytometry.Western blot was used to study the expression of miR-199a mimic-or inhibitor-treated cells,and the expression of p-p65,p65,and p-iκBα,iκBαand apoptotic proteins in rat primary cardiomyocytes induced by LPS.Results Compared with the control group,LPS inhibited the viability of primary rat cardiomyocytes(P<0.01).However,after LPS induction and simulant treatments,the viability of primary rat cardiomyocytes was more severely inhibited(P<0.05).Therefore,miR-199a may aggravate cardiomyocyte damage in myocarditis.Compared with the mimics,inhibition of miR-199a reduced LPS-induced rat primary cardiomyocyte viability(P<0.01).Compared with the NC group,LPS induced the release of TNF-αand IL-1β(P<0.05).Compared with the LPS group,miR-199a mimic aggravated the release of TNF-α(P<0.01)and IL-1β(P<0.05).However,after treatment with miR-199a inhibitors,the release of TNF-α(P<0.01)and IL-1β(P<0.05)was decreased.Flow cytometry and Western blot showed that 50nmol/L miR-199a mimetics aggravate dapoptosis of primary rat cardiomyocytes induced by LPS(P<0.05).This may be related to the further upregulation of caspase3 and bax expression through miR-199a mimetics and downregulation of bcl2 expression(LPS)induced the activation of the NF-κB pathway in primary rat cardiomyocytes,which was specifically manifested by upregulation of p-iκBαand p-p65 expression.However,compared with the LPS group,p-p65 and p-iκBαwere more upregulated after treatment with the mimics.After blocking miR-199a,expression of pp65 and p-iκBαwas downregulated compared with the mimic group.These findings indicate that treatment with mimics aggravate activation of the NF-κB signaling pathway in LPS-induced myocarditis in vivo.Further use of NF-κB pathway inhibitors showed that miR-199a mimics did not exacerbate the inhibitory effect of LPS on primary rat cardiomyocytes,which further suggested that miR-199a exacerbates cardiomyocyte damage through the NF-κB pathway.Conclusions miR-199a aggravates damage of myocardial cells in myocarditis,which is mainly mediatedthrough the NF-κB signaling pathway.