利多卡因通过降低微小RNA-181a表达抑制缺氧/复氧诱导的心肌细胞损伤

Lidocaine inhibits hypoxia/reoxygenation-induced cardiomyocyte damage by down-regulating microRNA-181a

目的 探讨利多卡因通过调控微小RNA-181a(miR-181a)对缺氧/复氧(H/R)诱导的心肌细胞H9C2损伤的影响。方法 培养H9C2细胞,建立H/R模型,作为H/R组;正常培养的细胞作为对照(Con)组。使用1.0、2.5、5.0、10.0、20.0μmol/L利多卡因处理H/R诱导的H9C2细胞,并分别设为1.0μmol/L组、2.5μmol/L组、5.0μmol/L组、10.0μmol/L组和20.0μmol/L组。将anti-miR-NC、anti-miR-181a分别转染至H/R诱导的H9C2细胞,记为H/R+anti-miR-NC组、H/R+anti-miR-181a组。将miR-NC、miR-181a分别转染至H/R诱导的H9C2细胞,再用20.0μmol/L利多卡因处理,分别记为H/R+miR-NC+20.0μmol/L组、H/R+miR-181a+20.0μmol/L组。采用MTT实验检测细胞活性;采用流式细胞术检测细胞凋亡;采用蛋白质印迹(Western blot)检测细胞半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)蛋白表达;采用实时荧光定量聚合酶链反应(RT-qPCR)检测miR-181a表达;检测丙二醛(MDA)含量及乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)活性;采用酶联免疫吸附实验(ELISA)检测炎性因子[肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)]的水平。结果 与Con组相比,H/R组细胞活性降低,差异有统计学意义(P<0.05)。与H/R组相比,5.0μmol/L组、10.0μmol/L组和20.0μmol/L组的细胞活性提高,差异有统计学意义(P<0.05)。因此,后续以20.0μmol/L利多卡因进行实验。与Con组相比,H/R组细胞凋亡率、Caspase-3蛋白表达升高,差异有统计学意义(P<0.05)。与H/R组相比,20.0μmol/L组细胞凋亡率、Caspase-3蛋白表达降低,差异有统计学意义(P<0.05)。与Con组相比,H/R组MDA含量、LDH活性以及炎症因子水平提高,SOD活性降低,差异有统计学意义(P<0.05);与H/R组相比,20.0μmol/L组MDA含量、LDH活性以及炎性因子水平降低,SOD活性提高,差异有统计学意义(P<0.05)。与H/R+anti-miR-NC组相比,H/R+anti-miR-181a组细胞miR-181a表达、凋亡率和Caspase-3蛋白表达降低,差异有统计学意义(P<0.05)。与H/R+anti-miR-NC组相比,H/R+anti-miR-181a组MDA含量、LDH活性以及炎性因子水平降低,SOD活性升高,差异有统计学意义(P<0.05)。与H/R+miR-NC+20.0μmol/L组相比,H/R+miR-181a+20.0μmol/L组细胞凋亡率、Caspase-3蛋白和MDA含量、LDH活性以及炎性因子水平升高,SOD活性降低,差异有统计学意义(P<0.05)。结论 利多卡因通过干扰miR-181a表达,抑制H/R诱导的心肌细胞H9C2损伤。

Objective To investigate the effect of lidocaine on cardiomyocyte H9C2 injury induced by hypoxia/reoxygenation(H/R) by regulating microRNA-181a(miR-181a).Methods H9C2 cells were cultured and an H/R model was established as H/R group;normal cultured cells were used as the control(Con) group.H/R-induced H9C2 cells were treated with 1.0,2.5,5.0,10.0 and 20.0 μmol/L lidocaine and were set as 1.0 μmol/L group,2.5 μmol/L group,5.0 μmol/L group,10.0 μmol/L group and 20.0 μmol/L group,respectively.Anti-miR-NC and anti-miR-181a were transfected into H/R-induced H9C2 cells,included in H/R+anti-miR-NC group and H/R+anti-miR-181a group,respectively.The miR-NC and miR-181a were transfected into H/R-induced H9C2 cells,and then treated with 20 μmol/L lidocaine,which were recorded as H/R+miR-NC+20 μmol/L group and H/R+miR-181a+20 μmol/L group,respectively.The cell activity was detected by MTT assay;flow cytometry was used to detect apoptosis;the expression of Caspase-3 protein was detected by Western blot;real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) was used to detect the expression of miR-181a;the content of malondialdehyde(MDA) and the activities of lactate dehydrogenase(LDH) and and superoxide dismutase(SOD) were detected;the levels of inflammatory factors [ tumor necrosis factor-α( TNF-α),Interleukin-1β( IL-1β),Interleukin-6(IL-6)] were detected by enzyme-linked immunosorbent assay(ELISA).Results Compared with the Con group,the cell activity in the H/R group was significantly decreased(P<0.05).Compared with the H/R group,the cell activity of 5.0 μmol/L group,10.0 μmol/L group and 20.0 μmol/L group was significantly increased(P< 0.05).Therefore,the experiment was conducted with 20.0 μmol/L lidocaine.Compared with the Con group,apoptosis rate and Caspase-3 protein expression in the H/R group were significantly increased(P< 0.05).Compared with the H/R group,the apoptosis rate and Caspase-3 protein expression in the 20.0 μmol/L group were significantly decreased(P<0.05).Compared with the Con group,MDA content,LDH activity and inflammatory factor levels in the H/R group were significantly increased,while SOD activity was significantly decreased in the H/R group;compared with the H/R group,MDA content,LDH activity and inflammatory factor level in the 20.0 μmol/L group were significantly decreased,and SOD activity was significantly increased(P< 0.05).Compared with the H/R + anti-miR-NC group,the expression of miR-181a,apoptosis rate and Caspase-3 protein in the H/R + anti-miR-181a group were significantly decreased(P< 0.05).Compared with the H/R + anti-miR-NC group,the MDA content,LDH activity and inflammatory factor levels in the H/R + anti-miR-181a group were significantly decreased,while SOD activity was significantly increased(P< 0.05).Compared with the H/R + miR-NC +20.0 μmol/L group,the apoptosis rate,the contents of Caspase-3 protein as well as MDA content,the activity of LDH and the level of inflammatory factors in the H/R + miR-181a + 20.0 μmol/L group were significantly increased,and the SOD activity was significantly decreased(P< 0.05).Conclusion Lidocaine inhibits H/R-induced cardiomyocyte H9C2 injury by interfering with the expression of miR-181a.