Maresin 1激活Nrf2信号通路减轻低氧/复氧诱导的心肌细胞损伤

Maresin 1 attenuates hypoxia/reoxygenation-induced injury in cardiomyocytes by activating Nrf2 signaling pathway

目的探讨Maresin 1(MaR1)对低氧/复氧诱导的心肌细胞损伤的影响和作用机制。方法培养小鼠HL-1心肌细胞,采用低氧孵育6 h和复氧12 h来构建低氧/复氧细胞模型。小鼠HL-1心肌细胞分为常氧对照组(常氧培养18 h)、低氧/复氧组(低氧培养6 h,然后复氧处理12 h)、低氧/复氧+溶剂组(加入溶剂对照二甲基亚砜预处理30 min后,进行低氧/复氧处理)、低氧/复氧+MaR1组(加入50 nmol/L的MaR1预处理30 min后,进行低氧/复氧处理)和低氧/复氧+MaR1+ML385组(加入50 nmol/L的MaR1和5μmol/L的ML385预处理30 min后,进行低氧/复氧处理)。采用细胞计数试剂盒-8(cell counting kit-8,CCK-8)方法检测细胞存活率。乳酸脱氢酶(lactate dehydrogenase,LDH)细胞毒性检测试剂盒检测细胞损伤。TUNEL试剂盒检测细胞凋亡。活性氧(reactive oxygen species,ROS)试剂盒法检测ROS水平。丙二醛(malondialdehyde,MDA)试剂盒法检测MDA含量。超氧化物歧化酶(superoxide dismutase,SOD)试剂盒法检测SOD酶活性。利用Western blotting检测核转录因子E2相关因子2(nuclear factor-erythroid 2-related factor 2,Nrf2)、血红素加氧酶-1(heme oxygenase-1,HO-1)和醌氧化还原酶1(quinone oxidoreductase 1,NQO1)的蛋白表达水平。结果与常氧对照组比较,低氧/复氧组心肌细胞存活率显著降低(P<0.01),凋亡心肌细胞数量显著增加(P<0.01),ROS产生水平显著升高(P<0.01),MDA含量显著增加(P<0.01),SOD酶活性显著降低(P<0.01)。与低氧/复氧组和低氧/复氧+溶剂组比较,低氧/复氧+MaR1组心肌细胞存活率显著上升(P<0.01),凋亡心肌细胞数量显著减少(P<0.01),ROS产生水平显著降低(P<0.01),MDA含量显著减少(P<0.01),SOD酶活性显著升高(P<0.01)。与常氧对照组比较,低氧/复氧组Nrf2蛋白在细胞核中的表达量显著增加(P<0.05),Nrf2下游基因HO-1和NQO-1表达水平显著提高(P<0.01)。与低氧/复氧组和低氧/复氧+溶剂组比较,低氧/复氧+MaR1组Nrf2蛋白在细胞核中的表达量显著增加(P<0.01),HO-1和NQO-1表达水平显著提高(P<0.01)。与低氧/复氧+MaR1组比较,低氧/复氧+MaR1+ML385组Nrf2蛋白在细胞核中的表达量显著减少(P<0.01),凋亡心肌细胞数量显著增加(P<0.01),ROS产生水平显著升高(P<0.01)。结论MaR1通过激活Nrf2信号通路减轻低氧/复氧诱导的心肌细胞损伤。

Objective To investigate the effect of Maresin 1(MaR1)on hypoxia/reoxygenation(H/R)-induced injury in cardiomyocytes and explore its potential underlying mechanism.Methods The mouse HL-1 cardiomyocytes were cultured for 6 h under hypoxic condition followed by 12 h reoxygenation to establish the H/R cell model.The mouse HL-1 cardiomyocytes were divided into five groups:normoxic control group(18 h normoxic culture),H/R group(6 h hypoxic culture and then 12 h reoxygenation),H/R+vehicle group(pretreatment with dimethylsulfoxide for 30 min and then H/R treatment),H/R+MaR1 group(pretreatment with 50 nmol/L MaR1 for 30 min and then H/R treatment),and H/R+MaR1+ML385 group(pretreatment with 50 nmol/L MaR1 and 5μmol/L ML385 for 30 min and then H/R treatment).Cell counting kit-8(CCK-8)assay was used to detect the cell survival rate.A lactate dehydrogenase(LDH)cytotoxicity assay kit was utilized to determine the cell injury.TUNEL assay was applied to examine the cell apoptosis.A reactive oxygen species(ROS)assay kit was utilized to measure the ROS level.A malondialdehyde(MDA)assay kit was used to assess MDA content.A superoxide dismutase(SOD)assay kit was used to evaluate SOD activity.Western blotting was used to detect the protein level of nuclear factor-erythroid 2-related factor 2(Nrf2),heme oxygenase-1(HO-1),and quinone oxidoreductase 1(NQO1).Results Compared with normoxic control group,the survival rate of cardiomyocytes was significantly decreased in H/R group(P<0.01),the number of apoptotic cardiomyocytes was significantly increased(P<0.01),the level of ROS was significantly upregulated(P<0.01),the content of MDA was significantly increased(P<0.01),and the activity of SOD was significantly decreased(P<0.01).Compared with H/R group and H/R+vehicle group,the survival rate of cardiomyocytes was significantly improved in H/R+MaR1 group(P<0.01),the number of apoptotic cardiomyocytes was significantly reduced(P<0.01),the level of ROS was significantly downregulated(P<0.01),the content of MDA was significantly decreased(P<0.01),and the activity of SOD was significantly increased(P<0.01).Compared with normoxic control group,the expression of Nrf2 in the nucleus was significantly increased in H/R group(P<0.05),and the expression of Nrf2 target genes,including HO-1 and NQO-1,was significantly upregulated(P<0.01).Compared with H/R group and H/R+vehicle group,the expression of Nrf2 in the nucleus was significantly enhanced in H/R+MaR1 group(P<0.01),and the expression of HO-1 and NQO-1 was also significantly increased(P<0.01).Compared with H/R+MaR1 group,the expression of Nrf2 in the nucleus was significantly reduced in H/R+MaR1+ML385 group(P<0.01),the number of apoptotic cardiomyocytes was significantly upregulated(P<0.01),and the level of ROS was significantly increased(P<0.01).Conclusion Maresin 1 attenuates H/R-induced injury in cardiomyocytes by enhancing the activation of Nrf2 signaling pathway.