依托咪酯通过调控miR‑214‑3p/TBL1XR1分子轴抑制脂多糖诱导心肌细胞炎症及凋亡

Etomidate inhibits lipopolysaccharideinduced cardiomyocyte inflammation and apoptosis by regulating miR2143p/TBL1XR1 molecular axis

目的探讨依托咪酯对脂多糖(lipopolysaccharide,LPS)诱导心肌细胞炎症、凋亡的影响及其可能作用机制。方法将体外培养的大鼠心肌细胞H9c2采用随机数字表法分为5组(每组3个复孔,每组实验重复3次):正常对照组(Con组)、脂多糖组(LPS组)、脂多糖+20μmol/L依托咪酯组(EL组)、脂多糖+50μmol/L依托咪酯组(EM组)、脂多糖+100μmol/L依托咪酯组(EH组)。Con组正常培养,LPS组加入含浓度1 mg/L LPS的杜氏改良培养基(dulbecco's modified eagle medium,DMEM)培养24 h,EL组、EM组、EH组分别使用不同浓度(20、50、100μmol/L)依托咪酯与1 mg/L LPS的DMEM培养液培养24 h。再次选取正常培养的心肌细胞采用随机数字表法分为两组(每组3个复孔,每组实验重复3次):脂多糖+100μmol/L依托咪酯+anti微RNA(microRNA,miR)NC组(antimiRNC组)和脂多糖+100μmol/L依托咪酯+antimiR2143p组(antimiR2143p组)。antimiRNC组和antimiR2143p组分别将antimiRNC和antimiR2143p转染至心肌细胞,加入含有100μmol/L依托咪酯与1 mg/L LPS的DMEM培养液培养24 h。ELISA法检测TNFα、IL6、IL1β水平,流式细胞术检测细胞凋亡率,实时荧光定量PCR(quantitative realtime PCR,qRTPCR)检测miR2143p与转导素β1X连锁受体蛋白质1(transducinβlike 1X related protein 1,TBL1XR1)的表达量,双荧光素酶报告实验验证miR2143p与TBL1XR1的靶向调控关系,Western blot法检测TBL1XR1、B淋巴细胞瘤2(Bcell lymphoma2,Bcl2)相关蛋白(Bcl2associated X protein,Bax)、Bcl2的表达量。结果与Con组比较,LPS组TNFα、IL6、IL1β、Bax水平以及细胞凋亡率、TBL1XR1 mRNA表达升高(P<0.05),Bcl2蛋白水平和miR2143p表达降低(P<0.05);与LPS组比较,EL组、EM组、EH组TNFα、IL6、IL1β、Bax水平以及细胞凋亡率、TBL1XR1 mRNA表达呈剂量依赖性降低(P<0.05),Bcl2蛋白水平和miR2143p表达呈剂量依赖性升高(P<0.05);EL组、EM组、EH组两两比较差异有统计学意义(P<0.05)。miR2143p可负向调控TBL1XR1的表达(P<0.05)。与antimiRNC组比较,antimiR2143p组TNFα、IL6、IL1β、Bax水平以及细胞凋亡率升高(P<0.05),Bcl2蛋白水平降低(P<0.05)。结论依托咪酯可通过调控miR2143p/TBL1XR1分子轴从而抑制LPS诱导的心肌细胞炎症及凋亡。

Objective To investigate the effects of etomidate on lipopolysaccharide(LPS)induced inflammation and apoptosis of cardiomyocytes and its possible mechanism.Methods According to the random number table method,rat cardiomyocytes H9c2 cultured in vitro were divided into five groups(n=3,and each experiment was repeated three times):a normal control(Con)group,a LPS group,a LPS+20μmol/L etomidate(EL)group,a LPS+50μmol/L etomidate(EM)group,and a LPS+100μmol/L etomidate(EH)group.The Con group was normally cultivated;the LPS group was cultured in dulbecco's modified eagle medium(DMEM)medium containing 1 mg/L LPS for 24 h;and the EL group,EM group,and EH group were treated with different concentrations(20,50μmol/L,and 100μmol/L)etomidate and 1 mg/L LPS in DMEM medium for 24 h.According to the random number table method,normal cultured cardiomyocytes were divided into two groups(n=3,and each experiment was repeated three times):a LPS+100μmol/L etomidate+antimicroRNA(microRNA,miR)NC(antimiRNC)group and a LPS+100μmol/L etomidate+antimiR2143p(antimiR2143p)group.The antimiRNC group and antimiR2143p group were transfected with antimiRNC or antimiR2143p to cardiomyocytes,respectively,before cultivation with DMEM medium containing 100μmol/L etomidate and 1 mg/L LPS for 24 h.The enzymelinked immunosorbent assay(ELISA)method was used to detect the levels of tumor necrosis factorα(TNFα),interleukin(IL)6,and IL1β,while flow cytometry was used to detect cell apoptosis rate.The quantitative realtime polymerase chain reaction(qRTPCR)was used to detect the expression of miR2143p and transducinβlike 1X related protein 1(TBL1XR1).The dual luciferase report test was used to verify the targeted regulation relationship between miR2143p and TBL1XR1.The expression of TBL1XR1 and Bcell lymphoma2(Bcl2)associated X protein(Bax),Bcl2 was measured by Western blot.Results Compared with the Con group,the LPS group presented remarkable increases in the levels of TNFα,IL6,IL1βand Bax protein,apoptosis rate and the amount of TBL1XR1 mRNA(P<0.05),and decreases in the level of Bcl2 and miR2143p protein(P<0.05).Compared with the LPS group,the levels of TNFα,IL6,IL1β,and Bax,apoptosis rate and the amount of TBL1XR1 mRNA in the EL,EM,and EH group decreased in a dosedependent manner(P<0.05),while the level of Bcl2 and miR2143p increased in a dosedependent manner(P<0.05),and statistical differences were found among the EL,EM,and EH groups(P<0.05).MiR2143p negatively regulated the expression of TBL1XR1(P<0.05).Compared with the antimiRNC group,the levels of TNFα,IL6,IL1β,and Bax,apoptosis rate in the antimiR2143p group increased(P<0.05),while the level of Bcl2 decreased(P<0.05).Conclusions Etomidate inhibits LPSinduced inflammation and apoptosis in cardiomyocytes through regulating the miR2143p/TBL1XR1 molecular axis.