右美托咪定联合Tol样受体4抑制剂对缺氧复氧心肌细胞凋亡和炎症反应的影响及其机制

目的探讨右美托咪定(DEX)联合Toll样受体4(TLR4)抑制剂TAK-242对缺氧复氧(H/R)心肌细胞凋亡和炎症反应的影响及其机制。方法心肌细胞H9C2分为对照(Con)组、H/R组(H/R损伤)、DEX组(1.0μmol/L DEX,再行H/R损伤)、TAK-242组(30μmol/L TAK-242,再行H/R损伤)和DEX+TAK-242组(30μmol/L TAK-242及1.0μmol/L DEX,再行H/R损伤处理)。各组细胞复氧培养6 h后,采用MTT法、流式细胞仪、试剂盒、酶联免疫吸附法检测细胞增殖、凋亡、乳酸脱氢酶(LDH)释放率、白介素1β(IL-1β)和肿瘤坏死因子-α(TNF-α)含量,Western blot检测B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、裂解的caspase-3(cleaved caspase-3)、TLR4和核因子B p65(NF-κB p65)蛋白表达。结果五组细胞凋亡率、LDH释放率、Bax、Bcl-2、cleaved caspase-3、TLR4、NF-κB p65蛋白表达水平、IL-1β、TNF-α含量比较差异均有统计学意义(F=316.938、330.004、839.933、169.750、378.365、476.535、298.527、99.219、293.498,P<0.05)。与Con组相比,H/R组细胞的凋亡率、LDH释放率、Bax、cleaved caspase-3、TLR4和NF-κB p65蛋白表达水平、细胞上清液中IL-1β和TNF-α含量均明显升高(均P<0.05),Bcl-2蛋白表达水平明显降低(均P<0.05)。与H/R组相比,DEX组、TAK-242组和DEX+TAK-242组的细胞凋亡率、LDH释放率、Bax蛋白、cleaved caspase-3、TLR4和NF-κB p65蛋白、IL-1β、TNF-α的表达水平均明显降低,Bcl-2蛋白表达水平明显升高(均P<0.05)。与DEX组、TAK-242组相比,DEX+TAK-242组的细胞凋亡率、LDH释放率、Bax、cleaved caspase-3、TLR4和NF-κB p65蛋白表达、IL-1β、TNF-α表达水平更低,Bcl-2蛋白的表达更高(均P<0.05)。结论DEX和TAK-242联合可协同抑制H/R引起的心肌细胞凋亡和炎症反应,其作用机制可能与协同抑制TLR4/NF-κB通路有关。

The effect and mechanism of dexmedetomidine combined with toll-like receptor 4 inhibitor on apoptosis and inflammation of hypoxia/reoxygenation cardiomyocytes

Objective To investigate the effect of dexmedetomidine(DEX)combined with toll-like receptor 4(TLR4)inhibitor TAK-242 on apoptosis and inflammation of cardiomyocytes induced by hypoxia/reoxygenation(H/R).Methods The cardiomyocytes H9 C2 were divided into the control(Con)group,the H/R group(H/R injury),the DEX group(1.0μmol/L DEX before H/R injury),the TAK-242 group(30μmol/L TAK-242 before H/R injury)and the DEX+TAK-242 group(1.0μmol/L DEX+30μmol/L TAK-242 before H/R injury).After 6 hours of cell reoxygenation in each group,MTT method,flow cytometry,kit and enzyme linked immunosorbent assay method were used to detect cell proliferation,cell apoptosis,lactic dehydrogenase(LDH)release rate,interleukin-1β(IL-1β)and tumor necrosis factor-a(TNF-α).Western blot was used to detect B-cell lymphoma/leukemia-2(Bcl-2),Bcl-2 related X protein(Bax),cleaved caspase-3,TLR4 and nuclear factor B p65(NF-κB p65)protein expression levels.Results The apoptosis rate,LDH release rate,Bax,Bcl-2,cleaved caspase-3,TLR4,NF-κB p65 protein expression levels,IL-1β,and TNF-αexpression levels among the five groups were statistically significant different(F=316.938,330.004,839.933,169.750,378.365,476.535,298.527,99.219,293.498;P<0.05).Compared with the Con group,the apoptosis rate,LDH release rate,Bax,cleaved caspase-3,TLR4 and NF-κB p65 protein expression level,IL-1βand TNF-αcontent in cell supernatant had increased significantly,and the expression level of Bcl-2 protein decreased significantly(all P<0.05).Compared with the H/R group,the apoptosis rate,LDH release rate,Bax protein,cleaved caspase-3 protein,TLR4 and NF-κB p65 protein and IL-1β,TNF-αexpression level of the DEX group,TAK-242 group and the DEX+TAK-242 group were significantly reduced,while the expression level of Bcl-2 protein increased significantly(all P<0.05).Compared with the DEX group and TAK-242 group,the cell apoptosis rate,LDH release rate,Bax protein,cleaved caspase-3,TLR4 and NF-κB p65 protein,IL-1β,TNF-αexpression level were lower in the DEX+TAK-242 group,and the expression of Bcl-2 protein was higher(all P<0.05).Conclusion The DEX combined with TAK-242 can synergistically inhibit the apoptosis and inflammation of cardiomyocytes induced by H/R,and its mechanism may be related to the co-inhibition of TLR4/NF-κB pathway.