多柔比星对乳鼠心肌细胞TLR4/NF-κB信号通路的影响及地塞米松的干预实验研究

Research on TLR4/NF-κB expression of cardiac cells and signal-transduction pathway of DOX in newborn rats and the intervention of dexamethasone

目的:观察多柔比星(doxorub ic in,DOX)对乳鼠心肌细胞Toll样受体4(toll-like receptor 4,TLR4)、核因子-κB(NF-κB)、肿瘤坏死因子-α(TNF-α)表达的影响,探讨多柔比星在心肌损伤中的可能发病机制及地塞米松(dex-am ethasone,DXM)对其干预作用。方法:采用体外培养乳鼠心肌细胞,用多柔比星制备心肌细胞损伤模型。随机分为3组:对照组,将心肌细胞悬液换用无血清培养基继续培养未加任何药物;DOX组,在培养液中加入多柔比星1 mg/L;DXM+DOX组,在加入DOX 1 mg/L前30 m in给予地塞米松32 mg/L。作用6 h后免疫组织化学法检测心肌细胞TLR4,TNF-α蛋白表达;分别作用2,6,24 h后用W estern b lot法对TLR4,NF-κB,TNF-α在心肌细胞的表达水平进行半定量分析;比色法测定培养液中乳酸脱氢酶(LDH)的活性。结果:DOX组心肌细胞TLR4,NF-κB,TNF-α表达较对照组明显增加(P<0.01);DXM+DOX组心肌细胞TLR4,NF-κB,TNF-α表达水平低于DOX组(P<0.01);与对照组比较,DOX组的LDH活性明显增高(P<0.01,6 h组);与DOX组比较,DXM+DOX组的LDH活性明显降低(P<0.01,6 h组);与对照组比较,DXM+DOX组的LDH活性稍高(P<0.05,6 h组)。结论:多柔比星作用于鼠心肌细胞,导致心肌细胞TLR4过度表达,激活其下游信号转导系统,表达细胞因子TNF-α,是其心肌细胞损伤的机制之一;DXM可以干预多柔比星心肌细胞TLR4及其下游信号转导NF-κB,TNF-α的过度表达,具有一定的心肌保护作用。

Objective： To observe the effect of doxorubicin（DOX） on the Toll-like receptor 4（TLR4）,nuclear factor-kappa B（NF-κB）,tumor necrosis factor-alpha（TNF-α）expression of cardiac cells in newborn rats,and to discuss the possible effects of doxorubicin on myocardial damage and the intervention effects and possible mechanism of dexamethasone（DXM）on doxorubicin-injured cardiac cells.Methods： Doxorubicin was administered to establish the injury model of primary cultured cardiomyocytes in newborn rats.Primary cultured cardiomyocytes were randomly divided into three groups,control group,myocardial cell suspension was continue cultivated with serum-free medium and no drugs put into;DOX group,with doxorubicin 1 mg/L in cultured fluid;DXM＋DOX group,with dexamethasone 32 mg/L and DOX 1 mg/L half an hour later in cultured fluid.The expression of TLR4,TNF-α protein in cardiac cells was checked with immunohistochemical after they were operated for 6 h.The semiquantitative analysis of TLR4,NF-κB,TNF-α protein was checked with Western blot after they were operated for 2 h,6 h and 24 h respectively.The activity of LDH was checked in cultured fluid with colorimetry. Results： The TLR4,NF-κB,TNF-α expression of the cardiac cells in DOX group were markedly increased when compared with control group（P〈0.01）.The TLR4,NF-κB,TNF-α expression of cardiac cells in DXM＋DOX group were considerably decreased compared with DOX group（P〈0.01）.Activity of LDH： Compared with control group,the activity of LDH was increased significantly in DOX group（P〈0.01,6 h group）.Compared with DOX group,the activity of LDH was decreased in DOX＋DXM group significantly（P〈0.01,6 h group）.Compared with control group,the activity of LDH was increased slighty in DOX＋DXM group（P〈0.05,6 h group）. Conclusion： TLR4 of cardiac cells under DOX was over expressed and activating the downstream signal-transduction system,making cytokine TNF-α expressed.This is one of the cardiac cells injuring mechanism.DXM could interfere the over expression of TLR4 in cardiac cells and its downstream signal-transduction NF-κB and TNF-α,thus to protect the cardiac muscle invariably.