【作者】 唐丹丽；

【导师】 曹洪欣；

【作者基本信息】 中国中医科学院 ， 中医基础理论， 2009， 博士

【摘要】 近年来研究表明,心肌缺血再灌注损伤的主要机制之一是过度的炎症反应,在心肌缺血再灌注损伤的过程中炎性细胞因子大量释放并发挥重要的作用。而NF-κB信号转导通路参与介导细胞增殖、炎症、凋亡等多种生理过程,对于心肌缺血再灌注损伤的发生、发展具有关键作用。因此,本研究以心肌缺血再灌注损伤中炎症反应过程为研究主线,NF-κB信号转导通路为切入点,以温阳益心活血化痰法为干预手段,从细胞及分子生物学水平揭示温阳益心活血化痰法对心肌缺血再灌注损伤的抗炎效应及作用靶点,为优化治疗方案,提高临床疗效提供实验依据。目的:探讨温阳益心活血化痰法对心肌缺血再灌注后NF-κB信号转导通路的干预作用,明确本法对心肌缺血再灌注炎症损伤的具体作用靶点,阐明其抗炎作用机理。方法:通过结扎大鼠心脏左前降支动脉建立心肌缺血再灌注损伤动物模型,运用透射电镜观察心肌组织超微结构改变;全自动分析仪连续监测法检测大鼠血清LDH-L、CK水平;免疫印迹(western-blot)法检测心肌NIK、IKKβ、IκBα蛋白表达;采用逆转录-聚合酶链式反应(RT-PCR)及免疫组化法检测心肌NF-κBp65mRNA及蛋白表达;酶联免疫吸试验(ELISA)测定血清IL-1β、IL-6及IL-10的含量;利用免疫组化方法观察心肌TNF-α和ICAM-1蛋白表达。结果:①温阳益心活血化痰法指导下创立的温心方可以显著改善心肌超微结构损伤,抑制LDH-L、CK的生成和释放,降低再灌注后心律失常发生率,对心肌缺血再灌注损伤有保护作用;②温心方能够抑制再灌注后心肌NIK、IKKβ表达,并同时上调IκBα蛋白表达,与模型组比较有显著性差异(P＜0.01);③温心方能明显抑制心肌NF-κBp65mRNA及蛋白表达,阻止其核移位,与模型组相比统计学有显著性差异(P＜0.01);④温心方能显著降低NF-κB调控的下游炎性细胞因子IL-1β、IL-6及TNF-α、ICAM-1的含量,并促进抑炎因子IL-10的表达,各项指标与模型组相比均有显著性差异(P＜0.01),且低剂量组明显优于高剂量组。结论:温阳益心活血化痰法能够抑制血清心肌酶CK、LDH的生成和释放、降低再灌注后心律失常发生率、减轻心肌超微结构损伤,对心肌缺血再灌注损伤有保护作用。而其所具备良好的抗再灌注后炎症损伤作用,可能是通过抑制NF-κB信号转导通路上游激酶的活化,从而阻止NF-κB核移位,下调其转录调控的炎性因子表达,同时促进抑炎因子释放而实现的。更多还原

【Abstract】 Recently,it has been reported that inflammatory response is one of the main mechanisms of myocardial ischemic reperfusion injury(MI/RI). A large amount of inflammatory cytokines are released and play an important role in the progression of MI/RI.NF-κB signal transduction pathway participates in vital physiological processes,such as cell proliferation、inflammation and apoptosis.Therefore,this research regards the inflammatory response of MI/RI as research main track and mainly focuses on NF-κB signal transduction pathway.The study used Wenxin compound as the intervening method to reveal anti-inflammatory effects and treatment target of warming yang and supplementingheart;promoting blood circulation and resolving phlegm(ab.as WSPR) on cellular and molecular level.Moreover,the research provides experimental evidence for optimizing treatment strategies and improving clinical effects.Objective:To investigate the intervention effect of WSPR on NF-κB signal transduction pathway of MI/RI,showing the anti-inflammatory treatment target andmechanism of WSPR.Method:MI/RI rat models were established by ligating the anterior branch of the left coronary artery and the myocardial ultrastructure at the ischemia region was observed under the transmission electron microscope.The content of CK and LDH in blood serum were measured with continuous monitoring using automatic blood chemistry analyzer.Besides, NIK、IKKβand IκBαprotein expression of myocardial tissues in rats were detected by western-blot;the gene and protein expression of NF-κBp65 were detected by immunohistochemistry and RT-PCR respectively.Moreover,the levels of serum IL-1β,IL-6 and IL-10 were measured by ELISA,and TNF-α, ICAM-1 expressions in myocardium were determined with immunohistochemistry.Results:①Wenxin compound could significantly improve myocardial ultrastructural changes and inhibit the release of LDH-L and CK,decrease the incidence rate of reperfusion arrhythmias;②Wenxin compound was able to inhibit the protein expression of NIK,IKKβand enhanced the expression of IKBα,the difference is significant compared to the model group(P＜0.01);③Compared with model group,the gene and protein expression and the nuclear translocation of NF-κBp65 in the Wenxin compound were obviously inhibited(P＜0.01);④Wenxin compound could significantly decrease the contents of NF-κB downstream inflammatory factors such as IL-1β、IL-6 and TNF-α,ICAM-1(P＜0.01) and simultaneously strengthen the expression of IL-10.The differences of all the indicators were significant comparing to the model group(P＜0.01) and the effect of low dose is superior to high dose.Conclusion:WSPR has cardioprotective effects against MI/RI in rats possibly by inhibiting the expression of Ck and LDH,and reducing arrhythmia and the injury of the myocardial ultrastructure.WSPR could protect myocardium from inflammatory damage during MI/RI,its mechanism might restrain the activation of upstream kinase of NF-κB signal transduction pathway and then inhibiting nuclear translocation of NF-κB,inhibiting the release of the inflammatory factors and promoting the expression of anti-inflammatory cytokines.更多还原