MyD88依赖性核因子-κB信号途径在心肌肥大发生过程中的调控作用

Role of MyD88-dependent nuclear factor-κB signaling pathway in the development of cardiac hypertrophy in vivo

目的探讨MyD88依赖性核因子κB(nuclearfactorkappaB,NFκB)信号途径在心肌肥大发生发展方面的作用。方法以缩窄SD大鼠升主动脉诱导的心肌肥大为模型,对MyD88介导的NFκB信号通路进行了分析。NFκB结合活性用EMSA的方法检测;IKKα/β和IκBα的磷酸化水平用Western印迹分析;并进一步将腺病毒携带的无功能MyD88(dnMyD88)转染到心肌组织中,观察阻断MyD88下游信号通路对心肌肥大发生发展的影响。结果缩窄升主动脉后,大鼠心脏重量(g)×100/体重(g)比值明显增加,缩窄3周后,与周龄配对的假手术组相比,升高了378%,(P<001),同时,心肌组织中ANP蛋白表达水平也明显增加(P<001)。升主动脉缩窄3周后,与周龄配对的假手术组相比心肌组织中的NFκB活性增加了1448%(P<001),同时心肌组织中IκBα和IKKα/β的磷酸化水平也明显增高(P<001)。转染Ad5dnMyD88到心肌组织中明显减轻心肌肥大,与升主动脉缩窄组相比心脏重量/体重比值下降了116%(P<001),ANP蛋白表达水平下降了362%(P<001)。转染Ad5dnMyD88明显抑制NFκB活性,与心肌肥大模型组相比下降了418%(P<005)。转染Ad5dnMyD88亦抑制增高的pIKBα和pIKKα/β水平,与心肌肥大模型组相比分别下降了267%和774%。结论MyD88依赖性NFκB信号途径是导致心肌肥大的一个新的信号途径,?

Objective To investigate the role of MyD88-dependent nuclear factor-κB (NF-(B) activation signaling pathway in the development of cardiac hypertrophy in vivo. Methods Dominant negative myeloid differentiation protein (dn-MyD) 88 fragment was inserted into pShuttle plasmid and then fused into adenovirus so as to construct Ad5-dn-MyD88. Some Sprague-Dawley (SD) rats underwent banding of aorta (aorta binding group) and some SD rats underwent sham operation (sham operation group). Part of the rats in the aorta banding group were transfected with Ad5-dn-MyD88 into the myocardium tissue (Ad5-dn-MyD88 transfection group) or adenovirus expressing dnMyD88 (Ad5-GFP) (control group) so as to determine the effect of blocking down stream of MyD88 signaling on the development of cardiac hypertrophy. Three days after the hearts of some rats from the 4 groups were collected and Western blotting was used to detect the expression of dn-MyD88 protein and fluorescent microscopy was used to detect the expression of GFP. Three weeks after the beginning of experiment the hearts were collected to calculate the heart weight/body weight (HW/BW) ratio and extract the plasma protein and nuclear protein. Electrophoretic mobility shift assay (EMSA) was used to determine the NFκB binding activity. Western blotting was used to examine the phosphorylation of IκBα and IKKα/β with appropriate specific anti-phospho antibodies. Results Flag and dn-MyD88 were effectively expressed 3 days after the transfection of Ad5-dn-MyD88 into the myocardium. Three weeks after the HW/BW ratio was 0.47±0.01 in the aorta banding group, significantly higher, by 37.8%, than that of the sham operation group (0.34±0.01,P<0.01), and was 0.41±0.02 in the Ad5-dn-MyD88 transfection group, significantly lower, by 11.58%, than that of the aorta banding group (P<0.01) ; the myocardial ANP protein expression level of the aorta binding group was significantly higher, by 43.5%, than that of the sham operation group (P<0.01) and 36.2% higher than that of the Ad5-dnMyD88 transfection group (P<0.01); the ANP/GAPDH in the aorta binding group was significantly higher than that of the sham operation group (P<0.01) and that of the Ad5-dn-MyD88 transfection group (P<0.01); the NF-κB binding activity in the myocardium of the aorta banding group was 9.94±1.58, significantly higher, by 144.8%, than that of the sham operation group (4.06±0.52, P<0.01) and significantly lower, by 41.8%, than that of the Ad5-dn-MyD88 transfection group (5.79±0.52, P<0.05); the phospho-(p-) IκBα level and p-IκBα/IκBα of the aorta binding group were significantly higher than those of the sham operation group (P<0.01) and significantly higher, by 26.7%, than that of the Ad5-dn-MyD88 transfection group (P<0.05); the p-IKKαβ/ IKKαβ in the myocardium of the aorta binding group was significantly higher, by 318.0%, than that of the sham operation group (P<0.01), and significantly higher, by 77.4%, than that of the Ad5-dn-MyD88 transfection group (P<0.01). Conclusion MyD88-dependent NFκB signaling is a novel pathway for inducing the development of cardiac hypertrophy in vivo and blocking MyD88 mediated signaling pathway attenuates the development of cardiac hypertrophy.