Bcl-2/Bad/mPTP通路介导14-3-3γ对抗脂多糖所致心肌损伤  

Bcl-2 /Bad /mPTP pathway mediates 14-3-3γ protecting against LPS-induced myocardial injury

作　　者：刘丹[1] 彭易安[1] 刘卓琦[2] 汤蕾[1] 尹东[3] 何明[1] 

机构地区：[1]南昌大学医学院药理学教研室,江西南昌330006 [2]南昌大学医学院生化教研室,江西南昌330006 [3]南昌大学第二附属医院江西省分子医学重点实验室,江西南昌330006

出　　处：《中国药理学通报》2013年第1期48-52,共5页Chinese Pharmacological Bulletin

基　　金：国家重点基础研究发展计划(973计划)资助项目(No2009CB 526405);国家自然科学基金资助项目(No81160402;81072632)

摘　　要：目的探讨Bcl-2/Bad/mPTP通路在14-3-3γ对抗脂多糖所致心肌损伤中的作用。方法构建pFLAG-14-3-3γ重组质粒,转染至原代乳鼠心肌细胞中,然后行LPS损伤处理。处理结束后,取培养液检测LDH活性,MTT比色法检测细胞存活率,流式细胞术检测细胞凋亡,线粒体肿胀实验检测mPTP开放,Western blot检测14-3-3γ、Bad、phospho-Bad以及线粒体Bcl-2蛋白表达。结果 LPS损伤使心肌细胞LDH活性升高、细胞存活率下降、凋亡细胞增加、mPTP开放加剧,转染pFLAG-14-3-3γ重组质粒后再行LPS损伤,则LDH活性下降、细胞存活率升高、mPTP开放与细胞凋亡减少,同时phospho-Bad蛋白表达增加,线粒体Bcl-2蛋白表达增加。结论 pFLAG-14-3-3γ能够对抗脂多糖所致的心肌细胞损伤,其机制可能与磷酸化Bad,释放Bcl-2至线粒体,抑制mPTP的开放有关。Aim To explore the role of Bcl-2/Bad/ mPTP pathway in 14-3-3r protecting against LPS-in- dueed cardiomyocyte injury. Methods pFLAG-14-3- 3rrecombinant plasmid was constructed and transfeet- ed into primary neonatal SD rat cardiomyocytes. After treatment of LPS injury, the activity of LDH was ana- lyzed in supernatant; cell viability was detected by MTT colorimetrie assay; cell apoptosis was determina- ted by flow eytometry; opening of mitochondrial perme- ability transition pore （mPTP） was detected by swelling of isolated cardiac mitochondria; the level of 14-3-3r, Bad, phospho-Bad protein in cell and the level of Bcl-2 protein in mitoehondria were analyzed by Western blot.Results LPS damage made LDH activity increase, cell viability decrease, cell apoptosis increase, and mVl＇P opening aggravate. After the transfection with pFLAG-14-3-3-r, LDH activity and cell apoptosis were decreased, cell viability was increased, mPTP opening was inhibited, and level of phospho-Bad was raised, while level of Bcl-2 in mitochondria was also increased. Conclusions pFLAG-14-3-3r can protect against LPS-induced cardiomyocyte damage, and the mecha- nism is related to phosphorylating Bad, releasing Bcl-2 to mitochondria, and inhibiting mPTP opening.

关 键 词：BCL-2 BAD 线粒体 MPTP 质粒转染 脂多糖 心肌 损伤 

分 类 号：R-332[医药卫生] R322.11