目的 研究p38MAPK/14-3-3γ通路在LPS预处理对抗缺氧/复氧(A/R)损伤中的作用.方法 采用原代培养乳鼠心肌细胞,建立A/R损伤模型及APC、LPS预处理保护模型.实验结束后测定p38MAPK 磷酸化蛋白表达及14-3-3γ蛋白表达,同时检测培养液中乳酸脱氢酶(LDH) 活性、四唑盐(MTT)比色试验测定细胞存活率、TUNEL法检测细胞凋亡、线粒体肿胀实验检测线粒体通透性转换孔(mPTP)开放、流式细胞仪检测线粒体膜电位.结果 LPS预处理对随后心肌A/R损伤有类似APC的保护作用,同时伴随p38 MAPK的磷酸化水平升高和14-3-3γ蛋白表达升高;给予SB203850特异性阻断p38MAPK通路,则14-3-3γ蛋白表达明显下调,并且心肌细胞LDH值升高,细胞存活率下降,心肌细胞的凋亡指数升高,mPTP开放加剧,线粒体膜电位降低.结论 LPS预处理对心肌细胞A/R损伤有保护作用,其机制可能是通过激活p38 MAPK,上调14-3-3γ的表达实现.%Aim To study the protective effect of LPS preconditioning on cardiomyocytes against anoxia/reox-ygenation( A/R ) injury through p38MAPK/14-3-3γ signal pathway. Methods The A/R and anoxia preconditioning APC/LPS preconditioning models of primary cultured neonatal rat myocytes were established. After treatment, the phosphor-p38MAPK and 14-3-3γ levels were detected by western blot. Meanwhile, the activity of LDH in culture medium was measured. Cell viability was detected by MTT. Apoptosis was analyzed by TUNEL. Opening of mitochondrial permeability transition pore( mPTP ) was determined by Ca2+-induced swelling of isolated cardiac mitochondria. Mitochondria membrane potential was determined by flow cytometry. Results LPS preconditioning had a protective effectrnagainst following A/R injury just like APC, and phos-pho-p38MAPK protein and 14-3-3γ protein were elevated. Once treated by p38MAPK specific inhibitor SB203850, 14-3-37 expression was inhibited; LDH was increased and viability was decreased; apoptotic index was heightened; mPTP opening was aggravated, and mitochondria membrane potential was reduced. Conclusions LPS preconditioning has a protective effect on cardiomyocytes against A/R injury. Its mechanism may be related to activating p38MAPK and up-regulating 14-3-3γ.
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