摘要
目的:探讨14-3-3γ对脂多糖(LPS)所致心肌损伤的作用与抑制Bax向线粒体移位的关系。方法:采用原代SD乳鼠心肌细胞,随机分为4组:对照组;LPS组,LPS10mg/L加至培养基中6h;pFLAG+LPS组,空载质粒pFLAG转染至心肌细胞,24h后处理同LPS组;pFLAG-14-3-3γ+LPS组,重组质粒pFLAG-14-3-3γ转染至心肌细胞,24h后处理同LPS组。四唑盐(MTT)比色法检测各组细胞存活率,全自动生化分析仪检测乳酸脱氢酶(LDH)、肌酸磷酸酶(CPK)值,流式细胞仪检测细胞凋亡率,Western blotting检测细胞14-3-3γ蛋白水平、细胞浆及线粒体的Bax蛋白水平。结果:LPS致心肌细胞损伤,与对照组相比,LPS处理使细胞存活率明显下降(P<0.01),LDH、CPK值明显升高(P<0.01),细胞凋亡率增加(P<0.01),促使Bax蛋白从胞浆向线粒体移位,转染重组质粒pFLAG-14-3-3γ使14-3-3γ在心肌细胞内高表达后可明显逆转LPS所致的损伤,上述各指标均有所改善,并抑制Bax蛋白向线粒体的移位。结论:14-3-3γ对抗LPS所致心肌细胞损伤与抑制Bax从胞浆向线粒体移位有关。
Objective： To investigate the protective effect of 14-3-3γ on cardiomyocyte against lipopolysaccharide （LPS） injury, and the relationship with the suppression of Bax translocation into mitochondria. Methods： The primary neonatal SD rat cardiomyocytes were randomly divided into 4 groups： control group, cardiomyocytes treated with 10 mg/L LPS for 6 h （LPS group）, pFLAG transfected into cardiomyocytes for 24 h （pFLAG＋LPS group） and pFLAG-14-3-3γ tnsfected into cardiomyocytes for 24 h （pFLAG-14-3-3γ＋LPS group）. After treatment, the cell viability was analyzed by methyl thiazoyl tetrazolium （MTF）, the activity of lactic acid dehydrogenase （LDH） and creatine phosphate kinase （CPK） were deteImined by auto-biochemistry analysator, apoptotic cells were measured by flow cytometry and levels of 14-3-3γ protein in cardiomyocytes, Bax protein in cytoplasm and mitochondria were detected by Western blotting. Results： Compared with control group, the cell viability was significantly reduced in cardiomyocytes treated with LPS （P 〈 0.01）, LDH and CPK activities were significantly increased （P 〈 0.01）, the apoptotic cell ratio was enhanced （P 〈 0.01） and then Bax translocation from cytoplasm to mitochondria was promoted. The elevated 14-3-3γ protein resulted by transfection of pFLAG-14-3-3γ reversed cardiomyocyte damage induced by LPS, and inhibited Bax translocation from cytoplasm to mitochondria. Conclusion： 14-3-3γ protects cardiomyocytes against LPS damage by inhibiting Bax translocation from cytoplasm to mitochondria.
出处
《天津医药》
CAS
北大核心
2012年第12期1222-1225,共4页
Tianjin Medical Journal
基金
国家自然科学基金资助项目(项目编号:81160402
81072632
81100104)
博士后基金资助项目(20110491496)
