自吞噬在脂多糖诱导HL-1心肌细胞损伤中的作用

目的 评价自吞噬在脂多糖(LPS)诱导HL-1心肌细胞损伤中的作用.方法 采用随机数字表法,将培养的HL-1细胞随机分为4组(n=15):正常对照组(C组)不予任何处理,继续培养24h；LPS组在细胞培养液中加入LPS(终浓度1 μg/ml)；自吞噬诱导剂纳巴霉素组(R组)在细胞培养液中加入纳巴霉素(终浓度0.2 μg/ml),孵育48 h时加入LPS(终浓度1 μg/ml)；自吞噬抑制剂三甲基嘌呤组(3-MA组)在细胞培养液中加入加入3-MA(终浓度10 mmol/L),孵育48 h时加入LPS(终浓度1μg/ml).各组孵育4h时测定自吞噬蛋白微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)表达水平,并观察线粒体超微结构,计算自吞噬体数量,测定线粒体光密度值、线粒体膜电位(JC-1).于孵育24h时测定细胞凋亡率和Caspase-3活性.结果 与C组比较,LPS组LC3Ⅱ表达水平、线粒体光密度值、细胞凋亡率和Caspase-3活性升高,自吞噬体数量增加,JC-1降低(P＜0.05)；与LPS组比较,R组LC3Ⅱ表达水平和JC-1升高,自吞噬体数量增加,线粒体光密度值、细胞凋亡率和Caspase-3活性降低,3-MA组LC3Ⅱ表达水平和JC-1降低,自吞噬体数量减少,线粒体光密度值、细胞凋亡率和Caspase-3活性升高(P＜0.05).R组线粒体超微结构损伤较LPS组减轻,3-MA组较LPS组加重.结论 自吞噬可减轻LPS诱导的HL-1心肌细胞损伤,机制可能与清除受损线粒体,改善线粒体功能,抑制细胞凋亡有关.

Role of autophagy in HL-1 cardiomyocyte injury induced by lipopolysaccharide

Objective To evaluate the role of autophagy in HL-1 cardiomyocyte injury induced by lipopolysaccharide( LPS).Methods Primary cultured HL-1 cardiomyocytes were randomly divided into 4 groups ( n =15each): normal control group( group C),LPS group,rapamycin( a autophagy inducer) group( group R) and 3-MA(a autophagy inhibitor) group.In group C cardiomyocytes were cultured continuously for 24 h.In group LPS cardiomyocytes were incubated with LPS (final concentration 1 μg/ml) for 24 h.In groups R and 3-MA,rapamycin and 3-MA was given 48 h before LPS (final concentration 1 μg/ml) incubation with final concentration of 0.2 μg/ml and 10 mmol/L respectively.The lipidated microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ ) expression,mitochondrial membrane potential,autophagosome number and optical density of mitochondria were determined,and ultrastructure of mitochondria was observed at 4 h of LPS incubation.Apoptosis rate and Caspase-3 activity were determined at 24 h of LPS incubation.Results LPS significantly increased LC3 Ⅱ expression,autophagosome number,optical density of mitochondria,apoptosis rate and Caspase-3 activity,decreased mitochondrial membrane potential in group LPS as compared with group C ( P ＜ 0.05).The LC3 Ⅱ expression,autophagosome number and mitochondrial membrane potential were higher,optical density of mitochondria,apoptosis rate and Caspase-3 activity lower in group R than in group LPS( P ＜ 0.05).The LC3 Ⅱ expression,autophagosome number and mitochondrial membrane potential were lower,optical density of mitochondria,apoptosis rate and Caspase-3 activity higher in group 3-MA than in group LPS (P ＜ 0.05).Mitochondrial histopathologic injury was reduced in group R and aggravated in group 3-MA as compared with group LPS.Conclusion Autophagy can reduce LPS-induced HL-1 cardiomyocyte injury by improving mitochondrial function and inhibiting apoptosis.