自吞噬在脂多糖诱导HL-1心肌细胞损伤中的作用

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

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1μg／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0．2μg／ml），孵育48h时加入LPs（终浓度1μg／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10mmol／L），孵育48h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。

Objective To evaluate the role of autophagy in HL-1 cardiomyocyte injury induced by lipopolysaccharide（ LPS）. Methods Primary cultured HL-1 eardiomyocytes were randomly divided into 4 groups （ n = 15 each） ： normal control group（ group C）, LPS group, rapamycin（ a autophagy inducer） group（ group R） and 3-MA（ a autophagy inhibitor） group. In group C eardiomyocytes 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 3Ⅱ （LC3Ⅱ ） expression, mitochondrial membrane potential, autophagosome number and optical density of mitoehondria were determined, and ultrastructure of mitoehondria 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 11 expression, autophagosome number,optical density of mitoehondria, apoptosis rate and Caspase-3 activity, decreased mitochondrial membrane potential in group LPS as compared with group C（ P 〈 0.05）. The LC3 Ⅱexpression, 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 LC3Ⅱexpression, 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.