细胞自噬在骨形成蛋白4促大鼠H9C2心肌细胞肥大中的作用及机制

Role of autophagy in the motivation of rat myocardial hypertrophy in H9c2 cells induced by bone morphogenetic protein 4 and its mechanism

目的骨形成蛋白4(bone morphogenetic protein 4,BMP4)可诱导大鼠H9C2心肌细胞肥大,为进一步寻求其调控机制,文中探讨细胞自噬在心肌肥大中的作用及其与细胞外信号调节激酶(extracellular signal regulating kinase,ERK1/2)的相互关系。方法将H9C2心肌细胞随机分为4组:对照组、BMP4组、BMP4+ERK1/2信号通路抑制剂(PD98059)组、BMP4+自噬抑制剂(3MA)组,PD98059与3MA终浓度分别为50μmol/L与5 mmol/L,阻断30 min后加入浓度为50μg/L的BMP4。各组继续培养30 min后提取总蛋白分别检测微管相关蛋白1轻链3(tiny tubes related proteins 1 light chain 3,LC3)与pERK1/2的表达;48h后观察细胞表面积、平均蛋白含量、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的蛋白表达水平。在倒置显微镜下观察细胞的形态,用Image J软件测量细胞表面积,BCA法测细胞总蛋白含量、Western blot检测LC3、ERK1/2、p-ERK1/2及α-SMA的蛋白表达。结果 30 min后,与对照组相比,BMP4组LC3、p-ERK1/2蛋白表达均显著增强[(1.54±0.05)vs(1.95±0.11),(0.94±0.04)vs(1.33±0.06),P<0.01],BMP4+3MA组及BMP4+PD98059组均无明显变化(P>0.05);与BMP4组相比,BMP4+PD98059组、BMP4+3MA组LC3表达与p-ERK1/2均显著降低(P<0.01);而BMP4+PD98059组与BMP4+3MA组比较表达均无明显变化(P>0.05)。48 h后,与对照组相比,BMP4组细胞表面积、平均蛋白含量、α-SMA的蛋白表达水平均显著增加[(644.1±15.01)μm2vs(745.6±14.43)μm2,(240.0±7.26)pg/cell vs(347.1±5.4)pg/cell,(1.22±0.06 vs 1.99±0.03),P<0.01];与BMP4组相比,BMP4+PD98059组、BMP4+3MA组细胞面积、平均蛋白含量、α-SMA的蛋白表达水平均显著减少(P<0.01),而BMP4+PD98059组与BMP4+3MA组比较差异均无统计学意义(P>0.05)。结论细胞自噬可能通过ERK1/2信号通路参与了BMP4促大鼠H9C2心肌细胞肥大,阻断细胞自噬或ERK1/2信号通路的激活,可能对心肌肥大有治疗作用。

Objective Bone morphogenetic protein 4（BMP4） induces rat myocardial hypertrophy in H9c2 cells,but the specific mechanism remains unclear.The study aimed to elucidate the role of autophagy in myocardial hypertrophy and its relationship with extracellular signal regulating kinase（ERK1/2） signal transduction pathway.Methods H9c2 cardiomyocytes were randomly divided into 4 groups： control group,BMP4 group,BMP4＋PD98059（ERK1/2 signal pathway inhibitor） group and BMP4＋3MA（autophagy inhibitor） group.With PD98059（50μmol/L） and 3MA（5mmol/L）blocking for 30 min,BMP4（50 μg/L） were added.Expressions of tiny tube related proteins 1 light chain 3（LC3） and p-ERK1/2 were detected after culturing for 30 min.48 h later,measurements were made on cell surface area,average protein content and α-smooth muscle actin（α-SMA） protein expression level.Cell morphology and size were measured respectively by inverted microscope and Image J software.Total protein content was detected by BCA,and western blot was used to measure the expressions of LC3,ERK1/2,p-ERK1/2 and α-SMA.Results 30 min later,the expressions of LC3 and pERK1/2 were significantly higher in BMP4 group than in control group [（1.54 ± 0.05） vs（1.95 ± 0.11）,（0.94 ± 0.04） vs（1.33 ±0.06）,P0.01] and no significant difference was found in other two groups.Compared with BMP4 group,significant decrease was found in BMP4＋PD98059 and BMP4＋3MA [（1.95 ± 0.11） vs（1.59 ± 0.08）,（1.36 ± 0.02）;（1.33 ± 0.06） vs（0.87 ± 0.05）,（0.95 ±0.15）,P 0.01],while no significant difference was found between BMP4＋PD98059 group and BMP4 ＋3MA group.48 h later,the cell area,protein content and α-SMA protein expression level are significantly lower in control group than in BMP4 group[（644.1 ±15.01） μm^2vs（745.6 ± 14.43） μm^2,（240.0 ±7.26） pg/cell vs（347.1 ± 5.4） pg/cell,（1.22 ± 0.06 vs 1.99 ± 0.03）,P 0.01],while the corresponding indexes were significantly higher in BMP4＋PD98059 and BMP4＋3MA compared with BMP4 group[（745.6 ±14.43） μm^2vs（645.0 ± 12.63） μm^2,（647.7 ±13.89） μm^2;（347.1 ±5.4） pg/cell vs（239.7 ± 3.39） pg/cell,（241.1 ±8.56） pg/cell;（1.99 ± 0.03） vs（1.13 ± 0.05）;（1.12 ± 0.02）,P0.01].No significant difference was found as to the indexes between BMP4＋PD98059 group and BMP4＋3MA group（P 0.05）.Conclusion Autophagy may participate in BMP4-induced rat myocardial hypertrophy in H9c2 cells through ERK1/2 signal transduction pathway.The clinical treatment of myocardial hypertrophy may benefit from the blocking of autophagy or ERK1/2 signal transduction pathway.