基于线粒体自噬探讨参苓白术散对COPD骨骼肌损伤细胞的保护作用机制

Protective mechanism of Shenling Baizhu powder on the injured cells of COPD skeletal muscle based on mitochondrial autophagy

目的通过PINK1/Parkin线粒体自噬信号通路探究参苓白术散对香烟烟雾(CSE)诱导的慢性阻塞性肺疾病(COPD)骨骼肌损伤细胞的保护作用机制。方法用L6大鼠成肌细胞作为研究对象,除正常组外,其余组分别用CSE诱导L6大鼠成肌细胞24 h后,中药干预组再用不同浓度的参苓白术散药液作用24 h。实验分组:①正常组(control)、②CSE模型组(CSE)、③参苓白术散低剂量组(SLB-L)、④参苓白术散中剂量组(SLB-M)、⑤参苓白术散高剂量组(SLB-H)。观察各组L6大鼠成肌细胞的氧化损伤水平及线粒体自噬水平,用荧光酶标仪法检测细胞中活性氧的水平;JC-1染色法检测活细胞内线粒体膜电位水平;ATP试剂盒检测线粒体能量代谢的变化;蛋白质印迹法(Western blot)检测PINK1、Parkin、微管相关蛋白1轻链3(LC3)蛋白以及p62的表达水平。结果与正常组相比,CSE模型组的L6大鼠成肌细胞活性氧含量增加(P<0.05),线粒体内ATP水平、膜电位水平均明显下降(P<0.05),自噬相关蛋白Parkin蛋白表达水平下调,LC3B、p62、PINK1水平上调(P<0.05)。参苓白术散药剂组与CSE模型组相比,活性氧的含量下降(P<0.05),线粒体产生ATP水平、膜电位水平显著上调(P<0.05),而自噬相关蛋白Parkin蛋白表达水平上调,LC3B表达水平进一步升高,而p62表达水平显著下调(P<0.05)。结论参苓白术散可通过PINK1/Parkin通路介导线粒体自噬来上调L6大鼠成肌细胞内线粒体自噬活性,改善线粒体功能,维持线粒体能量代谢,缓解CSE引起的氧化损伤,降低细胞的早期凋亡,从而提高骨骼肌肌力及耐力以达到保护COPD骨骼肌功能的作用。

Objective To explore the protective mechanism of ShenlingBaizhu powder on skeletal muscle injury cells in chronic obstructive pulmonary disease(COPD)induced by cigarette smoke(CSE)through PINK1/Parkin mitochondrial autophagy pathway.Methods L6 rat myoblasts were used as the study object.Except the normal group,the other groups were induced by CSE for 24 hours,and the intervention group was treated with different concentrations of ShenlingBaizhu powder for 24 hours.The experimental groups included the normal group(control),CSE model group(CSE),low dose group(SLB-L),medium dose group(SLB-M)and high dose group(SLB-H).The level of oxidative damage and mitochondrial autophagy in L6 rat myoblasts were observed.The level of reactive oxygen species(ROS)in myoblasts was detected by fluorescent enzyme labeling method.The level of mitochondrial membrane potential in living cells was detected by JC-1 staining method.The change of mitochondrial energy metabolism was detected by ATP kit.The levels of PINK1,Parkin,LC3 and p62 were detected by western blot.Results Compared with the normal group,the content of ROS was increased,the levels of ATP and membrane potential in mitochondria were decreased significantly,the expression level of autophagy-relatedParkin protein was decreased,and the levels of LC3B,p62 and PINK1 were increased in L6 myoblasts of CSE model group.Compared with CSE model group,the content of ROS was decreased,the levels of ATP and membrane potential in mitochondria were increased significantly,the expression level of Parkinwas increased,the expression levels of LC3Bwas increased further,and the expression level of p62 was decreased significantly in ShenlingBaizhu powder group.Conclusion Through PINK1/Parkin pathway,ShenlingBaizhu powder can upregulate mitochondrial autophagy activity in L6 rat myoblasts,improve mitochondrial function,maintain mitochondrial energy metabolism,alleviate oxidative damage caused by CSE,reduce early apoptosis of cells,so as to improve skeletal muscle strength and endurance and protect the function of COPD skeletal muscle.