竹荪多糖对亚砷酸钠诱导L-02细胞线粒体自噬的影响

Effect of dictyophora polysaccharide on mitophagy induced by sodium arsenite in L-02 cells

目的观察竹荪多糖(DIP)对亚砷酸钠(NaAsO_(2))诱导人肝细胞(L-02细胞)中PINK1/Parkin途径介导的线粒体自噬的干预作用。方法将处于对数生长期且状态良好的L-02细胞分为对照组、NaAsO_(2)组(10μmol/L)、DIP组(80μg/ml)、DIP+NaAsO_(2)组(80μg/ml DIP+10μmol/L NaAsO_(2))、活性氧(ROS)清除剂N-乙酰半胱氨酸(NAC)组(5 mmol/L)、NAC+NaAsO_(2)组(5 mmol/L NAC+10μmol/L NaAsO_(2))。蛋白免疫印迹法(Western blot)检测线粒体自噬相关蛋白p62、微管相关蛋白1轻链3(LC3)Ⅱ/LC3Ⅰ、PINK1、Parkin的表达水平,透射电子显微镜观察线粒体结构及自噬体,荧光探针法检测细胞内ROS水平。结果与对照组比较,NaAsO_(2)组p62、LC3Ⅱ/LC3Ⅰ、PINK1、Parkin蛋白表达量均较高(P均<0.05);与NaAsO_(2)组比较,DIP、DIP+NaAsO_(2)、NAC、NAC+NaAsO_(2)组p62、LC3Ⅱ/LC3Ⅰ、PINK1、Parkin蛋白表达量均较低(P均<0.05)。透射电子显微镜下,与对照组比较,NaAsO_(2)组L-02细胞线粒体损伤明显,自噬小体数量增多;与NaAsO_(2)组比较,DIP+NaAsO_(2)组线粒体肿胀程度减小,空泡变性减少,自噬小体数量减少。与对照组(33110.00±2191.28)比较,NaAsO_(2)组细胞内ROS水平较高(48000.00±2395.31,P<0.05);DIP+NaAsO_(2)组细胞内ROS水平(38670.00±2620.56)与NaAsO_(2)组比较明显降低(P<0.05),与对照组比较未见明显改变(P>0.05)。结论NaAsO_(2)可以诱导L-02细胞内PINK1/Parkin途径介导的线粒体自噬发生,DIP可以缓解NaAsO_(2)诱导的线粒体自噬。DIP可能通过对ROS的调控来影响NaAsO_(2)诱导的PINK1/Parkin途径介导的线粒体自噬。

Objective To observe the effect of dictyophora polysaccharide(DIP)on PINK1/Parkin pathway mediated mitophagy induced by sodium arsenite(NaAsO_(2))in human hepatocytes(L-02 cells).Methods The L-02 cells in logarithmic growth phase and in good condition were divided into control group,NaAsO_(2) group(10μmol/L),DIP group(80μg/ml),DIP+NaAsO_(2) group(80μg/ml DIP+10μmol/L NaAsO_(2)),N-acetylcysteine(NAC)group(5 mmol/L),and NAC+NaAsO_(2) group(5 mmol/L NAC+10μmol/L NaAsO_(2)).Western blotting was used to detect the expression levels of mitophagy related proteins p62,microtubule-associated protein 1 light chain 3(LC3)Ⅱ/LC3Ⅰ,PINK1,and Parkin.The mitochondrial stucture and autophagosomes were observed by transmission electron microscope,the fluorescent probe method was used to detect the expression level of intracellular reactive oxygen species(ROS).Results Compared with the control group,the protein expressions of p62,LC3Ⅱ/LC3Ⅰ,PINK1,and Parkin in NaAsO_(2) group were higher(P<0.05);compared with the NaAsO_(2) group,the protein expressions of p62,LC3Ⅱ/LC3Ⅰ,PINK1 and Parkin were lower in DIP,DIP+NaAsO_(2),NAC,and NAC+NaAsO_(2) groups(P<0.05).According to the transmission electron microscope,compared with the control group,the mitochondria of L-02 cells in NaAsO_(2) group were significantly damaged and the number of autophagosomes increased.Compared with NaAsO_(2) group,the degree of mitochondrial swelling,vacuolar degeneration and the number of autophagosomes decreased in DIP+NaAsO_(2) group.Compared with the control group(33110.00±2191.28),the intracellular ROS level in NaAsO_(2) group was higher(48000.00±2395.31,P<0.05);the level of intracellular ROS in DIP+NaAsO_(2) group(38670.00±2620.56)was significantly lower than that in NaAsO_(2) group(P<0.05),and there was no significant change compared with the control group(P>0.05).Conclusions NaAsO_(2) can induce PINK1/Parkin mediated mitophagy in L-02 cells.DIP can alleviate NaAsO_(2) induced mitophagy.DIP may affect PINK1/Parkin mediated mitophagy induced by NaAsO_(2) through the regulation of ROS.