氧化苦参碱通过COX-2/PINK1/Parkin信号通路介导的线粒体自噬对MG63骨肉瘤细胞的促凋亡机制

Mechanism of oxymatrine promoting the apoptosis of osteosarcoma MG63 cell line through mitophagy mediated by COX-2/PINK1/Parkin signaling pathway

目的基于环氧合酶2(COX-2)/PTEN诱导激酶1(PINK1)/帕金森病蛋白2(Parkin)信号通路介导的线粒体自噬研究氧化苦参碱对人骨肉瘤MG63细胞的促凋亡机制。方法取MG63细胞经2.0、4.0、8.0 mg/mL的氧化苦参碱和6μmol/L的5-氟尿嘧啶(5-FU)作用后,检测细胞凋亡率、凋亡相关蛋白[B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)]表达水平、线粒体膜电位降低比例、线粒体自噬水平以及PINK1、Parkin、微管相关蛋白1轻链3Ⅱ(LC3-Ⅱ)蛋白表达水平。采用PINK1小干扰RNA(PINK1 siRNA)干扰PINK1的表达,将细胞分为对照组、PINK1 siRNA组、氧化苦参碱组、PINK1 siRNA+氧化苦参碱组,检测细胞中PINK1、Parkin、LC3-Ⅱ蛋白表达水平和线粒体膜电位降低比例以及细胞凋亡率。采用慢病毒感染使COX-2过表达,将细胞分为对照组、氧化苦参碱组、COX-2组、COX-2+氧化苦参碱组,检测细胞中COX-2、PINK1和Parkin蛋白表达水平以及线粒体膜电位降低比例。结果经氧化苦参碱干预后,细胞凋亡率,Bax、PINK1、Parkin、LC3-Ⅱ蛋白表达水平,线粒体自噬水平和线粒体膜电位降低比例均显著升高(P<0.05),Bcl-2蛋白表达水平显著降低(P<0.05)。与氧化苦参碱组比较,PINK1 siRNA+氧化苦参碱组细胞中PINK1、Parkin、LC3-Ⅱ蛋白表达水平和细胞凋亡率以及线粒体膜电位降低比例均显著降低(P<0.05)。与氧化苦参碱组比较,COX-2+氧化苦参碱组细胞中COX-2蛋白表达水平显著升高(P<0.05),PINK1、Parkin蛋白表达水平和线粒体膜电位降低比例均显著降低(P<0.05)。结论氧化苦参碱可通过抑制COX-2表达,介导线粒体自噬信号通路PINK1/Parkin的过度活化,从而促进骨肉瘤细胞凋亡。

OBJECTIVE To study the mechanism of oxymatrine inducing apoptosis of osteosarcoma MG63 cell line based on mitophagy mediated by cyclooxygenase-2(COX-2)/PTEN-induced putative kinase-1(PINK1)/Parkinson disease protein-2(Parkin)signaling pathway.METHODS MG63 cells were treated with 2.0,4.0,8.0 mg/mL oxymatrine and 6μmol/L 5-fluorouracil,then the apoptotic rate,the expression of apoptosis-related proteins[B-cell lymphoma-2(Bcl-2),Bcl-2 related X protein(Bax)],the proportion of decrease in mitochondrial membrane potential,the level of mitophagy as well as the protein expressions of PINK1,Parkin,and microtubule-associated protein 1 light chain-3Ⅱ(LC3-Ⅱ)were detected.PINK1 small interfering RNA(siRNA)was adopted to intervene in the expression of PINK1,the cells were divided into control group,PINK1 siRNA group,oxymatrine group,and PINK1 siRNA+oxymatrine group;the protein expressions of PINK1,Parkin,and LC3-Ⅱ,the proportion of decrease in mitochondrial membrane potential(MMP)as well as apoptotic rate were detected.The lentivirus infection technique was used to overexpress COX-2,the cells were divided into control group,oxymatrine group,COX-2 group,and COX-2+oxymatrine group.The protein expressions of COX-2,PINK1,and Parkin,as well as the proportion of decrease in MMP were detected.RESULTS After being treated with oxymatrine,the apoptotic rate,the protein expressions of Bax,PINK1,Parkin,and LC3-Ⅱ,the level of mitophagy as well as the proportion of decrease in MMP were significantly increased(P<0.05),while the protein expression of Bcl-2 was significantly decreased(P<0.05).Compared with the oxymatrine group,the protein expressions of PINK1,Parkin,and LC3-Ⅱ,apoptotic rate and the proportion of decrease in MMP were significantly decreased in PINK1 siRNA+oxymatrine group(P<0.05).Compared with the oxymatrine group,the protein expression of COX-2 in the COX-2+oxymatrine group was increased significantly(P<0.05),while the protein expressions of PINK1 and Parkin as well as the proportion of decrease in MMP were decreased significantly(P<0.05)CONCLUSIONS Oxymatrine can mediate the overactivity of mitophagy based on the PINK1/Parkin signaling pathway by inhibiting COX-2 expression,thus promoting the apoptosis of the MG63 osteosarcoma cell line.