依达拉奉对朊粒蛋白106-126诱导分化后PC12细胞凋亡的保护作用

目的 从细胞凋亡改变程度观察自由基清除剂--依达拉奉对朊粒蛋白(PrP)106-126诱导的分化PC12细胞损伤的保护作用,从而寻找更有效的朊粒病治疗方法.方法 应用膜联蛋白V/碘化丙啶双标记法检测细胞凋亡.激光共聚焦、显微镜观察线粒体膜电位,免疫组织化学法检测Bcl-2、Bax表达,caspase-3/CPP32细胞凋亡检测试剂盒和Western印迹检测Caspase-3活性.不同剂量依达拉奉组和各对照组同一时间点采用t检验.不同组间比较采用单因素方差分析.结果 在空白对照组中,94.5%为正常细胞;经100μmol/L PrP106-126处理后.损伤细胞达82.1%,其中早期凋亡细胞比例占21.2%,晚期凋亡细胞和坏死细胞占60.9%;给予30及60μmol/L依达拉奉处理后,凋亡、坏死细胞分别下降至31.8%和15.2%.当分化后的PC12细胞经PrP106-126肽段处理72 h后,线粒体膜电位下降36.1%.而加依达拉奉则抑制线粒体膜电位下降,且效应呈剂量依赖性;15、30、45和60μmol/L依达拉奉作用下,线粒体膜电位分别为对照组的47.3%、73.3%、94.1%和97.3%.依达拉奉能增强Bcl-2的表达,但抑制Bax的表达.PrP106-126肽段感染分化后的PCI2细胞,Caspase-3活性增至空白对照组的(397.21±5.63)%,15、30和60 μmol/L依达拉奉处理后,Caspase-3活性分别降至(170.12±5.01)%、(120.67±6.02)%和(100.21±8.04)%;Western印迹也证实依达拉奉呈剂量依赖性地抑制Caspase-3活性.结论 依达拉奉对PrP106-126诱导的分化PC12细胞具有抗氧化损伤作用,其机制可能是减轻细胞凋亡.

The protective effect of edaravone on apoptosis of the differentiated PC12 cells induced by prion protein 106-126 peptide

Objective To explore the protection of free radical scavenger (edaravone) against apoptosis of the differentiated PC12 cells induced by prion protein (PrP) 106-126 peptide and develop more effective treatment of prion diseases. Methods Cell apoptosis was detected by Annexin-V and opidium ioide (PI) dual staining. The mitoehondrial membrane potential was observed by laser scanning confocal microscope. Expressions of Bcl-2 and Bax were determined by immunohistochemistry. The Caspase-3 activity was analyzed by Caspase-3/CPP32 colorimetrie assay kit and Western blot. The comparison between edaravone group and control group at the same time point was done using t-test. Comparison between groups was performed by single factor analysis of variance. Results In control group, the percentage of normal cells was 94.5 % ; after treated with 100 μmol/L PrP106-126 peptide, the percentage of damaged cells was 82. 1%, of which the percentage of early stage apoptosis was 21.2%,the late stage apoptosis and necrosis was 60.9%. The percentage of apoptosis/necrosis decreased to 31.80% and 15.2%, respectively after treated with 30 μmol/L and 60 μmol/L edaravone. After 72 hours of PrP106-126 peptide treatment, mitochondrial membrane potential in differentiated PC12 cell decreased 36.1 %. While adding edaravone inhibited the decrease of mitochondrial membrane potential, which was dose-dependent. After treated with 15, 30, 45 and 60 μmol/L of edaravone, the mitochondrial membrane potentials decreased to 47.3%, 73.3%, 94.1%, 97.3% of control group, respectively. Edaravone could enhance Bcl-2 expression, but inhibit Bax expression. The Caspase-3 activity of differentiated PC12 cells increased to (397.21±5.63)% of control group after infected with PrPI06-126 peptide. After treated with 15, 30 and 60 μmol/L of edaravone, the Caspase-3 activity decreased to ( 170.12±5.01) %, ( 120.67±6.02)% and (100.21±8.04)%, respectively. Western blot also found that edaravone inhibited Caspase-3 activity dose-dependently. Conclusions Edaravone shows anti-oxidative effect on differentiated PC12 cells infected with PrP 106-126 peptide. The mechanism could be alleviation of cell apoptosis.