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

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

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PCI2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60umol／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PCI2细胞经PrP106—126肽段处理72h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Bcl2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60umol／L依达拉奉处理后，Caspase3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。

Objective To explore the protection of free radical scavenger （edaravone） against apoptosis of the differentiated PC12 ceils 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 mitochondrial 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 Caspase3/CPP32 colorimetric 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 100μmol/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 31.8G and 15.2%, respectively after treated with 30μmol/L and 60μmol/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 60μmol/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 PrP106-126 peptide. After treated with 15, 30 and 60μmol/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.