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首都医科大学友谊医院麻醉科  缪慧慧     导师  田鸣

人参皂苷Rg1经线粒体抑制异氟醚诱导的Caspase-3活化

背景:随着全球老龄化社会的到来, 老年人的医疗质量和生存质量得到了全世界的广泛关注。随着医疗水平不断提高, 术后死亡率和各种严重并发症的发生率已明显下降。但是, 术后中枢神经系统并发症却越来越多地引起人们的重视，特别是老年人术后认知功能障碍(post-operative cognitive dysfunction, POCD)，严重者发展为老年痴呆症（Alzheimer’s disease,AD）所造成的医学及社会问题日趋严重。患者生存质量显著下降，包括丧失自理能力和需要特别的护理等等。然而目前却没有很好的治疗和预防POCD的药物。研究显示吸入麻醉药异氟醚会诱发线粒体功能障碍及凋亡效应蛋白caspase-3的活化,并由此导致学习及记忆功能受损。据报道人参皂苷Rg1具有神经保护作用。因此，本研究决定通过实验观察人参皂苷Rg1是否可以通过抑制线粒体功能障碍从而减轻异氟醚诱导的caspase-3活化。

方法：通过设置不同浓度12.5, 25和50µmol/L的人参皂苷Rg1以及不同作用时间12h和24h来观察人参皂苷Rg1对异氟醚诱导caspase-3活化的影响。实验选用人神经胶质瘤细胞(H4 naïve cell)和稳定转染淀粉样蛋白前体(amyloid precursor protein ,APP)基因的人神经胶质瘤细胞(H4-APP cell)。通过检测线粒体通透性转换孔(mitochondrial permeability transition pore,mPTP)以及5'-三磷酸腺苷(adenosine-5’-triphosphate , ATP)的水平观察线粒体功能。本研究采用蛋白印记分析(Western blot),化学发光法以及流式细胞技术。

结果：实验结果显示与对照组相比，异氟醚处理会诱发caspase-3活化及线粒体功能障碍。对于H4-APP细胞，加入50µmol/L的人参皂苷Rg1预处理12h能够抑制异氟醚诱导的caspase-3活化:4.02 vs. 7.23倍(^#P=0.03);增加线粒体ATP生成水平: 0.68 vs. 0.41倍 (^#P=0.02); 减少mPTP的开放程度：3.25 vs.1.39 (^#P=0.00)。对于H4-APP细胞，加入25和50µmol/L的人参皂苷Rg1预处理24h能够抑制异氟醚诱导的caspase-3活化: ：4.30和2.99 vs. 8.16倍(^#P=0.04,0.01);增加线粒体ATP生成水平: 0.72和0.89 vs. 0.13 倍 (^#P=0.01和0.00) 减少mPTP的开放程度：4.88和6.49 vs.1.37 (^#P=0.00和0.00)。对于H4 naïve细胞，加入25和50µmol/L的人参皂苷Rg1预处理24h能够抑制异氟醚诱导的caspase-3活化: 2.95和2.70 vs.4.57倍(^#P=0.04,0.02);增加线粒体ATP生成水平: 0.62和0.82vs. 0.25倍 (^#P=0.02和0.00); 减少mPTP的开放程度：4.51和5.98 vs.1.54 (^#P=0.00和0.00)。

结论：实验数据显示人参皂苷Rg1预处理可能通过抑制线粒体功能障碍而减弱异氟醚诱导的caspase-3活化。随着研究的深入，这些结果可能提示人参皂苷Rg1具有预防及治疗异氟醚诱导相关神经毒性的药理作用。

关键词：人参皂苷Rg1,异氟醚,神经毒性,线粒体功能障碍 

Ginsenoside Rg1 Attenuates Isoflurane-induced

caspase-3 Activation via Mitochondrial Pathway

Abstract

Backgroud As the aging society is coming, the quality of medical care and life have been focused on by the world.With the improvement of the clinic technique, the mortality and the incidence of serious complications decrease significantly. However, post-operative complications of CNS become more and more, especially the post-operative cognitive dysfunction (POCD) and Alzheimer’s disease (AD), which made the medical and social problems. The quality of life decline, including self-care ability lost and need of special clinic treatment. However, there is not good enough drug for treatment or pretreatment of POCD.The inhalation anesthetic isoflurane has been shown to induce mitochondrial dysfunction and caspase-3 activation, which may lead to learning and memory impairment. Ginsenoside Rg1 is reported to be neuroprotective. We therefore set out to determine whether ginsenoside Rg1 can attenuate isoflurane-induced caspase-3 activation via inhibiting mitochondrial dysfunction.

Methods We investigated the effects of ginsenoside Rg1 at concentrations of 12.5, 25, and 50µmol/L and pretreatment times of 12h and 24h on isoflurane-induced caspase-3 activation in H4 naïve and stably transfected H4 human neuroglioma cells that express full-length human amyloid precursor protein (APP) (H4-APP cells). For mitochondrial dysfunction, we assessed mitochondrial permeability transition pore (mPTP) and adenosine-5’-triphosphate (ATP) levels. We employed Western blot analysis, chemiluminescence, and flowcytometry.

Results Here we showed that isoflurene may induce caspase-3 activation and mitochondrial dysfunction compared with control group . Pretreatment with 50 µmol/L ginsenoside Rg1 for 12h in H4-APP cells attenuated isoflurane-induced caspase-3 activation: 4.02 vs. 7.23fold(^#P=0.03);increase the ATP level: 0.68 vs. 0.41倍 (^#P=0.02);reduce the opening of mPTP: 3.25 vs.1.39 (^#P=0.00). Pretreatment with 25 and 50µmol/L ginsenoside Rg1 for 24h in H4-APP cells attenuated isoflurane-induced caspase-3 activation: 4.30和2.99 vs. 8.16倍(^#P=0.04,0.01);increase the ATP level: 0.72和0.89 vs. 0.13 倍 (^#P=0.01和0.00) reduce the opening of mPTP: 4.88和6.49 vs.1.37 (^#P=0.00和0.00)。Pretreatment with 25 and 50µmol/L ginsenoside Rg1 for 24h in H4 naïve cells attenuated isoflurane-induced caspase-3 activation:2.95和2.70 vs.4.57倍(^#P=0.04,0.02); increase the ATP level: 0.62和0.82vs. 0.25倍 (^#P=0.02和0.00); reduce the opening of mPTP: 4.51和5.98 vs.1.54 (^#P=0.00和0.00)。

Conclusion These data suggest that ginsenoside Rg1 may ameliorate isoflurane-induced caspase-3 activation by inhibiting mitochondrial dysfunction. Pending further studies, these findings might recommend the use of ginsenoside Rg1 in preventing and treating isoflurane-induced neurotoxicity.

Key words:Ginsenoside Rg1, Isoflurane, Neurotoxicity, Mitochondrial dysfunction

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