人参皂苷 Rb1 通过抑制线粒体通透性转换孔开放来保护新生大鼠心肌细胞免受 CoCl2 诱导的细胞凋亡。
Ginsenoside Rb1 protects cardiomyocytes against CoCl2-induced apoptosis in neonatal rats by inhibiting mitochondria permeability transition pore opening.
机构信息
Department of Cardiology, the People's Hospital of Liaoning Province, Shenyang, China.
出版信息
Acta Pharmacol Sin. 2010 Jun;31(6):687-95. doi: 10.1038/aps.2010.52.
AIM
To investigate whether mitochondria permeability transition pore (mPTP) opening was involved in ginsenoside Rb1 (Gs-Rb1) induced anti-hypoxia effects in neonatal rat cardiomyocytes ex vivo.
METHODS
Cardiomyocytes were randomly divided into 7 groups: control group, hypoxia group (500 micromol/L CoCl(2)), Gs-Rb1 200 micromol/L group (CoCl(2) intervention+Gs-Rb1), wortmannin (PI3K inhibitor) 0.5 micromol/L group, wortmannin+Gs-Rb1 group, adenine 9-beta-D-arabinofuranoside (Ara A, AMPK inhibitor) 500 micromol/L group, and Ara A and Gs-Rb1 group. Apoptosis rate was determined by using flow cytometry. The opening of the transient mPTP was assessed by using co-loading with calcein AM and CoCl(2) in high conductance mode. Expression of GSK-3beta, cytochrome c, caspase-3 and poly (ADP-ribose) polymerase (PARP) was measured by using Western blotting. DeltaGSK-3beta was defined as the ratio of p-Ser9-GSK-3beta to total GSK-3beta.
RESULTS
CoCl(2) significantly stimulated mPTP opening and up-regulated the level of DeltaGSK-3beta. There was a statistically significant positive correlation between apoptosis rate and mPTP opening, between apoptosis rate and DeltaGSK-3beta, and between mPTP opening and DeltaGSK-3beta. Gs-Rb1 significantly inhibited mPTP opening induced by hypoxia (41.3%+/-2.0%, P<0.001) . Gs-Rb1 caused a 77.3%+/-3.2% reduction in the expression of GSK-3beta protein (P<0.001) and a significant increase of 1.182+/-0.007-fold (P=0.0001) in p-Ser9-GSK-3beta compared with control group. Wortmannin and Ara A significantly inhibited the effect of Gs-Rb1 on mPTP opening and DeltaGSK-3beta. Gs-Rb1 significantly decreased expression of cytochrome c (66.1%+/-1.7%, P=0.001), caspase-3 (56.5%+/-2.7%, P=0.001) and cleaved poly ADP-ribose polymerase (PARP) (57.9%+/-1.4%, P=0.001).
CONCLUSION
Gs-Rb1 exerted anti-hypoxia effect on neonatal rat cardiomyocytes by inhibiting GSK-3beta-mediated mPTP opening.
目的
探讨线粒体通透性转换孔（mPTP）开放是否参与人参皂甙 Rb1（Gs-Rb1）诱导的新生大鼠心肌细胞体外抗缺氧作用。
方法
心肌细胞随机分为 7 组：对照组、缺氧组（500 μmol/L CoCl2）、Gs-Rb1 200 μmol/L 组（CoCl2 干预+Gs-Rb1）、wortmannin（PI3K 抑制剂）0.5 μmol/L 组、wortmannin+Gs-Rb1 组、腺嘌呤 9-β-D-阿拉伯呋喃糖苷（Ara A，AMPK 抑制剂）500 μmol/L 组和 Ara A 和 Gs-Rb1 组。采用流式细胞术测定细胞凋亡率。采用共加载 calcein AM 和高电导模式 CoCl2 评估瞬时 mPTP 的开放。采用 Western blot 测定 GSK-3β、细胞色素 c、caspase-3 和多聚（ADP-核糖）聚合酶（PARP）的表达。定义 p-Ser9-GSK-3β与总 GSK-3β的比值为 DeltaGSK-3β。
结果
CoCl2 显著刺激 mPTP 开放并上调 DeltaGSK-3β 水平。细胞凋亡率与 mPTP 开放、细胞凋亡率与 DeltaGSK-3β、mPTP 开放与 DeltaGSK-3β之间存在统计学显著正相关。Gs-Rb1 显著抑制缺氧诱导的 mPTP 开放（41.3%+/-2.0%，P<0.001）。Gs-Rb1 导致 GSK-3β 蛋白表达降低 77.3%+/-3.2%（P<0.001），p-Ser9-GSK-3β 显著增加 1.182+/-0.007 倍（P=0.0001）。wortmannin 和 Ara A 显著抑制 Gs-Rb1 对 mPTP 开放和 DeltaGSK-3β 的作用。Gs-Rb1 显著降低细胞色素 c（66.1%+/-1.7%，P=0.001）、caspase-3（56.5%+/-2.7%，P=0.001）和裂解多聚 ADP-核糖聚合酶（PARP）（57.9%+/-1.4%，P=0.001）的表达。
结论
Gs-Rb1 通过抑制 GSK-3β 介导的 mPTP 开放对新生大鼠心肌细胞发挥抗缺氧作用。
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