Acta Pharmacologica Sinica (2010) 31:273–280; doi: 10.1038/aps.2010.9; published online 8 February 2010

Jun-rong ZHU¹, Yi-fu TAO^{1, *}, Shen LOU¹,  Zi-mei WU²

¹Department of Pharmacy, Nanjing First Hospital Affiliated to Nanjing Medical University, Nanjing 210006, China; ²School of Pharmacy, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand

Aim: To investigate the protective effects of ginsenoside Rb₃, a triterpenoid saponin isolated from the leaves of Panax notoginseng, on ischemic and reperfusion injury model of PC12 cells and elucidate the related mechanisms. 

Methods: PC12 cells exposed to oxygen and glucose deprivation (OGD) and restoration (OGD-Rep) were used as an in vitro model of ischemia and reperfusion.  3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) leakage were used to evaluate the protective effects of ginsenoside Rb₃.  Cellular apoptosis and mitochondrial membrane potential (MMP) were analyzed using flow cytometry.  Intracellular calcium ion concentration ([Ca²⁺]_i) was detected using fluorophotometer system.  Caspase-3, -8, and -9 activities were measured using assay kits with an ELISA reader.  Western blotting assay was used to evaluate the release of cytochrome c and expression of caspase-3, Bcl-2 and Bax proteins.

Results: It was shown that ginsenoside Rb₃ (0.1–10 μmol/L) significantly increased cell viability and inhibited LDH release in a dose-dependent manner on the ischemic model.  In addition, ginsenoside Rb₃ also significantly inhibited ischemic injury-induced apoptosis, [Ca²⁺]_i elevation, and decrease of MMP.  Meanwhile, pretreatment with ginsenoside Rb₃ significantly induced an increase of Bcl-2 protein expression and a decrease of cytosolic cytochrome c, cleaved-caspase 3 and Bax protein expression, the caspase-3, -8, and -9 activity were also inhibited. 

Conclusion: The results indicated that ginsenoside Rb₃ could markedly protected OGD-Rep induced ischemic injury and the mechanisms maybe related to its suppression of the intracellular Ca²⁺ elevation and inhibition of apoptosis and caspase activity.  Ginsenoside Rb₃ could be a promising candidate in the development of a novel class of anti-ischemic agent.

Keywords: ginsenoside Rb₃; PC12 cells; ischemic injury; apoptosis; oxygen and glucose deprivation