Protective effects of ginsenoside Rb(3) on oxygen and glucose deprivation-induced ischemic injury in PC12 cells.
OBJECTIVE
To investigate the protective effects of ginsenoside Rb(3), 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 Rb(3). Cellular apoptosis and mitochondrial membrane potential (MMP) were analyzed using flow cytometry. Intracellular calcium ion concentration ([Ca(2+)](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 Rb(3) (0.1-10 micromol/L) significantly increased cell viability and inhibited LDH release in a dose-dependent manner on the ischemic model. In addition, ginsenoside Rb(3) also significantly inhibited ischemic injury-induced apoptosis, [Ca(2+)](i) elevation, and decrease of MMP. Meanwhile, pretreatment with ginsenoside Rb(3) 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.


CONCLUSIONS
The results indicated that ginsenoside Rb(3) could markedly protected OGD-Rep induced ischemic injury and the mechanisms maybe related to its suppression of the intracellular Ca(2+) elevation and inhibition of apoptosis and caspase activity. Ginsenoside Rb(3) could be a promising candidate in the development of a novel class of anti-ischemic agent.
Zhu
Jun-rong
JR
Department of Pharmacy, Nanjing First Hospital Affiliated to Nanjing Medical University, China.
Tao
Yi-fu
YF
Lou
Shen
S
Wu
Zi-mei
ZM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D015687:Cell Hypoxia / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism; D019610:Cytoprotection; D036145:Ginsenosides / Q000494:pharmacology* / Q000627:therapeutic use; D005947:Glucose / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D010100:Oxygen / Q000378:metabolism*; D016716:PC12 Cells; D053723:Panax notoginseng / Q000737:chemistry; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D015427:Reperfusion Injury / Q000188:drug therapy*; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D036145:Ginsenosides; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C044463:ginsenoside Rb3; D045304:Cytochromes c; D007770:L-Lactate Dehydrogenase; D020169:Caspases; D005947:Glucose; D010100:Oxygen; D002118:Calcium