Ginsenoside Rb1 protects cardiomyocytes against CoCl2-induced apoptosis in neonatal rats by inhibiting mitochondria permeability transition pore opening.
OBJECTIVE
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).


CONCLUSIONS
Gs-Rb1 exerted anti-hypoxia effect on neonatal rat cardiomyocytes by inhibiting GSK-3beta-mediated mPTP opening.
Kong
Hong-liang
HL
Department of Cardiology, the People's Hospital of Liaoning Province, Shenyang, China. kh1339@sina.com
Li
Zhan-quan
ZQ
Zhao
Ying-jun
YJ
Zhao
Shu-mei
SM
Zhu
Li
L
Li
Tong
T
Fu
Yao
Y
Li
Hui-jun
HJ
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002316:Cardiotonic Agents / Q000494:pharmacology*; D053148:Caspase 3 / Q000032:analysis; D015687:Cell Hypoxia / Q000187:drug effects*; D002478:Cells, Cultured; D003035:Cobalt / Q000633:toxicity*; D045304:Cytochromes c / Q000032:analysis; D005434:Flow Cytometry; D036145:Ginsenosides / Q000494:pharmacology*; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D007150:Immunohistochemistry; D008929:Mitochondria, Heart / Q000187:drug effects* / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism*; D000083162:Mitochondrial Permeability Transition Pore; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011897:Random Allocation; D051381:Rats; D000077191:Wortmannin
D000730:Androstadienes; D002316:Cardiotonic Agents; D036145:Ginsenosides; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D047428:Protein Kinase Inhibitors; D003035:Cobalt; C442759:ginsenoside Rb1; D045304:Cytochromes c; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D038362:Glycogen Synthase Kinase 3; D053148:Caspase 3; C018021:cobaltous chloride; D000077191:Wortmannin