Astragaloside IV inhibits oxidative stress-induced mitochondrial permeability transition pore opening by inactivating GSK-3β via nitric oxide in H9c2 cardiac cells.
OBJECTIVE
This study aimed to investigate whether astragaloside IV modulates the mitochondrial permeability transition pore (mPTP) opening through glycogen synthase kinase 3β (GSK-3β) in H9c2 cells.


METHODS
H9c2 cells were exposed to astragaloside IV for 20 min. GSK-3β (Ser(9)), Akt (Ser(473)), and VASP (Ser(239)) activities were determined with western blot. The mPTP opening was evaluated by measuring mitochondrial membrane potential (ΔΨ(m)). Nitric oxide (NO) generation was measured by 4-amino-5-methylamino-2', 7'-difluorofluorescein (DAF-FM) diacetate. Fluorescence images were obtained with confocal microscopy.


RESULTS
Astragaloside IV significantly enhanced GSK-3β phosphorylation and prevented H(2)O(2)-induced loss of ΔΨ(m). These effects of astragaloside IV were reversed by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, the NO sensitive guanylyl cyclase selective inhibitor ODQ, and the PKG inhibitor KT5823. Astragaloside IV activated Akt and PKG. Astragaloside IV was also shown to increase NO production, an effect that was reversed by L-NAME and LY294002. Astragaloside IV applied at reperfusion reduced cell death caused by simulated ischemia/reperfusion, indicating that astragaloside IV can prevent reperfusion injury.


CONCLUSIONS
These data suggest that astragaloside IV prevents the mPTP opening and reperfusion injury by inactivating GSK-3β through the NO/cGMP/PKG signaling pathway. NOS is responsible for NO generation and is activated by the PI3K/Akt pathway.
Department of Internal Medicine, Hebei United University, Tangshan 063000, China.
Yonggui
Y
He
Jinkun
J
Xi
Huan
H
Zheng
Yidong
Y
Zhang
Yuanzhe
Y
Jin
Zhelong
Z
Xu
D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002867:Chromones / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006861:Hydrogen Peroxide / Q000494:pharmacology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D033681:Mitochondrial Membrane Transport Proteins / Q000187:drug effects / Q000378:metabolism*; D000083162:Mitochondrial Permeability Transition Pore; D009025:Morpholines / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D012503:Saponins / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D014315:Triterpenes / Q000494:pharmacology*
D002867:Chromones; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D009025:Morpholines; D012503:Saponins; D014315:Triterpenes; D009569:Nitric Oxide; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C052064:astragaloside A; D006861:Hydrogen Peroxide; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D051057:Proto-Oncogene Proteins c-akt; D017869:Cyclic GMP-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3