AstragalosideⅣPrevents the Mitochondrial Permeability Transition Pore Opening by Inhibiting Glycogen Synthase Kinase 3β via the NO/cGMP/PKG Pathway in Cardiac Cells
会议名称：《中国生理学会心血管生理学术研讨会》
会议日期：2011年
学科分类：1006[医学-中西医结合] 10[医学] 100602[医学-中西医结合临床]
基 金：the National Natural Science Foundation of China(No.30900494) the Medical Scientific Research key Project Plan of the Health Department of Hebei Province (No.20110168)
关 键 词：AstragalosideⅣ mPTP GSK-3β cGMP/PKG NO PI3K/Akt
摘 要：Aim:AstragalosideⅣ,a major ingredient of traditional Chinese herb Radix Astragali,has been widely used for treatments of cardiovascular diseases in China,but the exact cellular and molecular mechanisms underlying its action remain unclear.We aimed to test if astragalosideⅣmodulates mitochondrial permeability transition pore(mPTP) opening through glycogen synthase kinase-3β(GSK-3β) in H9c2 cardiac cells.Further more,the molecular mechanism was studied,by which Astragaloside IV inhibits GSK-3βactivity in this study.Methods:Rat heart tissue-derived H9c2 cells were purchased from ATCC.And cardiac H9c2 cells were exposed to HO(600μM) for 20 min to induce mitochondrial oxidant damage.Fluorescence dyes including tetramethylrhodamine ethyl ester(TMRE,red) and 4-amino-5-methylamino-2’, 7’-difluorofluorescein(DAF-FM,green) diacetate were used to image mitochondrial membrane potential(Δψm) and nitric oxide(NO),respectively.Fluorescence images were obtained with laser-scanning confocal microscopy.The red fluorescence was excited with a 543 nm line of He-Ne laser line and imaged through a 560 nm long-path filter.The green fluorescence was excited at 488 nm and imaged through a 525 nm long-path filter.The cell viability was assessed by propidium iodide fluorometry using a fluorescence reader.Cells in 12-well plates coated with laminin were incubated in standard Tyrode solution,and fluorescence intensity was measured at the excitation and emission wavelengths of 540 and 590 nm,respectively.H9c2 cells were exposed to astragalosideⅣfor 20 min,the GSK-3β(Ser),Akt(Ser),and VASP(Ser) activities were determined by measuring their phosphorylation statuses with Western blot. Results:Cardiac cells treated with different dose of astragalosideⅣ(30μM-80μM),and 50μM astragalosideⅣsignificantly increased GSK-3βphosphorylation at Ser(121.1±2.7%) and prevented oxidative stress-induced loss of mitochondrial membrane potential(MMP) in H9c2 cardiac cells(89.7±4.3%vs.49.7±6.3%in control).The GSK-3βinhibitor SB216763(3μM) and the specific mPTP closer cyclosporin A(0.2μM) mimicked the effect of astragalosideⅣby preventing the loss of MMP,indicating that astragalosideⅣmay prevent mPTP opening by inhibiting GSK-3β.These effects of astragalosideⅣwere reversed by the phosphatidylinositol 3-kinase(PI3K) inhibitor LY294002(10μM),the potent NO-sensitive guanylyl cyclase selective inhibitor ODQ(5μM),and the specific PKG inhibitor KT5823(1μM),implying that the PI3K/Akt/cGMP/PKG pathway is involved in the protective effect of astragalosideⅣ.In support,astragalosideⅣcould activate phosphorylation of Akt at Ser and VASP at Ser. AstragalosideⅣapplied at reperfusion reduced cell death in cells subjected to simulated ischemia/reperfusion,implying that astragalosideⅣcan prevent reperfusion injury.Further experiments showed that astragalosideⅣwas able to increase NO production(138.9±7.7%vs. 108.3±2.1%in control),an effect that was blocked by the nitric oxide synthase(NOS) inhibitor L-NAME(200μM)(97.8±7.4%) and LY294002(95.6±5.7%).Conclusions:Taken together, these data suggest that astragalosideⅣprevents the mPTP opening by inactivating GSK-3βthrough the NO/cGMP/PKG signaling pathway.NOS is responsible for NO generation and is activated by the PI3K/Akt pathway.