CAJ | 학술논문

目的探讨白藜芦醇是否通过抑制糖原合成酶激酶3β(GSK-3β)的活性进而阻止线粒体通透性转移孔(mPTP)的开放来发挥心脏保护作用,及其可能的信号转导机制.方法常规培养大鼠胚胎心脏组织来源的H9c2细胞株,实验随机分为对照组、白藜芦醇组、白藜芦醇+PKG抑制剂(KT5823)组和KT5823组.激光扫描共聚焦显微镜成像法测定细胞线粒体膜电位(△Ψm)的变化及细胞内一氧化氮(NO)的含量.SABC法检测白藜芦醇对H9c2细胞GSK-3β Ser9位点磷酸化的影响.Western blot法检测磷酸化GSK-3β、血管扩张刺激磷蛋白(VASP)的表达水平.结果共聚焦显微镜测定△Ψm结果显示,白藜芦醇组[减少到(81.9±1.1)％]能够抑制△Ψm的减少,与对照组[减少到(38.6±1.9)％]比较差异有统计学意义(P＜0.05).白藜芦醇+ KT5823组△Ψm[减少到(41.8±1.2)％]明显低于白藜芦醇组(P＜0.05).Western blot结果显示白藜芦醇组的磷酸化GSK-3β及VASP蛋白表达水平[分别为(160.6±3.6)％和(157.9±1.3)％]明显高于对照组(P＜0.01).白藜芦醇+ KT5823组的磷酸化GSK-3β及VASP蛋白表达水平[分别为(91.6±5.6)％和(98.0±0.5)％]明显低于白藜芦醇组(P＜0.01).KT5823组与对照组比较差异无统计学意义.SABC结果进一步显示,白藜芦醇组GSK-3β Ser9位点的磷酸化水平明显高于对照组(P＜0.01).共聚焦显微镜测定细胞内NO的结果显示,白藜芦醇组DAF-FM的绿色荧光强度与对照组比较差异无统计学意义[分别为(71.2±5.3)％和(77.0±5.4)％].结论白藜芦醇通过cGMP/PKG信号通路抑制GSK-3β活性,进而阻止mPTP的开放来保护心脏.NO可能未参与白藜芦醇的线粒体保护作用.
목적 탐토백려호순시부통과억제당원합성매격매3β(GSK-3β)적활성진이조지선립체통투성전이공(mPTP)적개방래발휘심장보호작용,급기가능적신호전도궤제.방법 상규배양대서배태심장조직래원적H9c2세포주,실험수궤분위대조조、백려호순조、백려호순+PKG억제제(KT5823)조화KT5823조.격광소묘공취초현미경성상법측정세포선립체막전위(△Ψm)적변화급세포내일양화담(NO)적함량.SABC법검측백려호순대H9c2세포GSK-3β Ser9위점린산화적영향.Western blot법검측린산화GSK-3β、혈관확장자격린단백(VASP)적표체수평.결과 공취초현미경측정△Ψm결과현시,백려호순조[감소도(81.9±1.1)％]능구억제△Ψm적감소,여대조조[감소도(38.6±1.9)％]비교차이유통계학의의(P＜0.05).백려호순+ KT5823조△Ψm[감소도(41.8±1.2)％]명현저우백려호순조(P＜0.05).Western blot결과현시백려호순조적린산화GSK-3β급VASP단백표체수평[분별위(160.6±3.6)％화(157.9±1.3)％]명현고우대조조(P＜0.01).백려호순+ KT5823조적린산화GSK-3β급VASP단백표체수평[분별위(91.6±5.6)％화(98.0±0.5)％]명현저우백려호순조(P＜0.01).KT5823조여대조조비교차이무통계학의의.SABC결과진일보현시,백려호순조GSK-3β Ser9위점적린산화수평명현고우대조조(P＜0.01).공취초현미경측정세포내NO적결과현시,백려호순조DAF-FM적록색형광강도여대조조비교차이무통계학의의[분별위(71.2±5.3)％화(77.0±5.4)％].결론 백려호순통과cGMP/PKG신호통로억제GSK-3β활성,진이조지mPTP적개방래보호심장.NO가능미삼여백려호순적선립체보호작용.
Objective To investigate the underlying mechanism of the protective effects of resveratrol on oxidant-induced mitochondrial damage in embryonic rat cardiomyocytes.Methods H9c2 cells,a permanent cell line derived from embryonic rat cardiac tissue,and then randomly divided into control group [PBS,cells exposed to H2O2 (600 μmol/L) for 20 min to induce mitochondrial oxidant damage],resveratrol group (0.01,0.1,1,5,10 and 20 μmol/L for 20 min at 20 min before exposing to H2O2),resveratrol plus inhibitor group (1 μmol/L KT5823 for 10 min at 10 min before 5 pmol/L resveratrol treatment) and inhibitor group (1 μmol/L KT5823 for 10 min).Mitochondrial membrane potential (△Ψm) was measured by staining cells with tetramethylrhodamine ethyl ester (TMRE) and the mitochondrial permeability transition pore (mPTP) opening was evaluated by measuring the decrease of TMRE fluorescence intensity.Immunofluorescence assay was used to observe GSK-3β phosphorylation.The phosphorylation of GSK-3β and VASP were determined by Western blot.To detect intracellular NO,cells were loaded with DAF-FM DA (specific fluorescent dye of NO) and imaged with confocal microscopy.Results Compared to the control group,resveratrol (0.01-5 μmol/L) attenuated H2O2-inducedmitochondrial damage reflected by attenuating the H2O2-induced TMRE fluorescence intensity decrease in a dose-dependent manner and the efficacy of 10 and 20 μmol/L resveratrol was significantly lower than that of 5 μmol/L resveratrol.Resveratrol also significantly upregulated the protein expression of VASP and increased GSK-3β Ser9 phosphorylation,which could lead the inactivation of GSK-3β.These effects of resveratrol could be significantly abolished by protein kinase G inhibitor KT5823,while KT5823 alone did not affect CSK-3β and VASP phosphorylation.Confocal microscopy showed that DAF-FM (specific NO indicator) was similar between resveratrol and control group,suggesting that resveratrol did not produce NO.Conclusions Resveratrol could attenuate oxidant-induced mitochondrial damage in embryonic rat cardiomyocytes by inactivating GSK-3β via cGMP/PKG signaling pathway independent of NO-related mechanism.