CAJ | 학술논문

目的:研究过氧化物酶体增殖物激活受体-α(PPAR-α)在病理性心肌肥大中的作用及其信号机制.方法: 应用Invitrogen's Stealth RNAi抑制心肌细胞PPAR-α的表达;采用[~3H]-亮氨酸掺入法和RT-PCR检查心肌细胞蛋白质合成和心房利钠因子(ANF) mRNA的表达;采用Western blotting法检测Akt/GSK3β的磷酸化表达;应用免疫荧光技术检测NFATc4的胞核移位.结果: (1)RSS304168 是最有效的PPAR-α RNAi,特异性地抑制了PPAR-α的表达.(2)非诺贝特预处理抑制了内皮素-1(ET-1)诱导的心肌细胞肥大(蛋白质合成和ANF mRNA的表达);RSS304168加强了ET-1的诱导效应,而且逆转了非诺贝特对心肌肥大的抑制效应.(3)非诺贝特降低了ET-1诱导的Akt/GSK3β的磷酸化表达,RSS304168增强了ET-1的诱导效应,ET-1和RSS304168的上述作用可被PI3K阻断剂LY294002所阻断;RSS304168逆转了非诺贝特对Akt/GSK3β的磷酸化表达的负性调控作用.(4)非诺贝特抑制了ET-1诱导的NFATc4的胞核移位;而RSS304168加强了ET-1的诱导作用,逆转了非诺贝特对NFATc4胞核移位的抑制作用.结论: PPAR-α激活可以通过PI3K/Akt/GSK3β-NFATc4通路抑制ET-1诱导的心肌肥大反应.
목적:연구과양화물매체증식물격활수체-α(PPAR-α)재병이성심기비대중적작용급기신호궤제.방법: 응용Invitrogen's Stealth RNAi억제심기세포PPAR-α적표체;채용[~3H]-량안산참입법화RT-PCR검사심기세포단백질합성화심방리납인자(ANF) mRNA적표체;채용Western blotting법검측Akt/GSK3β적린산화표체;응용면역형광기술검측NFATc4적포핵이위.결과: (1)RSS304168 시최유효적PPAR-α RNAi,특이성지억제료PPAR-α적표체.(2)비낙패특예처리억제료내피소-1(ET-1)유도적심기세포비대(단백질합성화ANF mRNA적표체);RSS304168가강료ET-1적유도효응,이차역전료비낙패특대심기비대적억제효응.(3)비낙패특강저료ET-1유도적Akt/GSK3β적린산화표체,RSS304168증강료ET-1적유도효응,ET-1화RSS304168적상술작용가피PI3K조단제LY294002소조단;RSS304168역전료비낙패특대Akt/GSK3β적린산화표체적부성조공작용.(4)비낙패특억제료ET-1유도적NFATc4적포핵이위;이RSS304168가강료ET-1적유도작용,역전료비낙패특대NFATc4포핵이위적억제작용.결론: PPAR-α격활가이통과PI3K/Akt/GSK3β-NFATc4통로억제ET-1유도적심기비대반응.
AIM: To investigate the role and signal mechanism of PPAR-α in the pathogenesis of cardiac hypertrophy. METHODS: Small interfering RNA (siRNA) was applied to efficiently silence the gene expression of PPAR-α in cardiac myocytes. [~3H] leucine incorporation assay was performed to measure protein synthesis. Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze the mRNA level of atrial natriuretic factor (ANF) and PPAR-α. Western blotting analysis was performed to investigate the levels of phosphorylation of protein kinase B (PKB/Akt) and glycogen synthase kinase 3β (GSK3β). Immunofluorescence analysis was used to examine the cellular localization of NFATc4. RESULTS: (1)RSS304168 was the most efficient stealth RNAi duplex to specifically inhibit PPAR-α expression. (2)RSS304168 significantly potentiated the ET-1-induced cardiomyocyte hypertrophy and enhanced ET-1-induced protein synthesis and ANF mRNA expression in cardiomyocytes. Moreover, RSS304168 completely reversed the inhibitory effects of fenofibrate on ET-1-induced protein synthesis and ANF mRNA expression. (3)RSS304168 enhanced ET-1-induced phosphorylation of Akt at Ser473 and GSK3β at Ser9. The effects of ET-1 or ET-1 combined with RSS304168 on phosphorylation of Akt/GSK3β were completely blocked by LY294002, a PI3K specific inhibitor. Fenofibrate markedly inhibited ET-1-induced phosphorylation of Akt/GSK3β while RSS304168 abolished these effects of fenofibrate. (4)Fenofibrate prevented the nuclear translocation of NFATc4 induced by ET-1 while RSS304168 abolished this effect of fenofibrate. CONCLUSION: Activation of PPAR-α inhibits ET-1-induced cardiomyocyte hypertrophy through blocking Akt/GSK3β-NFATc4 signaling pathways.