CAJ | 학술논문

目的探讨JAK/STAT3介导的信号途径在苦参碱对K562细胞增殖抑制作用中的分子机制.方法以不同浓度苦参碱处理K562细胞,Western blot法分析JAK/STAT3通路分子JAK2、STAT3及其磷酸化蛋白表达的改变;荧光实时定量RT-PCR及Western blot法分析STAT3下游调控基因Bcl-xL、Cyclin D1、c-Myc的表达;荧光实时定量RT-PCR及ELISA法检测K562细胞IL-6表达的改变.以IL-6预处理K562细胞,Western blot法分析苦参碱对细胞内JAK2、STAT3及其磷酸化蛋白表达的作用.结果 0.5 mg/ml苦参碱处理48 h,K562细胞内STAT3及JAK2总蛋白表达无明显变化,磷酸化STAT3(p-Tyr705 STAT3、p-Ser727 STAT3)和磷酸化JAK2 (p-JAK2)表达水平分别由处理前的0.690±0.119、1.150±0.263和0.670±0.137下降至0.370±0.024、0.700±0.172和0.049±0.057.苦参碱对STAT3下游调控基因Bcl-xL、Cyclin D1及c-Myc的转录及蛋白表达均有抑制作用,并可显著抑制IL-6的表达和分泌,0.5和0.8 mg/ml苦参碱处理后细胞培养上清中IL-6浓度分别为(10.74±1.83)pg/ml和(8.66±1.24) pg/ml,较处理前[(35.1±1.93) pg/ml]显著降低(P＜0.05).IL-6预处理K562细胞后,STAT3、JAK2、p-STAT3及p-JAK2表达均有增加,苦参碱可抑制IL-6对上述分子表达的上调.结论苦参碱抑制K562细胞增殖与JAK/STAT3介导的信号通路抑制有关,IL-6表达抑制可能是苦参碱调控JAK/STAT3通路活性的始动机制.
목적 탐토JAK/STAT3개도적신호도경재고삼감대K562세포증식억제작용중적분자궤제.방법 이불동농도고삼감처리K562세포,Western blot법분석JAK/STAT3통로분자JAK2、STAT3급기린산화단백표체적개변;형광실시정량RT-PCR급Western blot법분석STAT3하유조공기인Bcl-xL、Cyclin D1、c-Myc적표체;형광실시정량RT-PCR급ELISA법검측K562세포IL-6표체적개변.이IL-6예처리K562세포,Western blot법분석고삼감대세포내JAK2、STAT3급기린산화단백표체적작용.결과 0.5 mg/ml고삼감처리48 h,K562세포내STAT3급JAK2총단백표체무명현변화,린산화STAT3(p-Tyr705 STAT3、p-Ser727 STAT3)화린산화JAK2 (p-JAK2)표체수평분별유처리전적0.690±0.119、1.150±0.263화0.670±0.137하강지0.370±0.024、0.700±0.172화0.049±0.057.고삼감대STAT3하유조공기인Bcl-xL、Cyclin D1급c-Myc적전록급단백표체균유억제작용,병가현저억제IL-6적표체화분비,0.5화0.8 mg/ml고삼감처리후세포배양상청중IL-6농도분별위(10.74±1.83)pg/ml화(8.66±1.24) pg/ml,교처리전[(35.1±1.93) pg/ml]현저강저(P＜0.05).IL-6예처리K562세포후,STAT3、JAK2、p-STAT3급p-JAK2표체균유증가,고삼감가억제IL-6대상술분자표체적상조.결론 고삼감억제K562세포증식여JAK/STAT3개도적신호통로억제유관,IL-6표체억제가능시고삼감조공JAK/STAT3통로활성적시동궤제.
Objective To investigate the molecular mechanism of the growth inhibitory effect of matrine on K562 cells in JAK/STAT3 mediated signal pathway.Methods Western blot analyses were performed to investigate the differential expression of JAK2,STAT3,phospho-STAT3 (Tyr705 & Ser727) and phospho-JAK2 proteins after matrine treatment in K562 cells with or without human recombinant interleukin 6 (IL-6) pretreatment.The expressions of STAT3 response gene products such as Bcl-xL,Cyclin D1 and c-Myc,were investigated by Western blot and quantitative real time RT-PCR (qRT-PCR).Expression of IL-6,a potent upstream activating factor of JAK/STAT3 pathway,was analyzed by both real time qRT-PCR and ELISA.Results Western blot revealed that matrine treatment resulted in a strong down-regulation of phospho-STAT3 both in Tyr705 and Ser727 sites or phospho-JAK2 proteins expression without significant effects on the total STAT3 and JAK2 proteins.The expression of phospho-Tyr705 STAT3 and phospho-Ser727 STAT3 was decreased to 0.370±0.024 and 0.700±0.172 in K562 cells treated with 0.5 mg/ml matrine for 48 h,respectively,from 0.690±0.119 and 1.150±0.263 in control cells,accompanied with a dramatical down-regulation of phospho-JAK2 from 0.670±0.137 to 0.049±0.057 (P＜ 0.05).In addition,it was found that the expression of Bcl-xL,Cyclin D1,c-Myc was decreased both at the transcriptional and protein level in K562 cells after matrine treatment.Matrine treatment resulted in a significant decrease in the expression level of IL-6 in K562 cells from (35.1± 1.93) to (10.74±1.83) and (8.66± 1.24) pg/ml at the dose of 0.5 and 0.8 mg/ml,respectively (P＜0.05).Matrine treatment could diminish the up-regulation of STAT3,JAK2,phospho-STAT3 and phospho-JAK2 protein following pretreatment with IL-6 in K562 cells.Conclusion Matrine exerts its anti-leukemia effect by interfering with the JAK2/STAT3 signaling pathway.The inhibition of IL-6 expression may play a pivotal role in the disruption of JAK/STAT pathway by matrine.