CAJ | 학술논문

目的探讨胰岛素对烧伤血清致心肌细胞凋亡的保护作用及机制.方法通过烧伤血清作用的心肌细胞模型,用胰岛素及其信号通路抑制剂SB203580、LY294002进行干预.蛋白质免疫印迹法检测心肌细胞内活化型天冬氨酸特异性半胱氨酸蛋白酶3(cleaved-caspase-3)、Bax和磷酸化核转录因子-κB抑制因子α(p-IκBα)的表达;定量逆转录-聚合酶链反应(RT-PCR)检测肿瘤坏死因子-α(TNF-α)基因表达;Hoechst 33258核染色法检测心肌细胞凋亡.同时通过阻断实验,初步研究胰岛素保护心肌细胞作用的信号通路.结果胰岛素能够明显降低烧伤血清作用的心肌细胞cleaved-caspase-3(2.22±0.30比4.84±0.74,P＜0.01)、Bax(1.33±0.35比3.74±0.65,P＜0.01)和p-IκBα( 1.43±0.62比3.62±0.74,P＜0.01)的表达;显著抑制TNF-α表达(0.72±0.27比2.02±0.63,P＜0.01);降低心肌细胞的凋亡率[(9.4±3.4)％比(19.1±5.6)％,P＜0.01].阻断实验结果显示,胰岛素活化磷酯酰肌醇-3-激酶/蛋白丝氨酸/苏氨酸激酶(PI3K/Akt)通路的抑制剂LY294002能够抵消胰岛素的保护作用;而p38丝裂素活化蛋白激酶(p38MAPK)通路活化抑制剂SB203580则能够抑制烧伤血清引起的心肌细胞损害,与胰岛素作用相当.结论在烧伤血清作用的心肌细胞模型,胰岛素可能通过对PI3K/Akt和p38MAPK通路的调控发挥其对心肌细胞的抗炎、抗凋亡作用.
목적 탐토이도소대소상혈청치심기세포조망적보호작용급궤제.방법 통과소상혈청작용적심기세포모형,용이도소급기신호통로억제제SB203580、LY294002진행간예.단백질면역인적법검측심기세포내활화형천동안산특이성반광안산단백매3(cleaved-caspase-3)、Bax화린산화핵전록인자-κB억제인자α(p-IκBα)적표체;정량역전록-취합매련반응(RT-PCR)검측종류배사인자-α(TNF-α)기인표체;Hoechst 33258핵염색법검측심기세포조망.동시통과조단실험,초보연구이도소보호심기세포작용적신호통로.결과 이도소능구명현강저소상혈청작용적심기세포cleaved-caspase-3(2.22±0.30비4.84±0.74,P＜0.01)、Bax(1.33±0.35비3.74±0.65,P＜0.01)화p-IκBα( 1.43±0.62비3.62±0.74,P＜0.01)적표체;현저억제TNF-α표체(0.72±0.27비2.02±0.63,P＜0.01);강저심기세포적조망솔[(9.4±3.4)％비(19.1±5.6)％,P＜0.01].조단실험결과현시,이도소활화린지선기순-3-격매/단백사안산/소안산격매(PI3K/Akt)통로적억제제LY294002능구저소이도소적보호작용;이p38사렬소활화단백격매(p38MAPK)통로활화억제제SB203580칙능구억제소상혈청인기적심기세포손해,여이도소작용상당.결론 재소상혈청작용적심기세포모형,이도소가능통과대PI3K/Akt화p38MAPK통로적조공발휘기대심기세포적항염、항조망작용.
Objective To investigate the protective effects of insulin on burn serum-challenged cardiocyte apoptosis and its mechanism.Methods Bum-serum challenged cardiocytes were pretreated with insulin and inhibitors to pathway SB203580 and LY294002.The expression of cardiac myofilament proteins cleaved-caspase-3,Bax and phosphorylation nuclear factor-κB inhibitive factor α (p-IκBα) were examined by Western blotting.The mRNA expression of tumor necrosis factor-α ( TNF-α ) was determined by real-time reverse transcription-polymerase chain reaction (RT-PCR).Apoptosis of cardiocyte was observed after Hoechst 33258 staining.Further blocking experiments were used to investigate the cytoprotective pathway of insulin.Results Insulin could significantly decrease the expression of cleaved-caspase-3 (2.22 ±0.30 vs.4.84 ±0.74,P＜0.01),Bax (1.33 ±0.35 vs.3.74 ±0.65,P＜0.01),p-IκBα (1.43 ±0.62 vs.3.62 ±0.74,P＜0.01),TNF-α (0.72 ±0.27 vs.2.02 ±0.63,P＜0.01) and the cardiocyte apoptosis rate [(9.4 ± 3.4)％ vs.( 19.1 ± 5.6)％,P＜0.01 ] in cardiocytes challenged by burn serum.Further blocking experiments showed that LY294002,phosphatidylinositol-3-kinase (PI3K)/Akt activation inhibitor,could mitigate the protective effects of insulin.Meanwhile,SB203580,an inhibitor of p38 mitogen-activated protein kinase (p38MAPK) pathway,was able to inhibit cardiocyte injury challenged by burn serum,and it was as effective as insulin.Conclusion For cardiocytes challenged by bum serum,insulin may decrease inflammatory cytokine expression and apoptosis via regulating PI3K/Akt and p38MAPK pathway.