摘要
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-KB抑制因子仅(p-IKBα)的表达;定量逆转录一聚合酶链反应(RT—PCR)检测肿瘤坏死因子-α(TNF-α)基因表达;Hoechst33258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一3(2.22-I-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-IKBα(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(P13K/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P13K/Akt和p38MAPK通路的调控发挥其对心肌细胞的抗炎、抗凋亡作用。
Objective To investigate the protective effects of insulin on burn serum-challenged cardiocyte apoptosis and its mechanism. Methods Burn-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-KB inhibitive factor α (p-IKBα) 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-IKBα (1.43 + 0.62 vs. 3.62 + 0.74, P〈0.01), TNF-α (0.+ 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 burn serum, insulin may decrease inflammatory cytokine expression and apoptosis via regulating PI3K/Akt and p38MAPK pathway.
出处
《中国危重病急救医学》
CAS
CSCD
北大核心
2012年第4期197-200,共4页
Chinese Critical Care Medicine
基金
国家自然科学基金(30772250)
河南省洛阳市科技发展计划项目(1101051A)