低温致乳鼠心肌细胞损伤中Bim蛋白的作用及PI3K/Akt相关机制

Role of pro-apoptotic protein Bim and PI3K/Akt signal-transduction pathway mechanism in hypothermia-induced cardiomyocytes damage in neonatal rats

背景:寒冷刺激可引发心血管疾病或导致其加重,研究极端气候条件下心血管疾病的发病规律及其分子生物学机制对制定有效的防范和治疗措施有重要意义。目的:观察低温对心肌细胞凋亡率、乳酸脱氢酶活性及促凋亡蛋白Bim表达的影响,并从PI3K/Akt信号通路探讨低温影响心肌细胞中Bim表达的可能途径。方法:将体外培养的心肌细胞在4℃冷水浴中处理1h后,放入37℃CO2培养箱中继续培养0,4,8,12,16,24h,AnnexinV-FITC/PI双染检测细胞凋亡率,MTS/PMS法检测细胞存活情况,全自动生化分析仪测细胞培养基中乳酸脱氢酶活性。Western blot测定Bim在心肌细胞中的表达情况,并在Bim蛋白开始表达的时间点向培养的心肌细胞中加入PI3K/Akt阻断剂LY29004,观察心肌细胞凋亡率、乳酸脱氢酶活性及Bim、p-PI3K蛋白表达的变化。结果与结论:冷处理后,心肌细胞凋亡率增加、存活率降低、培养基中乳酸脱氢酶活性增高、Bim表达增加(P<0.05),均在冷处理后24h达到极点。加入LY29004后,心肌细胞凋亡率增加更明显,培养基中乳酸脱氢酶活性及Bim表达也有所增加,p-PI3K表达降低(P<0.05)。说明低温能够通过促进Bim表达诱导心肌细胞凋亡,而PI3K/AKT信号通路可能参与了Bim的诱导表达。

BACKGROUND：Cold stimulation can result in cardiovascular disease or aggravate cardiovascular disease.It is significant to research the occurrence patterns and molecular mechanism for preventing and treating cardiovascular diseases.OBJECTIVE：To explore the effect of hypothermia on the cardiomyoctyes apoptosis and the level of lactate dehydrogenase（LDH） activity,as well as the role of pro-apoptosis protein Bim and PI3K/Akt signal-transduction pathway.METHODS：Cardiomyoctyes were exposed to 4 ℃ cold environment for 1 hour,then cultivating in 37 ℃ CO2 incubator for 0,4,8,12,16,and 24 hours.The degree of apoptosis was measured by flow cytometer by Annexin V-FITC/PI;LDH activity was determined with automatic biochemistry analyzer.Expressions of Bim were determined by Western blotting.All the changes were observed in the nutrient medium after adding PI3K inhibator LY294002.RESULTS AND CONCLUSION：After hypothermia treatment,the cell apoptosis was significantly increased,the survival rate decreased,and the LDH activity and Bim expression increased（P 0.05）,which reached the peak at 24 hours.After adding LY29004,cell apoptosis increased notably,the LDH activity and Bim expression increased,but p-PI3K expression were declined（P 0.05）.It suggested that hypothermia can induce cardiomyoctyes apoptosis.The PI3K/Aktsignal-transduction pathway is likely to participate in Bim expressions.