褪黑素预处理对异氟醚处理人神经母细胞瘤细胞TRF1蛋白的影响

Effect of melatonin pretreatment on TRF1 protein in human neuroblastoma cells treated with isoflurane

中文关键词: 褪黑素  异氟醚  端粒体复制结合因子1  磷脂酰肌醇3激酶/蛋白激酶B

英文关键词: Melatonin  Isoflurane  Telomeric repeat-binding factor 1  Phosphoinositide 3-kinase/protein kinase B (PI3K/Akt)

中文摘要:

      
目的 观察褪黑素预处理对异氟醚处理人神经母细胞瘤细胞（SHSY-5Y）TRF1蛋白含量的影响。
方法 选择SHSY-5Y细胞进行培养，将细胞随机分为四组：对照组（C组）、异氟醚组（I组）、异氟醚+褪黑素组（IM组）、异氟醚+褪黑素+抑制剂组（IMB组），每组细胞浓度为4.8×106～8.0×106个/ml。C组未进行药物处理，正常培养。I组予1.5%异氟醚处理6 h。IM组予1 mmol/L褪黑素1 μl处理细胞11 h后，1.5%异氟醚处理6 h。IMB组予100 mmol/L磷脂酰肌醇3激酶（PI3K）（p110α）选择性抑制剂BYL719 1 μl处理30 min后，1 mmol/L褪黑素1 μl处理11 h，1.5%异氟醚处理6 h。采用RT-qPCR法检测蛋白激酶B（Akt）和端粒体复制结合因子1（TRF1）mRNA表达量，Western blot法检测Akt、p-Akt（Ser473）和TRF1蛋白含量。
结果 与C组比较，I组和IM组Akt和TRF1 mRNA表达量和Akt蛋白含量明显降低（P<0.05），IM组p-Akt和TRF1蛋白含量明显升高（P<0.05）；IMB组Akt和TRF1 mRNA表达量和Akt蛋白含量明显升高（P<0.05）。与I组比较，IM组TRF1蛋白含量明显升高（P<0.05）；IMB组Akt mRNA表达量和Akt蛋白含量明显升高，TRF1 mRNA表达量明显降低（P<0.05）。与IM组比较，IMB组Akt和TRF1 mRNA表达量和Akt蛋白含量明显升高，p-Akt（Ser473）和TRF1蛋白含量明显降低（P<0.05）。
结论 褪黑素明显上调异氟醚处理后SHSY-5Y细胞TRF1蛋白表达，其机制与PI3K/Akt通路有关。

英文摘要:

      
Objective To observe the effect of melatonin pretreatment on TRF1 protein content of isoflurane treated human neuroblastoma cells (SHSY-5Y).
Methods SHSY-5Y cells were cultured and randomly divided into four groups: control group (group C), isoflurane group (group I), isoflurane+melatonin group (group IM), and isoflurane+melatonin+inhibitor group (group IMB). The cell concentration of each group was (4.8-8.0) × 106 cells/ml. Group C was cultured normally without drug treatment. Group I was treated with 1.5% isoflurane for 6 hours. Group IM was treated with 1 mmol/L melatonin 1 μl for 11 hours and then treated with 1.5% isoflurane for 6 hours. Group IMB was treated with 100 mmol/L phosphoinositide 3-kinase (PI3K) (p110α) selective inhibitor BYL719 1 μl for 30 minutes, then treated with 1 mmol/L melatonin 1 μl for 11 hours, and 1.5% isoflurane for 6 hours. The expression of protein kinase B (Akt) and telomeric repeat binding factor 1 (TRF1) mRNA were detected by RT-qPCR, and the protein levels of Akt, p-Akt (Ser473), and TRF1 were detected by Western blot.
Results Compared with group C, the expressions of Akt and TRF1 mRNA and the level of Akt protein were significantly decreased in groups I and IM (P < 0.05); the levels of p-Akt (Ser473) and TRF1 protein were significantly increased in group IM (P < 0.05); the expression of Akt mRNA and Akt protein were significantly increased and the expression of TRF1 mRNA was significantly decreased in group IMB (P < 0.05). Compared with group I, the expression of TRF1 protein was significantly increased in group IM (P < 0.05); the expression of Akt and TRF1 mRNA and the levels of Akt protein were significantly increased in group IMB (P < 0.05). Compared with group IM, the expression of Akt and TRF1 mRNA and the levels of Akt protein were increased, whereas the levels of p-Akt (Ser473) and TRF1 protein were significantly decreased in group IMB (P < 0.05).
Conclusion Melatonin pretreatment significantly up-regulates the expression of TRF1 protein in SHSY-5Y cells after isoflurane treatment, whose mechanism may be related to PI3K/Akt signaling pathway.