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

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4.8×10^(6)~8.0×10^(6)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1μl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BYL7191μl处理30 min后,1 mmol/L褪黑素1μl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。

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)×10^(6)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 1μl 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 BYL7191μl for 30 minutes,then treated with 1 mmol/L melatonin 1μl 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.