摘要
目的:研究PI3K抑制剂联合地塞米松对H_(2)O_(2)-TNFα-U937细胞模型激素敏感性的影响及其作用机制。方法:采用过氧化氢(H_(2)O_(2))刺激巨噬细胞系U937,建立H_(2)O_(2)-TNFα-U937细胞氧化应激-激素耐受模型(简称H_(2)O_(2)-TNFα-U937细胞模型),将磷酸肌醇3激酶(PI3K)抑制剂(BEZ235或LY294002)单独或联合地塞米松(Dex)干预该模型。使用PCR与ELISA方法比较其抑制IL-8基因与蛋白质的表达水平,组蛋白去乙酰化酶(HDAC)-2试剂盒检测HDAC2活力改变,并通过Western blot检测AP-1、NF-κB磷酸化水平。结果:在H_(2)O_(2)-TNFα-U937细胞模型中,Dex抑制该细胞模型释放IL-8的能力降低,并且单用PI3K抑制剂(BEZ235或LY294002)不能明显抑制IL-8释放(P<0.05),而经PI3K抑制剂(BEZ235或LY294002)联合Dex干预后细胞对激素的抵抗得到明显改善(P<0.05)。BEZ235而不是LY294002部分逆转了细胞核蛋白HDAC2的活力,但两种抑制剂均能相应降低炎症转录因子NF-κB、AP-1的磷酸化水平。结论:PI3K抑制剂联合Dex能改善氧化应激诱导的U937细胞对激素不敏感性,其机制可能与PI3K抑制剂能部分恢复因氧化应激作用导致的HADC2活力和降低NF-κB、AP-1蛋白磷酸化水平有关。
Objective:To investigate the effect and mechanism of PI3K inhibitor combined with dexamethasone on H_(2)O_(2)-TNFα-U937 cell model to improve its corticosteroid insensitivity.Methods:The macrophage cell U937 stimulated by hydrogen peroxide(H_(2)O_(2))was used to establish the H_(2)O_(2)-TNFα-U937 oxidative-stress steroid insensitive cell models,this model was treated with the PI3K inhibitor(BEZ235/LY294002)alone or combined with dexamethasone(Dex).After PI3K inhibitor-treated alone or combined with Dex,polymerase chain reaction(PCR)and enzyme-linked immunosorbent assay(ELISA)were used to compare the IL-8 gene and protein expression,HDAC2 kit was used to detect the HDAC2 activity,Western blot was used to detect the phosphorylation level of AP-1 and NF-κB protein.Results:In the H_(2)O_(2)-TNFα-U937 cell model,the ability of Dex in inhibiting the releasing of IL-8 was reduced,PI3K inhibitor(BEZ235/LY294002)treated alone cannot inhibit the releasing of IL-8 and even lower than Dex(P<0.05).Whereas the PI3K inhibitor combined with Dex obviously improved the corticosteroid insensitivity,in contrast to PI3K inhibitor-treated alone(P<0.05),only BEZ235 could partially reverse the HDAC2 activity,but both inhibitors could correspondingly reduce the phosphorylation levels of inflammatory transcription factors NF-κB and AP-1.Conclusion:PI3K inhibitor combined with Dex may restore glucocorticoid insensitivity of U937 cells caused by oxidative stress.The mechanism may be related to partial restoration of the HADC2 activity and reduction of phosphorylation levels of NF-κB and AP-1 protein after PI3K inhibitor treatment in oxidative stress culture medium.
作者
曾瑜真
杜开锋
金建军
闵智慧
毛若琳
陈智鸿
ZENG Yu-Zhen;DU Kai-Feng;JIN Jian-Jun;MIN Zhi-Hui;MAO Ruo-Lin;CHEN Zhi-Hong(Department of Pul-monary and Critical Care Medicine,Zhongshan Hospital,Fudan University,Shanghai 200032,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2021年第12期1409-1413,共5页
Chinese Journal of Immunology
基金
国家自然科学基金(No.81470211,No.81970023)
上海市卫健委课题(201840288)。
关键词
激素不敏感
氧化应激
HDAC2
PI3K抑制剂
Corticosteroid insensitivity
Oxidative stress
Histone deacetylase-2
PI3K inhibitor
