摘要
目的:探讨肿瘤坏死因子α诱导蛋白8样分子2(TIPE2)对脂多糖(LPS)活化的巨噬细胞凋亡的影响及作用机制。方法:采用LPS(1.0 mg/L)处理RAW264.7细胞24 h,RT-qPCR检测IL-6、TNF-α、TIPE2 mRNA表达,Western blot检测TIPE2蛋白表达。采用LPS处理稳定过表达或敲降TIPE2细胞系24 h,CCK-8检测细胞活力,Annexin V-FITC/PI检测细胞凋亡率,Tunel染色检测细胞凋亡指数,Western blot检测凋亡相关蛋白cleaved Caspase-8、Bcl-2、Bax、CHOP、cleaved Caspase-3及Akt、p-Akt^(Ser473)蛋白表达。采用类胰岛素生长因子(IGF-1,100μg/L)预孵育过表达TIPE2细胞系2 h,LPS共处理24 h,CCK-8检测细胞活力,Western blot检测凋亡相关蛋白表达。结果:LPS可活化巨噬细胞,上调IL-6、TNF-αmRNA表达,下调TIPE2 mRNA和蛋白表达(P<0.05)。过表达TIPE2明显降低活化巨噬细胞活力,升高凋亡率和凋亡指数,促进cleaved Caspase-8、Bax、CHOP、cleaved Caspase-3蛋白表达,抑制Bcl-2、p-Akt^(Ser473)蛋白表达(P<0.05);敲降TIPE2作用与之相反。此外,Akt磷酸化激动剂IGF-1可有效减弱TIPE2对活化巨噬细胞活力及凋亡相关蛋白的作用(P<0.05)。结论:TIPE2可能通过抑制Akt磷酸化,进而激活死亡受体途径、线粒体途径和内质网应激途径,促进LPS活化的巨噬细胞凋亡。
Objective:To explore effects of tumor necrosis factor-α-induced protein 8-like 2(TIPE2)on lipopolysaccharides(LPS)-activated macrophages apoptosis and its molecular mechanism.Methods:RAW264.7 cells were incubated with LPS(1.0 mg/L)for 24 h. Expressions of IL-6,TNF-α and TIPE2 mRNA were detected by RT-qPCR and protein level of TIPE2 was detected by Western blot. Cells overexpression or knockdown of TIPE2 were treated with LPS for 24 h. Cell viability was determined by CCK-8,apoptosis rate and index were determined by Annexin V-FITC/PI and Tunel staining,protein of cleaved Caspase-8,Bcl-2,Bax,CHOP,cleaved Caspase-3,Akt and p-Akt^(Ser473) were measured by Western blot. Cells overexpression of TIPE2 were pretreated with insulin-like growth factors(IGF-1,100 μg/L)for 2 h and co-treated with LPS for another 24 h. Cell viability was measured by CCK-8,apoptosisrelated proteins expressions were detected by Western blot.Results:LPS activated macrophages,increased IL-6 and TNF-α mRNA expressions,and decreased expressions of TIPE2 mRNA and protein(P<0.05). Overexpression of TIPE2 significantly reduced viability of activated macrophages,increased apoptosis rate and index,promoted protein levels of cleaved Caspase-8,Bax,CHOP and cleaved Caspase-3,and inhibited Bcl-2 and p-Akt^(Ser473) expressions(P<0.05). However,knockdown of TIPE2 showed opposite effect. Akt phosphorylation agonist IGF-1 effectively reduced effects of TIPE2 on cell viability and apoptosis related proteins(P<0.05).Conclusion:TIPE2 may promote LPS-activated macrophages apoptosis via death receptor pathway,mitochondrial pathway,and endoplasmic reticulum stress pathway which due to inhibition of Akt phosphorylation.
作者
丛丽
谢小林
刘素娟
向丽萍
符晓华(指导)
CONG Li;XIE Xiaolin;LIU Sujuan;XIANG Liping;FU Xiaohua(Key Lab of Study and Discovery of Small Targeted Molecules of Hunan Province,Hunan Normal University,Changsha 410013,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2022年第2期135-141,共7页
Chinese Journal of Immunology
基金
国家自然科学基金项目(82100490)
湖南省自然科学基金项目(2020JJ5381)
湖南省卫生健康委科研课题项目(202103011683)。