摘要
目的探究去泛素化酶在单核细胞驯化免疫中的作用及机制。方法Q-PCR检测单核细胞驯化免疫中去泛素化酶相关基因的表达情况;ELISA法检测去泛素化酶对驯化的单核细胞分泌肿瘤坏死因子-α(TNF-α)水平的影响;双荧光素酶报告基因检测系统测定去泛素化酶对NF-κB信号通路的影响。双荧光素酶报告基因检测系统测定:UCHL1对NF-κB信号通路的影响、UCHL1与NF-κB信号通路关键信号分子之间的作用及UCHL1影响NF-κB信号通路所依赖的酶活性;ELISA法检测UCHL1对单核细胞驯化作用中TNF-α分泌水平的影响。结果去泛素化酶USP2、UCHL1、OTUB2和OTUD4在单核细胞驯化免疫中的表达量明显升高;去泛素化酶抑制剂可明显抑制驯化单核细胞中TNF-α的分泌水平;USP2、UCHL1、OTUB2、PSMD14等可明显激活NF-κB信号通路,通过上述实验筛选出对NF-κB影响最为明显的UCHL1进行后续研究。UCHL1的表达明显影响NF-κB信号通路的激活;UCHL1的表达促进了由IκB-α、IKK-α或IKK-β介导的NF-κB的激活;UCHL1主要通过其泛素水解酶活性调控NF-κB信号通路;UCHL1抑制剂可明显减弱单核细胞的驯化作用。结论自身免疫病中免疫复合物可以驯化单核细胞增强其敏感性,这种作用可通过UCHL1调节NF-κB信号通路的激活实现。
Aim To investigate the role and mechanism of deubiquitinating enzymes in trained immunity of monocytes.Methods Q-PCR was used to detect the expression of deubiquitinating enzyme-related genes in trained monocytes,the effect of deubiquitinating enzyme on the secretion of tumor necrosis factor-α(TNF-α)in trained 0monocytes was detected by ELISA,and the effect of deubiquitinating enzyme on the NF-κB signaling pathway was determined by dual luciferase reporter assay system.The dual luciferase reporter assay system was also used to determine the effect of UCHL1 expression on NF-κB signaling pathway,the interaction with the NF-κB pathway,and the effect of enzyme activity on the NF-κB pathway.ELISA was used to verify the effect of UCHL1 on the secretion level of TNF-αin trained immunity.Results The results showed that the expression levels of USP2,UCHL1,OTUB2 and OTUD4 significantly increased in training of monocytes.The deubiquitinating enzyme inhibitor could significantly inhibit the secretion of TNF-αin trained monocytes.USP2,UCHL1,OTUB2 and PSMD14 could significantly activate the NF-κB signaling pathway,and UCHL1,which had the most significant effect on NF-κB,was selected for further investigation.It was shown that UCHL1 expression could affect the activation of NF-κB pathway;UCHL1 promoted the activation of NF-κB induced by IκB-α,IKK-αor IKK-β,and the regulation of UCHL1 on the NF-κB pathway depended mainly on its ubiquitin hydrolase activity.Furthermore,UCHL1 inhibitors could greatly reduce the trained immunity of monocytes.Conclusion The immune complexes of patients with autoimmune diseases can be domesticated monocytes that increase their sensitivity by regulating the UCHL1-activated NF-κB pathway.
作者
王洪民
马子涵
潘雅颖
高晓明
龚方苑
WANG Hong-min;MA Zi-han;PAN Ya-ying;GAO Xiao-ming;GONG Fang-yuan(School of Biology and Basic Medical Sciences,Soochow University,Suzhou Jiangsu 215123,China)
出处
《中国药理学通报》
CAS
CSCD
北大核心
2024年第4期637-645,共9页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 31570868)。
作者简介
王洪民(1987-),女,硕士,助理研究员,研究方向:自身免疫病,E-mail:hmwang@suda.edu.cn;共同第一作者:马子涵(1990-),女,硕士生,研究方向:自身免疫病,E-mail:mazihan0827@sina.com;通信作者:高晓明(1962-),男,博士,教授,博士生导师,研究方向:自身免疫病,E-mail:xmgao@suda.edu.cn;通信作者:龚方苑(1984-),女,博士,副教授,硕士生导师,研究方向:肿瘤免疫及自身免疫病,E-mail:gongfangyuan@suda.edu.cn。
