组蛋白去乙酰化酶抑制剂tubastatin A促进嗜肺军团菌感染RAW264.7细胞自噬的机制  

Mechanism of histone deacetylase inhibitor tubastatin A promoting autophagy of Legionella pneumophila infected RAW264. 7 cells

作　　者：杨晓辰 曹秀琴[2] 盛琳琳 杨志伟[1] YANG Xiaochen;CAO Xiuqin;SHENG Linlin;YANG Zhiwei(Department of Pathogenic Biology and Immunology,School of Basic Medicine,Ningxia Medical University,School of Basic Medicine,Ningxia Medical University,Yinchuan 750004,China;Ministry-of-Educatin Key Laboratory of Fertility Preservation and Maintenance,School of Basic Medicine,Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学基础医学院病原生物学与免疫学系,宁夏银川750004 [2]宁夏医科大学生育力保持省部级共建教育部重点实验室,宁夏银川750004

出　　处：《细胞与分子免疫学杂志》2021年第8期693-701,共9页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(82060362);2019年宁夏自然科学基金(2019AAC03082)。

摘　　要：目的探讨组蛋白去乙酰化酶6(HDAC6)在嗜肺军团菌干扰巨噬细胞自噬中的作用及机制。方法采用(10、5、2.5)μmol/L tubastatin A(TubA)处理RAW264.7巨噬细胞,CCK-8法检测RAW264.7巨噬细胞的增殖活性确定TubA的半数抑制浓度(IC50)。建立嗜肺军团菌感染RAW264.7巨噬细胞感染模型,实验分为无TubA组(每组设细胞对照组、灭活菌组、活菌组),(10、5、2.5)μmol/L TubA处理组(每组均设细胞对照组、灭活菌组、活菌组)。嗜肺军团菌感染6、12、24、48 h,收集各组细胞。细菌增殖实验检测嗜肺军团菌在RAW264.7巨噬细胞内增殖的情况;pmCherry-C1-EGFP-LC3B质粒转染RAW264.7小鼠巨噬细胞检测各组内自噬流变化;实时定量PCR和Western blot法检测组蛋白去乙酰化酶6(HDAC6)、P62、微管相关蛋白1轻链3(LC3)、α微管蛋白(α-tubulin)、含缬酪肽蛋白(p97/VCP)、热休克蛋白90(HSP90)、HSP70、热休克转录因子1(HSF1)和纤维型肌动蛋白(F-actin)的mRNA及蛋白表达水平。结果TubA的IC50为50μmol/L。与RAW264.7细胞正常对照组相比,加入TubA后,嗜肺军团菌在小鼠巨噬细胞内增殖明显减少。在无HDAC6抑制剂组中,与正常对照组相比,活菌组比灭活菌组对自噬流的抑制作用更强。在HDAC6抑制剂TubA组中,活菌组的绿色荧光亮点均减少,自噬流增加。嗜肺军团菌的灭活菌和活菌分别作用于RAW264.7巨噬细胞6、12、24、48 h,与正常对照RAW264.7细胞相比,TubA嗜肺军团菌干扰组HDAC6、α-tubulin、p97/VCP、P62 mRNA和蛋白表达均降低。结论嗜肺军团菌干扰RAW264.7巨噬细胞自噬与HDAC6/P62/LC3B和HDAC6/p97/HSF1信号通路有关。Objective To investigate the role of HDAC6 in the interference of Legionella pneumophila on the autophagy of macrophages and its mechanism. Methods RAW264.7 macrophages were treated with 10 μmol/L, 5 μmol/L, and 2.5 μmol/L tubastatin A(TubA). CCK-8 assay was used to detect the proliferative activity of RAW264.7 macrophages, and the half maximal inhibitory concentration(IC50) of TubA was determined. A model of RAW264.7 macrophages infected with Legionella pneumophila was established and divided into TubA free groups(further divided into cell control group, inactivated bacteria group, and live bacteria group) and TubA treatment groups(10 μmol/L, 5 μmol/L, 2.5 μmol/L, each further divided into cell control group, inactivated bacteria group, and live bacteria group). The cells were collected at 6, 12, 24, and 48 h after Legionella pneumophila infection. The bacterial proliferation assay was conducted to detect the proliferation of Legionella pneumophila in RAW264.7 macrophages;RAW264.7 macrophages were transfected with pmCherry-C1-EGFP-LC3B plasmid to detect autophagic flux changes in each group;real-time quantitative PCR and Western blot were used respectively to detect the mRNA and protein expression levels of histone deacetylase 6(HDAC6), sequestosome 1(SQSTM1/P62), microtubule associated protein 1 light chain 3(LC3), α-tubulin, valosin containing protein(p97/VCP), heat shock protein 90(HSP90), HSP70, heat shock transcription factor 1(HSF1), and filamentous actin(F-actin). Results IC50 of TubA was 50 μmol/L.Compared with those in the RAW264.7 normal control group, the proliferation of Legionella pneumophila in mouse macrophages was significantly reduced after the addition of TubA. In the groups without the HDAC6 inhibitor, the live bacteria group had a stronger inhibiting effect on autophagic flux than the inactivated bacteria group compared with the normal control group. In the TubA groups with the HDAC6 inhibitor, the green fluorescence bright spots decreased and the autophagic flux increased in the live

关 键 词：嗜肺军团菌 组蛋白去乙酰化酶6(HDAC6) Tubastatin A(TubA) 

分 类 号：R965[医药卫生—药理学] R392.1[医药卫生—药学]