组蛋白去乙酰化酶6调控鞭毛重组蛋白A干扰巨噬细胞自噬

HDAC6 Regulates Flagellar Recombinant Protein A Interference Macrophage Autophagy

目的观察组蛋白去乙酰化酶6(HDAC6)调控嗜肺军团菌鞭毛重组蛋白A(rflaA)对小鼠巨噬细胞自噬相关因子表达的影响,分析其可能的作用机制。方法采用支气管肺泡灌洗法提取C57BL/6J与HDAC6-/-C57BL/6J两种小鼠巨噬细胞,用小鼠巨噬细胞表面标记物F4/80鉴定;纯化rflaA,CCK-8法检测其对小鼠巨噬细胞半数抑制浓度(IC50),筛选最佳作用浓度用于后续实验;rflaA分别干预C57BL/6J及HDAC6-/-C57BL/6J小鼠巨噬细胞6、12和24 h,RT-qPCR、Western blot及免疫荧光检测各组自噬相关因子自噬效应蛋白1(Beclin1)、自噬相关蛋白5(ATG5)、自噬微管相关蛋白轻链3(LC3)、SQSTM1蛋白(P62)的表达水平。结果根据CCK-8结果计算,IC50为0.41μg·μL^(-1),最佳作用浓度为0.041μg·μL^(-1)用于后续实验;RT-qPCR、Western blot、免疫荧光结果显示,rflaA干预C57BL/6J小鼠巨噬细胞后,与0 h相比,6、12、24 h HDAC6的表达水平升高,LC3、ATG5表达水平均降低,P62的表达水平升高,Beclin1的表达水平先降低后升高(P均<0.05);rflaA干预HDAC6-/-C57BL/6J小鼠巨噬细胞后,与0 h相比,6、12、24 h LC3、ATG5、Beclin1表达水平均升高,P62的表达水平降低(P均<0.05);在相同的时间点,与C57BL/6J小鼠巨噬细胞各组相比,HDAC6敲除后各组细胞ATG5、LC3表达水平升高,Beclin1先升高后降低,P62降低(P均<0.05)。结论HDAC6促进rflaA抑制小鼠巨噬细胞自噬,HDAC6可能通过影响微管蛋白乙酰化干扰巨噬细胞自噬。

Objective To observe the effect of HDAC6 on the expression of autophagy-related factors in mouse macrophages by regulating Legionella pneumophila flagellar recombinant protein A(rflaA),and to analyze its possible mechanism.Methods C57BL/6J and HDAC6-/-C57BL/6J mice macrophages were extracted by bronchoalveolar lavage,and identified by mouse macrophage surface marker F4/80.The rflaA was purified and its half inhibitory concentration(IC50)on mouse macrophages was detected by CCK-8 method to screen the optimal concentration for subsequent experiments.C57BL/6J and HDAC6-/-C57BL/6J mice macrophages were co-cultured with rflaA for 6,12 and 24 h,respectively.The expression levels of autophagy-related factors Beclin1,ATG5,LC3 and P62 were detected by RT-qPCR,Western blot and immunofluorescence.Results According to the results of CCK-8,the IC50 was calculated to be 0.41μg·μL^(-1),and the opti mal concentration was 0.041μg·μL^(-1) for subsequent experiments.The results of RT-qPCR,Western blot and immunofluorescence showed that after rflaA was co-cultured with C57BL/6J mouse macrophages,compared with 0 h,the expression level of 6,12 and 24 h HDAC6 increased,the expression levels of LC3 and ATG5 decreased,the expression level of P62 increased,and the expression level of Beclin1 decreased first and then increased(P all<0.05).After rflaA was co-cultured with HDAC6-/-C57BL/6J mouse macrophages,compared with 0 h,the expression levels of LC3,ATG5 and Beclin1 increased at 6,12 and 24 h,and the expression level of P62 decreased(P all<0.05);At the same time point compared with each group of C57BL/6J mouse macrophages,the expression levels of ATG5 and LC3 in each group of cells after HDAC6 knockout increased,Beclin1 increased first and then decreased,and P62 decreased(P all<0.05).Conclusion HDAC6 promotes rflaA to inhibit macrophage autophagy in mice.HDAC6 may interfere with macrophage autophagy by affecting tubulin acetylation.