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  • Title: [Mechanism of histone deacetylase inhibitor tubastatin A promoting autophagy of Legionella pneumophila infected RAW264.7 cells].
    Author: Yang X, Cao X, Sheng L, Yang Z.
    Journal: Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi; 2021 Aug; 37(8):693-701. PubMed ID: 34236029.
    Abstract:
    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 bacteria group. After the RAW264.7 macrophages were treated with inactivated and live Legionella pneumophila for 6, 12, 24, and 48 h, the mRNA and protein expression levels of HDAC6, α-tubulin, p97-VCP, and P62 decreased in the TubA group interfered with the Legionella pneumophila compared with the RAW264.7 normal control group. Conclusion The interference of Legionella pneumophila on the autophagy of macrophages is associated with the signal pathways of HDAC6/P62/LC3B and HDAC6/p97/HSF1.
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