没食子酸对卡介苗感染巨噬细胞的免疫调控OA

[Objective]To explore the host immunomodulatory effect and its molecular mechanism of gallic acid(GA)in Bacillus Calmette-Guérin(BCG)infected macrophages,and to provide experimental evidence for host-directed anti-tuberculosis research against Mycobacterium tuberculosis(Mtb).[Methods]The minimum inhibitory concentration(MIC)of GA against BCG was determined by resazurin colorimetric assay.Bacterial growth-curve test and MTT colorimetric assay were used to evaluate the effects of GA on BCG proliferation and invasion.A BCG-infected RAW264.7 macrophage model was established,including infection control group(Ctrl),isoniazid(INH)group,and low-,medium-and high-dose GA groups.Flow cytometry combined with colony-plate counting was performed to detect intracellular BCG survival.Cell apoptosis was measured by Annexin V-APC/7-AAD double staining,and qRT-PCR was used to detect the mRNA expression of apoptosis-related genes Caspase-3 and Bcl-2.Macrophage polarization phenotypes were analyzed by flow cytometry.Intracellular reactive oxygen species(ROS)were detected using DHE fluorescent probe.The Griess assay was applied to determine supernatant nitric oxide(NO)concentration.ELISA was used to detect the levels of TNF-α,IFN-γ,IL-6 and IL-10 in cell culture supernatant.[Results]The MIC of GA against BCG was 1 250 μmol/L.GA inhibited BCG proliferation and invasion in a concentration-dependent manner(P<0.05).Safe concentrations of 20,40 and 80 μmol/L were selected for subsequent cellular experiments.Thirty-six-hour GA pretreatment markedly reduced the intracellular BCG bacterial load(P<0.05).GA exerted bidirectional regulation on macrophage apoptosis:it inhibited apoptosis at the early-stage infection and promoted apoptosis at the late-stage infection.GA induced M1-type polarization in early infection and facilitated M2-type polarization in late infection(P<0.05).Meanwhile,GA decreased intracellular ROS and NO levels,down-regulated the secretion of pro-inflammatory cytokines and up-regulated the secretion of anti-inflammatory cytokine IL-10(P<0.05).[Conclusion]GA inhibits BCG proliferation and invasion in vitro.It remodels host immune microenvironment via bidirectional regulation of macrophage apoptosis and polarization as well as anti-oxidative and anti-inflammatory effects,showing potential value for host-directed anti-mycobacterial therapy.