Enterovirus 71 can induce autophagy and apoptosis of THP-1 macrophages / 南方医科大学学报

OBJECTIVE@#To investigate enterovirus 71 (EV71)-induced of autophagy, apoptosis and the related signaling pathways in THP-1 macrophages.@*METHODS@#THP-1 macrophages were infected with EV71 at the multiplicity of infection (MOI) of 0.1 for 2, 8 or 16 h, and the cell proliferation and toxicity were analyzed using CCK-8 kit. The intracellular viral nucleic acid in THP-1 macrophages were detected by fluorescence quantitative PCR, and the ultrastructural changes of the cells were observed using transmission electron microscopy. Cell apoptosis induced by EV71 infection was detected using Hoechst 33342 staining and AnnexinV/PI double staining. Western blotting was performed for analysis of changes in autophagy and apoptosis of the cells and in the expressions of the related proteins. The effect of EV71 infection on apoptosis of THP-1 macrophages incubated with 3-MA and Ac-DEVD-CHO inhibitor for 2 h was assessed using Western blotting.@*RESULTS@#EV71 infection significantly lowered the cell survival rate of THP-1 macrophages at 2, 8 h and 16 h after the infection ( < 0.05). The total copy number of viral nucleic acid in THP-1 macrophages incubated with EV71 increased significantly and progressively over time ( < 0.01). Intracellular autophagosomes and virions could be seen in EV71-infected THP-1 macrophages. The total apoptotic rate of the infected cell also increased significantly over time ( < 0.01). EV71 infection significantly increased LC3 conversion (LC3-Ⅱ/ LC3-I) and the expression of cleaved caspase 3 protein and decreased the protein expressions of p62, Bcl-2 and caspase-3 ( < 0.01) without causing obvious changes in cleaved caspase-8 (>0.05). 3-MA significantly inhibited the EV71-induced autophagy of THP-1 macrophages and reduced LC3 conversion (LC3-Ⅱ/LC3-I) and p62 protein expression at 8 h after EV71 infection ( < 0.01). Compared with DMSO, Ac-DEVD-CHO significantly inhibited EV71-induced apoptosis of THP-1 macrophages (15.5% 7.7%, < 0.01).@*CONCLUSIONS@#EV71 not only can infect and replicate in THP-1 macrophages, but also induces autophagy and cell apoptosis possibly by activating LC3/p62 autophagy pathway and caspase apoptosis pathway.

Experiment of THP-1 macrophage foam-cell formation through mTOR signal pathway activation induced by inflammatory stress / 中国药理学通报

Aim To investigate if LPS increases the sterol regulatory element binding proteins ( SREBPs ) cleavage-activating protein ( SCAP )-SREBP2 expres-sion by activation of mTOR signal pathway in THP-1 macrophages , upgrading LDLr level , causing foam-cell formation .Methods THP-1 macrophages were incu-bated in serum free medium in the absence of 5 mg?L-1 LDL alone , or 5 mg? L-1 LDL plus 200 μg? L-1 LPS, or 5 mg? L-1 LDL plus 200 μg? L-1 LPS plus 10 μg? L-1 rapamycin .Morphological examination of macrophages was performed with Oil Red O staining . Expression changes of LDLr , SREBP2, SCAP, S6K1 and mTOR mRNA were detected by real time quantita-tive polymerase chain reaction ( PCR ) .Western blot was used to analyze protein expression changes of LD-Lr, S6K1 and mTOR.Translocation of SCAP-SREBP2 complex from the endoplasmic reticulum ( ER ) to the Golgi was determined by confocal microscopy .Results LPS enhanced transformation of THP-1 macrophages into foam cells by increased uptake of lipid as evi-denced by Oil Red O assay .LPS increased mRNA lev-els of LDLr, SREBP2, SCAP, S6K1 and mTOR ( P <0.05) .Rapamycin reduced the mRNA levels of LDLr , SREBP2,SCAP,S6K1 and mTOR induced by LPS ( P<0.05 ) .Western blot demonstrated that LPS also caused over-expression of protein of LDLr , S6K1 and mTOR(P<0.05).Rapamycin reduced the expression of protein of LDLr, S6K1 and mTOR induced by LPS ( P <0.05 ) .Confocal microscopy demonstrated LPS caused an escape of SCAP-SREBP2 complex from the ER to the Golgi .Rapamycin inhibited the translocation of SCAP-SREBP2 complex from the ER to the Golgi . Conclusions Inflammatory stress increases SCAP/SREBP2 expression by activation of mTOR signal path-way, resulting in an escape of SCAP-SREBP2 complex from the ER to the Golgi , furthermore elevating LDLr expression and causing foam-cell formation .Rapamy-cin reverses the activation of mTOR signal pathway and decreases lipid deposition in THP-1 macrophages in-duced by LPS .

Panton-valentine leukocidin induce THP-1 macrophages IL-8 and IL-6 response through nuclear TLR4/NF-κB pathway / 中华微生物学和免疫学杂志

Objective To investigate the influence of panton-valentine leucocidin (PVL) on expression of Toll like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) signals and IL-8,IL-6 in THP-1 macrophages,and to study the mechanism of PVL-related lung tissue damage.Methods THP-1 cells were cultured in the presence of 100 nmol/L phorbol-12-myristate 3-acetate (PMA) for 48 h to induce monocytemacrophage differentiation.rPVL-F and rPVL-S were induced and expressed from the recombinant plasmid,respectively purified with chromatographic column. After that,THP-1 macrophages were incubated with rPVL,and then ELISA was performed to test expression of IL-8 and L-6 in supernatants fluid; RT-PCR was performed to detect expression of IL-8,L-6 and TLR4 ; NF-κB was analyzed by Western blot and immunohistochemistry method.Results PVL was able to induce expression of IL-8 and IL-6 in THP-1 macrophages in time-and concentration-dependent manners.PVL could also significantly promote the activation of TLR4/NF-κB signals.Conclusion PVL can activate the expression of TLR4/NF-κB signals,and increased the high expression of inflammatory cytokines.Maybe it's the mechanism of action of PVL exerts the function of lung tissue damage.