TLR4 regulated immune responses in Mycobacterium tuberculosis-infected macrophages through the NF-κB/TGF-β signaling pathway
Objective To explore the role of toll-like receptor 4(TLR4)in the NF-kappa B(NF-κB)/transforming growth factor-β(TGF-β)signaling pathway in regulating immune responses in macrophages infected by Mycobacterium tuberculosis,and to explore the mechanism of action.Methods The RAW264.7 cell line was cultured in vitro.All cells were divided into four groups:TLR4 overexpression plasmid(OE-TLR4 group),blank plasmid(Empty vector group),TLR4 small interfering RNA(si-TLR4 group)and control si-NC(si-NC group).TLR4 overexpression plasmid,blank plasmid,TLR4 small interfering RNA and its corresponding control si-NC were transfected into mouse mononuclear macrophages(RAW264.7),and the cells were subsequently infected with Mycobacterium tuberculosis strain H37Rv.Clonogenesis assay was used to detect the number of bacterial colonies.CCK-8 assay was used to detect cell viability.ELISA kits were used to detect the levels of IL-1β,IL-6 and TNF-α in the supernatant.Western blot assay was used to detect the expression of NF-κB and MAPK signaling pathway related proteins(IκB,p-IκB,SMAD2,p-SMAD2)levels.Results TLR4 expression levels were significantly higher in RAW264.7 cells infected with Mycobacterium tuberculosis strain H37Rv than in uninfected cells.Compared with the Empty vector group,the OE-TLR4 group had a higher number of cellular bacterial colonies(t=-10.61,P＜0.05),lower cell viability(t=-1.861,P＜0.05),IL-6,IL-1 β,TNF-α,p-IκB and p-SMAD2 showed a significantly higher expression(P＜0.05),the differences were statistically significant.Compared with the si-NC group,the si-TLR4 group had fewer cellular bacterial colonies(t=-5.425,P＜0.05),higher cell viability(t=-2.250,P＜0.05),IL-6,IL-1 β,TNF-α,p-IκB and p-SMAD2 with significantly lower expression levels(P＜0.05),the differences were statistically significant.Conclusions TLR4 could promote immune responses and cell viability in Mycobacterium tuberculosis-infected macrophages and was associated with activation of NF-κB and TGF-β signaling pathways.
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