High mobility group nucleosome binding protein 1(HMGN1)induces activation of mouse BV2 microglia and upregulates their pro-inflammatory mediator expression by activating TLR4/MyD88/NF-κB p65/IKK-β signal pathway
Objective To explore the effects and mechanism of high-mobility group nucleosome-binding protein 1(HMGN1)on the inflammatory response of mouse BV2 microglia.Methods BV2 cells were incubated with recombinant HMGN1 at different concentrations(0,100,200,500,1000,2000 ng/mL)for 6 hours,and the morphological changes were observed under a microscope.The mRNA levels of tumor necrosis factor α(TNF-α),interleukin-6(IL-6),interleukin-1β(IL-1β)and monocyte chemotactic protein 1(MCP-1)were detected by real time quantitative PCR.Microglial cells were then randomly divided into a control group,model group,inhibitor group and antagonist group.The cells in the model group were treated with 500 ng/mL HMGN1,while the antagonist group was treated with 500 ng/mL TAK-242(resatorvid),a Toll-like receptor 4(TLR4)antagonist,in addition to HMGN1.Real time quantitative PCR and immunofluorescence were used to detect the expression of M1/M2 markers in the four groups,and Western blot analysis was used to measure the protein expression levels of inducible nitric-oxide synthase(iNOS),TLR4,myeloid differentiation factor88(MyD88),nuclear factor κB p65(NF-κB p65)and inhibitor of NF-κB(IκB)kinase β(IKK-β).Results After the treatment of HMGN1,the morphology of BV2 cells changed significantly,showing an amoeba-like appearance.The mRNA levels of TNF-α,IL-6,IL-1β and MCP-1 increased with the HMGN1 concentration,with a statistically significant difference compared to the 0 ng/mL HMGN1 group.At the same time,the mRNA level of iNOS,a M1 phenotype marker,increased with the HMGN1 concentration,while the level of CD206,a M2 phenotype marker,decreased with HMGN1 concentration,showing a statistically significant difference compared to the 0 ng/mL HMGN1 group.Compared with the model group,the mRNA level of M1 phenotypic marker iNOS in the antagonist group was significantly lower,and the level of M2 phenotypic marker CD206 was significantly higher.The results of immunofluorescence cytochemistry also showed that the expression of M1 phenotypic marker iNOS in the antagonist group was lower.The results of Western blot suggested that the protein expression levels of iNOS,TLR4,MyD88,NF-κB p65 and IKK-β decreased significantly in the antagonist group.Conclusion HMGN1 may induce the activation of BV2 microglial cells by upregulating pro-inflammatory mediators through activating the TLR4/MyD88/NF-κB p65/IKK-β signaling pathway.
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