Nuclear Transcription Factor ?B Participates In Lps-induced Microglial Inflammation By Regulating GGH1 Expression

Objective: The purpose of this experiment is to study the effect of nuclear factor ?B on GCH1 and downstream inflammatory factors in LPS-induced BV2 microglial inflammation.Therefore,the effect of microglia inflammation on neuropathic pain and its mechanism are further explored,and a theoretical basis is provided for the treatment of neuropathic pain.Methods: In this experiment,mouse BV2 microglial cells in a good growth state were induced by lipopolysaccharide(LPS)to establish a model of neuroinflammatory pain microglia.Divided into five groups by different intervention methods: normal microglia group(Control group),microglia + LPS(LPS group),microglia + NC siRNA + LPS(Control siRNA + LPS group),microglia + NF-?B siRNA + LPS(NF-?B siRNA + LPS group),microglia + NF-?B siRNA + GCH1 siRNA + LPS(NF-?B siRNA + GCH1 siRNA + LPS group).IF was used to detect the expression of GCH1 protein in each group;Western Blot was used to detect the expression of GCH1 at the protein level;RT-PCR and ELISA were used to detect pro-inflammatory factors(IL-1?,IL-6,TNF-?)and anti-inflammatory factor(IL-10)expression at the mRNA level.Results: Compared with the control group,the density of LPS-induced microglia decreased,and the morphological cell body gradually became smaller and branched,and the cell process extended like amoeba.GCH1 fluorescence intensity of BV2 cells in LPS group and Control siRNA + LPS group was significantly higher than that of Control group(P <0.001);compared to LPS group,GCH1 fluorescence intensity of NF-?B siRNA + LPS group was significantly reduced(P <0.001).Compared with the Control group,the mRNA and protein expression of GCH1 in the LPS group and the Control siRNA + LPS group were significantly increased(P <0.001),and the expression of proinflammatory factors(IL-1?,IL-6,TNF-?)was significantly increased(P < 0.01),the expression of anti-inflammatory factor(IL-10)was significantly reduced(P <0.001);compared with the LPS group,the expression of GCH1 mRNA and protein of NF-?B siRNA + LPS was reduced(P <0.05),IL-1?,The expression of IL-6 and TNF-? was significantly reduced(P <0.01),and the expression of IL-10 was significantly increased(P <0.01).Compared with the LPS group,the expression of GCH1 mRNA and protein in the NF-?B siRNA + GCH1 siRNA + LPS group was significant.The expression levels of IL-1?,IL-6 and TNF-? were significantly reduced(P <0.01),and the expression levels of IL-10 were significantly increased(P <0.001).Compared with GCH1 siRNA + LPS,the expression of IL-1?,IL-6 and TNF-? of NF-?B siRNA + GCH1 siRNA + LPS was significantly reduced(P <0.01),and the expression of IL-10 was significantly increased(P <0.001).Conclusion: The pathophysiological mechanism of NP is complex,and more and more evidence indicates that microglial inflammatory response plays an important role in central sensitization and NP development and maintenance.This study shows that in the inflammatory response model of BV2 microglia induced by LPS,the transcription and expression levels of GCH1 in this model significantly increased;microglial cells were transfected with NF-?B siRNA and GCH1 siRNA,and then constructed with LPS In the inflammatory response model,the transcription and expression of GCH1 are inhibited,and the production of IL-1?,IL-6 and TNF-? produced by microglia is significantly suppressed,while the production of IL-10 is significantly enhanced,indicating that NF-?B signaling The pathway can participate in the inflammatory response of microglia by positively regulating GCH1,increasing pro-inflammatory factors(IL-1?,IL-6 and TNF-?)and reducing anti-inflammatory factors(IL-10).The results of this study indicate that NF-?B may participate in microglial inflammatory response by regulating GCH1 expression and affecting the expression of downstream inflammatory factors,leading to the occurrence of NP,which will provide new ideas for clinical working treatment of NP.