Effect Of GCH1 Inhibitor DAHP On LPS-induced Microglial Autophay

Objective: To investigate the effect of GCH1 inhibitor DAHP on the expression of autophagy in BV2 microglia induced by LPS.Methods: In this study,mouse BV2 microglia with good growt-h in logarithmic growth phase were selected as the research object,and lipopolysaccharide(LPS)was used to establish a neuroinflamm-atory pain cell model.The microglia were divided into the following four groups according to different intervention methods: normal control group,LPS-induced activation group,LPS induction DAHP group,DAHP group.The expression of GCH1,LC3 B and Beclin1 at the mRNA level was detected by RT-PCR.The expression of GCH1,LC3 B and Beclin1 at the protein level was detected by Wes-tern Blot.Results:1.The morphological characteristics of BV2 microglia without intervention were characterized by small round or oval shape,intact cell structure,good growth state,adherent growth,and BV2 microglial morphology after LPS intervention.The characteristi-cs change greatly,and most of the cells tend to become larger and irregularly branched from smaller round and oval.The morphology of BV2 microglia after DAHP intervention was not obvious before morphological changes.2.The results of RT-PCR showed that compared with the nor-mal group,the mRNA expression of GCH1,LC3 B and Beclin1 in the LPS-induced activation group was significantly increased,and the difference was statistically significant.Compared with the LPS-i nduced activation group,the LPS+DAHP group could significantly reduce GCH1.The mRNA expression of LC3 B and Beclin1 was statistically significant.Compared with the normal group,the DAHP group could decrease the mRNA expression of GCH1,and the diffe-rence was statistically significant,while the mRNA expression of Beclin1 and LC3 B had no significant difference.3.The results of Western Blot showed that compared with the normal group,the expression levels of GCH1,LC3 B and Beclin1 in the LPS-induced activation group were significantly increased,an-d the difference was statistically significant.The LPS-induced DAHP group was significantly more effective than the LPS-induced activation group.The protein expression of GCH1,LC3 B and Beclin1 was significantly decreased.The expression of GCH1 was decre-ased in the DAHP group compared with the normal group.The dif-ference was statistically significant,but the protein expressio n of LC3 B and Beclin1 was not significantly different.Conclusion: In the neuroinflammatory pain model induced by LPS-induced BV2 microglia activation,the expression level of GCH1 in BV2 microglia after LPS intervention was significantly in-creased,and the neuroinflammatory pain cell model induced by LPS to induce microglia activation was successfully established.In this cell model,the expression levels of LC3 B and Beclin1 were si-gnificantly increased,indicating that LPS induced autophagy in BV2 microglia;GCH1 inhibitor DAHP can reduce the mRNA and protein expression levels of GCH1 in normal BV2 microglia,and also decreased.LPS induced the expression of GCH1 in activated microglia and down-regulated the expression levels of autophagyrelat-ed proteins LC3 B and Beclin1 mRNA,indicating that DAHP can inhibit the autophagy level of neuroinflammatory pain cell model and reduce cell over-self Microglia damage caused by phagocytosis.The result-s of this study indicate that GCH1 may participate in the pathogen-esis of neuropathic pain by regulating microglia autophagy pathway,which will provide a new target for clinical treatment of neuropathic pain.