Study Of The Effect And Mechanism Of GLI1 DNA Methyltransferases On Regulating Inflammation And Bone Destruction In Rheumatoid Arthritis

Objective:Rheumatoid arthritis(RA)is a serious chronic inflammatory bone destruction disease.M1 polarization of macrophages and over activation of osteoclasts play an important role in promoting inflammation and bone resorption.Zinc finger transcription factor GLI1 is an essential effector of Hedgehog(Hh)signaling.Studies have shown that GLI1 is involved in a variety of inflammation related and bone metabolism related diseases.However,the effects of GLI1 on RA and its mechanism are not clear.In this study,we will explore the regulatory mechanism of GLI1 on macrophage polarization and osteoclast activation,clarify its role in the development of RA disease,and provide a theoretical basis for clinical application of GLI1 as a therapeutic target for RA.Methods:In vitro,bone marrow-derived macrophages(BMM)and RAW264.7 cells were used as research objects.M1 macrophages and osteoclasts were induced by lipopolysaccharide(LPS)/interferon-γ(IFN-γ)and receptor activator of nuclear factor kappa B ligand(RANKL)respectively.Drug intervention of GLI1 specific inhibitor GANT58,DNMTs specific inhibitors and overexpression intervention of lentivirus transfection were used to intervene the induction of Ml macrophages and osteoclasts(n=3-6).The phenotype change and mechanism in vitro were studied by means of western blot(GLI1,DNMT1,DNMT3a,NFATc1,CTSK,MMP9),real-time quantitative reverse transcription-polymerase chain reaction(RT-qPCR),immunofluorescence staining and tartrate resistant acid phosphatase(TRAP)staining.In vivo,the mixture of type Ⅱ collagen and adjuvant was injected into the tail of DBA mice(6 mice in each group)to establish collagen induced arthritis(CIA)model,and GANT5 8(specific inhibitor of GLI1)was used as a therapeutic drug.The tissue samples of knee joint and ankle joint of mice were collected for Micro-CT analysis,histomorphology staining,tissue TRAP staining,immunohistochemistry staining and other methods to explore the therapeutic effect and mechanism in vivo.Results:The expression of GLI1 changes with the polarization of macrophages and the induction of osteoclasts.During M1 macrophage induction,the expression of GLI1 was increased.GLI1 inhibition reduced the formation of M1 macrophages and the release of proinflammatory cytokines by reducing the expression of DNMT1 while LV-DNMT1 overexpression transfection could reverse this situation.At the same time,the decrease of GLI1 expression inhibited the formation of osteoclasts by down regulating the expression level of DNMT3a.The results of in vivo experiments showed that GANT58,a GLI1 specific inhibitor,could significantly alleviate the arthritis symptoms of CIA mice and reduce the formation of osteoclasts in tissues,thus reducing the degree of bone destruction eventually.Conclusion:Down regulation of GLI1 expression can inhibit the formation of proinflammatory M1 macrophages and the over activation of osteoclasts through the co direction regulation of DNMTs,thus alleviating the bone destruction caused by arthritis.This study may provide a new potential therapeutic target for the treatment of RA.