Study on the Inhibitory Effect of Cl-amidine on Ml Macrophage Polarization through JAK3-STAT5 in Anky-losing Spondylitis

LI Ru-lin; DU Zhuang-wen; WANG En-liang

doi:10.13820/j.cnki.gdyx.20232016

Abstract：Objective To investigate the impact of Cl-amidine on M1 macrophage polarization and its clinical implications in ankylosing spondylitis(AS).Methods A total of 60 AS patients were selected and categorized into low AS disease and high AS disease groups based on disease activity scores,with 30 healthy subjects included as the control group.ELISA was employed to measure serum PAD4,iNOS,and bone formation marker BGP levels,and Pearson correla-tion coefficient analysis was conducted to assess the correlation of PAD4,iNOS,and BGP.Flow cytometry was used to de-tect the proportion of M1 macrophages(CD68+CD86+)in peripheral blood from the three groups.THP-1 monocytic cell lines were cultured,induced into M1 macrophages in vitro,and treated with Cl-amidine during incubation.The groups included MO,M1,and M1+Cl-amidine.Western blot was employed to examine PAD4 and iNOS protein expres-sion levels in each group.RNA-seq was used to detect differentially expressed genes in M1 and M1+Cl-amidine cells,followed by KEGG pathway analysis.Additionally,Western blot was performed to measure the protein expression levels of JAK3,p-JAK3,STAT5,and p-STAT5.Results In comparison to the control group,the low AS disease group showed elevated PAD4 and iNOS levels,decreased BGP levels,and an increased proportion of M1 macrophages.Com-pared to the low AS disease group,the high AS disease group exhibited higher PAD4 and iNOS levels,lower BGP levels,and an increased proportion of M1 macrophages.PAD4 levels were positively correlated with iNOS levels,and both PAD4 and iNOS levels were negatively correlated with BGP levels.In comparison to the MO group,the M1 group showed in-creased expression of PAD4 and iNOS proteins.Compared to the M1 group,the M1+Cl-amidine group displayed 163 upregulated genes and 272 downregulated genes.The upregulated genes were mainly involved in the JAK-STAT signaling pathway.The M1+Cl-amidine group exhibited decreased protein expression of iNOS,p-JAK3/JAK3,and p-STAT5/STAT5 compared to the M1 group.Conclusion PAD4 and M1 macrophage levels are positively correlated with the sever-ity of AS.The PAD4 inhibitor Cl-amidine may suppress macrophage polarization toward M1 through the JAK3-STAT5 signaling pathway,providing insights into the research on clinical treatment for AS.