环鸟苷酸-腺苷酸合酶抑制剂RU.521通过调控JAK1/STAT3信号通路抑制类风湿关节炎巨噬细胞极化OA

OBJECTIVE To investigate the effect of cyclic GMP-AMP synthase(cGAS)inhibitor RU.521 on lipopolysaccharide(LPS)induced RAW264.7 macrophage polarization and collagen-induced arthritis(CIA)in mice and the molecular mechanism.METHODS ① Cell experiments:RAW264.7 cells were divided into a cell control group,LPS group,LPS+RU.521 0.75,1.5,and 3.0 μmol·L-1 groups,and LPS 100 ng·L-1 with tofacitinib 100 nmol·L-1 group.Except the control group,all these groups were stimulated with LPS 100 ng·L-1 and simultaneously treated with corresponding drugs for 24 h.Western blot was used to measure cellular cGAS levels,and the concentration of RU.521 that significantly inhibited LPS-induced cGAS expression was selected for subsequent mechanistic studies.Real-time quantitative PCR was used to measure cytoplasmic mtDNA levels and cGAS mRNA expres-sions.Flow cytometry and immunofluorescence were adopted to detect reactive oxygen species(ROS)levels and mitochondrial membrane potential.Cluster of differentiation 86(CD86)expressions were detected via immunofluorescence.RT-qPCR was employed to detect inducible nitric oxide synthase(iNOS),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α)mRNA levels.cGAS,iNOS,and argi-nase-1(Arg-1)protein expression levels were determined by Western blotting.The proportion of M1/M2 macrophages was calculated via flow cytometry to analyze the macrophage phenotype.Western blotting was used to detect cGAS,PTEN-induced kinase 1(PINK1),ubiquitin-binding protein(p62),E3 ubiquitin ligase(Parkin),and microtubule-associated protein 1A/1B-light chain 3(LC3),autophagy-related proteins,and Janus kinase 1(JAK1)/signal transducer and activator of transcription 3(STAT3)pathway-related protein expressions.② Animal experiments:Mice were divided into a normal control group,model group,model RU.521 group,and model tofacitinib group.Except the normal control group,these groups were intradermally injected at the root and back of tails with an emulsion of chicken typeⅡcollagen and complete Freund's adjuvant on days 0 and 21.On day 28,the model was considered successful when the average arthritis score of the model group was significantly higher than that of the normal control group.From day 29,the model RU.521 group received RU.521 at 5 mg·kg-1 intraperitoneally every 2 days while the model tofacitinib group received tofacitinib at 10 mg·kg-1 orally every day for 4 weeks.Arthritis scores,paw swelling,and body weight of the mice were monitored regularly.On day 66,joint tissues were collected for HE staining to observe pathological changes in the knee joint before peritoneal macrophages were isolated for RT-qPCR detection of cGAS,TNF-α,and IL-6 mRNA expressions.Flow cytometry was used to analyze the proportion of M1 macrophages while Western blotting was employed to detect the phosphorylation levels of JAK1/STAT3 proteins.RESULTS ① Cell experi-ments:Compared with the cell control group,cytoplasmic mtDNA levels,cGAS mRNA expressions and ROS levels were increased in the LPS group while mitochondrial membrane potential was decreased.CD86,iNOS,as well as iNOS,IL-6,TNF-α mRNA expressions were increased while the Arg-1 expres-sion was decreased.The M1/M2 ratio became higher,but expressions of cGAS,Parkin,PINK1 and p62 were upregulated.The LC3-Ⅱ/LC3-Ⅰratio was reduced,and the JAK1/STAT3 pathway was acti-vated.Compared with the LPS group,RU.521 inhibited cGAS expressions,reduced ROS levels,restored mitochondrial membrane potential,downregulated CD86,iNOS,IL-6,and TNF-α expressions,upregulated Arg-1,decreased the M1/M2 ratio,inhibited cGAS as well as PINK1,p62,and Parkin expressions,restored the LC3-Ⅱ/LC3-Ⅰratio,and suppressed the phosphorylation activation of JAK1 and STAT3.② Animal experiments:Compared with the normal control group,the mice in the model group showed significantly increased scores of knee arthritis,significantly elevated mRNA expression levels of cGAS,TNF-α,and IL-6 in peritoneal macrophages,a significantly increased proportion of M1-type cells,and significantly increased phosphorylation levels of JAK1/STAT3.Compared with the model group,RU.521 could alleviate knee joint damage,reduce the mRNA expression levels of cGAS,TNF-α and IL-6 in peritoneal macrophages and the proportion of M1-type cells while inhibiting phos-phorylation of the JAK1/STAT3 pathway.CONCLUSION RU.521 can inhibit macrophage M1 polariza-tion and alleviate CIA in mice by regulating JAK1/STAT3 signaling pathway,which is a potential treat-ment strategy for RA.