关键词: RA, 中性粒细胞, CIA小鼠, NETs

Abstract: Objective: To explore whether down-regulation of neutrophils can alleviate the disease severity of collagen-induced arthritis (CIA) mice, and to analyze the differences in transcriptome expression after the formation of neutrophil extratrapping networks (NETs), providing a theoretical basis for the follow-up research on the effect of NETs on RA disease progression. Methods: DBA1 mice were used to construct a CIA model, and anti-Ly6G monoclonal antibody was used to knock down neutrophils in CIA mice after secondary collagen boosting immunization (once a week), and the intervention was continued for 8 weeks. After 8 weeks, the mice were sacrificed to collect the lungs, spleens, and hindlimbs of mice for histopathological observation; RA peripheral blood neutrophils were collected by magnetic bead sorting, and neutrophil nuclei were stained with DAPI dye, and kept at room temperature. After standing under light for 15 min, the morphological characteristics of neutrophils were observed under microscope; RA peripheral blood neutrophils were collected, stimulated with different concentrations (8 nmol/L, 16 nmol/L, 32 nmol/L) of PMA, cultured for 2 h, and stained with NETs markers. The optimal concentration of stimulating neutrophils to form NETs was observed under a fluorescent inverted microscope; Samples were collected for transcriptome sequencing after stimulation of neutrophils using PMA to analyze changes in transcriptome expression after formation of RA NETs. Results: Down-regulation of neutrophil expression partially alleviated the pathological changes in the joint synovium, lung and spleen of CIA mice; 8 nmol/L PMA stimulated RA neutrophils for 2 h, 37°C, 5 % CO₂ culture; Differential analysis screened out 2742 differential genes(P≤0.05),including 1579 up-regulated differential genes [log₂(RA_control/RA_PMA＞0)] and 1163 down-regulated differential genes [log₂(RA_control/RA_PMA＜0]. 340 significantly different genes (log₂|RA_control/RA_PMA|≥2) were identified and displayed by PPI network construction; GO, KEGG and GSEA KEGG pathway analysis of 340 differential genes were enriched respectively as follows: immune response and signal receptor activity In the regulation of cytokines, the mutual regulation pathway between cytokines, the interaction of cytokine receptors, apoptosis and Toll-like receptor signaling pathways. Conclusion: Down-regulation of neutrophils can alleviate the pathological changes of joint synovium, lung and spleen in CIA mice; the formation of NETs in RA is related to immune regulation, apoptosis and the interaction between cytokines, indicating that the formation of NETs may be involved in the above The immune process plays an important role in the progression of RA disease.

Key words: RA, Neutrophil, CIA mice, NETs