The Mechanism Of Tree Shrew Chemokine CXCL8-CXCR1Signaling Pathway In Rheumatoid Arthritis

AimsRheumatoid Arthritis (RA) is a group of complex human autoimmune diseases. Few studies related to the tree shrew-based RA pathological animal models have been successfully studied. Chemokine CXC subfamily CXCL8and its cognate receptors CXCR1/CXCR2regulate the different signaling pathways that play the multiple roles in the development of RA. However, the molecular mechanisms of chemokines in the tree shrew remains little understood. In the dissertation, we cloned the tree shrew chemokine CXCL8and CXCR1genes, study their basic structure and functions and explore the molecular menchanisms of CXCL8-CXCR1in the Collagen-induced Arthritis (CIA) tree shrew model.Methods1) To Clone tree shrew’s CXCL8and CXCR1genes, and to future analysis the homology of gene sequences using the bioinformatics-based software. Besides, the mRNA expression of tree shrew’s CXCL8, CXCR1and CXCR2in various tissues and cells were detected by real time PCR.2) Testing the CXCL8concentration in peripheral blood and the lymphocytes proportion of CXCR1/CXCR2-expressed by using the human CXCL8ELISA kit and PE-conjugated mouse anti-human CXCR1/CXCR2reagent.3) With the aid of recombinant human CXCL8protein, the function of tree shrew’s CXCR1could be investigated through the cell transmigration-induced assay. In addition, three kinds of receptor-specific antagonists Reparixin, SB225002and SB265610have adopted to block the CXCR1-regulated cell migration.4) Creating collagen-induced arthritis tree shrew model, and to analysis the feature of this model:clinical score, symptoms, immunohistochemistry and X-ray radiography in the following areas.5) To measure CXCL8and CXCR1expression change in CIA tree shrew. Furthermore, Reparixin (15mg/kg) for the treatment of CIA tree shrew, the therapeutic effect of Reparixin to be proved by testing the myeloperoxidase (MPO) activity and clinical score. Results and Conclusion1)We cloned tree shrews’CXCL8and CXCR1genes. CXCL8and CXCR1proteins had the relatively conservative molecular structure, and shared the highest homologous with those of primates. The expression of CXCR1/CXCR2was broadly spectrum in different tissues and cells of tree shrew.2) We successfully detected the endogenous expression of tree shrew’s CXCL8and CXCR1using human CXCL8and CXCR1antibodies.3) The recombinant human CXCL8protein was able to induce lymphocytes migration, the three receptor-specific inhibitors Reparixin, SB225002and SB265610blocked the human CXCL8-induced migration process in vitro. This result implied that chemokine tsCXCL8-CXCR1axis maybe plays a pivotal role in pathological conditions.4) Conducted the Collagen-induced Arthritis (CIA) tree shrew model by immunized with bovine type Ⅱ collagen mixed in complete Freund’s adjuvant, its clinical score was similar to that of CIA mouse. HE staining and X-ray displayed mononuclear cells infiltrated into the cartilage and cartilage destruction.5) The concentration of CXCL8in peripheral blood was increased obviously, and the percentage of CXCR1-positive lymphocytes is up-regulation, inrespectly. Reparixin treated and reduced MPO activity in CIA tree shrew, and decreased the concentration of CXCL8and the percentage of expressed CXCR1-positive lymphocytes.Above research result indicates that tree shrew chemokine CXCL8and CXCR1not only have conservative structure, function also similar to human. CXCL8-CXCR1signaling pathway involved in the peripheral lymphocytes-mediated RA pathogenesis in tree shrew. It is suggested that CXCL8-CXCR1signaling pathway plays a pivotal in the occurrence and development of tree shrew’s RA pathogenesis.