Experimental Research On The Effect Of Immunoregulators CH2b In Arthritis Induced By Mixed Peptides Derived From Glucose-6-phosphate Isomerase In DBA/1mice

ObjectiveTo identification an animal model of rheumatoid arthritis(RA)in DBA/1 mice induced by immunodominant mixed peptides derived from glucose-6-phosphate isomerase(GPI).On this basis, to explore the intervention effects of immune modulators CH2 b with a thiazolidin-4-one ring in RA mice.To investigate the effects of the frequency and function of iNKT cells, and the influence on Th1 / Th2, Th17 / Treg immune imbalance.Discussing the possible recognition mode and activate signaling pathways of iNKT cells on CH2 b.It will be significant to provide a new theoretical basis of the pathogenesis and treatment methods of RA.Method1. Establishment and identification of RA mice model.Mixed peptide fragments of hGPI325-339+hGPI469-483 were fully emulsionized with complete Freund's adjuvant, DBA/1 mice were given subcutaneous injection of the immunogens and then pertussis toxin was used to strengthen the immunity. Compare to the healthy and the ones induced by single peptides hGPI325-339, weight change, ankle joint swelling in mice were observated. The joint tissues stained with hematoxylin and eosin(HE)was used to evaluate the inflammatory cells. Fluorescence-activated cell sorting( FACS)was used to detect the frequency changes of CD4+T and iNKT cells. Cytometric bead array( CBA) was used to analyze the levels of serum cytokines IL-4, IFN-?,TNF- ?, IL-6.2. The intervention mechanism of immune regulators CH2 b on RA mice and the possible recognition mode and activate signaling pathways of iNKT cells on CH2 b.RA mice were intervened with CH2 b, ? galactosyl ceramide(?-GalCer) as a positive control, the changes of body weight, ankle joint symptoms scores were observed.The joint tissues stained with HE was used to evaluate the changes of inflammatory cells.FACS was used to detect the frequency changes of iNKT cells. Western Blot were used to detect the expression of T-Bet, GATA-3 involved.3. Detection the influence of CH2 b on immune balance disorders of Th1 / Th2, Th17 / Tregin RA mice.RA mice were intervened with CH2 b, ?-GalCer as a positive control, CBA was used to analyze the levels of serum cytokines IL-4, IFN-?,TNF-?,IL-6 in each period in each group.PCR were used to detect the expression of T-bet mRNA, GATA-3 mRNA, ROR-?t mRNA,Foxp3 mRNA of each group in the liver tissue.Result1.Compared with the healthy and the mice treated with single peptide, the increase in the body weight of the mice treated with mixed peptide fragments was slow. The hind paw of the RA mice treated with mixed peptide fragments first began to appear redness and swelling on8 th day, then accentuated gradually to the limbs, the swelling reached peak on 14 th d, and was then gradually relieved. Inflammatory cells infiltration were found in histomorphological observation, ankle joint was given priority to monocytes and lymphocytes, but plasma cells with a small amount. The inflammatory effect of mixed peptides was more obvious than that of the single one(P<0.05).2.?-GalCer and CH2 b could improve the body weight growth and the joint swelling( P<0.05).The inflammatory cells infiltration were decreased.The frequency of iNKT cells in peak were increased(P<0.05). Compared with healthy controls,the expression of T-bet was obviously increased in model group on the 14 th day(P < 0.05).But the expression of GATA-3 in groups of ?-GalCer and CH2 b were obviously increased(P < 0.05), and the expression of T-bet was no statistically significant difference compared with the healthy(P > 0.05).3. The levels of TNF- ?, IL-6, IFN-? in serum were significantly decreased by ?-GalCer and CH2b(P < 0.05)on Day 14.Compared with model, the expression of GATA-3 mRNA increased obviously(P < 0.05). The levels of ROR-?t mRNA had no obvious difference(P > 0.05) in each group. T-bet mRNA expression in model group was obviously increased on the 14 th day(P < 0.05), but Foxp3 mRNA in groups of ?-GalCer and CH2 b were increased(P < 0.05).Conclusion1.Rheumatoid arthritis induced by mixed GPI peptides in DBA/1 mice is more closed to the RA patients in immunological characteristics, especially in the immune pathological aspects of CD4+T and iNKT cells, and it will be used as a new tool of studying the mechanism and immunological intervention.2.The immune modulators CH2 b by activating iNKT cells GATA-3 activation pathways could improve inflammatory conditions of arthritis induced GPI mixed peptides, it may be a new potential tools on the targeted therapy application of RA.3.CH2 b could correct the immune balance disorder of Th1 / Th2, Th17 / Treg in RA mice,maintain immune tolerance. CH2 b will have a potential clinical application value.