Study On The Effect Of 78c On Collagen Induced Arthritis In Mice

Objective In this study,we constructed a collagen induced arthritis(CIA)model to observe the drug intervention effect of CD38 inhibitor 78c on CIA mice,and analyzed the mechanism of 78c action by in vitro cellular assay.To explore the potential value and clinical significance of CD38 inhibitors in rheumatoid arthritis(RA).Methods 18 C57BL/6J mice were randomly divided into normal control group,CIA control group,and CIA treatment group.CIA treatment group were injected intraperitoneally with 78c 20μg/g,normal control group and CIA control group were injected intraperitoneally with the same amount of solvent twice a week for 3 weeks and the toe thickness of each group was measured.The histopathological changes of the joints of the mice in each group were observed by hematoxylin-eosin(HE)staining;the biochemical and routine blood parameters of the mice in each group were observed by automatic biochemical analyzer and hematocrit analyzer;the lymphocyte subtypes and cytokine concentrations in the peripheral blood of the mice in each group were measured by flow cytometry;the frequency of spontaneous movement and upright movement of the mice in each group were observed by Oxylet system.Peripheral blood samples were collected from 10 healthy volunteers,and peripheral blood single nucleated cells were extracted using Ficoll separation;CD4~+T cells,CD38~+NK cells,and CD38~+NK-like T cells were sorted using sorting magnetic bead kit.The above cells were randomly divided into group A(CD4~+T cells),group B(CD4~+T cells co-cultured with CD38~+NK cells),group C(CD4~+T cells co-cultured with CD38~+NK-like T cells),group D(CD4~+T cells co-cultured with 78c pretreated CD38~+NK cells),group E(CD4~+T cells co-cultured with 78c pretreated(CD4~+T cells co-cultured with CD38~+NK-like T cells after 78c pretreatment).Lymphocyte subtypes and cytokine concentrations were detected by flow cytometry.Results In the CIA treated group,compared with the CIA control group,the swelling of the toe was reduced and there was some recovery in joint movement,and the thickness of the toe was significantly reduced(F=41.38,P<0.05);WBC,Neu and PLT in peripheral blood were reduced(F=5.04 to 15.43,P<0.05);and TP in plasma was increased(F=7.50,P<0.05);The proportions of NK cells,CD38~+NK cells,CD38~+cells and CD38~+B cells in peripheral blood were significantly lower(F=5.42 to 10.21,P<0.05)and the proportion of Treg cells was significantly higher(F=10.50,P<0.05);serum concentrations of IFN-γ,IL-6 and TNF-αwere lower(F=4.28 to 8.41,P<0.05)and increased IL-10 concentrations(F=7.64,P<0.05);a significant decrease in serum Th1/Th2 ratio(F=7.67,16.33,P<0.05);and an increase in the frequency of spontaneous and upright movements(F=20.63,12.96,P<0.05).Pathological findings showed that the CIA treated group showed reduced synovial hyperplasia and reduced inflammatory cell infiltration compared to the CIA control group.After 78c treatment,the inflammatory state of the mice was alleviated,suggesting that 78c may have a therapeutic effect on CIA.The results of the comparison of lymphocyte subtypes showed that the Th17/Treg ratio and Th1/Th2 ratio were reduced under group D compared to group B(F=28.93 to65.10,P<0.05).This result indicated that the role of CD38~+NK cells in promoting Th1and Th17 lymphocyte production was inhibited after 78c treatment,thereby restoring Th17/Treg and Th1/Th2 phenotypes and related cytokines to normal levels and regulating the balance of Th17/Treg and Th1/Th2 cells.Compared to group A,group B showed a significant decrease in the Treg/CD4~+T ratio(F=14.10,P<0.05).This observation suggests that CD38~+NK cells can inhibit the differentiation of CD4~+T cells to Treg cells;Comparison of cytokine concentrations showed that IFN-γ,IL-6,TNF-α,IL-10 and IL-17A concentrations were decreased in group D compared with group B(F=6.39 to14.06,P<0.05);IFN-γ,IL-6 and TNF-αconcentrations were increased in group B compared with group A(P<0.05);IL-6 and IL-10 concentrations were increased in group C(P<0.05);IL-10 concentrations decreased in group D(P<0.05).These results suggest that among CD4~+T cells,the addition of CD38~+NK cells or CD38~+NK-like T cells inhibited the differentiation of CD4+T cells to Treg,thereby elevating the levels of inflammation-related factors,whereas after 78c treatment,CD38~+NK cells lost their inhibitory effect on CD4~+T cells,allowing IFN-γ,IL-6,TNF-α,IL-10,IL-17A and other inflammatory factors were reduced.Conclusion 78c has the effect of mitigating CIA.It may restore the immune balance by reducing the proportion of CD38~+NK cells,affecting the secretion of inflammatory factors and promoting the differentiation of CD4~+T cells into Treg cells.CD38 inhibitors have good clinical application prospects in alleviating the effects of RA and have the potential to be used as RA therapeutic drugs.