Effect Of Massoia Lactone On Immune Regulation Based On JAK/STAT Pathway

ObjectiveBased on our previous research on the effect of Cordyceps militaris mycelium extract on hepatitis,we studied the immunomodulatory effects of C-10 Massoia Lactone(C-10ML).It provides new possibilities for the research of autoimmune diseases.1.To investigate the regulation and mechanism of C-10ML on Helper T lymphocytes(Th)through inducing T lymphocyte proliferation by mitogen.2.To further confirm the regulation and mechanism of C-10ML in vivo through the OVA mice model.3.To investigate the therapeutic action of C-10ML in arthrophlogosis through collagen-induced arthritis(CIA)model.Method1.The inhibitory effect of C-10ML on lymphocyte proliferation was detected by cell proliferation and toxicity test kit.2.The effect of C-10ML on the proportion of helper T cell subsets was analyzed by flow cytometry.3.The contents of cytokines IFN-gamma,IL-17,IL-2 and IL-10 in cell supernatant and serum were detected by enzyme linked immunoabsorbent assay(ELISA),and the changes of specific antibodies in serum after modeling were also detected.4.The expression of transcription factors in helper T cell subsets was detected through real-time quantitative polymerase chain reaction.5.The changes of target proteins in JAK/STAT signaling pathway was detected through protein imprinting assay.6.In vivo,OVA model was established and after drug intervention,investigating the effects of the pesticide effect of c-10ml on cellular immunity and humoral immunity,meanwhile investigate its action mechanism through cell level,transcription level,cytokine level and target protein expression.7.To establish CIA model and after drug intervention,the arthritis score,cell-specific proliferation,serum specific antibody content and morphological data were used to verify the efficacy of C-10ML on arthritis.Result1.C-10ML showed significant inhibitory effect on mitogen induction and antibody-induced T lymphocyte proliferation in vitro(p<0.001),and had a dose-effect relationship.2.Mechanism of action of C-10ML immunoregulation(1)At the cellular level,C-10ML can reduce the ratio of Thl.(2)At the transcriptional level,it has a significant inhibitory effect on Thl-specific transcription factor T-bet(p<0.001),and has a dose-effect relationship;however,it has little effect on other Th cell subset transcription factors.(3)At the cytokine level,C-10ML had a significant inhibitory effect on the inflammatory factor IL-2 and IFN-y secreted by Thl type(p<0.001)at a dose of 24 hours,and has a dose-effect relationship.(4)Analysis of the expression levels and phosphorylation levels of the target proteins STAT1,STAT5 in the JAK/STAT pathway.C-10ml has the most significant effect on the phosphorylation of STAT1 protein at 6 hours,and can significantly inhibit the phosphorylation of STAT1,with a dose-effect relationship3.The effect of C-10ML on cellular immunity and humoral immunity in OVA mice(1)After OVA immunization challenged mice,the content of OVA-specific antibodies in the blood of mice was significantly increased(p<0.001),and the antibody content in the C-10ML administration group was significantly decreased(p<0.001).(2)After OVA immunization challenged mice,the mouse lymphocytes were specifically induced by OVA in vitro,and the proliferation rate of the model group was significantly higher than that of the normal group(p<0.001),while the C-10ML administration group and the model group were compared.There was a significant inhibition of proliferation(p<0.001).4.Mechanism of immunomodulatory effects of C-10ML on OVA mice(1)At the cellular level,the C-10ML administration group can effectively reduce the ratio of Thl and simultaneously increase the ratio of Treg for the model group.(2)At the transcriptional level,compared with normal group,the model group could promote T-bet expression(p<0.001)and inhibit GATA3 and Foxp3 expression(p<0.001),but had no effect on RoRyt(p>0.05).The C-10ML administration group was significantly reversed(p<0.001).(3)At the cytokine level,the IFN-y content in the serum of the model group was significantly increased(p<0.001),and the secretion of IFN-y was effectively inhibited by drug intervention(p<0.001).Under the induction of OVA,the secretion of IL-2 in the model group was significantly increased(p<0.01),and the administration group had significant inhibition compared with the model group(p<0.01).(4)At the protein level,the drug-administered group was able to inhibit STAT1phosphorylation and promote STAT5 phosphorylation compared to the model group.5.Therapeutic effect of C-10ML on CIA model miceIn the C-10ML administration group,the arthritis score was significantly reduced in the model group(p<0.05).After C-10ML intervention,the pathological changes of the diseased joint tissues in the mice were significantly reduced in morphology,and only mild synovial membranes appeared around the articular cartilage.Hyperplasia,inflammatory cell infiltration also improved significantly.The C-10ML administration group also exhibited significant on cellular immunity as well as humoral immune effects.Result1.C-10ML has immunosuppressive activity against T lymphocyte proliferation in vitro.2.The significant selective inhibition of C-10ML on Thl cells may be related to STAT protein.3.C-10ML has inhibitory effects on cellular and humoral immunity in vivo,and its mechanism is the same as that in vitro.4.C-10ML can improve arthritis and relieve symptoms in CIA mice.