Regulation Effect Of Koumine On Activation And Proliferation Of The Progenitor Splenocytes And The Polarization Migration Of Th Cells As Well As The Expression Of TSPO In Mice

Rheumatoid arthritis?RA?is a chronic,inflammatory synovial pathologic disease,which is not effective.Antigen dependence on the excessive activation of CD4⁺T lymphocyte proliferation and polarization migration is considered the key links of the pathogenesis of RA,by adjusting the Th1/Th2 cells in the body or the balance of Th17/Treg cells can regulate the body's immune status,and control the onset and progression of the disease of RA.Koumine is the active ingredient of indole alkaloids in Gelsemium elegans Benth.,which is a traditional medicinal plant.Koumine is the most abundant composition and exerts relatively low toxicity.The early stage of the research work on koumine RA resistance and its mechanism of action research,found in animal level koumine can significantly inhibit spleen Th1/Th2,RA animal model of Th17/Treg cells and abnormally elevated serum related cytokines,prompt its polarization of Th cell migration may have a regulatory role,further research found that koumine start on Th cell polarization migration has significant inhibitory effect on immune cell proliferation.However,it is necessary to further study and confirm its effect on the polarization migration of Th cells,and its action mechanism,especially the target and action mode,is not yet clear,and it remains to be further studied and explored.In addition,the early stage of the research study found that mitochondria 18 kDa transfer protein?TSPO?hook kiss element are important targets in the body,but if it has a koumine RA effect on resistance,and its expression of the bias in Th cell polarization situation is unclear.As a result,this paper first proved in primitive culture mice spleen cells,the proliferation of T cell activation and the influence of the cell cycle,cell level and in the validation koumine on Th cell polarization offset adjustment,finally observe TSPO expression of the bias in Th cell polarization and koumine on its expression.This study will help to further clarify the molecular mechanism for the treatment of RA,and the potential for the development of a new drug to treat RA.1 Inhibitory effect of koumine on T cell activation and proliferation in mice1.1 Inhibitory effect of koumine on the proliferation of spleen cells in mice induced by mixed lymphocytes culturePreparation of healthy BALB/c mice spleen cell suspension,using mitomycin c treatment as a donor to stimulate cells,at the same time,the preparation of health C57BL/6 mice spleen cells suspension as receptor response,on the basis of the given koumine?4,0.8,0.16,0.032 mmol/L?trained 48 h,observe the mixed culture of the lymphocyte proliferation inhibition,the result shows an concentration dependence of koumine can inhibit mixed culture lymphocyte proliferation,IC₅₀0 for 1.448 mmol/L.1.2 Inhibitory effect of koumine anti-CD3 and anti-CD28 induced T cell proliferation in miceTo observe koumine of T cell receptor?TCR?whether the initial T cell proliferation induced by crosslinking has inhibitory effect,competitive TCR antibodies to stimulate T cells simulation TCR/CD3+CD28 cues inducing initial T cell activation.Normal BALB/c mice spleen cells and to get the initial T cell immune magnetic beads purification,co-culture with anti CD3 and anti CD28 antibody after 96 h,join the concentration gradient of koumine?15,3,0.6,0.12,0.024,0.0048 mmol/L?48 h,continue to cultivate with BrdU incorporation method to detect cell proliferation.The results suggest that anti-CD3+anti-CD28 antibody can effectively induce the proliferation of initial t-cells.Koumine can significantly inhibit anti CD3+CD28antibody induction of initial T cell proliferation and function is dose dependent IC₅₀0 for3.253 mmol/L,with koumine of mixed lymphocyte reaction inhibition level of IC₅₀0 was close to koumine cell toxicity level,comprehensive performance,both prompt koumine first signal to the T cell activation and the second signal has a certain inhibitory effect,but its function is weak,could not koumine main function of inhibiting proliferation of T cell activation.1.3 Inhibitory effect of koumine on the proliferation of spleen lymphocytes in mice induced by Con AIn order to further explore koumine of Con A induced mice spleen lymphocyte proliferation inhibition and preparation of normal BALB/c mice spleen cells suspension,use fluorescent dyestuff CFSE handle after inoculation on 96-well cell culture plates,join Con A induced cell proliferation,while adding a concentration gradient of koumine?5000,1250,312.5,78.125,19.531,78.125?mol/mL?trained 48 h,with fluorescent tags anti CD3 antibodies labeled APC,flow cytometry to detect fluorescent signals.In Con A induced proliferation of mice spleen cells to koumine process,the results found with the koumine dosage increased,CFSE method to detect the fluorescence signal peak gradually reduce the number of figure in proliferation,prompt koumine can significantly inhibit mouse spleen lymphocyte proliferation induced by Con A,and the early stage of the team to BrdU mixing method to detect koumine of Con A induced mice spleen cell proliferation inhibition results are consistent.1.4 Effects of koumine on the proliferation cycle of spleen cells in mice induced by Con AIn the preparation of BALB/c mice,the spleen cell suspension was induced to proliferate with Con A,while the gradient concentration of kouimne?93.75,187.5,375?mol/mL?was cultured for 48 h after PI staining,and the flow cytometry was detected.The G0/G1 phase signal peak shape of each treatment group was similar to the visual observation group,while the signal peak of S phase showed the trend of uplifting with the increase of the concentration of the crocus,and the change trend of G2/M signal peak was the opposite.According to G0/G1 cell cycle-S-G2/M phase in the process of each phase of the cell relative amount of change,the above results suggest koumine also assumes the concentration dependence block Con A induced mice spleen cell proliferation of the cell cycle process,make its blockade in S phase.2 Regulation effect of koumine level induced Th-cell polarization migrationPreparation of mice spleen suspension,with Con A in vitro culture,at the same time joined the concentration gradient of kouminw?20000,5000,1250,312.5,78.125,19.531,78.125?mol/mL?,continuous culture after 48 h,mice with BD company Th1/Th2/Th17 CBA kit,flow cytometry instrument FACSVerse detection,FCAP software data read,according to the standard curve fitting,the test results into conversion for inflammatory cytokine expression level.The results showed that compared with the control group,the signal of IL-10,TNF-?,IFN-?,IL-6 and IL-4 in the model group showed a right deviation,indicating a high expression trend of IL-10,TNF-?,IFN-?,IL-6 and IL-4 induced by Con A.Compared with the model group,the signal deviation of koumine subgroup showed different degree of restorative change with the increase of the concentration of koumine.Values measured in standard curve for each cytokine expression level,koumine of Con A induced mice spleen cells IL-17A,TNF-?,IFN-?,IL-6,IL-4,IL-10 and IL-2 changes in the level of cytokine expression are different degree of inhibition effect.It was found that the increase of IFN-?and IL-4 ratio in the model group had a significant inhibitory effect,suggesting that it had a moderating effect on the polarization deviation of Th1/Th2 cells induced by the dissociation level.Koumine,can significantly reduce the model group cells the expression of IL-17A level,prompt the inhibits Th0 cells into Th17 cells polarization,IL-17A/IL-10 ratio to measure relative relationship of Th17/Treg cells,koumine can significantly inhibit model group IL-17A/IL-10 ratio increases,the prompt koumine,of in vitro level induced polarization of Th17/Treg cells migration also has a regulatory role.In addition,the increase in the expression level of TN-?and IL-6 in mice induced by Con A-induced splenocytic proinflammatory cytokines in mice was also significantly inhibited.In Con A model group expression levels of IL-10 and IL-2,give koumine,after processing,the high concentration of koumine processing group of IL-10?1.25-20 mmol/L for koumine?and IL-2?0.3125-20 mmol/L for koumine?levels did not show significant changes,compared with Con A model group had no significant difference.However,the treatment group of the low-concentration hook snakin can significantly increase the expression of IL-10?4.88-312.5?mol/mL?and IL-2?4.88-78.13?mol/mL?.So the early stage of the comprehensive research on the overall level of animal observed results,can confirm koumine of Th1/Th2,polarization migration has significant regulatory role of Th17/Treg cells,can restore the RA onset Th1/Th2,Th17/Treg cells and its downstream inflammatory/anti-inflammatory cytokine balance between state and give play to effect of treatment of RA.3 Expression of TSPO reduction in Th cell polarization-induced polarization reducedPreparation of mouse spleen cell suspension inoculation in RPMI 1640 medium containing Con A and 10%serum,and give the final concentration respectively when vaccination for 500,50?mol/mL koumine processing,at the same time set up culture contains 10%serum RPMI 1640 medium as the control group only and does not contain the blank group,serum RPMI 1640 medium after 48 h after continuous culture extracted proteins of each cell.Results showed that inoculation in serum of mice spleen cells in RPMI 1640 medium containing 10%TSPO expression level was significantly higher than that of blank group,while the amount of Con A induced the expression of model group TSPO further increases,significantly higher than that of blank group and the control group.The expression level of?-actin protein in the spleen cells of Con A model was similar to that of other groups,while the expression level of TSPO was significantly lower than that of Con A model group and control group.Prompt TSPO high expression during Th cell polarization migration,koumine after processing the expression level down,the results support the anti-inflammatory effect of koumine,but koumine in regulating when Th cell polarization TSPO lower expression actually comes from the direct effect of koumine or secondary to koumine anti-inflammatory effects,further research is needed to clarify.The above results indicate:1.Koumine of significant inhibitory effect on the activation of immune cell proliferation can make the stagnation of the cell cycle in S phase,the dependence of T cell activation antigen beginning which identify antigen one signals with two also has a certain inhibition;2.There is a significant regulatory effect on the polarization shift of Th cell in the Th1/Th2,Th17/Treg cell subgroups and the equilibrium state of the downstream proinflammatory/anti-inflammatory cytokines;3.The expression level of TSPO can be down-regulated when the TSPO expression level is observed at Th cell polarization deviation.