| Rheumatoid arthritis(RA)is a chronic progressive autoimmune disease with unclear etiology and high prevalence,and the current treatments against RA remain unsatisfactory or accompanied with the emergence of side effects.Thus,there still exists an urgent need for novel anti-RA agent with high efficiency and low toxicity.Gelsemium elegans Benth.(G.elegans)has been widely used in Chinese folk medicine as a kind of medical plant,from which a variety of active ingredients have been isolated.Koumine is the most abundant monomeric alkaloid with relatively lowertoxicity in Chinese G.elegans and has great value for the development of new drugs.The previous work of our group showed that koumine had significant therapeutic effects on RA via attenuation of the increase in the expression of proinflammatory cytokines such as tumor necrosis factor-?(TNF-?),interleukin-1?(IL-1?),etc.But its specific mechanism needs further investigations.The pathogenesis of RA is very complex,in which a variety of immune cells and related cytokines may be involved.Among these cells,antigen dependent activation of T cells especially T helper(Th)cells is considered as the key point of the origination and progress of the disease.During the pathogenesis of RA,Th cells develop activation,proliferation and polarization shift,and the balance of the network system consisting of the cell subsets and associated inflammatory cytokines is broken,causing activation of other effect cells,release of inflammatory mediators and activation of certain degrading enzymes,further leading to the inflammatory process of RA and bone destruction.By regulating the polarization state of Th1/Th2 or Th17/Treg cells,the balance of the network system comprised of different Th cells and their cytokines can be restored to achieve the control of the pathogenesis and progression of RA.Taking Th cell polarization as the breakthrough point,the current thesis was described along the course and the pathway of key effector molecules involved in the pathogenesis to explore the regulation mechanism of koumine on Th cell polarization against RA,so as to provide new targets and pathways for novel antirheumatic drug research & development(R&D),as well as to establish the theoretical foundation for the development of koumine as an innovative drug with independent intellectual property rights,and additionally to contribute to the further comprehensions of the pathogenesis of RA.The main content of this paper is as follows:1 Effects of koumine on regulation of serum Th cytokine imbalance in animal models of rheumatoid arthritis1.1 Therapeutic effect of koumine on mice with type?collagen induced arthritis and its regulatory effect on serum inflammatory cytokine levels in the modelType?collagen-induced arthritis(CIA)model was developed in C57BL/6 mice.Immunized mice received koumine(0.4,2,or 10 mg·kg-1 per day)or vehicle by gastric gavage for 10 consecutive days,starting 22 days from the first type?collagen(C?)injection.A group of immunized mice receiving methotrexate(1 mg·kg-1 every other day)during the same period were included as a positive control.Clinical evaluation was performed prior to the immunization,1 day prior to the treatment and on alternate days after the initiation of koumine/methotrexate treatment through hind paw thickness,arthritis index(AI)score and paw withdraw thermal latency(PWTL).Serum samples were collected 1 h after the completion of treatmen from 6 mice randomly selected in each group,and then assayed for cytokine levels by Procarta Plex Multiplex Luminex Immunoassay andenzyme-linked immunosorbent assay(ELISA).Koumine inhibited increases in hind paw thickness,AI score and thermal hyperalgesia in a dose-dependent manner in mice with CIA,reflecting the therapeutic effect.The detection results of cytokine levels showed that koumine canreverse the imbalance of Th1/Th2,Th17/Treg cytokines,characterized by the inhibition of proinflammatory cytokine levels(IFN-?,IL-1?,IL-2,IL-6,IL-12,TNF-? for Th1 cytokines as well as IL-17 A for Th17)and the levels of positive regulation cytokines for Th1 and Th17.1.2 Therapeutic effect of koumine on rats with adjuvant induced arthritis and its regulatory effect on serum Th cytokine levels in the modelLewis rats were immunized for the development ofadjuvant induced arthritis(AIA)model for RA.Clinical evaluation was performed through volume and mechanical withdraw threshold(MWT)of the injected and contralateral hind paws respectively,as well as the AI score.According to the features of the model,two schemes were designed,comrising acute and chronic treatments of koumine for further clarification about its regulatory effect on Th-cytokine imbalance.For the acute treatment,immunized rats received koumine(0.6,3,or 15 mg·kg-1 per day)or vehicle by gastric gavage 1 h before the induction and daily for the next 9 days.A group of immunized rats receiving indometacin(2.5 mg·kg-1 per day)during the same period served as positive control.Clinical evaluation was performed prior to the immunization and on alternate days after the initiation of koumine/indometacin treatment.Serum samples collected before the induction and after the completion of treatment were assayed for cytokine levels.And for the chronic treatment,drugs were administrated by gastric gavage for 10 consecutive days,starting 14 days from the injection,clinical evaluation was performed prior to the immunization,2 days prior to the treatment and on alternate days after the initiation of koumine/indometacin treatment.Serum samples for cytokine level determination were collected before the induction,2 days prior to the treatment and after the completion of treatment.Acute treatment of koumine did not show significant inhibition for the development of joint swelling and AI score,but it does-dependently inhibited mechanical allodynia of the injected hind paw.No significant differences of cytokine levels were found in all groups before immunization,only elevated levels of IL-17 A were detected on 9 days after immunization,other major effector cytokine levels of Th cell subsets such as IL-1?,IL-2,IL-4,IL-6,IL-10,IFN-? and TGF-? were not changed significantly during the acute phase,and the acute treatment of koumine had no effect on them,either.However,the chronic treatment of koumine alleviated mechanical allodynia of both of the injected and the contralateral hind paws,and reduced the score of AI in a dose-dependent manner.The detection results of cytokine levels showed that chronic treatment of koumine attenuated the increase levels of IL-1?,IFN-? and IL-17 A in rats with AIA,resulting in a reverse effect on IFN-?/IL-4,IFN-?/IL-10 and IL-17A/TGF-? ratios,suggesting the regulating effect on serum Th cytokine levels in the animal model of RA.2Effects of koumine on regulation of Th-cell polarization in rats with CIAWistar rats were immunized for the establishment of CIA model.Immunized rats received koumine(0.6 or 15 mg·kg-1 per day)or vehicle by gastric gavage for 10 consecutive days,starting 21 days from the first C?injection.Clinical evaluation was performed prior to the immunization,1 day prior to the treatment and on alternate days after the initiation of koumine/vehicle treatment through measurements of edema,and evaluation of articular nociception.Splenocyte suspension samples collected after the completion of treatment were assayed for Th cell percentages by flow cytometry.The results showed that koumine alleviated swelling and pain in hind paws and ankle joints of the rats with CIA.Increased Th1/Th2 and Th17/Treg ratios were detected in splenocytes of rats with CIA,suggesting the imbalances of Th-cell polarization,and they were both attenuated by the administration of koumine.Combined with the effect of koumine on imbalance of Th cytokines in RA models,the resultsdemonstrated that koumine may play a regulatory role on Th-cell polarization,so as to reverse the imbalance of Th1/Th2,Th17/Treg cytokines,restore the balance of the network system with different Th cell subsets and cytokines,which may be one of the main mechanisms for the therapeutic effect of koumine against RA.3Inhibitory effects of koumine on proliferation ofsplenocytes in mouseIn order to further clarify the effects of koumine on activation and proliferation of immune cells at initiation of Th-cell polarization,the proliferative response of mouse splenocytes to different mitogens were studied following treatment with multiple doses of koumine by 5-bromodeoxyuridine(Brd U)incorporation assay,and the cytotoxicity of koumine was examinedby CCK-8 method invitro.The results showed that koumine had inhibitory effects on the proliferation of mouse splenocytes to concanavalin A(Con A),lipopolysaccharides(LPS)and phytohaemagglutinin(PHA),in which the effects on proliferations to Con A and PHA were more sensitive(50%inhibiting concentration: 101.3 ?mol/L and 95.28 ?mol/L,respectively),with lower concentrations than the cytotoxic range,demonstrating that koumine may have relatively selective inhibit effect on the proliferation of T cells especially CD4+ T cells,this effect may be one of the reasons of the regulatory effect of koumine on Th-cell polarization.In summary,these results demonstrate that kouminemay inhibit the activation and proliferation of immune cells by selective action on T lymphocytes,then subsequentlyadjust the polarization shift of different Th cell subsets and thedownstream imbalance of pro/anti-inflammatory cytokines,so as to recover the balance stateof the network system with different Th cell subsets and cytokines,which may be an importantmechanism for theantirheumatic effect of koumine. |
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