Mechanism Research Of Loureirin B And Its Derivative In The Prevention And Treatment Of Rheumatoid Arthritis

Rheumatoid arthritis（RA）is a multi-joint disease mainly invoving small joints of hands and wrists.Its clinical manifestations are joint swelling and hyperalgesia of the infected joints with persistent and recurrent lesions,and high frequency of disability.At present its exact pathogenesis remains unknown,which poses a great challenge to the effective prevention and treatment of RA in clinic.Anti-inflammatory and analgesic pain are still the predominant treatment of prevention and treatment of RA.The discovery of autoantibodies in serum of patients gradually revealed that RA belonged to the autoimmune disease and laid a theoretical basis on its immunosuppressive therapy.The pathogenesis of autoimmune diseases mainly involves in abnormal activation and proliferation of effector memory T cells(T_EMs),which is closely related to the overexpression of Kv1.3,a voltage-gated potassium channel on TEM cell membrane.Drug blocking or knocking out Kv1.3 channel showed a strong therapeutic effect on RA model animals,suggesting that Kv1.3 is a new target for the discovery of specific RA immunosuppressive drugs.Severe joint pain is one of the main clinical symptoms of RA patients,relieving joint pain can significantly improve the life quality of RA patients.Analgesic effect of Morphine is significant,but with addictive side effects.Accordingly,it is vital to find a non-addictive drug that significantly improve the clinical symptoms of RA patients.Various types of voltage-gated sodium channels are expressed on mammalian primary sensory neurons.Tetrodotoxin sensitive（TTX-S）sodium channel is an important target protein for the development of non-addictive analgesic drugs.Obviously,if the active substance can block the Kv1.3 and TTX-S sodium channels simultaneously,the dual pharmacological effects of immunosuppression and analgesia can be produced,which promises to be an excellent lead compound for the development of prevention and treatment of RA drugs.Our previous study showed that Loureirin B（LrB）can block the TTX-S sodium channel in mammalian primary sensory neurons,resulting in a good analgesic effect and further study manifested that LrB can inhibit exogenous and endogenous Kv1.3 channels in mammals.Previous studies suggested that LrB is likely to have a good effect on the prevention and treatment of RA.Hence,in this study we planned to synthesize LrB and LrB acetyl product（Acetylized Loureirin B,AcLrB）with structural stability.Moreover,the therapeutic effects of synthetic LrB and AcLrB on RA rats have been systematically studied and the cellular and molecular mechanisms of both compounds against RA have been explored.In the first part of this study,patch clamp experiments were performed to examine the modulation of Kv1.3 channels by synthetic LrB and AcLrB.Electrophysiological results showed that LrB could block Kv1.3 channel current exogenously expressed on HEK-293T cell membrane in a concentration-dependent manner,with half maximal inhibitory concentration(IC₅₀)of Kv1.3,6.33±0.49μM.AcLrB blocks Kv1.3 more effectively than LrB,with IC₅₀ of 2.24±0.15μM.In addition,the selectivity of AcLrB to block Kv1.3channel was also stronger than that of LrB,and the blocking efficiency of AcLrB was reduced about 10-fold and 100-fold respectively for the Kv1.1 homologues Kv1.1 and Kv1.2.The results of this part suggest that the synthetic LrB retains a good pharmacological activity of blocking the Kv1.3 channel.After being acetylated,the modified structure of LrB is more stable and the efficiency and selectivity of blocking Kv1.3 have been improved in a certain extent.In the second part of the present study,patch clamp technique is still used to detect the regulatory effects of synthetic LrB and AcLrB on pain related voltage-gated sodium channels in mammals.The results showed that both LrB and AcLrB could block the TTX-S sodium channel on the membrane of dorsal root ganglion cells in mice,and IC₅₀ was 6.14±0.51 M and 13.69±0.56 M respectively.Nav1.7 is the major TTX-S sodium channel subtype on the primary sense neurons of mammalian.This study further examined the regulatory effect of LrB and AcLrB on Nav1.7 exogenously expressed on the HKE-293T cell membrane.The results showed that LrB and AcLrB showed a similar blocking effect on Nav1.7,and IC₅₀ was3.97±0.08μM and 4.35±0.16μM respectively.The results of this part showed that similar to the activity of previously natural extracted LrB,the synthesized LrB can also efficiently block the TTX-S sodium channel on the primary sense neurons of mammalian,and LrB after acetylated modification still retained similar blocking activity against sodium channel.The first two parts of the research showed that LrB and its acetylated derivative AcLrB could block Kv1.3 and TTX-S sodium channels at the same time,which strongly suggested that they may have good pharmacological effects against RA.In the third part of this study,RA rat model was induced by bovine type II collagen,and the effect of LrB and AcLrB on the prevention and the control of RA rats has been tested.Behavioral experiments showed that both LrB and AcLrB could effectively reduce the behavioral score of RA rats,relieve the joint swelling and increase the pain threshold of RA rats,either in the prevention group or in the treatment group.CT examination showed that both LrB and AcLrB could effectively alleviate or improve the bone and joint hyperplasia of RA rats in some extent.Results of HE showed that both LrB and AcLrB could reduce the inflammatory infiltration of the ankle joint in RA rats and reduce the synovium hyperplasia and cartilage lesion of the ankle joint.The results of ELISA test showed that both LrB and AcLrB could significantly reduce the concentration of IL-6,IL-1βand other inflammatory cytokines in the serum of the RA rats in the drug treatment group.The results of flow cytometry counting test showed that both LrB and AcLrB significantly inhibited the proliferation of total T cells and CD4⁺T cell subsets in peripheral blood of RA rats.The results of RT-PCR test showed that in the RA rat model group,mRNA expression levels of inflammatory cytokines IL-1 beta,IL-6 and potassium channel Kv1.3 in the peripheral blood mononuclear cells（PBMCs）were significantly higher than that of the rats in blank group;compared with the model group,the mRNA expression of IL-1β,IL-6 and Kv1.3 in the LrB and AcLrB treatment group significantly decreased.The results of this study strongly suggested that both LrB and its acetylated derivative AcLrB have good effect of analgesia and immunosuppression on RA model rats.In the fourth part of this study,taken Jurkat T cells activated by phytohemagglutinin（PHA）and PBMCs activated by concanavalin A（ConA）as cell models,combined with ELISA kit,RT-PCR assay and flow cytometry,the cellular and molecular mechanisms of immune suppression of LrB and AcLrB were explored,and the in vitro cytotoxicity of AcLrB and LrB was tested.The results showed that LrB and AcLrB could significantly inhibit the release of cytokine IL-2 and reduce Kv1.3 mRNA and protein expression in Jurkat T cells activated by PHA.For rat PBMC activated by ConA,both LrB and AcLrB can significantly inhibit the release of inflammatory cytokines IL-2 and IL-17,and significantly reduce the expression levels of Kv1.3 mRNA and protein.CCK8 kit was used to detect the toxicity of LrB and AcLrB to rat PBMC.The results showed that LrB and AcLrB in high concentration of even10μM did not have obvious toxic effects on rat PBMC.The results of this study suggested that the immunosuppressive effects of LrB and AcLrB may be related to the reduction of the Kv1.3 expression level in the activated T cells.Integrating all above parts,this study revealed the LrB and its acetylated derivative AcLrB directly blocked Kv1.3 channel on the T cell membrane,and reduced the Kv1.3 expression level of activated T cell,thus inhibited the proliferation of activated T cell and the release of inflammatory cytokines,thereby effectively weaken the autoimmune response in RA rats;on the other hand,that LrB and AcLrB effectively blocked the TTX-S sodium channel was likely to the important pharmacological effects that resulted in the increased pain threshold of RA rats.The synergetic effect of immunosuppression and analgesia from LrB and AcLrB revealed both compounds possessed potential medicinal value in the prevention and control of RA.