Therapeutic Effect And Mechanism Study Of Kv1.3-mediated Sanguis Draxonis On Autoimmune Disease

Studies showed that the high expression of Kv1.3 channel on the cell membrane caused the abnormal activation and proliferation of effector memory T lymphocytes(T_EM),which might lead to the occurrence of many autoimmune diseases.By gene knockout or pharmacological blockade of Kv1.3 channel,T_EM cell activation and proliferation were inhibited,and the clinic condition of autoimmune model animals was improved effectively.Therefore,Kv1.3 channel was considered to be a new target for the treatment of autoimmune diseases,and the development of selective Kv1.3 channel blockers for the treatment of autoimmune diseases was research focus nowadays.The immunomodulatory activity of SD was proposed to involve inhibition of both classical?CP?and alternative?AP?pathways of complement system as well as proliferation of activated T-cells.Through whole cell patch clamp experiment,present study first tested the effect of sanguis draxonis?SD?and its main active component loureirin B?Lr B?on Kv1.3 current and Jurkat T lymphocyte membrane potential,then examined the IL-2 secretion from Jurkat T cells using an ELISA kit and measured the intracellular free calcium concentration of Jurkat T cells with calcium ion imaging techniques.Both SD and Lr B inhibited heterologous and endogenous Kv1.3 currents,produced a membrane depolarization and inhibited IL-2 release in activated Jurkat T cells.In addition,application of Lr B reduced Ca²⁺influx in Jurkat T cells.Furthermore,point mutations in the selective filter region significantly reduced the inhibitory effect of Lr B on Kv1.3.The results of these experiments provided evidence that Lr B is a channel blocker of Kv1.3 by interacting with amino acid residues in its selective filter region.Direct inhibition of Kv1.3 in T cells by SD and Lr B might be the cellular and molecular basis of SD mediated immunosuppression.In order to increase the activity of Lr B which was an important active component of SD to block Kv1.3 channel,present research synthesized a series of Lr B's derivates by structure modification and electrophysiological activity assay.Compared to Lr B,its acetylation product?being abbreviated as ES?possessed a much stronger inhibitory effect on Kv1.3 with a 2.1±0.39?M half inhibitory concentration.The effect of ES on the Kv1.1 and Kv1.2currents was also tested.Electrophysiological results showed that the blocking effect of ES on Kv1.1 and Kv1.2 was weaker than Kv1.3,which indicated that ES had better selectivity for Kv1.3 channels.Strong blocking effect of ES on Kv1.3 channel suggested that ES might have therapeutic effect on autoimmune diseases.Using the cerebral spinal cord homogenate of Sprague-Dawley rats to immunized themselves,present work successfully constructed the experimental autoimmune inflammatory disease?EAE?rat model which were used to evaluate the therapeutic effect of ES by behavioral score.The results showed that ES reduced the behavioral scores of EAE rats,and effectively alleviated the clinic condition of EAE rats,which suggested the potential therapeutic effect of ES on autoimmune diseases.From molecular,cellular and animal level,this research revealed that Kv1.3 channel mediated the immunosuppression of sanguis draxonis.In the future,we will further optimize the structure of loureirin B to develop its desirable derivatives with stronger inhibiting effect and better selectivity on Kv1.3 channel.Also,to further verify the role of ES in the treatment of autoimmune diseases,we will use morphological and immunological indexes to evaluate the therapeutic effect of ES on EAE rats.