Target Identification And Validation Of Britanin As An Effective Prevention And Treatment For Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a central nervous system chronic inflammatorydemyelinating disease with a high recurrence, persistent, and disability, and the exactpathogenesis is not clear. Experimental autoimmune encephalomyelitis (EAE), which isthe simulation model of multiple sclerosis in rodents, can be used to study thepathogenesis of multiple sclerosis and the drug therapy. Because of the mechanism of MSis unknown, and lack of effective drug therapy, a clear mechanism for research and aneffective research for new drugs was urgent need.Britanin is a sesquiterpene lactone, which was isolated from Inula Britannica in theprevious studies in our lab. A lot of literatures show that this class of compounds has aclear anti-inflammatory activity in vitro; our previous screening for anti-NO productionalso shown that britianin could reduce the produce of Nitric oxide. We used Britanin forthe prevention and treatment of EAE, found that it has a better therapeutic effect fordelayed the occurrence and reduced the degree of severity of the disease.In spite of some studies about the mechanism of Britanin had been done, but therestill are many questions left to solve. In this study, we used affinity chromatography forsearching the target which britanin treat for EAE and uncovering the mechanism and thetarget.In this study, britanin was modificated, and two biotinylated products BX-4andBX-8was synthesized as affinity chromatography probes. Their chemical structures wereidentified by MS and NMR, and purity was analysised by HPLC, both of them met therequirements. Furthermore, we explored the synthesis route of the probes, found a fastand simple synthetic route. The products can be quickly obtained by this route.The probes were tested for inhibitory activities against LPS-induced NO productionin RAW264.7macrophages. We found that the activities of BX-4and BX-8weredecreased, their IC50values was5.82μM and23.00μM respectively, fell by8-fold and31-fold compared with Britanin (IC50=0.74μM). They inhibited NO production under5μM and10μM concentration, with statistical significance, shown that the probe can beused. At the same time we also found that a water solubility linker could make a moredegree reduction to ability. Cytotoxic experiments show that BX-4exists cytotoxic athigher concentrations, but under the concentration of5μM and10μM found no cytotoxicity, BX-8was not cytotoxicity even under high concentration. The studiesfurther exclude the influence of cytotoxic to the ability.According to the principle that the hydroxyl group on Britanin can couple with theresins, we used CNBr-activated Sepharose4B and Epoxy-activated Sepharose6B forexploring for affinity chromatography before we used the synthesized biotinylated probes.Because of the short linker of CNBr-activated Sepharose4B, and the solubility linker inEpoxy-activated Sepharose6B, we did not get a nice result in the end. This indicated thatthe linker was very important in this experiment. Based on the principle that avidincoated on the surface of magnetic beads can bind biotin on the probes by a high affinity,we fixed the probe on the beads surface. After incubated with cell lysate for enough time,and then washed to remove the non-specific protein repeatedly, the binding proteins wereeluted with denaturing conditions, separated by gel. We found8differences bands, andchose the5greatest difference bands for identification by mass spectrometry. Weselected6proteins from the identified proteins: Traf2and NCK-interacting protein kinase(TNIK), Serine/threonine-protein kinase N2(PKN2), Ras-related C3botulinum toxinsubstrate2, Myelin basic protein, Calmodulin, Protein MICAL-2for validation.The docking result shows that there is an interaction between the-methylene-γ-lactone in Britanin and kinase TNIK, PKN2. The kinase experiment wasused to verify the interaction; by the mothod of ATP competition and the ATPconcentration were9μM and20μM respectively. It shown a low activity, their IC50valueswere greater than10μM, which shown a weak or low affinity between Britaninand this two kinases. Vimentin, an interested protein, which was came from the proteomecomparison of band8, it is a cytoskeletal protein which associated with inflammation andimmune responses in recent studies. At the same time, it is a marker of EAE onset. Weused western blot to verify interaction between BX-4and vimentin, and this interactionwas blocked when pre-incubated the lysate with Britanin. Therefore, a further study wasneeded to investigate the interaction mode and biological function.The study was expected to provide effective small molecule for the treatment ofEAE, provide more theoretical basis for the pathogenesis of EAE, and provide moreevidences for the anti-inflammatory mechanisms of sesquiterpene lactones.