The Therapeutic Potential of Bilabolide on Experimental Autoimmune Encephalomyelitis(EAE) Mice

Background:Multiple sclerosis（MS） is a chronic immune-mediated inflammatory demyelinating disease of the central nervous system（CNS）. Migration and infiltration of activated CD4+ T cells and macrophages into the CNS,where together with tissueresident microglia,result in a neuroinflammation and severe demyelination of MS and its animal model experimental autoimmune encephalomyelitis（EAE）. Bilobalide（BB）,a novel compound isolated from Ginkgo biloba,effectively exhibited its neuroprotective capacity,including suppression of cell death and inflammatory inhibition. Here,we explored that BB had therapeutic effects in EAE model. Object:To investigate whether BB contributes to neuroprotection of myelin sheath and whether it has a capacity of immunomodulatory. Methods:EAE was induced by MOG35-55 in C57 BL/6 mice,which were treated with BB on day 3 post immunization until day 27. Clinical scores were observed and pathological changes of spinal cords were detected by Luxol fast blue（FLB）,hematoxylin/eosin（H&E） staining and immunohistochemical method. The expression of relevant cytokines was detected by ELISA. Western blot was performed to detect the levels of iNOS,Arg-1,TLR4/MyD88/NF-kB,etc. Splenic mononuclear cells were isolated from spleens of EAE mice on day 9 after immunization with MOG35-55 and re-stimulated with MOG35-55 peptides,which were then treated with/without BB followed by an observation of flow cytometry and ELISA. Results:Compared with the EAE group,BB alleviated the severity of disease in EAE mice. The mean onset days in EAE group were（11.00±0.44） and that in BB group were（13.71±0.74）,which showed that BB could delay the onset of disease in EAE mice（P<0.01 vs. EAE group）. H&E and LFB staining showed that the infiltration of inflammation cells in CNS was（1 367±173.8） and accumulated optical density of myelin sheath was（64 053±6 529）,which was（176.5±49.87） and（80 013±10 470） in BB（20 mg·kg-1） treatment group（P<0.001 and 0.01,respectively）. CD4+ T cells（147.30±33.50） and CD68+ macrophages（1 226±160） were found in the spinal cords of EAE mice,while BB significantly reduced infiltration of CD4+ T cells（32.67±8.84） and CD68+ macrophages（139.50±18.86）,P<0.001 and 0.01,respectively. Compared with the EAE mice,BB inhibited the activation and proliferation of peripheral lymphocytes re-stimulated by MOG35-55（P<0.001） and production of inflammatory cytokines INF-γ,TNF-α,IL-17,IL-6 and iNOS（P<0.05）,but increased the expression of IL-10 and Arg-1（P<0.001）. The levels of TLR-4 was（0.79±0.10）,MyD88 was（0.37±0.02）,and p-NF-κB was（0.26±0.02） in CNS in EAE group. Correspondingly,BB group was（0.54±0.09）,（0.14±0.02） and（0.08±0.01）(P<0.01,respectively). BB decreased the expression of TNF-α and IL-1β（P<0.05,respectively） and promoted the expression of IL-10 and TGF-β（P<0.05,respectively） in spinal cords. In addition,compared with the EAE group,BB significantly inhibited the expression of iNOS（P<0.001） and increased the expression of Arg-1（P<0.05） in CNS. The number of Iba-1+iNOS+ cells in the EAE group was（55.67±5.78）,and that in the BB group was（28±2.08）(P<0.01). The number of Iba-1+ Arg-1+ cells was（22.33±4.70） and（40.33±6.33）(P<0.05) in each group respectively. And BB also down-regulated inflammatory cytokines NO,TNF-α,and IL-1β in LPS-induced BV2 microglia（P<0.05）,and promoted BV2 cell proliferation（P<0.05）. What's more,the number of CD4+ Cleave-caspase-3+ cells in the EAE group was（95.67±12.44）,and that in the BB group was（8.66±2.02）(P<0.001). BB inhibited the production of apoptotic proteins Bax and Cleave-Caspase-3（P<0.01）,but increased the expression of antiapoptotic protein Bcl-2（P<0.05） and reduced myelin loss（P<0.01）. Furthermore,in MOG recall experiment,compared with EAE group,BB inhibited CD4+IFN-γ+,CD4+IL-10+ and CD4+TGF-β+ T cells（P<0.01,0.1 and 0.01,respectively）,but without effects on CD4+IL-17+ and CD4+CD25+ T cells. Meanwhile,BB increased CD11 b+CD16/32+ and CD11 b+IL-10+ macrophages（P<0.01 and 0.001,respectively） though without effects on CD11 b+IL-12+ and CD11 b+CD206+ macrophages. Similarly,data of ELISA showed that BB down-regulated the expression of INF-γ,IL-1β,IL-6 and IL-17（P<0.01,0.01,0.05 and 0.01,respectively）. Conclusions:BB can effectively alleviate clinical symptoms and reduce myelin loss in EAE model possibly by inducing reactive T cell tolerance,and inhibiting the infiltration of immune cells in spinal cord. Simultaneously,BB induces the polarization of M2 macrophage/microglia and inhibiting inflammatory responses. Ultimately,the improvement of inflammatory microenvironment mediated via BB treatment reduces oligodendrocyte apoptosis and protect neurons against inflammation. However,the accurate mechanism of BB action and the feasibility of clinical application in the prevention and treatment of demyelination remain to be further explored.