The Anti-inflammatory And Immunoregulatory Effect Of Bilabolide On Experimental Autoimmune Encephalomyelitis Mice

Objective:To investigate the anti-inflammatory,immunomodulatory effects on myelin sheath and oligodendrocytes of Bilabolide.Methods:1.C57BL/6 female mice were subcutaneously injected at four point with MOG_35-55plus CFA to establish EAE model.On days 3 to 27 post immunization?p.i.?,BB was intraperitoneally injected.Clinical scores were observed by two investigators at blind until day 27.Subsequently mice executed on day 28,splenic mononuclear cells?MNCs?were dissociated and cultured to investigate immune response.Spinal cords were removed to observe the pathological change of demyelination and inflammatory infiltration.Histochemical staining?H&E,LFB?was performed to observe the infiltration of inflammatory cells and demyelination;Immunofluorescence staining was performed to observe the infiltration of inflammatory CD4⁺T cells and CD68⁺macrophages,microglia activation and its phenotypic shift and the interaction of them as well as myelin sheath.ELISA was performed to exam the levels of pro-and anti-inflammatory cytokine expression?INF-?,TNF-?,IL-1?,IL-17,IL-6,IL-10 and TGF-??both in spinal cord and cultured MNCs.Western blot was performed to analyze the expression of related proteins from MNCs and spinal cord,including iNOS,Arg-1,TLR4,MyD88,NF-?B,Bax,Cleave-caspase-3 and Bcl-2.2.BV2 microglia induced by LPS were used to establish inflammatory cell model.After intervention by BB for 24 to 48 hours,cell activity and the expression of inflammatory cytokines?NO,TNF-?and IL-1??were observed by MTT,CCK-8 and ELISA assay,respectively.3.C57BL/6 female mice were injected subcutaneously with MOG peptide.After 9days immunized,the spleens were taken aseptically to isolate the spleen mononuclear lymphocytes for vitro studies.MOG-specific encephalitis lymphocytes were stimulated by MOG_35-55 for 48 hours,cell viability was detected by MTT.The secretions of pro-or anti-inflammatory cytokines,including INF-??TNF-??IL-1??IL-17?IL-6?IL-l0 and TGF-?,were determined by ELISA.The expressions of TLR4,MyD88 and NF-?B were observed by Western blot.Results:1.Compared with the EAE group,BB alleviated the severity of disease in EAE mice.The mean times of onset in EAE group is 11.000±0.436 and BB group is 13.710±0.738,which showed that BB can delay the onset of disease in EAE mice?p<0.01 vs.EAE group?.H&E and LFB staining showed infiltration of inflammation cells in EAE mice was 1367±173.800 and accumulated optical density of myelin sheath was 64053±6529,which was was 176.500±49.870 and 80013±10470 in BB?20mg/kg?treatment group,respectively?p<0.001 and 0.01?.These indicated that BB reduced the infiltration of spinal inflammatory cells and the loss of myelin sheath.reduces infiltration of inflammatory cells and myelin loss in CNS.2.A large number of CD4⁺T cells?147.300±33.500?and CD68⁺macrophages?1226±160?were found in the spinal cord of EAE mice,while CD4⁺T cells?32.670±8.838?and CD68⁺macrophages?139.500±18.860?were significantly reduced in the BB group?p<0.001 and 0.01,respectively?.BB can reduce the infiltration of CD4⁺T cells and CD68⁺macrophages in the spinal cord of EAE mice.Compared with the model group,BB inhibited the activation and proliferation of peripheral lymphocytes in presence of MOG?p<0.001?and production of inflammatory cytokines including INF-??TNF-??IL-17?IL-6 and iNOS in peripheral lymphocytes?p<0.05?,but increased the expression of anti-inflammatory cytokines IL-10 and Arg-1?p<0.001?.3.BB can inhibit the TLR signaling pathway and expression of TLR-4,MyD88 and p-NF-?B in EAE mice.The relative expression of TLR-4 in the EAE group was 0.788±0.095,MyD88 was 0.377±0.024,and p-NF-?B was 0.265±0.028.Correspondly,BB treatment groups were 0.542±0.096,0.148±0.027 and 0.081±0.012,respectively?p<0.01,respectively?.The expression of TNF-?and IL-?was decreased by BB treatment?p<0.05?.At the same time,BB promoted the expression of anti-inflammatory cytokines IL-10and TGF-??p<0.05?.4.In addition,BB promoted the transformation of activated microglia from M1-type to M2-type.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?.The number of Iba-1⁺iNOS⁺cells in the EAE group were 55.670±5.783,and that in the BB group was 28±2.082?p<0.01?.The number of Iba-1⁺Arg-1⁺cells in the EAE group were 22.330±4.702,and that in the BB group were 40.330±6.333?p<0.05?.5.BB reduced the release of inflammatory cytokines NO,TNF-?,and IL-?in LPS-induced BV2 microglia?p<0.05?,and promoted cell proliferation?p<0.05?.6.BB inhibited the production of apoptotic proteins Bax and Cleave-Caspase-3?p<0.01?,but increased the expression of anti-apoptotic protein Bcl-2?p<0.05?and reduced myelin loss?p<0.01?.The number of CD4⁺C-Caspase-3⁺cells in the EAE group were95.670±12.440,and that in the BB group were 8.667±2.028?p<0.001?.BB significantly promoted the survival of CNPase⁺oligodendrocytes by inhibiting the expression of apoptotic proteins and upregulating the expression of anti-apoptotic proteins?p<0.01,respectively?.7.Compared with the model group,BB inhibited the proliferation of MOG-specific encephalitis lymphocytes?p<0.05?.The expression of NO,IFN-?,IL-?,IL-6 and IL-17was inhibited?p<0.05?.And BB inhibited the inflammatory signaling pathway of TLR-NF-?B.Conclusion:BB could inhibit inflammatory reaction which includes down-regulating pro-inflammatory cytokines and up-regulating anti-inflammatory cytokines,the proliferation of myelin specific lymphocyte and modify peripheral immune response.BB could also inhibit the microglia activation and regulate its phenotype.Especially BB protects myelin sheath through decreasing apoptosis proteins.And BB may have certain potential of playing a positive role for the treatment of multiple sclerosis.