Study On The Effect And Mechanism Of The Novel Chalcone Derivative MW-9 On Experimental Autoimmune Encephalomyelitis

Purpose:Based on the screening results of the anti-inflammatory activity in the early stage of our research group,the anti-inflammatory effect of MW-9 and the mechanism of prevention and treatment of multiple sclerosis were further revealed in the preliminary study of novel chalcone derivatives,which it provided scientific basis for the development of a new chalcone derivatives MW-9 candidate drug with independent intellectual property rights.Method:1.In vitro study,the mouse macrophage cell line Raw264.7 inflammatory cell model and primary macrophage model were constructed,MTT assay was used to detect the cytotoxic effect of MW-9 on inflammatory cells;Griss method was used to detect the effect of MW-9 on the production of NO in inflammatory cell model;and the phosphorylation of NF-?B signaling pathway induced by MW-9 was measured by Western blot assay.2.In vivo experiments,the model of experimental autoimmune encephalomyelitis（EAE）induced by MOG35-55 was established to assess the effects on EAE,the clinical score was used to the effects of MW-9 on EAE,and the degree of spinal cord pathological injury in mice was observed by HE staining.The concentration of IL-17A,IFN-?and TGF-?in the cell culture supernatant were detected by ELISA assay;The expression of cytokines and chemokines mRNA in spinal cord tissues of EAE mice were detected by q-PCR assay and Microar ray.Flow cytometry was used to detect the proportion of lymphocyte surface markers（CD25,CD44,CD11b,Gr1）and T cell subsets（Th17,Th1 cells and Treg cells）.3.In vitro Th17 differentiation experiment,CD4⁺T cell cells were sorted by immunomagnetic beads negative selection.Under the condition of Th17 polarization,the naive T cells were induced to differentiate into Th17 cells.After MW-9intervention,the expression of Th17 cells（CD4⁺IL-17A⁺）was detected by flow cytometryResult:1.The results of in vitro experiments showed that the new chalcone derivative MW-9 significantly inhibited the production of NO,and it’s CC₅₀and IC₅₀ were11.24?g/ml and 5.37?g/ml,respectively.Western blot assay showed that MW-9 drug intervention significantly inhibited the phosphorylation of NF-?B.2.Compared with the model group,MW-9 significantly decreased the clinical score of EAE mice,improved the pathological damage and inflammatory cells infiltration of spinal cord lesions;The results of ELISA assay showed that the levels of IFN-?and IL-17A in the lymphocyte culture supernatant in the MW-9 intervention group were lower than those in model group,and the difference of IL-17A was significant（P<0.05）.The results of Flow cytometry showed that MW-9 treatment significantly decreased the proportion of CD11b⁺Gr1⁺neutrophils（P<0.05）,and significantly down-regulated the expression of Th17 cells（CD4⁺IL-17A⁺）（P<0.05）.However,there was no significant effect on the proportion of Th1 cells(CD4⁺IFN-?^（10）)and Treg cells（CD4⁺Foxp3⁺）（P>0.05）.3.The results of microarray showed that the cytokines and chemokines in the spinal cord of the MW-9 intervention group were significantly down-regulated in the model group.The results of real-time PCR showed that the mRNA expression of inflammatory cytokines and chemokines in the spinal cord of mice in MW-9intervention group was lower than that in the model group,and the transcription factors STAT1,STAT4 and CCR2 were significantly different（P<0.05）.4.MW-9 drug intervention significantly inhibited the differentiation of naive T cells into inflammatory Th17 cells,and the difference was significant（P<0.05）.Conclusion:1.The novel chalcone derivative MW-9 has significant anti-inflammatory effect,and its mechanism may be related to the inhibition of NF-?B pathway phosphorylation.2.The novel chalcone derivative MW-9 has significant anti-multiple sclerosis effect,and its mechanism may be related to inhibition of Th17 cell differentiation and down-regulation of chemokine expression.