Mechanism Of Baicalein In The Treatment Of Experimental Autoimmune Encephalomyelitis

Objective: Multiple sclerosis(MS)is an autoimmune disease of the central nervous system mediated by T cells.Experimental autoimmune encephalomyelitis(EAE)is the most common animal model used to study MS.Baicalein(BAI),the main component of Scutellaria baicalensis,is a dietary supplement,which has been proved to have anti-inflammatory,neuroprotective and immunomodulatory effects.This study was to explore the protective effect of BAI on EAE mice and its related mechanism.Methods: 1.Twenty C57BL/6 female mice were randomly divided into 4 groups:Control group,EAE group,EAE+BAI group and EAE+DEX group.The changes of body weight,neurological function score,pathological section staining and immunofluorescence were used to detect inflammatory cell infiltration and demyelination in the spinal cord of mice in each group.2.Immunofluorescence was used to detect the expression of i NOS,Iba-1 and Arg1 in the spinal cord of mice.The m RNA expression levels of i NOS,CD86,IL-18,IL-12p35 and IL-12p40 in spinal cord tissues of mice in each group were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction(q RT-PCR).Western blot was used to detect the expression of Iba-1 and i NOS in the spinal cord and the activation of STAT1 in mouse spleen and RAW264.7 cells after BAI treatment.The secretion of M1-related inflammatory factors in the supernatant of mouse spinal cord tissue /RAW264.7 cell line was detected by flow cytometry and mouse M1 macrophage kit.Detection of STAT1 activation in mouse spleen and RAW264.7 cells induced by BAI by Western blot.3.The binding ability of BAI to STAT1 and the effect of BAI on p-STAT1 signal transduction of macrophages during M1 polarization were detected by computer simulation of molecular docking,pull-down,Westernblot and immunofluorescence.4.Flow cytometry and q RT-PCR were used to detect the number of Th1/17 cells and the m RNA expression levels of related cytokines in EAE model mice after BAI intervention.Western blot was used to detect the expression of JAK/STATs signaling pathway related proteins and related proinflammatory cytokines.The binding ability of BAI to STAT1/3 and the effect of BAI on p-STAT1/3 signal transduction were detected by computer simulation of molecular docking,pull-down,Western blot and immunofluorescence.Results: 1.Compared with EAE group,the pathological injury of EAE+BAI group was slighter and the trend of weight loss was significantly improved.2.Compared with the EAE group,the number of Iba-1 positive microglia in the spinal cord tissue of EAE+BAI group was significantly reduced.The expression of i NOS was also weakened.The m RNA levels of i NOS,CD86,IL-12p35,IL-12p40 and IL-18 in the EAE+BAI and EAE+DEX groups were significantly lower than those in the EAE group.Compared with the EAE group,the protein expressions of Iba-1 and i NOS in the spinal cord tissues of EAE+BAI and EAE+DEX groups were significantly decreased.The secretion of CXCL1,IL-12p70 and IL-18 in the spinal cord of EAE group was significantly up-regulated,while it was significantly downregulated in the EAE+BAI group and EAE+DEX group.In vitro results showed that results showed that after BAI intervention,the morphological changes of cells induced by LPS were significantly inhibited.Flow cytometry showed that BAI inhibited the upregulation of mean fluorescence intensity and percentage of CD86.After LPS stimulation,the secretion of IL-1β,IL-6,IL-12p40,IL-12p70,IL-18 and TNF-α was significantly up-regulated,while BAI inhibited the LPS-induced secretion of pro-inflammatory factors.3.In vivo,p-STAT1 was up-regulated in spinal cord tissues of EAE mice,but significantly down-regulated in EAE mice treated with BAI.The results of in vitro experiments showed that LPS and IFN-γ could significantly up-regulate p-STAT1 in RAW264.7 cells,and BAI could significantly down-regulate the phosphorylation of STAT1 protein induced by LPS and IFN-γ.Molecular docking,pull-down assay and co-localization results showed that BAI could bind to STAT1.Moreover,it also inhibited the nuclear translocation of p-STAT1 in RAW264.7 cells.4.It was found that BAI can significantly reduce the percentage of Th1 and Th17 cells in vivo,while BAI reduce the m RNA and protein expression of IFN-γ and TNF-α,and the m RNA expression of IL-17 A in the spinal cord of EAE mice.The protein expression of p-STAT1 and p-STAT3 in spleen cells of EAE mice was significantly increased,and decreased after BAI treatment.Further study showed that the transcription levels of transcription factors T-bet and RORγt were also significantly decreased in EAE+BAI group,which may be caused by the inhibition of JAK-STAT1-mediated Th1 differentiation and JAK-STAT3-mediated Th17 differentiation by BAI.Through molecular docking,pull-down and immunofluorescence experiments confirmed that BAI had the ability to target STAT1/3 and immobilate p-STAT1/3 in the cytoplasm to block the transduction of JAK/STATs signaling,thereby inhibiting the inflammatory response of Th1/17 cells.Conclusion: To sum up,BAI treatment significantly improved the clinical symptoms and pathological changes of EAE mice.BAI effectively down-regulated the number of activated microglia / macrophages and inhibited their M1 polarization.In addition,BAI inhibited the secretion of inflammatory factors in Th1 and Th17 cells,so BAI could improve the symptoms of EAE mice.The protective effect of BAI on EAE mice may be through targeting STAT1 to inhibit the polarization of microglia /macrophages to M1 in EAE mice.On the other hand,it may inhibit Th1 and Th17 inflammation in EAE mice by targeting STAT1/3.