Study Of Inhibitory Effects Of The Matrine Derivate MASM On Astrocyte Reactivity And Experimental Autoimmune Encephalomyelitis In Mice And Its Mechanism

Objective:In this study,we investigated the effect of MASM on AST activation in vitro and on autoimmune encephalomyelitis model mice in vivo,and further explored underlying mechanism,providing an experimental basis for clinical application.Methods:(1)Culture and identification of primary mouse astrocytes: The brains of the neonatal mice were digested by 0.25% trypsin and 1%DNase I within 3 days.After filtering and centrifuging,the supernatant was discarded.The cells were re-suspended and inoculated in polylysine-coated cell culture flask and then incubated at 37℃ in a incubator with 5% CO2.With fluid change every 3 to 4 days,the cells fused into monolayer.Finally,Real-time PCR and immunofluorescence were used to identify the purity of these cells.(2)Cytotoxicity AST activity was detected by Cell Counting Kit-8(CCK-8)method to determine the range of MASM concentration;(3)The AST is divided into the following groups: Control group,model group,5μM MASM group,10μM MASM group,20μM MASM group,Control group without any stimulant,the other four groups were treated with 100ng/ m L Lipopolysaccharide to induce AST activation.(4)The effect of LPS induced AST release of cytokines,m RNA levels of IL-6,TNF-α,IL-1β,MCP-1 and i NOS and IL-10 were detected by real-time PCR.(5)TNF-α and NO content in cell supernatants were quantified by ELISA and Griess assay kit.(6)The effect of astrocyte conditioned medium on the migration of mononuclear macrophages was determined by Boyden chamber.(7)m RNA levels of marker genes H2-T23,H2-D1 and Serping1 of A1 astrocytes were detected by real-time PCR.(8)Fluorescence Aβ42 phagocytic assay was used to detect the changes of AST phagocytic ability.(9)m RNA levels of AST phagocytic receptors Mertk,Megf10 and Ax1 were detected by real-time PCR.(10)Thbs1,Sparcl1,Gpc4 and Gpc6 of AST synaptic factors,m RNA levels were detected by real-time PCR.(11)EAE model induced by MOG35-55: complete culture medium made of MOG35-55 was injected subcutaneously at 4points on the spine of mouse,and pertussis toxin was injected intravenously into the tail of mice on day 0 and day 1.From day 8,MASM(3mg/kg)was intragastric administration daily until the end of the experiment.Daily scores and body weight were recorded.(12)Hematoxylin-eosin staining(HE)was used to check the pathological changes in the spinal cord.(13)Luxol Fast Blue staining(LFB)was used to observe the changes of demyelination in the spinal cord.(14)Bioeischowsky silver staining was used to detect the loss of axons in the spinal cord tissue.(15)m RNA levels of TNF-α,IL-6,IL-1β,IL-10,Mc P-1 and i NOS in the brain and spinal cord were detected by real-time PCR.(16)The expression of Glial fibrillary acidic protein(GFAP)in the brain and spinal cord was detected by immunohistochemistry.(17)GFAP protein expression levels in spinal cord and brain tissues were detected by Western blot.(18)The expression of Aquaporin 4(AQP4)in spinal cord was detected by immunofluorescence.(19)The expression levels of C3 and GFAP in spinal cord and brain tissues were detected by immunofluorescence double-staining.(20)The expression of P65,P-P65,STAT3 and P-STAT3 in AST was detected by Western blot.(21)p-P65 and P-STAT3 in spinal cord were detected by immunohistochemistry.Results:1.MASM inhibits LPS induced astrocyte inflammation:MASM could decrease the m RNA levels of TNF-α,IL-6,IL-1β,IL-10,MCP-1 and i NOS.MASM concentration-dependent reduced AST releases to TNF-α and NO.MASM attenuated the migration effect of ACM on RAW246.7 cells.2.MASM inhibits AST differentiation:MASM inhibited the m RNA levels of A1 astrocytes marker genes H2-T23,H2-D1 and Serping1,they were induced by LPS,TNF-α,IL-1α and TNF-α combined with IL-1α.3.MASM ameliorates the phagocytosis and pro-synapse function of AST activization:MASM improved the phagocytosis of AST in a concentration-dependent way.MASM concentration-dependent improved m RNA levels Mertk.MEGF10 and Axl.MASM increased the m RNA levels of Thbs1,Sparcl1,Gpc4 and Gpc6 in a concentration-dependent way.4.MASM(3 mg/kg)can effectively ameliorates EAE:MASM(3 mg/kg)can effectively improve EAE clinical score and weight loss;11.MASM obviously ameliorates the inflammatory reaction 、 demyelination and axon loss of EAE.MASM decreased the m RNA levels of inflammatory mediators in the brain and spinal cord of EAE mice.5.MASM inhibits the activation of astrocytes in spinal cords and improves the functions of BBB:MASM administration remarkably decreased the GFAP-positive astrocytes and inhibited morphological changes in the brain and spinal cord.MASM reduced the expression of GFAP in the brain and spinal cord.MASM reduced AQP4 expression in EAE mice.MASM reduced the number of A1 type astrocytes in EAE mice.6.MASM inhibited the phosphorylation of NF-κB and STAT3: MASM inhibited the increase of p-P65 and P-STAT3 at the cellular level and the spinal cord of EAE mice.Conclusion:MASM can inhibit the activation of AST,reduce the release of cytokines,inhibit the transformation of A1 type astrocytes,increase the expression of synaptic forming factors and phagocytic receptors,and restore the impaired phagocytic ability of AST.MASM inhibits inflammation,alleviates demyelination and axon loss,and ameliorates hind limb paralysis of EAE.MASA may inhibit NF-κB pathway and AST activation to prevent A1 formation by which myelin demyelination was inhibited or myelin regeneration was promoted,thereby improving the effect of EAE.