| Objective:This study was aimed to investigate the effects of Radix Astragali extract(RAE)and Total Flavonoids of Astragalus(TFA)on the pathogenesis of experimental autoimmune encephalomyelitis in mice,an anmimal model of multiple sclerosis(MS),and to explore the possible mechanisms,which may provide theoretical basis of Astragalus and its active ingredients in clinical MS treatment.Method:1.Extraction and detection of RAE: RAE was extracted by boiling method.The contents of total saponins,polysaccharides and flavonoids in RAE were determined by ultraviolet spectrophotometry with astragaloside IV,glucose and rutin used as reference standards.2.Establishment of EAE model,administration and detection of related indexes of pathological mechanisms: Female C57BL/6 mice were induced by MOG35-55 to establish experimental autoimmune encephalomyelitis(EAE)animal model.Mice were randomly divided into normal control group,model(EAE)group,RAE(125,250,500 mg kg-1)groups,total flavonoids of Astragalus(TFA 25,50,100 mg kg-1)groups,and positive group(methylprednisolone,MPD 20 mg kg-1).RAE/TFA groups were given respective dosages of drugs two days prior to EAE induction and continuously for 23 days.The neurological score and body weight were recorded in a double-blind manner;HE and LFB staining were conducted to assess the inflammatory infiltration and demyelination of spinal cord;IHC staining were used to detected the expression of Iba1 and GFAP protein in spinal cord;ELISA were used to detect the serum IL-6 and TNF-? levels;Flow cytometry was used to detect the percentage of CD4,CD11 b,CD11c,Th1,and Th17 positive cells in spleen;Quantitative-PCR method was used to analyze the expression levels of inflammation-related genes in spinal cord;Western blotting assay was performed to analyze the protein expression of NF-?B/MAPK signaling pathway protein,PI3K/AKT signaling pathway protein and apoptosis related protein in spinal cord.3.Establishment of BV-2 cell inflammation model,administration and detection of inflammation-related indexes: BV2 cells induced by LPS(200 ng/ml)were treated with TFA(100,200,300 ?g/ml)and CAL(25,50,100 ?M),respectively;Cell viability was detected by CCK-8 kit;Secretion of nitric oxide(NO)in the medium was detected by Greiss method;Cell proliferation was detected by Ed U staining;IL-6and TNF-? secretion was detected by ELISA kits;Gene expression of inflammatory factors was detected by quantitative-PCR method;Nucleus translocation of NF-?B was detected by ICC method;Transcriptional activity of NF-?B was detected by dual-luciferase reporter gene assay kit;Proteins involved in inflammation,NF?B signaling pathway and MAPK signaling pathway were detected by western blotting approach.Results:1.Effects and mechanisms of RAE on EAE mice: RAE could effectively improve the severity of EAE,reduce the weight loss,and delay the onset of the disease;RAE could effectively attenuate the inflammatory infiltration and demyelination of spinal cord in EAE mice;RAE could significantly decrease the secretion of IL-6 in EAE mice;RAE could significantly reduce the proportion of CD4,CD11 b,CD11c positive cells;RAE(125 mg·kg-1)could significantly down-regulate the protein expressions of NF-?B pathway,Iba1 and Bax,and up-regulate the protein expressions of cleaved-caspase3 and PI3K/AKT pathway in spinal cord.2.Effects and mechanisms of TFA on EAE mice: TFA could effectively improve the severity of EAE,reduce the weight loss,and delay the onset of the disease;TFA could effectively attenuate the inflammatory infiltration and demyelintion of spinal cord in EAE mice;TFA could significantly decrease the secretion of IL-6 and TNF-? in EAE mice;TFA could significantly mitigate the proportion of CD11 b,CD11c,Th1 and Th17 positive cells;TFA could significantly reduce the expression of Iba1 and GFAP in spinal cord.TFA(50 mg/kg)could significantly down-regulate the gene expressions of i NOS,COX2,IL-1?,TNF-?,and CD11 b.TFA(50 mg/kg)could significantly down-regulate the protein expressions of NF-?B pathway,i NOS,COX2,Iba1,and GFAP in spinal cord.3.Effects and mechanisms of TFA on BV-2 cells induced by LPS: TFA(100,200,300 ?g/ml)could significantly decrease the secretion of NO and it had no cytoxicity on BV2 cells;TFA could significantly decrease the secretion of IL-6 and TNF-? in cell supernatant;TFA could significantly down-regulate the gene expression of i NOS,COX2,IL-1?,TNF-? and CD11b;TFA(300 ?g/ml)could significantly inhibit the nuclear translocation and transcriptional activity of NF-?B;TFA could down-regulate the protein expression of i NOS,COX2,and the phosphorylation of AKT,JNK,and NF-?B pathway proteins.4.Effects and mechanisms of CAL on BV-2 cells induced by LPS: CAL(25,50,100?M)could significantly decrease the secretion of NO and inhibit the cell proliferation;CAL could dose-dependently decrease the secretion of IL-6 and TNF-? in cell supernatant;CAL could dose-dependently down-regulate the gene expression of i NOS,COX2,IL-6,TNF-?,and CD11b;TFA(100 ?M)could significantly inhibit the nuclear translocation and transcriptional activity of NF-?B;TFA could significantly down-regulate the protein expression of i NOS,COX2,and the phosphorylation of AKT,JNK,and NF-?B pathway.Conclusion:1.RAE could effectively relieve the severity of EAE mice,reduce the inflammatory infiltration and demyelination of the central nervous system.The action mechanism of RAE might be mediated through restraining the activation of peripheral inflammatory cells,suppressing neuroinflammation and inhibiting neuronal apoptosis.2.TFA could effectively alleviate the neurological damage in EAE mice,reduce the inflammatory infiltration and demyelination of the central nervous system.The action mechanism of TFA might be mediated through restraining the activation of NF-?B signaling pathway and peripheral inflammatory cells,and suppressing neuroinflammation.3.TFA and CAL could effectively alleviate the activation of BV2 cells induced by LPS,which was probably mediated through NF?B /MAPK signaling pathway. |
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