Isolation,Purification And Functional Verification Of Immunosuppressive Components Of Capparis Spinosa L.

Autoimmune diseases(AID)are immune disorders caused by various causes,which cause an immune response to autoantigens and cause multiple system damage.It is a chronic inflammatory disease that causes great harm to people’s health.Capparis spinosa L.is a medicinal plant commonly used in the treatment of rheumatoid arthritis and gout and other immune diseases.Dendritic cells(DCs)act as professional antigen presenting cells in the body,and their maturation status and type of secreted cytokines determine the nature of the immune response.The ethanol extracts of Capparis spinosa fruits were fractionated by organic solvent,and further purified by cation exchange resin column and Sephadex-LH20 column chromatography to obtain CSFA and CSES.The effect of CSFA and CSES on the maturation of murine bone marrow-derived dendritic cells(DCs),including the effect of alone or combination with lipopolysaccharide(LPS)on the expression of surface molecules and cytokine secretion of DCs;DC incubated with fluorescently labeled dextran(FITC-dextran)and detect the effects of co-treatment of CSFA(C.spinosa fruit alkaloids)、CSES(C.spinosa fruit ethanol extracts,by Sephadex-LH20)and LPS on DC phagocytosis;DC morphology was observed by inverted microscope;Na?ve mice were injected with CSES and LPS by footpad,24 hours later,blood was collected from the eyeballs,draining lymph nodes were isolated,the DC maturation and cytokines were detected by flow cytometry and ELISA;the changes of DC-activated T cells were detected by mixed lymphocyte reaction(MLR).Western blot assay was performed to analyze the activation of key molecules in mitogen-activated protein kinases(MAPK)and nuclear factor-kappa B(NF-κB)signaling pathways;HPLC-MS identified major compounds in CSES and analyzed the types of compounds that may have immunosuppressive effects.The major results were as the following:(1)CSFA and CSES significantly suppressed lipopolysaccharides(LPS)-induced DC maturation.2 mg/mL and 3 mg/mL CSFA combined with 40 ng/mL LPS significantly reduced the expression of DCs surface molecules CD80,CD86,MHC IIand secretion of cytokines TNF-α,IL-12p40;1 mg/mL,2 mg/mL and 3 mg/mL CSFA significantly increased the secretion of IL-10;1 mg/mL and 1.5 mg/mL CSES combined with 40 ng/mL LPS significantly decreased the expression levels of DC surface molecules CD40,CD80,CD86,MHC II and secretion of cytokines TNF-α,IL-1β,but increased IL-10 secretion and the effects were more significant than the aqueous fraction.(2)CSFA and CSES combined with LPS to treat DCs showed small,round morphology and less synaptic compared to untreated DCs,which is typically immature DC morphology,while significantly increasing the ability of the ability of phagocytosis,significantly inhibiting the ability of DCs to stimulate T cell proliferation.(3)CSES significantly inhibited LPS-induced DC maturation in vivo,decreased secretion of IL-6 and TNF-α in serum,increased secretion of IL-10,and the phosphorylation of key molecules in MAPK and NF-κB signaling pathway induced by LPS were inhibited by CSES.(4)The flavonoids,erpenoids,phenols and organic acids in CSES were identified by HPLC-MS.Among them,flavonoids,terpenes and organic acids may have anti-inflammatory and anti-immune effects.In summary,CSES and CSFA inhibit LPS-induced DC maturation in vitro.CSES inhibit LPS-induced DC maturation in vivo,and CSES significantly reduced the ability of LPS-induced DC to stimulate the proliferation of T cells.DC maturation induced by LPS was inhibited by decreasing the activation of key molecules in MAPK and NF-κB signaling pathways.This results provide a theoretical basis for the development of a new safe and efficient immunosuppressive agent.