Research Of Saponin From Periploca Forrestii Schltr Ameliorates Oxazolone-induced Atopic Dermatitis Via Inhibiting Macrophage Activation

Objective Here,we aimed to assess the effects of Periploca forrestii schltr saponin（PFS）on oxazolone-induced atopic dermatitis（AD）and M2 macrophage polarization.Methods The PFS was identified by HPLC-MS/MS and the active ingredient periplocin was determined.In vivo experiment: AD model was established by repeated stimulation of the ears of BALB/c mice 34 d with oxazolone.From the 14 th day onwards,the positive drug group and the drug-administered group were given hydrocortisone（HC）and PFS for 20 days.Mice’s ear swelling thickness and clinically scored was measured during the modeling period.H＆E,toluidine blue and immunohistochemical staining（including Nitric Oxide Synthase（NOS）2 and Arginase（Arg）1）were detected in the histopathology of mice.The effects of PFS on the expression of inflammatory factors and macrophage-associated factors in AD mice were detected by ELISA,Western blot and RT-PCR.In vitro experiments:Macrophage colony-stimulating factor（M-CSF）was used to stimulate bone marrow-derived macrophages（BMDM or M2 macrophages）in bone marrow cells of BALB/c mice.LPS was used to induce M2 macrophages to be polarized into M1 macrophages.MTT assay was used to determine the effect of different doses of PFS on the activity of BMDM cells.Immunofluorescence staining and Western blot were used to detect the effect of PFS on the expression of phenotypic characteristic factors related to LPS-induced M2 macrophage polarization.Results: The results of HPLC-MS/MS detection showed that the active ingredient periplocin of PFS was 0.4%（g/g）.In vivo results: PFS significantly reduced the degree of ear swelling,clinical score,epidermal thickening and inflammatory cell infiltration in AD mice;PFS down-regulated the expression of NOS2 and Arg1 in AD mice ear tissue.PFS significantly decreased the expression levels of plasma Ig E and inflammatory mediators,and down-regulated the expression of inflammatory cytokines and macrophageassociated factor proteins and genes in AD mice.In vitro results: PFS had a certain proliferative effect on BMDM and did not produce cytotoxicity in 0.2-20μg/m L by the experiment;Immunofluorescence staining showed that LPS induced M1 macrophage differentiation and CD68 factor production,and intervented with the polarization of M2 macrophages to M1 macrophages.PFS inhibited the expression level of LPS-induced M2 macrophage polarization-associated proteins.Conclusions: In an oxazolone-induced AD mouse model,PFS treatment can improve AD inflammatory response.PFS produces anti-inflammatory effects by regulating inflammatory mediators and inhibiting the expression of related macrophage factors in AD mice.In vitro LPS-induced expression of related factors in M2 macrophage polarization to inhibit the production of inflammatory M1 macrophages.Therefore,PFS may be an effective drug to treat AD inflammation by regulating macrophage activation.