| Objective(s):Patchouli alcohol(PA)is the main medicinal ingredient of the Chinese herb Pogostemon cablin(Blanco)Benth,a tricyclic sesquiterpenoid with immunomodulatory,anti-inflammatory and other biological activities.To elucidate the therapeutic effects and mechanisms of PA in rheumatoid arthritis(RA).We will investigate in vivo the effect of PA on the improvement of collagen-induced arthritis(CIA)in mice;in vitro the effect of PA on the regulation of inflammatory factor secretion in synovial fibroblasts(MH7A);and the effect of PA on the regulation of bone marrow-derived macrophage(BMDM)polarization.Method(s):Male C57BL/6 mice were used to establish CIA model.The mice were divided into blank group(Normal),model group(Vehicle)and treatment group(PA).HE staining and toluidine blue staining were performed to observe the histopathological changes of the ankle joints of mice in each group.The relative concentration of anti-collagen II(CII)antibody in plasma was determined by ELISA.The expression of ankle inflammatory factors,NF-κB pathway-related proteins,MAPK pathway-related proteins and macrophage polarization-related proteins was detected by WB.2.MTT assay was used to detect the effects of different doses of PA on the viability of MH7 A cells.Cell mobility was measured by wound healing assay.MH7 A cells were treated with TNF-α and gradient concentration PA and divided into blank group,model group and different dose groups of PA,then The expression of ankle inflammatory factors(IL-6,TNF-α),NF-κB pathway-related proteins was detected by WB 3.M-CSF was used to induce BMDM,and MTT assay was used to detect the influence of different doses of PA on the viability of BMDM.After PA interfered with LPS treated BMDM,detected the expression of polarimetric related proteins in macrophages by WB.Result(s):1.PA reduced the degree of ankle swelling,clinical scores and splenic index in CIA mice,and PA treatment decreased cartilage damage and inflammatory cell infiltration in the ankle joints of CIA mice;PA reduced plasma levels of anti-CII-specific Ig G,Ig G2 a and Ig M antibodies in CIA mice(P<0.05);PA administration inhibited IL-6,TNF-α in the ankle joints of CIA mice,and PA administration inhibited macrophage-associated factor NOS2,CD11 b,and increased CD163 expression in the ankle joints of CIA mice.Further PA administration inhibited the expression of NF-κB signaling pathway proteins p-IκBα,p-p65,MAPK signaling pathway proteins p-JNK,p-ERK1/2,p-p38 in the ankle joints of CIA mice(P<0.05).2.MTT assay results showed that compared with blank group,there was no significant difference in the activity of MH7 A cells treated with PA in the concentration less than 100μmol/L;PA inhibited migration of MH7 A cell induced by TNF-α.Compared with blank group,the levels of inflammatory cytokines IL-6 and TNF-α in MH7 A cells were increased in model group.Compared with model group,the levels of IL-6,TNF-α and p-IκBα and p-p65 in MH7 A cells treated with PA(5,10,20,50μmol/L)were decreased(P<0.05).3.Bone marrow cells were induced to differentiate into macrophages(BMDM)by M-CSF.MTT assay showed that PA had no significant toxicity to BMDM when concentrations were less than100μmol/L,and WB experiment showed that PA could inhibit NOS2 expression and increase CD206 expression in BMDM stimulated by LPS.Conclusion(s):PA can regulate humoral antibody production in CIA mice,regulate the polarization level of ankle joint macrophages,inhibit the activation of NF-κB and MAPK signaling pathways,reduce the level of inflammatory factors,and alleviate the inflammatory injury of CIA mice.PA can inhibit MH7 A migration,reduce the expression of inflammatory factors and inhibit the activation of NF-κB signaling pathway in MH7 A cells.PA inhibited the polarization of M1 macrophages induced by LPS.The results in vivo and in vitro indicated that PA had anti-inflammatory and immunosuppressive effects on CIA,which provided a data reference for further study on the effect of PA on RA. |
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