Xanthatin Reduces Allergic Inflammation By Inhibiting TSLP And NK Cell Differentiation

Xanthatin,a main component of Xanthium sibiricum Patrin ex Widder,is mainly used for the treatment of cold,rheumatism,rubella and itching,especially in the treatment of allergic rhinitis.Previous research data suggested that xanthatin possessed anti-tumor and anti-bacterial efficacy while its anti-allergy activity remains to be further investigated.Meanwhile,the growing incidence of allergic diseases urges us to develop potent therapeutic approaches against them.Therefore,this study aimed to explore the role of xanthatin in allergic diseases and its mechanism on allergic inflammation,through which we might lay the foundation for further research on allergy.Based on previous study,this study mainly focuses on the following aspects:1.The effect of xanthatin on allergic inflammationAn allergic contact dermatitis(ACD)mice model was induced by FITC,and different concentrations of xanthatin were administered to investigate the efficacy of this ingredient.The results showed that xanthatin could significantly inhibit ear swelling,alleviate ear pathological changes in the ACD model,and reduce the levels of IL-4,IL-13 and IL-5 in ear homogenate.Mice model of Th2 allercgic asthma was induced by house dust mite(HDM).Eosinophil(EOS)in peripheral blood(PB),serum IgE,and the levels of IL-4?IL-5,IL-13 in bronchoalveolar Lavage Fluid(BALF)and lung homogenates were detected to investigate the efficacy of xanthatin.The results showed that xanthatin could markedly inhibit the number of EOS in mice PB and the production of IgE in serum.Pathological sections of the lung portrayed that xanthatin could remarkably alleviate inflammatory cell infiltration.The type 2 cytokines IL-4,IL-5 and IL-13 in BALF and lungs also significantly reduced after the administration of xanthatin.2.The effect of xanthatin on three main potential cells involved in allergic inflammationAfter demonstrating the effects of xanthatin on allergic inflammation in vivo,we further explore the underlying mechanism of it.Three cells that are mainly involved in allergic inflammation:murine macrophage RAW264.7,mouse spleen lymphocytes and epithelial cells were screened.The effect of xanthatin on the proliferation of these cells and the production of TNF-a after stimulation of LPS was detected by MTT and ELISA,respectively.The proliferation of xanthatin on mouse splenic lymphocytes and ConA and LPS-induced lymphocytes were detected by MTT assay;Poly(I:C)&TNF-a were applied to induce TSLP production in 16HBE and HaCaT cells,and the effect of xanthatin on TSLP production in epithelial cells was observed.The results showed that xanthatin reduced the production of LPS-induced TNF-a in RAW264.7 cells,but it had no significant effect on the proliferation of T lymphocytes and B lymphocytes in the spleen of mice under activated condition.In 16HBE and HaCaT cells,xanthatin could decrease the expression of TSLP induced by poly(I:C)&TNF-a stimulation.In addition,the potent concentration of xanthatin to down-regulate epithelial cell-derived TSLP is lower than that to exert its anti-TNF-? efficacy in RAW264.7,suggesting that epithelial cells might be the main effector cells of xanthatin.3.Changes of key pro-inflammatory cytokines and NK typing in the early stage of allergic diseaseBecause xanthatin has a significant inhibitory effect on epithelial cells-derived key pro-inflammatory cytokine TSLP in vitro,we subsequently established an initial stage of HDM-induced mice allergic asthma and observed the inflammatory changes and the expressions of pro-inflammatory cytokines.And the results showed that xanthatin administration significantly alleviated the inflammatory changes,and markedly reduced the expressions of TSLP and IL-33 in the lungs and BALF of mice.Intriguingly,the proportion of NK2 in the lung tissue of model mice increased,suggesting that bronchial epithelial cells might produce a large number of pro-inflammatory key cytokines,and induce NK cells to transform into NK2 type subsequently.4-Effect of TSLP on NK cells differentiation and the role of xanthatin on itA change in NK typing was observed when bronchial epithelial cells produced high levels of pro-inflammatory cytokines TSLP and IL-33 in vivo,indicating that the pro-allergic priming factor might induce NK cell differentiation.To verify this,we observed the effects of IL-33 or TSLP on NK cell phenotype transformation in mouse peripheral blood mononuclear cells(PBMC)and NK92MI cells.The effects of xanthatin in NK92MI and mouse PBMC on NK cells and their phenotypic transformation were observed under TSLP stimulation.And the results showed that different concentrations of IL-33 induced negligible NK cells typing change.In sharp contrast,different concentrations of TSLP significantly elevated IL-13 expression in NK92MI cells.And particularly,NK2 subset was dominant after the stimulation of TSLP at 20 ng/mL.NK and NK2 ratios were also up-regulated after stimulation with TSLP in mouse PBMC while NK1 subset showed a downward trend.Incubation of xanthatin with TSLP-stimulated NK92MI prominently decreased IL-13 levels and increased IFN-y levels,which further induced NK cells to differentiate into NK1 type.Besides,xanthatin reduced the percentage of NK cells in mouse PBMC stimulated by TSLP,decreased the proportion of NK2 and increased the proportion of NKl.To investigate the mechanism of TSLP-induced NK cell typing,the effect of TSLP on the activation of STAT3 was explored in vitro,and its mechanism on NK typing was explored.It mainly included:(1)Exploring the changes of p-STAT3 under the stimulation of TSLP in NK92MI in different concentrations and at different time point,and the effect of xanthatin on it was also demonstrated.(2)The activation of p-STAT3 was inhibited,and the transformation of NK cells stimulated by TSLP in mouse PBMC was detected subsequently.The results indicated that the stimulation of TSLP at 100 ng/mL for 24h could markedly activate STAT3.And after the treatment of p-STAT3 inhibitor on PBMC 2h before TSLP stimulation,the NK2 differentiation induced by TSLP were significantly reversed.Conclusion:(1)Xanthatin could significantly alleviate mice allergic dermatitis and allergic asthma,which suggested that it possessed anti-inflammation efficacy in allergic diseases.(2)Epithelial-derived TSLP could induce NK cells to differentiate into NK2 subsets,and further secrete Th2 cytokines which promote inflammatory responses by activating STAT3.(3)Xanthatin could decrese the expression of TSLP and reverse TSLP-induced NK2-type transformation and exert anti-inflammatory efficacy.