The Mechanisms Of YPFS Attenuating Th2 Cell-mediated Allergic Inflammation Based On The Signal Pathway Of TSLP

Allergic diseases,referred to as hypersensitivity disease becomes worldwide public concerned health problem.With the development of modern industry and the pollution of the living environment,the prevalence of allergic diseases has progressively increased over recent decades.It is difficult to cure completely because of its recurrence,so it becomes a research hotspot in recent years.Yupingfengsan(YPFS),mainly used for cold,is also used in the treatment of allergic diseases such as asthma,allergic rhinitis and urticaria etc.Previous studies of our laboratory have shown that the extract of YPFS can inhibit Th2-type allergic contact dermatitis induced by FITC,and referring to researches in the recent years,we found that thymic stromal lymphopoietin(TSLP)is a newly discovered important initiation factor of Th2-type allergic diseases.This enlighten us to establish a mouse model of TSLP production based on Th2 cell-mediated allergic inflammation to further study the effect of YPFS on the initial stage in Th2-type allergic inflammation,and to explorer regulation mechanism of YPFS on TSLP in Th2 cell-mediated allergic inflammation.Methods and Results:1.Establishment of animal models:Th2-type allergic contact dermatitis mouse models(ACD model):The mice were topically sensitized on the shaved abdomens with 1.5%FITC solution on dayO and day1(induction phase),on day6 the mice were elicited on its ears skin with 0.5%FITC solution(excitation phase),and the ear swelling was measured to evaluate the allergic inflammationA mouse model of TSLP production based on Th2 cell-mediated allergic inflammation(TSLP production model):the skin of the ear was sensitized on two consecutive days with 0.5%FITC solution causing the production of TSLP.The purpose of successfully establishing TSLP production model is to provide a reliable method for exploring mechanisms of drugs at the initial stage of Th2-type allergic diseases.Different from the mouse ACD mode,ear thickness of TSLP production model appeared no statistically increase,and the thymus index and spleen index had no detectable changes as well.This indicates that at initial stage of Th2 cell-mediated allergic inflammation,characterization of inflammation is not yet clear,and immune organs(thymus,spleen)are not yet involved in ACD pathological process,or the change can not be observed from its index.While the level of TSLP was significantly increased at the initial stage of sensitization,and its receptor TSLPR also showed high expression.Therefore a mouse model of TSLP production was successfully established based on Th2 cell-mediated allergic inflammation.2.The effect of YPFS on TSLP at initial stage of Th2 cell-mediated allergic inflammation.In this part we observed the effect of YPFS on TSLP at initial stage of Th2 cell-mediated allergic inflammation.By using ELISA,Real-time PCR and Western-blotting,the mRNA and protein levels of TSLP and its receptor TSLPR were examined respectively.The main purpose of this part is to explore whether YPFS extract can regulate the production of TSLP and its receptor TSLPR.YPFS could inhibit the production of TSLP at initial stage of Th2 cell-mediated allergic inflammation.TSLP protein and mRNA levels in the mouse ear tissue were significantly downregulated,and also there was a marked downregulating effect on TSLPR mRNA and protein levels;These showed that YPFS had inhibitory intervention role at initial stage of Th2 cell-mediated allergic inflammation.3.The effect of YPFS on TSLP related pathway factors at initial stage of Th2 cell-mediated allergic inflammationThe purpose of this part is to explore the pathway of YPFS regulateing TSLP expression.We mainly observed the effect of YPFS on TSLP production pathway,using Real-time PCR and Western-blotting method,investigating the effect of YPFS on TSLP production related factors,including TLRs?NF-?B and TLRs adapter proteins,which are MyD88 and TIRAP.Furthermore,the expression of Notch signaling pathway,which believed to negatively regulate TSLP production,were examined.MRNA expression of TLR2,TLR3,TLR4 and TLR8 in ear tissues of TSLP production model were significantly downregulated by YPFS,and mRNA expression of TLR9 and NF-?B also appears a downward trend,although there is no statistically difference.Protein expression of the adapter protein of TLRs(TIRAP and MyD88)also appears a downward trend.YPFS could downregulate mRNA expression of Notchl in ear tissues,upregulate its protein expression;and could upregulate mRNA expression of Jagged1,but have no significant effect on protein expression of Jagged1.Conclusion:The inhibition of YPFS on Th2-type allergic inflammation might be associated with the regulation of TSLP expression which can trigger Th2-type inflammation.Its mechanism maybe related with downregulating the expression of TLRs and its adapter protein that can regulate expression of TSLP and TSLPR.