| The prevention and treatment of allergic diseases are the hot potato which are being discussed all over the world. Modern medicine has the advantage of alleviating the allergic symptoms, but it cannot prevent the recurrence. Yupingfengsan, a traditional Chinese prescription, can alleviate allergic symptoms effectively and reduce the risk of recurrence on clinical. Astragalus membranaceus (Astragalus membranaceus (Fisch.) Bunge) is the principal component in this formula. We found out that Calycosin, the main active part of Astragalus membranaceus, was able to inhibit the allergic inflammation. Moreover, the effect has kicked in just during the induction phase of delayed type hypersensitivity. Thus, the exploration of the anti-allergy function of Calycosin and the discovery of the underlying mechanism will bring great help for clarifying the active components and the function mechanism of Yu ping feng san one step further. This paper includes the following four parts:(1) The discovery of the regulation of TJs by TLRs:Pattern recognition receptors (PRRs) are the main cell molecules activator expressed in epithelial cells. TLRs, as a member of PRRs can activate inflammatory signals through different pathways and then accelerate the inflammatory progress by releasing inflammatory factors. We detected the expression of ZO-1 and Occludin, the key members of epithelial tight junctions (Tight Junction, TJ) after stimulating HaCaT cells with TLRs ligand for 24h. We also measured the expression of TSLP and IL-33 which are proinflammatory cytokines. Purposed to figure out the pathological mechanism of the regulation of the epithelial barrier function and the release of inflammatory cytokines caused by TLRs.(2) Exploration on the mechanism of Calycosin in a ACD mice model:Using a 2-day ACD model sensitized by FITC which represents the induction phase, we detected the expression of TLR4 as well as its downstream adapter proteins. The connection between epithelial cells was measured via an electron microscopy and the expression of Occludin and ZO-1 were observed. At the same time, we explored the expression of proinflammatory cytokine TSLP, IL-33, the activation of NF-?B signal pathway and the effect Calycosin has.(3) The study of the effect and mechanism in a recurrence ACD-model administrated with Calycosin:We established a recurrence ACD-model to examine whether Calycosin is able to alleviate the inflammatory symptoms and then discovered the potential mechanism. Calycosin was administrated for 10 days during the remission stage. Then we ignited the inflammation again 24 hours after the latest administration. Ear swelling was relieved dramatically. Pathological changes of ear tissues were examined. We also detected the expression level of IL-4, IL-5, IL-9 and IL-13 in the treated ear. We measured the level of tight junction proteins including Occludin and CLDN-1, TLR4, TLR8, MyD88, TIRAP, TAK1 and NF-?B to evaluate whether Calycosin can regulate the expression of these proteins.(4) The study of the mechanism on the production of initial inflammatory factors in a LPS-induced HacaT cell model administrated with Calycosin:We used LPS, the ligand of TLR4 to induce the deficiency of the cell membrane barrier and see if Calycosin could fix the damage. After stimulating HaCaT cells for 24h using LPS, we detected the expression of TLR4 as well as its downstream proteins, Occludin, ZO-1 and the activation of NF-?B signal pathway. At the same time, we studied the effect which Calycosin had on both protein and gene levels.The results showed that:(1) After the human keratinocytes HaCaT incubating with TLRs (2,3,4,8,9) ligands for 24h hours, it turned out that LPS, the TLR4 ligand could reduce the expression of ZO-1 and Occludin notably which are the two key members of the TJs. TSLP and IL-33 levels were also increased significantly comparing with the control group. Further studies showed that the expression of the downstream proteins of TLR4 also showed a rising trend and NF-?B signal path was activated. The addition of inhibitions of NF-?B signal path could reverse the lack of the cell membrane barrier and the release of proinflammatory cytokines caused by LPS. All the results suggested that at first LPS acted on the TLRs which reacted the MyD88, TIRAP and TAK1, and then the NF-?B was ignited that leaded to the over expression of TSLP and IL-33 mediating by the TJs.(2) Calycosin could not only reduce the expression of TLR4 and its downstream proteins, such as MyD88, TIRAP and TAK1, but also narrow the pathological intercellular space between epithelial cells of the otic epithelial tissues caused by allergens. It was found out that Calycosin can significantly reduce the expression of TSLP and IL-33 both on gene and protein level in a ACD model induced by FITC. While in-depth study showed that Calycosin had a certain influence on NF-?B inflammatory signal pathway. In summary, Calycosin reserved the high level expression of TLR4-MyD88-TAK1 in a ACD mice model, suggesting that TLRs signal pathway is activated. The higher expression of TLR4 may be one of the crucial factors in ACD model, and it is correlated with our previous findings in Human keratinocytes.(3)We used different dosage regimen to compare the effect between Calycosin and dexamethasone. The ear swelling was alleviated dramatically while the mice were administrated with Calycosin during the recurrence phase until 24h before the second incitement. At the same period, we found the expression of Th2 inflammatory cytokines such as IL-4?IL-5?IL-9 and IL-13 have been reduced. However, positive drugs seemed invalid in this recurrence ACD-model. The results suggested that Calycosin's effect would related with the adjustment of the pathological state of mice, not by controlling the symptoms of inflammatory changes directly. Focused on the recurrence period, it turned out that the expression of TJs such as ZO-1 and Occludin had a falling tendency comparing with control group. Meanwhile, according to the overexpression of TLR4-MyD88-TAK1 and NF-?B, the TLR4 signal pathway was supposed to be activated and the overexpression of TLR4 could be an important factor. We found that Calycosin was able to decrease the protein level which suggested that the mechanism Calycosin inhibits the recurrence of ACD could be associated with NF-?B signal pathway mediating by TLR4-MyD88-TAK1.(4) We used LPS, the ligand of TLR4 to induce the barrier deficits in human keratinocytes intervented by Calycosin and attempted to clarify the role of Calycosin on repairing epithelial barrier function. After stimulating HaCaT cells for 24h with LPS, TLR4 expression was significantly increased comparing with the control group, However, Calycosin inhibits the expression of TLR4 in cells under stimulation with LPS. Subsequently, we examed the expression of MyD88, TIRAP and TAK1 protein levels in LPS-induced cells, it showed that Calycosin could significantly reduce the expression of these TLR4-related proteins to some extent. The Immunofluorescence results showed LPS could also reduce the expression of Occludin and ZO-1, Calycosin can reverse these effects. The expression of TSLP and IL-33 was increased dramatically under the LPS-stimulation on both protein and gene level, while different levels of Calycosin could decrease to varying degrees. Calycosin was also able to inhibit the activation of NF-?B signal pathway. These results suggested that Calycosin may interfere the production of TLR4 and its downstream associated proteins by inhibiting the activation of NF-?B signal pathway resulting in the repair of epithelial barrier which decreased the expression of pro-inflammatory cytokines.Conclusion:(1) The activation of NF-?B mediated by TLR4-TJs signal pathway can not only regulate TJs in human keratinocytes, but also lead to the defect in epithelial barrier which caused the release of TSLP and IL-33.(2) Calycosin can reduce many inflammatory symptoms including ear swelling, lymphocyte infiltration and ear histopathological changes in a mice ACD-recurrence model. Calycosin can ease the process of inflammatory by promoting the expression of ZO-1 and Occludin which leaded to the expression of TSLP and IL-33. In addition, The mechanism may be associated with the inhibition of NF-?B signal pathway mediated by TLR4-MyD88-TAK1 and the intervention of the release of inflammatory cytokines which eased the symptoms in a recurrence stage. |
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