| Object:This study based on the national natural science foundation of the year 2012,“the mechanism of regulating the neurogenic inflammation of allergic rhinitis?AR?via the axonal reflection of the Catgut implantation at acupoint?CIAA?”and the year2014,The immunomodulatory mechanism of the nasal mucosal immune system of AR via the“Sensory neuropeptide—nasal mucosa dendritic cells”pathway of the CIAA,and we did more deeper study.CIAA has been confirmed to regulate the neurogenic inflammation of the nasal mucosa via suppressing the release of neuropeptides such as SP,VIP,CGRP and so on.Thus,torelieve AR symptoms.However,it is still unclear how CIAA to regulate the mechanism of allergenic inflammation.In order to further exploreAR mechanism,this experiment was divided into three parts.Part ?:Internal animalexperiment:We put forward the hypothesis of the neural immune linkage regulated mechanism,TheAR rat model was established via OVA?egg albumin?sensitization method.We observed whether the CIAA have an intervention effect on the neuroimmune substance of the peripheral nasal mucosa.Among them,the neuropeptides,mainly SP,which were firstly suppressed by CIAA in the peripheral nasal mucosa,and then did they also have different effects on the activity of mast cell?MC,mast cell?in advance?The MC activity changes were observed by the upstream membrane receptor TLR2,TLR4 protein,the downstream transcription regulation of NF-kBp65 protein,and MC degranulation,etc.Part ?:Similarly,based on the AR rat model,We observed whether the CIAA have an intervention effect on the neuroimmune substance of the central hippocampal region.The expression levels of SP,ox-42?microglia?,GFAP?astrocytes?,TNF-a and IL-6 in hippocampal tissues were observed.Based on the study of partI and partII,We observed whether there may be a network information of"nose-brain axis"mechanism between in the peripheral nasal mucosa and the central hippocampus neuroimmune regulation substance network.Part ?:in vitro cell experiment:We put forward the“Acupuncture points-nasal mucosa nerve-dendritic cells"coupling hypothesis,That is,the CIAA via the acupoint Yingxiang—Trigeminal nerve—Butterfly palatine ganglion—Nasal mucosal nerve path—dendritic cells?DC?.and SP were firstly suppressed by CIAA,which directly effect onthefunction of DC Chemotactic,mature,etc.And then,withLaunching the three immune defenses of the mucosal immune system,Finally,the nasal mucosa inflammation was alleviated and AR symptoms were relieved.In this part,This study only discussed the intervention and expression of SP acted on DC cell.Methods:1.AR rats in vivo experiment:60 SD rats were randomly divided into 6 groups:blank control group,AR model group,budesende treatment group,sham CIAA group,CIAA for 1 week group,and CIAA for 2 weeks.In addition to the blank control group,All the other groups used the OVA sensitization method to make AR animal model.After the model is successful established,The C1 and C2 groups were subjected to the bilateral acupoint Yingxiang?LI20?,respectively for one week and two weeks.The Budesonide group were intranasally administered with Budesonide?2.5?g/nasal cavity?daily for 14 consecutive days,the Sham group included sole acupuncture at point Ying xiang?LI20?in the absence of catgut implantation,The model group maintained OVA sensitization,The control group gave saline nasal drops.Observation contents:1)behavior scores of AR model and effects of CIAA on AR model;2)HE staining of the nasal mucosa in each group;3)Serum sIgE,IL-4 and IFN-rlevels by ELISA detection in each group;4)the expression level of SP and NF-kBp65 by immunohistochemical method of the nasal mucosal tissues in each group;5)the protein expression differences of TLR2 and TLR4 on the surface of mast cell membrane by immunofluorescence double dye method in each group;6)the protein expression differences of TLR2 and TLR4 in mast cell membrane by Westernblot method in each group;7)the difference in the rate of degranulation of mast cells by toluidine blue staining in each group.2.AR rats in vivo experiment:Using the same model,grouping and intervention methods according to the method 1,based on that,Observation contents:1)TNF-a and IL-6 levels of hippocampus tissues by ELISA detection in each group;2)the expression level of SP,GFAP and OX-42 by immunohistochemical method of the hippocampus tissues in each group.3.In vitro mouse cell experiment:1)the mouse DC cells was resuscitated,transferred and frozen,and finally the cells wereidentified as DC cells;2)study the toxicity of SP(10-10 m-10-4m concentrations)on DC cells in 12,24,48,and 72h;3)the expression of CD40,CD80,CD86 and CD11c in mouse DC cell were analyzed by flow cytometry;4)the expression of SP on DC cells of TLR2 and TLR4 receptors by fluorescence quantitative methodResults:1.The research results of CIAA on peripheral nasal mucosal nerve immune linkage mechanism of allergic rhinitis.?1?Effects of CIAA on animal behavior:compared with model group and the sham CIAAgroup,CIAAgroup and budesonide group can obviously improve the AR symptoms,and with obvious statistical significance?P<0.01?.and CIAA for two weeks improved more obvious than CIAA for one week,with obvious statistical significance?P<0.01?.?2?Effects of CIAA on HE staining of the nasal mucosa:in the AR model group and the sham CIAA group,the structural integrity of nasal mucosa tissues is impaired;the ciliated epithelium is particularly irregular with disordered arrangements,also showing various degrees of fall-off,edema,and trafficking of eosinophils and lymph cells.Compared with the model group and the sham CIAA group,The inflammatory response of C2 group and budesonide group turn mild.?3?Effects of CIAA on sIgE,IL-4 and IFN-?level:model group and the sham CIAA group showed no significant difference?P>0.05?,the results showed that the sIgEand IL-4 was significantly higher,IFN-?was significantly lower.However,compared with model group and the sham CIAA group,The sIgE and IL-4 levels?P<0.01?were significantly reduced,and the IFN-r level was up-regulated?P<0.05?in both the C2and the budesonide group.?4?Effect of CIAA on SP and NF-kBp65 expression:model group and the sham CIAA group showed significantly higher?P>0.05?,However,compared with model group and the sham CIAA group,The SP and NF-kBp65 levels?P<0.01?were significantly reduced in C2 group and budesonide group.?5?Effects of CIAA on TLR2 and TLR4 expression:TLR2 shows green light,TLR4shows red,DAPI dye shows blue.model group and the sham CIAA group showed significantly higher?P>0.05?,However,the C2 group and budesonide group significantly reduced?P>0.05?.However,from the data trend,it was more obvious that the TLR2 protein was down-regulated in the C2 group,and the TLR4 protein was significantly reduced in the budesende group.?6?Effects of CIAA on the cytosolic expression of TLR2 and TLR4:model group and the sham CIAA group showed significantly higher?P>0.05?,However,the C1?C2group and budesonide group significantly reduced?P>0.05?.?7?Effects of CIAA on the degranulation rates of mast cells:the model group and the sham CIAA group showed significantly higher?P>0.05?,However,the C1?C2group and budesonide group significantly reduced?P>0.05?.theC2 and budesonide group was lower than that the model group or sham group?P<0.01?.2.The research results of CIAA on neural immunoregulation mechanism in the central hippocampus of allergic rhinitis rats.?1?Effects of CIAA on TNF-?and IL-6 level:the model group and the sham CIAA group showed significant increase?P>0.05?,However,compared with model group and the sham CIAA group,the C2 and the budesonide group significantly reduced?P<0.01?.?2?Effects of CIAA on SP,GFAP,and OX-42 expression:the model group and the sham CIAA group showed significant increase?P>0.05?,However,compared with model group and the sham CIAA group,the C2 group significantly reduced?P<0.05?.3.The intervention and expression of SP on mouse DC cell lines.?1?The mouse DC cell were cultured,and the cell morphology was observed in an inverted optical microscope?100 times,200 times,400 times?,which were fully proved to be mouse DC cells.?2?There was no significant toxicity in SP with different concentrations from0.1nM?10-10m?to 100mM?10-4m?on DC cells 12,24,48,and 72h.?3?AfterSP on DC cells 24 hours,and the cell morphology was observed in an inverted optical microscope?100 times,200 times,400 times?,which were fully proved to be no obvious change.?4?SP can effectively stimulate the DC cells,mainly stimulate CD86,However,stimulate CD40 lower in 24h,after 24 hours till 48 hours,the effect of stimulating CD40 was stronger.?5?Different concentration of SP on DC cells 24h and 48 h,which can promote the expression of TLR2 and TLR4 receptor,and improve the TLR4 receptor stronger than receptors TLR2,time and dose was positively related.Conclusion:1.SP was Preemptived inhibition by CIAA,Furthermore,it may be possible to Preemptiveregulatetheconductionofinflammatorysignals ofMC-TLR2/4-NF-kBp65 in the nasal mucosa,to achieve neuronal immune linkage regulation,and finally relieve AR symptoms.2.CIAA can downregulated the expression of SP,ox-42,GFAP and TNF-a and IL-6 in the hippocampus to relieve the AR behavioral symptoms.From the peripheral nasal mucosa and central nervous immune endocrine network regulation,On the one hand,which provide a preliminary theoretical basis of possible"nose-brain axis"theory,On the other hand,which provide scientific experimental basis for the early clinical observation of the hippocampal effect characteristics of AR,and promote further research.3.CCK8 method was used to detect DC cytotoxicity and flow cytometry was used to detect the expression of CD40,CD80,CD86 and CD11c in DC cells.fluorescence quantitative method was used to TLR2 and TLR4 receptors,the study confirmed thatSP is not toxic to DC within the working concentration range,and can effectively stimulate the expression of CD40,CD80,CD86 and CD11c,and alsopromotes the expression of TLR2 and TLR4 receptors,and the effect of TLR4receptor is stronger than that of TLR2 receptor.Moreover,the promotion effect was positively correlated with time and dose. |
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