| Object:（1）To verify whether the animal model of allergic rhinitis of lung deficiency type established in this experiment is reliable?（2）In this model,does the"nose-brain"axis pathway exist?How to position and define?Is the degeneration of nasal mucosa and hippocampus in step?（3）If we don’t choose Yingxiang point to bury the thread,is it still effective?If we choose Baihui,lung and spleen acupoints to embed thread,can it also have curative effect?（4）After 3 weeks of acupoint catgut embedding,can the degeneration in the nasal mucosa and hippocampus be reversed?What is the specific effect?Has it been over treated?Do we need further treatment?（5）After 3 weeks of acupoint catgut embedding,to record the concentrations of Ig E and IL-4,filling the gap of previous scholars.If OX-42 in the hippocampus will continue to decline?Does NKB have an immune response in the nasal mucosa?Can TLR-2 be expressed in hippocampus?Methods:（1）Group:45 SPF grade SD rats were purchased and randomly divided into 3 groups,15 rats in each group.Group A is buried wire group（mould before embedding wire）,group B is model group（mould without embedding wire）,group C is blank group（without mould and embedding wire）.（2）Fumigation:self made fumigation molding box and fumigation moxibustion strip.When smoking each time,insert 2 moxibustion strips into the holes on the side wall of the carton.Only 6 rats are put in the fumigation box for 30 minutes each time.Smoke twice a day at least 3 hours apart.Smoked for 14 days.（3）Sensitization:（1）Two groups of AB were injected with ovalbumin sensitizing solution for five times every other day.In the basic sensitization stage,continuous fumigation was needed,twice a day.（2）Enhance sensitization.Once a day,three drops were given to each nostril in AB groups.After 30 minutes of nasal drip,atomization begins,once a day,45 minutes each time.For 7 days.（3）Maintain sensitization.After the intensive sensitization,the rats in group A were treated with acupoint catgut embedding for 3 weeks,during which group A was continuously sensitized by nasal drip.In group BC,normal saline was used.（4）Catgut embedding at acupoint:Catgut Embedding at acupoint in group A,Baihui acupoint,bilateral lung and spleen acupoint were selected.A disposable thread embedding needle with the specification of 0.9 was used to insert the No.000 catgut with the length of3-5 mm into the needle tip.Quickly pierce the skin,with a sense of falling about 5 mm deeper,going to the best buried place between the skin and muscles.The embedding time is3 weeks,during which alcohol is used for disinfection once every 3 days.（5）Record behavioral characteristics:from the first day of basic sensitization,the phenomenon of scratching nose,running nose and sneezing of three groups of rats were recorded and scored according to the standard.Observe once a day for 30 minutes,and calculate the mean value and standard deviation of each group.（6）Test:（1）HE staining:the nasal mucosa,lung tissue and brain tissue of rats were stained with HE and the images were collected.（2）ELISA:the OD mean of Ig E and IL-4were measured.（3）Immunohistochemistry:NKB in the nasal mucosa and OX-42 in the hippocampus were detected.（4）Immunofluorescence:TLR-2 in the nasal mucosa and hippocampus of rats was detected simultaneously.（7）Statistics:SPSS 17.0 statistical software was used to analyze the data.The"mean±standard deviation"was used to express the data.When P<0.05,the difference was statistically significant.Results:（1）Before and after smoking.（1）Before smoking:no abnormality was found in the three groups.（2）After fumigation:the diet of AB groups was reduced,the drinking water was increased,the performance of avoiding,hugging,burying,closing eyes,sleeping and curling appeared,which are the manifestation of Qi deficiency.Group C was normal.（2）Before and after sensitization.（1）Before sensitization:no abnormality was found in the three groups.（2）After sensitization:symptoms such as scratching nose,rubbing eyes,scratching ears and itching nose appeared in AB groups.Group C was normal.（3）Before and after molding.（1）Respiratory rate increase:1.73±0.16 in group A,1.64±0.15 in group B,1.32±0.12 in group C.PAB=0.084,PAC<0.001,PBC<0.001。（2）Weight and mass increase:36.13±3.52 in group A,34.53±3.07 in group B,42.93±3.59in group C.PAB=0.205,PAC<0.001,PBC<0.001.（4）"Stage total score"of behavioral score.（1）After basic sensitization:3.54±0.26 in group A,3.48±0.25 in group B,2.23±0.29 in group C.PAB=0.544,PAC<0.001,PBC<0.001.（2）After intensive sensitization:5.27±0.29 in group A,5.33±0.20 in group B,1.94±0.20 in group C.PAB=0.442,PAC<0.001,PBC<0.001.（3）At the end of maintenance sensitization:5.94±0.22 in group A,5.07±0.23 in group B,2.03±0.18 in group C.PAB<0.001,PAC<0.001,PBC<0.001.（5）"End point factor score"of behavioral score.（1）Sneezing:1.89±0.22 in group A,1.65±0.16 in group B,0.57±0.14 in group C.（2）Runny nose:2.00±0.15 in group A,1.61±0.27 in group B,0.73±0.13 in group C.（3）Grasping nose:2.05±0.30 in group A,1.77±0.29 in group B,0.68±0.16 in group C.The pairwise comparison of the above factors are:PAB<0.001,PAC<0.001,PBC<0.001.（6）HE staining.（1）Nasal mucosa:in the model group,the cilia were lodging,the epithelium of mucosa was destroyed,and the goblet cells were highly hypertrophied!The blood vessels in the lamina propria were dilated with a large number of eosinophils and lymphocytes infiltration.In the embedding group,the goblet cells were slightly to moderately hypertrophied,the nuclei of cone cells and spindle cells were deeply stained,the allergic reaction was reduced,and the immune response was active.（2）Lung tissue:bronchocilia edema in model group,even lodging.Small alveoli collapse,even adhesion.Large alveoli are emphysematous and even ruptured.The walls of alveoli were in a state of shrinkage.A large number of red blood cells were found in the alveolar stroma,indicating obvious hyperemia.The widened stroma indicates the proliferation of fibrous tissue.In the catgut embedding group,the cilia of bronchus were not well arranged and presented as a insufficient dome.（3）Hippocampal area:in the model group,the cells were obviously heteromorphic,the nuclei were pyknosis,the staining was deepened,suggesting different degrees of degeneration,even necrosis.Glial cell proliferation and lymphocyte infiltration can be seen around the arcuate zone.In the embedding group,normal cells and heteromorphic cells coexisted,suggesting that the immune response was taking place.（7）ELISA.（1）Ig E（μg/ml）:3.114±0.202 in group A,4.189±0.439 in group B,2.295±0.303 in group C.PBC<0.001,indicating the success of AR animal model.PAB<0.001,indicating that the acupoint catgut embedding has obvious curative effect in 3 weeks.PAC<0.001,indicating that although the acupoint catgut embedding has curative effect 3weeks later,the allergic reaction in the body is still continuing,and it is possible to cure by prolonging the catgut embedding time.（2）IL-4（pg/ml）:14.228±0.651 in group A,18.608±2.403 in group B,11.401±1.229 in group C.PAB<0.001,PAC<0.001,PBC<0.001,with the same meaning as Ig E.（8）Immunohistochemistry.（1）NKB（OD mean）in nasal mucosa:0.1895±0.0027 in group A,0.2031±0.0074 in group B,0.1801±0.0038 in group C.PAB<0.001,PAC<0.001,PBC<0.001,with the same meaning as Ig E.（2）OX-42（OD mean）in hippocampus:0.1728±0.0016 in group A,0.1810±0.0046 in group B,0.1674±0.0025 in group C.PAB<0.001,PAC<0.001,PBC<0.001,with the same meaning as Ig E.（9）Immunofluorescence.（1）TLR-2 in nasal mucosa:the positive expression of TLR-2 in model group was concentrated in epithelial layer,lamina propria and whole layer of nasal mucosa.In the embedding group,the positive expression decreased in the above layers.（2）TLR-2 in hippocampus:in model group,the red staining was large and the cells were disordered.The blue stained nuclei become larger and blacker,indicating that the cells are in a state of decay.In the embedding group,the red staining of intercellular substance disappeared.The red staining of some cells is no longer a whole circle.It is suggested that the decline of cells,and metabolism is increasing,while the expression of TLR-2 is decreasing.Conclusion:（1）The animal model of allergic rhinitis of lung deficiency type established by this experiment is reliable and can be duplicated.This model can cause the degeneration of peripheral nasal mucosa and central hippocampal area at the same time,which shows that the"nose brain"axis is objective,and the hypothesis that its mechanism is located in hippocampal area and qualitative in neuropeptide is tenable.（2）For the acupoint catgut embedding of the animal model of allergic rhinitis of lung deficiency type（Do not choose Ying Xiang acupoint）,the group of acupoints Baihui,lung Shu and spleen Shu were selected instead,and the curative effect was also reliable.After acupoint catgut embedding,through the vertical and horizontal comparison of ELISA,immunohistochemistry and immunofluorescence,we can see that the degeneration of nasal mucosa and hippocampus is in step.（3）After 3 weeks of acupoint catgut embedding therapy,the degenerative changes in the nasal mucosa and hippocampus can be reversed.This experiment provides the basis of behavior,pathology and histology.And all the evidences showed that although the indexes of the embedding group were significantly less than those of the model group(PAB<0.001),there was a significant gap between the embedding group and the blank group(PAC<0.001),which indicated that the effect of the embedding for three weeks was not good enough,and it was necessary to continue the treatment.（4）Detection of Ig E and IL-4 in serum is a classical method to study allergic diseases.However,in the past,relevant scholars only compared this concentration one week,two weeks and four weeks after acupoint catgut embedding.At present,there is no data of three weeks after acupoint catgut embedding.This experiment has been improved to make it clear that the changes during this period are consistent and there is no inflection point.（5）This experiment proved that:after 3 weeks of acupoint catgut embedding treatment,the immune response of NKB on the nasal mucosa was objective,and OX-42still had a downward trend in the hippocampal region,and TLR-2 would show positive expression simultaneously in the nasal mucosa and hippocampal region.The definiteness of these results is the long cherished wish of relevant scholars in the past. |
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