In Vivo And In Vitro Research On The Regulation And Control Of Allergic Rhinitis By Maju Babu Ointment Based On The Balance Of SfTSLP/1fTSLP "percutaneous Treatment Of Lung"

Previous experiments have shown that although the blood concentration of "percutaneous treatment of lung" of Majie cataplasm is lower than that of oral administration group,it has better curative effect,which may be achieved through the specific regulatory pathway between lung and skin.TSLP is a key common factor in inflammatory immunity of lung and skin.Two subtypes of TSLP,lfTSLP and sfTSLP,play roles in promoting inflammation and inhibiting inflammatory response respectively.Based on this,we propose the following hypothesis:Majie cataplasm may regulate the balance of sfTSLP/lfTSLP between lung and skin,and then reduce Th2 inflammation,so as to achieve the good effect of "percutaneous treatment of lung".Objective1.To reveal the changes of sfTSLP/lfTSLP balance between lung and skin in allergic rhinitis.2.To evaluate the effect of Majie cataplasm on the balance of sfTSLP/lfTSLP in vivo and in vitro,and confirm that TSLP is the key factor in the therapeutic effect of "percutaneous treatment of lung ".3.To explore whether Majie cataplasm can affect the epithelial immune system by regulating the balance of sfTSLP/lfTSLP,and partly reveal the modern biological connotation of the theory of "lung governing fur".Method1.Forty SPF grade female C57BL/6J mice aged 6 weeks,weighing 21-25g,were randomly divided into 4 groups with 10 mice in each group:blank group(K),blank+sfTSLP pretreatment group(KS),model group(M),model+sfTSLP pretreatment group(MS).In the basic sensitization stage,0.1ml OVA basic sensitization solution was intraperitoneally injected into the model group and model+sfTSLP pretreatment group on day 0,7 and 14.The blank group and blank+sfTSLP pretreatment group were injected with PBS of equal volume.In the stage of sensitization and administration,the mice in the pretreatment group were treated with sfTSLP polypeptide solution nasal drip for 1 hour before each challenge and sensitization,40?l/mouse,and the other groups were not treated.After 1 hour of pretreatment,OVA sensitization solution(40?l/mouse)was injected into the nose of model group and model+sfTSLP pretreatment group,and the blank group and blank+sfTSLP pretreatment group were given equal volume of PBS nasal drip.On the 22nd and 30th days(7 days after stimulation and sensitization,and the last challenge sensitization),the number of scratching nose in 1 min was observed and the general state of mice was recorded.The nasal and lung tissues of mice were stained with he,the levels of serum IgE and TSLP were detected by ELISA,and the expression of TSLP,TNF-?,IL-4,IL-5 and IL-13 genes in lung tissue were detected by RT-PCR.2.Four 4-week-old female New Zealand white rabbits,weighing 2.3-2.5kg,were fed adaptively for 3 days.Freund's complete adjuvant was used for the first immunization,and the incomplete Freund's adjuvant was used for the enhanced immunization.Each rabbit was immunized with 4 parts every two weeks.The injection volume of each site was 200?l in the first immunization and 100?l in each part of the enhanced immunization.The titer of the antiserum was detected by indirect ELISA.The specificity of antiserum was detected by dot blot after purification.3.human bronchial epithelial cell line 16HBE cells were cultured in DMEM high glucose medium+10%fetal bovine serum(FBS)+1%penicillin streptomycin mixture,and logarithmic growth phase 16HBE cells were cultured.CCK8 method was used to detect the effect of different concentrations of Majie cataplasm on the activity of 16HBE cells.After cell count,the cell concentration was adjusted to 1*106/ml,inoculated with 6 hole plates per hole 2ml and 12 orifice plates.After 12 hours of inoculation,the cells were observed to have adherent growth under the microscope,then the original cell culture medium was discarded,and the cell culture medium mixed with different concentrations of OVA solution or HDM solution was replaced according to the experimental groups,and the culture was continued for 6-24 hours.RT-PCR was used to detect the content of lfTSLP and sfTSLP in allergic rhinitis cell model.Result1.The number of nose scratching in the model+sfTSLP pretreatment group was significantly less than that in the model group(P<0.01),and the general activity state was better than that in the model group;there was no obvious abnormality in the blank group and blank+sfTSLP pretreatment group.Compared with the model group.HE staining model+sfTSLP pretreatment group showed significant improvement,no obvious thickening of pseudostratified ciliated columnar epithelium and hyperplasia of mucus gland,only mild edema and few inflammatory cell infiltration were found in the surrounding tissues.The growth of nasal mucosa in blank group and blank+sfTSLP pretreatment group was normal;the lung tissue of HE staining model+sfTSLP pretreatment group was normal Compared with the model group,the edema of the surrounding tissues of bronchial mucosa was significantly reduced,and the number of inflammatory cells was significantly reduced.Occasionally,there were slightly congested and swollen alveoli,and a small amount of inflammatory cells mainly composed of lymphocytes were found in the alveoli.The status of bronchial mucosa epithelium and alveolar tissue in blank group and blank+sfTSLP pretreatment group were normal.Compared with the blank group,the levels of serum IgE and TSLP,the gene expression of TSLP,TNF-?,IL-4,IL-5 and IL-13 in lung tissue were significantly higher in the model group than in the blank group(P<0.01);compared with the model group,the levels of serum IgE and TSLP in the model+sfTSLP pretreatment group were significantly lower(P<0.01)but there was no significant difference between the blank group and the blank+sfTSLP pretreatment group Righteousness.2.In the first group,the titer of rabbit a antiserum was 0 K and that of rabbit B antiserum was 0 K;the titer of rabbit a antiserum was 0 K and that of rabbit B antiserum was 0 K.In the second group,the titer of rabbit serum C was 0 K,and that of rabbit D was 0 K.dot blot was negative.3.When the concentration of water decoction of Majie cataplasm was lmg/ml,the activity of 16HBE cells was slightly higher than that of the normal group(P<0.01).Under this concentration,the decoction of Majie cataplasm could promote the growth of cells,which could be used in the follow-up study of Majie cataplasm.The effective content of TNF-? was not detected in the supernatant of the two cell models detected by ELISA kit,and the effective signal of sftslp and lftslp gene expression was not detected by multiple RT-PCR detectionConclusion1.Sftslp has a good protective effect on bronchial and nasal mucosa of OVA induced allergic rhinitis mice,and sftslp has no obvious effect on normal mice2.Sftslp can significantly reduce the content of Th2 related inflammatory factors in OVA induced allergic rhinitis mice,and reduce the Th2 type inflammatory reaction mediated by TSLP(lftslp).3.The preparation of polyclonal antibody against lftslp and the establishment of cell model of allergic rhinitis were explored.