| Objective:Studies have shown that thymic stromal lymphopoietin(TSLP)is an important initiator of Th2 type inflammatory response in allergic diseases,which activates a variety of antigen-presenting cells(APCs).OX40 L is expressed on B cells,dendritic cells and other apcs,and enhances the secretion of a variety of cytokines.Based on TSLP/OX40 L,this study used HE section staining,ELISA,immunohistochemistry,RT-q PCR and other detection methods to investigate the effect of thermal moxibustion on the changes of nasal mucosa micro environment and Th2/ILC2 s inflammatory response in allergic rhinitis model rats,and to provide a basis for the clinical application and efficacy of thermal moxibustion.Methods:In this study,Ovalbumin(OVA)sensitization method was used to establish a rat model of allergic rhinitis.Thirty-six Specific pathogen free(SPF)male Sprague-Dawley rats,aged 6 weeks,weighing 180-220 g,were selected.Thirty SD rats were randomly divided into blank control group(n=6)and model group(n=24).The model group was given 0.3 mg OVA/m L of normal saline solution +30 mg AL(OH)3 solution for 14 days after adaptive feeding.From the 15 th day,5%concentration of OVA solution was applied to each rat by nasal drops of 50 μL each side for seven consecutive days.The control group rats were treated with the same amount of normal saline instead of OVA solution.The behavioral scores of rats were observed and recorded within 30 minutes after intranasal instillation.After modeling,they were randomly divided into a model group(n=6),a positive control group(n=6)and a moxibustion treatment group(n=12)by using the RAND function of Excel table according to their body weight.Positive control group received fluticasone propionate 50μL nasal drip on each side with microsyringe.The model group was treated with the same amount of normal saline instead.The moxibustion treatment groups were treated with suspended moxibustion at "Feishu",once a day,40 min each time.During the intervention period,the model group and the others intervention group were given long-term slow nasal drops of 5% OVA solution 25 μL(side / day)every other day to maintain sensitization,and the blank control group was given the same amount of normal saline instead of operation for 21 days.In the moxibustion treatment group,according to the changes in tail temperature and body temperature after the intervention period,the rats with thermal moxibustion sensation were defined as the thermal moxibustion group,and those without thermal moxibustion sensation were defined as the traditional moxibustion group.Keep an eye on the rats’ living conditions during the experiment and the rats were fasted for 8 hours before the last intervention,and 5% OVA solution was dropped through the nose at 1 hour after the last intervention.The rats were anesthetized within 30 minutes after the completion of the operation,and peripheral blood and nasal mucosa were collected and part of serum was separated for subsequent detection.1.The study of animal behavior and nasal mucosal microenvironment in rats with allergic rhinitis under the intervention of thermal moxibustion: the behavioral scores of rats in each group were recorded before and after intervention,and the data were compared.Nasal mucosa tissues were fixed with 4% paraformaldehyde,embedded in paraffin,and sectioned.HE staining was used to observe the changes of mucosal microenvironment and the distribution of inflammatory cells.2.To investigate the effect of thermal moxibustion on Th2/ILC2 s inflammatory response in allergic rhinitis rats: the serum levels of Ig E,TNF-α,IL-4,IL-10,IL-25,IL-33 and IL-37 were detected by ELISA.The expression of IL-10,IL-25,IL-33 and IL-37 in nasal mucosa was detected by immunohistochemistry.3.The effect of thermal moxibustion on the expression of TSLP and its synergistic factor OX40 L in allergic rhinitis rats: the content of TSLP in serum of allergic rhinitis rats was detected by ELISA,the expression of TSLP protein in nasal mucosa was detected by immunohistochemistry,and the transcription of TSLP m RNA and OX40 L m RNA was detected by real-time fluorescence quantitative method.Results:1.Effects of thermal moxibustion intervention on animal behavior in rats with allergic rhinitis:Thermal Moxibustion therapy can effectively improve allergic symptoms in rats with allergic rhinitis.Before modeling,the rats in each group had normal morphology and clean hair.After modeling,the model rats appeared sneezing,scratching the face or whole body,messy hair falling off,or even dyspnea,listlessness,and so on.Before the intervention,the behavioral score of the model group(6.50±1.14)was significantly different from that of the blank control group(2.50±1.05)(P <0.01),indicating that the model was successfully established.After 21 days of intervention,the behavioral scores of each treatment group were lower than those before intervention,and compared with the model group,the behavioral scores of each treatment group were significantly different(P < 0.01).The scores of the thermal moxibustion group with acupoint sensitization during moxibustion were higher than those of the traditional moxibustion group,and the difference was statistically significant(P < 0.05).2.Effects of thermal moxibustion intervention on local microenvironment of nasal mucosa in rats with allergic rhinitis:Thermal moxibustion can effectively improve the local microenvironment of nasal mucosa in rats with allergic rhinitis.The pathological observation of rat mucosa after HE staining showed that the structure of nasal mucosa in the blank group was complete,the mucosal epithelial layer was not thickened and obviously stratified with the lamina propria,the capillaries were not dilated,the mucosa was not edema,peeling,and there was no obvious inflammatory cell infiltration.In the model group,the mucosal epithelial layer was thickened,the structure was disordered and mixed with the lamina propria without continuous arrangement,and a large number of orange areas in the cytoplasm of eosinophils were seen,indicating infiltration.The epithelial layer of the positive control group was slightly thicker than that of the blank group,there was telangiectasia,the structure of the lamina propria was normal,and a small amount of inflammatory cell infiltration was seen.In the traditional moxibustion group,the mucosal epithelial layer was thickened obviously,the layers were clear but the structure was disordered,there was slight telangiectasia,and inflammatory cell infiltration was observed in the lamina propria of the mucosa.In the thermal moxibustion group,there was no thickening of the mucosal epithelial layer,slight telangiectasia,slightly separation from the lamina propria of the mucosa,and a small number of eosinophils were expressed,which were less than those in the positive control group and the traditional moxibustion group.3.Effects of thermal moxibustion on Th2/ILC2 s inflammatory response in allergic rhinitis rats:Serum ELISA results showed that: The expression of Th2 inflammatory factors such as Ig E,TNF-α,IL-4 and ILC2 s related inflammatory promoting factors IL-25 and IL-33 in the serum of the model group were significantly increased(P < 0.01).After 21 days of moxibustion intervention,the expression of Th2 type inflammatory factors such as Ig E and TNF-αand ILC2 s inflammatory promoting factors IL-25 and IL-33 in the Heat-sensitization group were significantly lower than those in the model group,suggesting that thermal moxibustion has a benign regulatory effect on Th2 and ILC2 s related pro-inflammatory factors in the serum of allergic rhinitis rats.IL-10 and IL-37 are anti-inflammatory factors.The serum level of IL-10 in the blank control group was higher than that in the sensitized group at the steady state,suggesting that the serum level of IL-10 in the model group decreased after the exogenous allergen stimulation,indicating that the secretion of anti-inflammatory factors IL-10 in various target cells was inhibited.After treatment,the serum level of IL-10 in the positive control group and the thermal moxibustion group was significantly higher than that in the model group(P < 0.01).The expression of IL-37 in the blank control group was low,and the expression of IL-37 in the model group was higher than that in the blank control group.The serum levels of IL-37 in the positive control group,thermal moxibustion group and traditional moxibustion group were higher than those in the blank control group,and the difference was statistically significant(P < 0.01).Compared with the model group,the serum IL-37 concentration of the thermal moxibustion group and the positive control group had a certain statistical difference(P < 0.05).The results of immunohistochemistry showed that the expression of inflammatory factors such as IL-25,IL-33 and TSLP in the nasal mucosa of the thermal moxibustion group,the positive control group and the traditional moxibustion group decreased after treatment.The positive control group and the thermal moxibustion group were better than the traditional moxibustion group in the expression of positive areas.The expression of anti-inflammatory cytokines such as IL-10 and IL-37 increased after intervention.4.Effects of thermal moxibustion on TSLP expression and co-factor OX40 L in rats with allergic rhinitis:ELISA showed that there was a significant difference in serum TSLP content between the blank control group and the model group(P < 0.05),which indicating that the serum TSLP content of the rats was significantly increased after allergic stimulation,suggesting that the model was successfully established.After treatment,compared with the model group,the content of TSLP in the positive control group,thermal moxibustion group and traditional moxibustion group decreased,indicating that the treatment was effective,and the differences were statistically significant(P <0.05).At the same time,compared with the positive control group,the thermal moxibustion group had a thermal sensitization effect after moxibustion with heat-sensitive moxa stick,and the serum TSLP concentration was lower in the thermal moxibustion group(P < 0.05).The positive control group and the traditional moxibustion group treated AR rats with different intervention measures,the change degree of serum TSLP concentration statistically speaking,the two groups have the same effect.The expression of TSLP protein in nasal mucosa was detected by immunohistochemistry,and the results were consistent with the trend of ELISA.Real-time fluorescence quantitative results showed that compared with the blank control group,the expression of TSLP m RNA in the model group was significantly increased(P < 0.01).The expression of TSLP m RNA in the positive control group and thermal moxibustion group was significantly lower than that in the model group (P < 0.01).At the same time,the expression of m RNA in the three groups was also lower than that in the blank control group,which was not consistent with the trend of serum TSLP concentration.The thermal moxibustion group had a significantly lower expression of TSLP gene than the positive control and traditional moxibustion groups(P < 0.05).The expression of OX40 L m RNA was consistent with that of TSLP m RNA,and the expression of OX40 L gene in the model group was increased(P <0.05).Compared with the model group,the treatment group 3 significantly decreased(P < 0.01);There was no significant difference in OX40 L gene expression among the positive control group,thermal moxibustion group and traditional moxibustion group.Conclusion:The anti-allergic effect of thermal moxibustion therapy on allergic rhinitis is through inhibiting the secretion of thymic stromal lymphopoietin TSLP secreted by mucosal epithelial cells after allergen challenge,inhibiting the activation of various antigen presenting cells(APC),and reducing the expression of TSLP and OX40 L to regulates the expression and secretion of Th2 and its mirror cells ILC2 s type inflammatory cytokines.1.Thermal moxibustion therapy can effectively improve the allergic symptoms of allergic rhinitis rat model.2.Thermal moxibustion therapy regulates the infiltration of inflammatory cells in nasal mucosa of allergic rhinitis rats and inhibits the immune response.3.Thermal moxibustion therapy can reduce the serum levels of Th2 type inflammatory cytokines such as Ig E,TNF-α,IL-4 and its mirror cell ILC2 s type inflammatory cytokines IL-25 and IL-33 in allergic rhinitis rats,and increase the serum levels of anti-inflammatory factors such as IL-10 and IL-37,so as to alleviate the mucosal allergic symptoms.4.Thermal moxibustion inhibits the protein expression of pro-inflammatory factors such as IL-25 and IL-33,and promotes the secretion of anti-inflammatory factors IL-10 and IL-37 in nasal mucosa of rats with allergic rhinitis.5.Thermal moxibustion inhibits the synthesis and activation of TSLP and OX40 L in TSLP/OX40 L pathway,and inhibits the expression of downstream inflammatory proteins in allergic rhinitis rats. |
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