| Asthma is a chronic disease characterized by airway inflammation,airway remodeling and airway hyperresponsiveness.Compared with Western Medicine,Traditional Chinese Medicine has great advantages in the clinical diagnosis and treatment of asthma disease,as it has fewer side effects,stable curative effect and is more acceptable to the population.The external treatment of Traditional Chinese Medicine has a long history,especially the acupoint application method,which has been repeatedly verified in clinical practice and has curative effect.The Majie cataplasm developed by our team with the combination of compound acupoint application method is composed of Ephedra,almond,corydalis corydalis,white mustard seed and ginger.It is made with the dosage form technology of Barbu.Based on the treatment theory of "transdermal treatment of lung",the Majie cataplasm has a certain relieving effect on asthma attacks by skin application.The team's previous research found that the peak blood volume of Majie cataplasm was only a quarter of that of oral dosing at the same raw dose,but its efficacy was comparable to that of oral dosing,suggesting that Majie cataplasm not only regulates asthma disorders through the skin,but that there may be a more rapid pathway between the skin and the lungs,allowing Majie cataplasm to regulate this pathway and exert a therapeutic effect.Previous studies have shown that neuroimmune regulation plays a certain role in the treatment of asthma by Majie cataplasm.Activation of TRPA1 in the TRP ion channel can cause the release of inflammatory neuropeptides,causing the generation of inflammatory cells,leading to neurogenic inflammation,and inducing the aggravation of asthma.On this basis,it is proposed that Majie cataplasm is likely to affect the expression of TRPA1 and CGRP in lung tissue,reduce the release of neuropeptides,relieve neurogenic inflammation and play a good role in the treatment of asthma.ObjectivesTo explore whether Majie cataplasm can affect TRPA1 and CGRP,play an anti-inflammatory role,relieve airway hyperresponsiveness,and then play a role in the treatment of asthma by affecting immune response.Methods1.Preparation of Majie cataplasm.2.The mice were divided into a blank control group,an OVA model group,an OVA Majie cataplasm group and an TRPA1 antagonist group.The asthmatic model of experimental animals was established by OVA with two stages of basic sensitization and nasal stimulation.Basic sensitization:On days 0,7 and 14,all groups except the blank control group were injected intraperitoneally with 0.1 mL of a basal sensitization suspension(containing 0.05 mg OVA and 1 mg aluminium hydroxide adjuvant)and the blank control group was injected intraperitoneally with an equal amount of PBS solution.Nasal drip stimulation:From day 15 to day 29 of the experiment,for a total of 15 days,all groups except the blank control group were stimulated with a nasal drip of OVA suspension(40 ?L/mouse),while the mice in the blank control group received a nasal drip of the same volume of PBS.Other interventions:no interventions were administered to the blank control and OVA model groups starting on day 15 of the experiment.Mice in the OVA Majie cataplasm group had their dorsal hair shaved with a shaver and were given Majie cataplasm(square cream patches cut in advance to a side length of 0.45 cm)to apply on their backs,one patch per day,with each patch intervening for 24 hours.In the TRPA1 antagonist group,the TRPA1 antagonist HC-030031 was administered by gavage on day 20 of the experiment at 0.2 mL/each(200 mg/kg)and on days 21,22 and 23 of the experiment at 0.1mL/each(200 mg/kg),twice a day.3.Sample collection:taken on day 29 of the experiment after excitation and administration.Blood was taken from mouse eyes and samples were centrifuged in a centrifuge at 4?,12000 r/min for 20 minutes to remove the upper layer of serum.The right and left lungs were removed and placed in 4%tissue fixative and lyophilized tubes respectively.Skin tissue from the back of the mouse was stripped and placed in lyophilisation tubes.4.HE staining of mouse lung tissue,behavioural evaluation of asthmatic mice,Elisa assay for serum IgE.5.Elisa assay for serum histamine,IL-4,IL-5,airway resistance test.6.Western-Blot assay for TRPA1 and CGRP in lung tissues and TRPA1 in skin tissues.Results1.The behavioural evaluation of asthmatic mice showed that the number of nasal scratches and sneezing episodes were significantly higher in the OVA model group compared to the blank control group(P<0.001).Compared with the OVA model group,the number of nasal scratches and sneezes and the degree of asthma attacks were reduced in the OVA Majie cataplasm groups(P<0.05;P<0.01).HE staining showed that the blank control group had intact tissue morphology,no inflammatory cell infiltration and no inflammatory exudate in the alveolar septum,while the OVA model group showed thickened tracheal wall,large inflammatory cell infiltration in the interstitial space and large adhesions in the alveolar wall.A small amount of inflammatory cell infiltration was seen in the OVA Majie cataplasm group and TRPA1 antagonist group,with a small amount of inflammatory exudate in the alveolar septum and significantly reduced tissue structure compared to the OVA model group.Elisa assay for serum IgE in mice:serum IgE levels were significantly increased in the OVA model group compared to the blank control group(P<0.001).Compared with the OVA model group,the serum IgE levels of mice in the OVA Majie cataplasm group and the TRPA1 antagonist group were significantly lower(P<0.001).2.Airway resistance test results show that:At a concentration of 0 mg/mL of acetylcholine chloride,airway resistance values were reduced in the blank control group compared to the OVA model group(P<0.01),and reduced in the OVA Majie cataplasm group compared to the TRPA1 antagonist group,with no statistically significant difference.When the concentration of acetylcholine chloride was 12.5 mg/mL,compared with the OVA model group,the airway resistance value of the blank control group was decreased(P<0.01),and the airway resistance value of the other two groups was decreased compared with the OVA model group,the difference was not statistically significant.When the concentration of acetylcholine chloride was 25 mg/mL,compared with the OVA model group,the airway resistance value of the blank control group was significantly decreased(P<0.01),and the airway resistance value of the other two groups was decreased compared with the OVA model group,the difference was not statistically significant.When the concentration of acetylcholine chloride was 50 mg/mL,compared with the OVA model group,the airway resistance value of the blank control group was significantly decreased(P<0.001),and the airway resistance value of the OVA Majie cataplasm group and the TRPA1 antagonistic group were significantly decreased(P<0.001).When the concentration of acetylcholine chloride was 100 mg/mL,compared with the OVA model group,the airway resistance value of the blank control group was significantly decreased(P<0.001),and the airway resistance value of the other two groups was decreased,the difference was not statistically significant.Elisa detection of serum histamine:Compared with the blank control group,the serum histamine content in the OVA model group was significantly increased(P<0.001).Compared with the OVA model group,the serum histamine content in the OVA Majie cataplasm group and the TRPA1 antagonistic group was significantly decreased(P<0.001).Elisa detection of serum IL-4:Compared with blank control group,serum IL-4 content in OVA model group was significantly increased(P<0.001).Compared with the OVA model group,the serum IL-4 content of mice in OVA Majie cataplasm group and TRPA1 antagonistic group was significantly decreased(P<0.001).Elisa detection of serum IL-5:Compared with blank control group,serum IL-5 content in OVA model group was significantly increased(P<0.001).Compared with the OVA model group,the serum IL-5 content of mice in OVA Majie cataplasm group and TRPA1 antagonistic group was significantly decreased(P<0.001).3.Western Blot analysis of TRPA1 expression in the lung tissue of mice in each group showed that compared with the blank control group,the expression of TRPA1 in the lung tissue of mice in the OVA model group was increased(P>0.05);Compared with OVA model group,the expression of TRPA1 in lung tissue of mice in OVA Majie cataplasm group and TRPA1 antagonistic group showed a decreasing trend(P>0.05;P>0.05).Western Blot analysis of CGRP expression in lung tissue of mice in each group showed that compared with the OVA model group,the expression of CGRP in lung tissue of mice in blank control group was significantly decreased(P<0.001);The expression of CGRP in lung tissue of mice in OVA Majie cataplasm group and TRPA1 antagonistic group was significantly decreased(P<0.01).Immunofluorescence:There was almost no TRPA1 expression in the lung tissue of mice in blank control group,and only a small amount of CGRP expression was found in the interstitial space between trachea and tissues.A large number of TRPA1 and CGRP were expressed in the lung tissues of mice in the OVA model group,mostly clustered around the trachea,and the expression of CGRP was aggregated.A small amount of TRPA1 and CGRP were expressed in the lung space and around the trachea in OVA Majie cataplasm group.TRPA1 and CGRP were only slightly expressed in the lung tissue space of TRPA1 antagonistic mice,and almost no CGRP was expressed around the trachea.Conclusions1.Majie cataplasm has a good effect on asthmatic model mice.2.Majie cataplasm has the effect of anti-inflammatory reaction and relieving airway hyperresponsiveness.3.Majie cataplasm plays a role in the treatment of asthma by affecting the expression of TRPA1 and CGRP,inhibiting the release of inflammatory neuropeptides and reducing the neurogenic inflammatory response. |
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