| Purpose: The Airway hyperresponsiveness is a characteristic feature of asthma.As a recent emphasis on traditional Chinese medicine therapy,effect and mechanism of herbal acupoint patch in treating airway inflammation of bronchial allergic asthma are reported in papers.But the research of the therapy on airway hyperresponsiveness of bronchial allergic asthma is rare.In this study,the effects of herbal acupoint patch of Baijiezitufa on airway hyperresponsiveness in asthmatic is reflected by the expiratory resistance,inspiratory resistance and lung compliance of guinea pigs with bronchial asthma under methacholine attack.To explore the mechanism,the level of key factors of the TGF-? signal pathway regulating airway epithelial barrier is detected.Further,the spatial distribution between acupoint and lung of key factors of neuro-immune crosstalk is researched to find the inner mechanism.?Material and method:1 Animal model preparing: Ussing ovalbumin to sensitizate and attack guinea pig to prepare an allergic asthma model.The general observation,lung tissue biopsy and other indicators are observed to find the differences between guinea pigs normal group and asthma model group.The effects of acupoint application on the airway sensitivity of asthmatic guinea pigs were investigated by methacholine challenge test while the expiratory resistance,inspiratory resistance and lung compliance are observe as main indexes.2 The expression of TGF-?,MMP9,TRPV1 and Pan-Cadherin in lung tissue were detected by Western Blot.The activity of matrix metalloproteinases in lung tissue was detected by gelatin zymography.The phosphorylation level of Erk1/2 and P38 in lung tissue was detected by western blot.3 Immunohistochemistry,immunofluorescence assay and immunoblotting were used to detect the changes of NGF,SP and TRPV1 in the acupoint skin and lung tissue after acupoint application.Results:1.1 Animal modeling and effects of herbal acupoint application on airway hypersensitivity.1.1 Preparation of ovalbumin sensitized guinea pig model,and acupoint patching are markedly successful.1.1.1 Animal general observation shows: animals of modelling group appear significan symptoms of asthmatics when inhale OVA,such as cyanosis in nose,mouth and ears,nodding while breathing,restlessness and other,showing the copying of the modeling is successful.1.1.2 Histopathology of lung tissue HE staining showed that the structure of bronchial tissue in asthmatic group was unclear,showing a large number of eosinophils,lymphocyte infiltration,airway smooth muscle thickening,stenosis,visible phlegm thrombosis,alveolar wall thickening.Compared with the asthma group,the application of the treatment group in inflammatory cell infiltration,bronchial wall smooth muscle thickening,alveolar wall edema thickening and so on are improved.1.2 Baijiezitufa acupoints patching take adjustment of methacholine-induced asthmatic guinea pig airway hypersensitivity.1.2.1 Inspiratory resistance: guinea pigs of asthmatic model compared to the ones of normal group,the mean value of inspiratory resistance and AUC of inspiratory resistance were increased at 0.125 mg/ml,0.25 mg/ml and 0.5 mg/ml methacholine stimulation,,the results have significantly difference(p<0.01).Guinea pigs of HAP group compared to the ones of asthmatic model group,the mean value of inspiratory resistance and AUC of inspiratory resistance were decreased at 0.125 mg/ml,0.25 mg/ml and 0.5 mg/ml methacholine stimulation,,the results have significantly difference(p<0.01)1.2.2 Exhalation resistance: guinea pigs of asthmatic model,the mean value of exhalation resistance and AUC of exhalation resistance were increased compared to the ones of normal group at 0.25 mg/ml and 0.5 mg/ml methacholine stimulation,,the results have significantly difference(p<0.01).Guinea pigs of HAP group compared to the ones of asthmatic model group,the mean value of exhalation resistance and AUC of exhalation resistance were decreased at 0.125 mg/ml,0.25 mg/ml,0.5 mg/ml methacholine stimulation,,the results have significantly difference(p<0.01)1.2.2 Lung compliance: guinea pigs of asthmatic model,the mean value of exhalation resistance and AUC of lung compliance were decreased compared to the ones of normal group at 0.25 mg/ml and 0.5 mg/ml methacholine stimulation,,the results have significantly difference(p<0.01).Guinea pigs of HAP group compared to the ones of asthmatic model group,the mean value of exhalation resistance and AUC of exhalation resistance were decreased at 0.125 mg/ml,0.25 mg/ml,0.5 mg/ml methacholine stimulation,,the results have significantly difference(p<0.01)1.2.4 duration of airway resistance peak: compared with asthma model group,acupoint application group at 0.125 mg / ml,airway resistance peak duration decreased,the results were significantly different(p<0.05).2 Protective effect of TGF? / MAPK / MMPs pathway on allergic asthma in guinea pig airway epithelial barrier-related proteins.2.1 The expression of transforming factor ? in lung tissue was detected by Western blotting.The expression of TGF? in lung tissue of guinea pigs was detected by Western Blot.The results showed that the relative expression of TGF? / ? in the model group was significantly higher than that in the normal group(p<0.05).The relative expression of TGF? / ? in the model group was significantly lower than that in the model group Differences(p<0.05).2.2 The phosphorylation of Erk1 / 2 and p38 in lung tissue was detected by Western blotting.The results showed that the phosphorylation level of Erk1 / 2 and p38 MAPK in the model group was significantly higher than that in the normal group(p<0.05).The phosphorylation of p38 MAPK in the acupoints was significantly lower than that in the model group(p<0.05).The results showed that there was significant difference between the two groups(p<0.05).2.3 Effects of HAP on Metalloproteinase Activity,Metalloproteinase 9 and Its Inhibitors in Lung Tissue:2.3.1 The activity of metalloproteinases in lung tissue: the activity of MMP9 and MMP2 in asthmatic model group was significantly higher than that in normal group,and the results were significant(p<0.05).The MMP9 and MMP2 protease activities in the acupoints group were significantly lower than those in the model group(p<0.05).2.3.2 The expression of MMP9 and TIMP1 on the in lung tissue was detected by Western Blot.The results showed that the relative expression of MMP9 in the model group was significantly higher than that in the normal group(p<0.05).The relative expression of MMP9 in the acupoint group was significantly lower than that in the model group(p<0.05).The relative expression of TIMP1 in model group was significantly higher than that in normal group(p<0.05).The relative expression of TIMP1 in acupoint application group was slightly lower than that in model group,but the result was not significant(p<0.05).2.4 The effect of acupoint application on the adhesion of connective protein in lung tissue of asthmatic guinea pigs:Western Blot was used to detect the expression of Pan-Cadherin in lung tissue of guinea pigs.The results showed that the relative expression of Pan-Cadherin in the model group was significantly higher than that in the normal group(p<0.05).The relative expression of Pan-Cadherin in the acupoint application group was significantly higher than that in the model group Significant difference(p<0.05).3.Mechanism of nerve-immune cascade related factors in HAP.3.1 The effect of HAP on the skin of guinea pigs acupoints:3.1.1 general observation results show that the white mustard coating after acupoints,see the skin slightly red,slightly swollen reaction,the next day see the sticky parts of the hair off,to the next paste,the acupressure department fur has been all grown,but slightly shorter than the surrounding,showing acupuncture points on the local skin irritation.3.1.2 pathological sections showed that the normal group and asthma group guinea pigs in the skin of the skin section showed epidermis,dermis and skin appendages no significant difference between the epidermal spine and hairy connective tissue sheath composed of two to three cells,Part of the asthma group showed cut off the stratum corneum and sebaceous glands slightly hyperplasia;asthma group guinea pig acupoints can be seen in the epidermis and hairy root connective tissue sheath thickening,nucleus pyknosis part of the nucleus disappeared,dermis visible lymphocytes,neutrophils Cells and other inflammatory cell infiltration.Showing acupuncture points after the local skin irritation caused by the inflammatory response.3.2 The effect of acupoint application on NGF content in viscera and acupoints: 3.2.1 The effect of Baijiezi Tufa on the content and localization of NGF,SP and TRPV1 in lung tissue:3.2.1.1 immunofluorescence results showed that the normal group of guinea pigs had weak lung NGF or SP expression,Airway endothelial cells in the bronchial tissue in the occasional positive expression;asthmatic model group in the bronchial part of the airway epithelial cells,subepithelial stromal cells and bronchial tissue around the strong positive NGF or SP signal,optical density index(IOD)(p<0.05).The positive cells of acupoint sticking group were mainly concentrated in the surrounding tissues of bronchial tissue,and the strong positive expression of airway epithelial cells in asthmatic group was almost the same as that of normal group(p<0.05)Disappeared,NGF or SP fluorescence signal IOD value compared with the model group was significantly lower,the results were significantly different(p<0.05).Immunohistochemical results showed that there were weakly positive TRPV1 receptors in the lung tissue of normal guinea pigs,and distributed in the epithelial cells of epithelial cells and subcutaneous stromal cells.The asthmatic model of airway epithelium and laryngeal infiltration(p<0.05).Compared with the asthmatic model group,the expression of TRPV1 receptor was down-regulated and the average optical density(p<0.05)was significantly higher than that of the normal group(p<0.05)Compared with the model group,there was significant difference(p<0.05).The expression of NGF in lung tissue of asthmatic model group was significantly higher than that in normal group(p<0.05).The expression of NGF in lung tissue of asthmatic model group was significantly higher than that in normal group(p<0.05)The relative expression of NGF was significantly lower than that of asthmatic group(p<0.05).The relative expression of TRPV1 in the lung tissue of asthmatic group was slightly higher than that of the normal group(p<0.05).The relative content of TRPV1 receptor in the exposed group was significantly lower than that in the asthma group,and the results were significant Sexual differences(p<0.05).The effects of acupoint application on the contents and localization of NGF,SP and TRPV1 in the skin of guinea pigs were observed.The results of immunohistochemistry showed that the epidermal spines,hairy connective tissue sheaths,some of the cells in the dermal tissue had positive NGF expression.The expression of NGF positive cells was similar to that of the normal group,and the area ratio of the positive positive area was slightly higher than that of the normal group(p<0.05).The acupoint application group was similar to the expression of the first two groups.The positive area was enlarged with the thickening of the sheath layer and the connective tissue sheath of the hairy root,and the dermal layer was infiltrated Most of the inflammatory cells had positive expression of NGF.Compared with asthma group,the positive area rate was significantly increased,and the results were significantly different(p<0.05).The expression of the positive SP in the skin of the normal group was similar to that of the normal group,but the positive cells in the various parts of the epidermis were similar to those in the normal group.(p<0.05).The positive area of the positive area was significantly higher than that of the normal group(p<0.05),and the positive area had a significant difference(p<0.05)In the skin,not only the acupoints,sebaceous glands and hairy connective tissue sheaths with strong positive expression,and in dermal cells with a large number of cells showed SP positive,compared with the asthma group,the positive area rate was significantly increased,the results were significant(p<0.05).There was no significant difference in the optical density between the positive region and the model group(p<0.05).The positive rate of TRPV1 was 16.5% ± 1.1% in the epidermis of guinea pigs,the sphenoid connective tissue,the sebaceous glands and the dermis of the guinea pigs were 16.5% ± 1.1%.The asthmatic guinea pig spikes,hairy connective tissue sheaths,sebaceous glands The expression of TRPV1 cells was slightly decreased,but the expression of sheath tissue in root connective tissue increased slightly,and the positive area rate was not significantly different from that of normal group(p<0.05).The guinea pig was not only the acupoints and the connective tissue(p<0.05),and the number of positive cells in the dermis layer was also increased,and the positive area rate was significantly higher than that of the asthma group(p<0.05).Conclusion:1.HAP for have reduced AHR in allergic asthma guinea pigs.2.HAP can inhibit the expression of matrix metalloproteinases(MMPs)and inhibit the enzymatic activity by inhibiting the phosphorylation of TGF-? and its downstream Erk1/2 and p38 MAPK,and thus protect the airway epithelial barrier-related proteins.3.HAP can regulate the spatial distribution of the key of factors neuro – inflammation between the acupuncture points and organs. |
PDF Full Text Request