Application of compound fresh bamboo juice in guinea pig asthma model

Abstract

The invention discloses an application of a compound fresh bamboo juice in a guinea pig asthma model, and relates to the field of medicines, a Hartley albino guinea pig is adopted to construct an OVA induced asthma model, and the model is verified to be successfully constructed through evaluation indexes; evaluating the efficacy of the successfully constructed guinea pig asthma model by using different doses of compound fresh bamboo juice, and evaluating the efficacy of the guinea pig asthma model by scoring guinea pig capsaicin stimulation cough inducing times, alveolar lavage fluid leukocyte number, inflammatory factor content, lung tissue hydroxyproline content and behavioral signs. And evaluating the treatment effect of the compound fresh bamboo juice through Western blot detection, lung histopathology and immunofluorescence staining indexes. In a guinea pig asthma model, high-dose and medium-dose groups of the compound fresh bamboo juice can be used for remarkably reducing the number of cough, reducing the number of white blood cells in alveolar lavage fluid and the level of inflammatory factors and inhibiting pulmonary fibrosis, and a good treatment effect is shown. The results provide powerful support for potential application of the compound fresh bamboo juice in asthma treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the application of compound fresh bamboo sap in OVA-induced guinea pig asthma model. BACKGROUND

[0002] Bronchial asthma is a chronic inflammatory disease of the airway, which is caused by a variety of cells (such as eosinophils, mast cells, T lymphocytes, neutrophils, airway epithelial cells, etc.) and cell components, and is related to airway epithelial damage and shedding, airway wall thickening and mucosal edema, and airway hyperresponsiveness. Since the pathogenesis of the disease is not clear, the commonly used clinical treatment drugs are leukotriene receptor antagonists, adrenal cortical hormones, and antiallergic drugs. These drugs can achieve the purpose of treatment by reducing immune response, reducing inflammatory response, and expanding airway, but adverse reactions may occur when used, and rebound phenomenon is easy to occur. With the vigorous development of traditional Chinese medicine, it is found that the adverse reactions of traditional Chinese medicine in the treatment of allergic asthma are smaller. Compound fresh bamboo sap, as a traditional cough-relieving and phlegm-removing Chinese medicine, has been proved to have anti-inflammatory and expectorant effects, but its specific treatment mechanism and efficacy data in asthma model are still lacking systematic research.

[0003] The current research has the following problems: (1) the effect of compound fresh bamboo sap on OVA-induced asthma model has not been determined, especially lacking systematic evaluation in guinea pigs, a classic asthma model; (2) existing researches mainly focus on simple cough-relieving and phlegm-removing effects, and the effect and dose-effect relationship of the drug in regulating airway inflammation, inhibiting pulmonary fibrosis and other key pathological processes of asthma are insufficient; (3) the influence of the drug on inflammatory factors (such as IL-4, IL-6) and lung tissue pathological changes in asthma model lacks quantitative data support, which limits its precise application in the treatment of asthma.

[0004] With the development of animal model technology, OVA-induced guinea pig asthma model has become an important tool for studying asthma because it can simulate the airway hyperresponsiveness and inflammatory infiltration of human asthma. However, the specific application method and efficacy characteristics of compound fresh bamboo sap in this model have not been determined. SUMMARY

[0005] The present application aims to at least solve one of the problems in the prior art, and provides the application of compound fresh bamboo sap in OVA-induced guinea pig asthma model. The technical solution of the present application is as follows: The application of compound fresh bamboo sap in OVA-induced guinea pig asthma model comprises the following steps: S1: Hartley albino guinea pigs are used to construct OVA-induced asthma model.

[0006] Reagent preparation: Ovalbumin (OVA) complex solution (sensitization): 100 ug OVA powder and 100 mg aluminum hydroxide (AI(OH)3) adjuvant were added to 1 mL of normal saline (NS) to prepare an OVA complex aluminum hydroxide solution; 1% ovalbumin solution (challenge): 1.0 g of OVA powder was added to 100 ml of NS to prepare a 1% OVA solution.

[0007] Modeling process: On the 1st and 14th day of the experiment, 100 ug / mL OVA was injected intraperitoneally for sensitization, and from the 20th to the 28th day, 0.4% OVA solution was used for aerosol challenge (2d / time).

[0008] Verification of whether the model is successfully constructed: The success of the asthma model was verified by behavioral scoring of guinea pigs, determination of the number of coughs induced by capsaicin stimulation, evaluation of body weight changes, white blood cell count in bronchoalveolar lavage fluid, detection of inflammatory factors, and determination of hydroxyproline content.

[0009] S2: The model successfully constructed was subjected to efficacy evaluation using compound fresh bamboo juice.

[0010] Experimental grouping: The experiment was divided into 7 groups: blank group, control group, model group, dexamethasone group, fresh bamboo juice low-dose group, fresh bamboo juice medium-dose group, and fresh bamboo juice high-dose group, with 70 guinea pigs in each group.

[0011] Group administration: From the 15th to the 28th day of the experiment, drug intervention was started (modeling and drug intervention were performed simultaneously). The dexamethasone group was given 0.5 mg / kg dexamethasone by gavage. Compound fresh bamboo juice was prepared into a solution for gavage administration. The dosage for guinea pigs was calculated based on the daily medication amount for a 70 kg adult and the body surface area, i.e., the fresh bamboo juice high-, medium-, and low-dose groups were given 7.8 ml / kg, 3.9 ml / kg, and 1.95 ml / kg, respectively. The model group and the blank group were given an equal volume of normal saline by gavage once a day for 14 days, and OVA aerosolization was performed 1 hour after drug administration.

[0012] 24 hours after the last administration, the guinea pigs were placed in a closed glass aerosolization box, and 1 x 10 -4 mol / L capsaicin was aerosolized and inhaled for 60 seconds. After turning off the aerosolizer, the guinea pigs remained in the aerosolization box for another 60 seconds. Then the aerosolization box was opened, and the number of coughs within the next 2 minutes was recorded.

[0013] Detection index: By comparing the capsaicin stimulation cough frequency of the model group and the administration group, the number of white blood cells in the alveolar lavage fluid, the content of inflammatory factors (IL-4, IL-6), the content of lung tissue hydroxyproline (HYP), the behavior score, the relative expression amount of VEGFA, TNF-A, IFN-c, TGF-beta1, Smad2 / 3, p-Smad2 / 3, Smad4, Smad7, TLR4, MyD88, IKKb, p-IKKb, NF-kB in lung tissue detected by Western blot, lung histopathology examination (HE staining, Masson staining) and immunofluorescence staining (siglece-f, p-nf-kb p65) indexes, the efficacy is evaluated.

[0014] The beneficial effects of the present application are: the present application takes Hartley albino guinea pigs as the research object, and establishes a technical system for evaluating the efficacy of compound fresh bamboo juice in the OVA-induced guinea pig asthma model. The experimental results show that the compound fresh bamboo juice (especially the medium and high doses) can significantly reduce the cough frequency of the model guinea pigs, reduce the level of inflammatory factors, and inhibit pulmonary fibrosis, and the effect and dose-effect relationship of treating asthma are clear. These results provide strong experimental support for the potential application of compound fresh bamboo juice in the treatment of asthma. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The present application adopts Hartley albino guinea pigs to construct the OVA-induced asthma model flow chart; Figure 2 The weight change results of the guinea pigs in each group of the present application; Figure 3 The capsaicin stimulation cough frequency results of the guinea pigs in each group of the present application; Figure 4 The total number of white blood cells in the alveolar lavage fluid of the guinea pigs in each group of the present application; Figure 5 The IL-4 and IL-6 content results of the alveolar lavage fluid of the guinea pigs in each group of the present application; Figure 6 The IL-4 and IL-6 content results of the serum of the guinea pigs in each group of the present application; Figure 7 The IL-4 and IL-6 content results of the lung tissue of the guinea pigs in each group of the present application; Figure 8 The hydroxyproline content results of the lung tissue of the guinea pigs in each group of the present application. DETAILED DESCRIPTION

[0016] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0017] This section will describe in detail specific embodiments of the present invention. Preferred embodiments of the present invention are shown in the accompanying drawings. The purpose of the drawings is to supplement the textual description with graphics, so that people can intuitively and vividly understand each technical feature and overall technical solution of the present invention, but they should not be construed as limiting the scope of protection of the present invention.

[0018] The compound fresh bamboo sap used in this embodiment was produced and supplied by Jiangxi Jiuhua Pharmaceutical Co., Ltd. (Approval number: National Drug Approval Number Z36020947; Drug Production Batch Number: 07230204). The drug type is traditional Chinese medicine, and the prescription type is over-the-counter (OTC). The main ingredients are: fresh bamboo sap, houttuynia cordata, loquat leaf, platycodon grandiflorus, raw pinellia ternata, ginger, and peppermint oil (or peppermint oil). The excipients are sucrose, purified water, and sodium benzoate.

[0019] 1. Construct an OVA-induced guinea pig asthma model and improve its model evaluation indicators. In animal asthma model research, the applicability of different species varies. Guinea pigs, due to their significant sensitization to allergens such as ovalbumin (OVA), can stably simulate the core pathological features of human asthma, including airway hyperresponsiveness, eosinophil infiltration, and elevated inflammatory factors. Furthermore, their respiratory physiological characteristics are close to those of human airway response mechanisms, making them an internationally recognized classic model animal for asthma research. In contrast, rats and mice have smaller airway structures, making it difficult to accurately assess macroscopic symptoms such as airway spasm. While larger animals such as dogs and monkeys have physiological characteristics closer to humans, their high breeding costs and long modeling periods make them unsuitable for initial drug efficacy screening. Therefore, this experiment selected Hartley albino guinea pigs as the research subject to ensure the stability of the model and its relevance to human asthma pathology.

[0020] like Figure 1 The specific modeling steps are as follows: Experimental animals: Hartley albino guinea pigs, male, 4-5 weeks old, weighing 200-250g, were purchased from Ganzhou Animal Husbandry and Fisheries Research Institute. The animals were housed in IVC cages at a temperature of 20-26℃ and humidity of 40%-70%, with alternating light and dark conditions for 12 hours. They had free access to Speforg guinea pig pellets and water. The experiment began after one week of acclimatization.

[0021] Group design: The animals were randomly divided into 7 groups, with 70 animals in each group. The groups were: blank group, control group, model group, dexamethasone group (positive control), low-dose compound fresh bamboo sap group, medium-dose compound fresh bamboo sap group, and high-dose compound fresh bamboo sap group.

[0022] Sensitization treatment: On day 1 and day 14 of the experiment, guinea pigs in the model group, dexamethasone group and each compound fresh bamboo sap dosage group were intraperitoneally injected with 1 mL of 100 μg / mL OVA solution (containing 100 mg aluminum hydroxide adjuvant, OVA purchased from Shanghai Yuanye Biotechnology Co., Ltd., purity 98%); the control group was injected with an equal volume of physiological saline; the blank group was not treated.

[0023] Activation treatment: From day 20 to day 28 of the experiment, the model group, dexamethasone group and each compound fresh bamboo sap dosage group were activated by nebulization using 0.4% OVA solution (dissolved in physiological saline) through the Ruiward HY-JSE01 aerosol nebulization exposure box for 1 minute each time, once every 2 days; the control group was nebulized with an equal volume of physiological saline; the blank group was fed normally and did not receive nebulization treatment.

[0024] Record weight: During the experiment, the weight of each group of guinea pigs (blank group, control group, model group, dexamethasone group, compound fresh bamboo juice / medium / high dose group) was measured weekly. The initial weight (day 0) and the weight data at 7 days, 14 days and 21 days after the intervention were recorded, and the weight change curve was plotted.

[0025] The results are as follows: Through observation, guinea pigs exhibit coughing by stretching their necks forward, opening their mouths, and twitching their abdomens, until they develop symptoms of airway spasm such as deeper and faster breathing. As shown in Figure 2, compared with the blank group and the control group, the guinea pigs in the model group had slow weight gain and even weight loss at some time points, suggesting that the growth of the OVA-induced asthma model was limited due to factors such as airway inflammation and impaired feeding. according to Figures 3 to 8 It can be seen that, compared with the control group and the blank group, the number of coughs induced by capsaicin in the model group of guinea pigs increased significantly, the number of white blood cells in the bronchoalveolar lavage fluid increased significantly, the levels of inflammatory factors such as IL-4 and IL-6 in the bronchoalveolar lavage fluid, serum and lung tissue increased significantly, and the content of HYP in lung tissue also increased significantly. All of the above indicators reflect the typical characteristics of the asthma model.

[0026] 2. Pharmacodynamic study of compound fresh bamboo sap in an OVA-induced guinea pig asthma model. Using an OVA-induced guinea pig asthma model as the research subject, this study investigated the therapeutic effect of compound fresh bamboo sap at different doses to explore the dose-response relationship. The daily dosage was calculated based on the body surface area of ​​guinea pigs and used as the reference for the dosage of the compound fresh bamboo sap. The dosages were divided into low-dose (1.95 ml / kg), medium-dose (3.9 ml / kg), and high-dose (7.8 ml / kg) groups. A model group, control group, blank control group, and dexamethasone group (0.5 mg / kg, positive control) were also included.

[0027] Experimental subjects: Hartley albino guinea pigs, male, 4-5 weeks old, weighing 200-250g, 70 in each group. After successful modeling, guinea pigs that met the behavioral scores and cough frequency criteria were selected for drug efficacy evaluation.

[0028] Experimental procedure: 1) Administration method On days 1 and 14 of the experiment, each guinea pig was intraperitoneally injected with 100 μg / mL OVA, while the control group was injected with an equal volume of saline. From days 20 to 28, each guinea pig was nebulized with 0.4% OVA solution for 1 minute, twice a day. The blank control group was given physiological saline instead. From days 15 to 28 of the experiment, drug intervention was initiated (modeling and drug intervention were carried out simultaneously). The dexamethasone group was given 0.5 mg / kg dexamethasone by gavage. The high, medium, and low dose groups of fresh bamboo sap were administered at doses of 7.8 ml / kg, 3.9 ml / kg, and 1.95 ml / kg, respectively. The model group and the blank control group were given an equal volume of physiological saline by gavage once a day for 14 days. OVA nebulization was performed 1 hour after drug administration.

[0029] 24 hours after the last administration, the guinea pigs were placed in a sealed glass nebulizer and 1×10⁻⁶ doses were administered. -4 The guinea pig was inhaled with capsaicin at a concentration of mol / L for 60 seconds. After turning off the nebulizer, the guinea pig remained in the nebulizer chamber for another 60 seconds. Then the nebulizer chamber was turned on, and the number of coughs in the guinea pig over the next 2 minutes was recorded.

[0030] 2) Behavioral characteristics score When the OVA nebulization model was initiated on day 20 of the experiment, the asthmatic animals were assessed behaviorally: trembling or head nodding was scored as 1 point, coughing or deepening of breathing as 2 points, rhythmic contraction-like panting as 3 points, and falling as 6 points. The most severe behavioral sign was used as the score.

[0031] 3) Animal culling After 28 days of the experiment, the guinea pigs were euthanized after the last administration of the drug and disinfected with 75% alcohol for 2-3 minutes.

[0032] 4) Serum collection Arterial blood was collected from the abdominal aorta of a guinea pig using a 5ml syringe, and serum was extracted by centrifugation.

[0033] 5) Cell classification and counting in bronchoalveolar lavage fluid (BALF): The limbs were secured on a clean bench, and the trachea was gradually exposed. Sterile saline was injected into the lungs at appropriate pressure after ligating one side of the hilum and inserting an endotracheal tube. The lungs were observed to expand and turn pale. The injection was left for 20-30 seconds. 10 mL of BALF was collected from each guinea pig and stored at 4°C. 200 μL of the mixed BALF was transferred to a cell counting chamber to count the total number of cells. The remaining solution was then distilled at 1500 r·min⁻¹. -1 Centrifuge for 10 min, resuspend the centrifuged cell pellet in Hanks' solution, smear the suspension, dry it and place it on a staining plate, add Wright's Giemsa staining solution to cover it, then rinse the smear and air dry it. Under an optical microscope, classify and count the cells according to their morphology.

[0034] 6) ELISA method for measuring inflammatory factors IL-4 and IL-6 in BALF, serum, and lung tissue were measured using the ELISA method, and the experimental procedures were strictly performed according to the instructions of the enzyme-linked immunosorbent assay kit.

[0035] 7) Determination of hydroxyproline in lung tissue A hydroxyproline (HYP) assay kit was used. Lung tissue (50 mg) was hydrolyzed, and the pH was adjusted to 6.5 according to the manufacturer's instructions. The tissue hydrolysate was then treated with 60 mg of activated charcoal, centrifuged for 10 min, and the supernatant was used to measure the absorbance at 550 nm using a UV spectrophotometer.

[0036] 8) Lung tissue pathological examination The left lung was fixed in neutral formaldehyde, rinsed with water for 4 h, and then dehydrated sequentially with 70%, 80%, 90%, and 100% ethanol. After dehydration, the vitreous tissue was encapsulated in xylene, embedded in paraffin, and cut into 5 μm sections using a microtome. HE staining and Masson staining were performed according to the kit instructions.

[0037] 9) Western blot The relative expression levels of vascular endothelial growth factor A (VEGFA), tumor necrosis factor A (TNF-A), interferon c (IFN-c), transforming growth factor β1 (TGF-β1), Smad2 / 3, phosphorylated (p)-Smad2 / 3, Smad4, Smad7, Toll-like receptor 4 (TLR4), myelodifferentiation factor 88 (MyD88), NF-κB kinase b inhibitor (IKKb), p-IKKb, and NF-κB were detected using Western blot analysis. Guinea pig lung tissue was collected, minced, divided, and centrifuged. Cytoplasmic and nuclear proteins were extracted using a protein extraction kit. The resulting protein solutions were analyzed using a BCA protein assay kit. Then, 20 μL of protein solution (20–40 μg) was added to an SDS-PAGE apparatus for electrophoresis. After completion, the samples were transferred to a PVDF membrane and blocked with 5% skim milk solution or 3% bovine serum albumin solution. The PVDF membrane was treated with primary antibody, secondary antibody, and ECL luminescence reagent. The optical density values ​​of the protein bands were analyzed using a Gel-Pro analyzer.

[0038] 10) Immunofluorescence staining Tissue samples were soaked in goat serum and then incubated overnight at 4°C with rabbit anti-Siglece-F antibody and mouse anti-p-nf-kb p65 antibody. Afterwards, the samples were heated at room temperature for 90 minutes with 1:2000 diluted FITC-labeled goat anti-mouse IgG and CY3-labeled goat anti-rabbit IgG. Finally, the samples were stained with DAPI and washed three times with PBS.

[0039] Experimental test results: a. Number of coughs: Figure 3 The results showed that, compared with the model group, the behavioral scores of guinea pigs in the medium and high dose fresh bamboo sap groups and the dexamethasone group were significantly reduced (P<0.05), and the number of coughs within 2 minutes after capsaicin stimulation was significantly reduced (P<0.01). The high dose group had similar effects to the dexamethasone group.

[0040] b. Analysis of bronchoalveolar lavage fluid (BALF): Figure 4 The results showed that the total number of white blood cells and the proportion of eosinophils in the model group were significantly higher than those in the control group (P<0.001); the medium and high doses of fresh bamboo sap significantly reduced the total number of white blood cells and the proportion of eosinophils (P<0.05), suggesting that it can reduce airway inflammatory infiltration.

[0041] c. Inflammatory factor detection: Figures 5-7The results showed that the detected indicators in the model group were significantly higher than those in the control group (P<0.001); the medium and high dose fresh bamboo sap groups could significantly downregulate the levels of IL-4 and IL-6 in BALF, serum and lung tissue (P<0.05) in a dose-dependent manner, indicating that it can inhibit the release of asthma-related inflammatory factors.

[0042] d. Determination of hydroxyproline (HYP) content in lung tissue: Figure 8 The results showed that the HYP content in the model group was significantly higher than that in the control group (P<0.001); the medium and high dose fresh bamboo sap groups could significantly reduce the HYP content (P<0.05), further confirming its inhibitory effect on pulmonary fibrosis.

[0043] In summary, compound fresh bamboo sap has a significant therapeutic effect on OVA-induced guinea pig asthma model, reducing airway hyperresponsiveness, inhibiting the release of inflammatory factors and pulmonary fibrosis, with better effects at medium and high doses, showing a certain dose-response relationship.

[0044] 3. Using Hartley albino guinea pigs as the research subject, this study explored the application prospects of compound fresh bamboo sap in the treatment of asthma in pets and humans. The above-mentioned experiments have demonstrated that compound fresh bamboo sap can significantly improve airway hyperresponsiveness, reduce inflammatory infiltration and inhibit pulmonary fibrosis in OVA-induced guinea pig asthma models, suggesting its potential application value in the treatment of asthma in pets (such as guinea pigs and other small pets) and humans.

[0045] In summary, this invention includes the following: (1) Based on the weight (200-250g) and age (4-5 weeks) of Hartley albino guinea pigs, precise dosage regimens of 1.95ml / kg, 3.9ml / kg, and 7.8ml / kg were designed to provide a reference for drug administration in animals of different weights. (2) An OVA-induced guinea pig asthma model was constructed, and a model evaluation index system with behavioral scores, capsaicin-induced cough frequency, bronchoalveolar lavage fluid white blood cell count, inflammatory factor levels, lung tissue pathology, and hydroxyproline content as the core was improved. (3) The OVA-induced guinea pig asthma model was treated with compound fresh bamboo sap, and its effect on improving key pathological processes of asthma was clarified. (4) A specific method for evaluating the efficacy of compound fresh bamboo sap based on multi-dimensional indicators (behavioral, inflammatory factors, histopathology, etc.) was established.

[0046] Compared with existing studies, this invention has the following advantages: (1) It establishes a standardized method for constructing an OVA-induced guinea pig asthma model and a multi-index evaluation system. The model has high stability and can accurately simulate the core pathological features of asthma. (2) It evaluates the efficacy of the drug from multiple levels, including behavioral (coughing, airway spasm), humoral (inflammatory factors), and histopathological (inflammatory infiltration, fibrosis), and comprehensively reflects the therapeutic effect of compound fresh bamboo sap on asthma. (3) By setting different dose groups, the dose-effect relationship of compound fresh bamboo sap was clarified (medium and high doses have more significant effects), providing experimental basis for the selection of clinical drug dosage. (4) This invention is the first to systematically verify the anti-asthmatic effect of compound fresh bamboo sap in an OVA-induced guinea pig asthma model, providing experimental support for its application in the treatment of pet asthma and human asthma.

[0047] Without causing conflict, those skilled in the art can freely combine and use the above-mentioned additional technical features.

[0048] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various other corresponding changes and modifications based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of the present invention.

Claims

1. The application of compound fresh bamboo sap in an OVA-induced guinea pig asthma model, characterized in that... Includes the following steps: S1: An OVA-induced asthma model was constructed using Hartley albino guinea pigs. The success of the asthma model was verified by measuring the behavioral scores of the guinea pigs, the number of coughs induced by capsaicin stimulation, the weight change, the white blood cell count in the bronchoalveolar lavage fluid, the detection of inflammatory factors, and the determination of hydroxyproline content. S2: The efficacy of compound fresh bamboo sap was evaluated in the successfully constructed guinea pig asthma model.

2. The application according to claim 1, characterized in that, In step S1, the Hartley albino guinea pigs are male, weighing 200-250g and aged 4-5 weeks.

3. The application according to claim 1, characterized in that, In step S1, the behavioral scoring includes 1 point for tremor or nodding, 2 points for cough or deepening of breathing, 3 points for rhythmic contraction-like shortness of breath, and 6 points for falling, with the most severe sign being scored; the bronchoalveolar lavage analysis includes total white blood cell count and differential count; the inflammatory factor detection includes IL-4 and IL-6 levels in BALF, serum, and lung tissue.

4. The application according to claim 1, characterized in that, In step S2, the method for evaluating drug efficacy specifically includes: After acclimatizing the guinea pigs, they were administered compound fresh bamboo sap via gavage at doses of 1.95 ml / kg, 3.9 ml / kg, and 7.8 ml / kg, once daily for 14 days. One hour after administration, OVA nebulization challenge was performed. The efficacy was evaluated by comparing the behavioral signs scores, capsaicin-induced coughing frequency, white blood cell count in bronchoalveolar lavage fluid, IL-4 and IL-6 inflammatory factor content, hydroxyproline content in lung tissue, relative expression levels of various proteins such as VEGFA and TNF-A in lung tissue, and the results of lung tissue pathological staining and immunofluorescence staining.

5. The application according to claim 4, characterized in that, The efficacy evaluation indicators include the number of coughs induced by capsaicin stimulation, the number of white blood cells in bronchoalveolar lavage fluid, the content of inflammatory factors IL-4 and IL-6, the content of hydroxyproline in lung tissue, behavioral signs scores, the relative expression levels of VEGFA, TNF-A, IFN-c, TGF-β1, Smad2 / 3, p-Smad2 / 3, Smad4, Smad7, TLR4, MyD88, IKKb, p-IKKb, and NF-κB in lung tissue detected by Western blot, lung tissue pathological examination, and immunofluorescence staining indicators.

6. The application according to claim 1, characterized in that, The compound fresh bamboo sap was able to reduce the number of coughs, lower the level of inflammatory factors, and inhibit pulmonary fibrosis in an OVA-induced guinea pig asthma model.

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