支气管哮喘动物模型 z-bo 2021-01-12 282

　　Background: Grape is widely used as a natural dietary supplement due to its unique chemical composition and high nutritional value. Fruits are polyphenols, anthocyanins, flavanols, stilbene (resveratrol), phenolic acid, protein, fat, vitamins (C, A), minerals (calcium, boron, phosphorus, etc.), water, carbohydrates And a good source of fiber. The medicinal value of plants has long been recognized by folk medicine. Regarding the inflammatory process of chronic airway diseases such as asthma and chronic obstructive pulmonary disease, the pathology of the disease is directly related to the increase in the production of reactive oxygen species in the lungs. Some clinical studies have shown that supplementing with antioxidants is beneficial for adults with mild to moderate asthma. Therefore, oxidative damage plays an important role in the pathogenesis of bronchial asthma, so it may be a potential target for asthma treatment. Asthma is a chronic inflammatory disease of the airway, which is characterized by reversible contraction of the tracheobronchial tree and high responsiveness to various stimuli to the airway, such as environmental allergens, respiratory infections, cold air, exercise and certain drugs. In humans, allergic asthma is mainly caused by type I allergic reactions, which reflect increased sensitivity to the production of immunoglobulin E (IgE) from exogenous allergens, and its secretion and differentiation depend on CD4 + auxiliary. T cells (Th2 type). The cytokines produced by activated Th2 cells such as interleukin (IL)-3, IL-4, IL-5, and IL-13 in turn promote IgE cell production, mast cell growth (IL-4) and eosinophilia in B cells IL-5 plays a role in the survival of granulocytes. IL-4 and IL-13 stimulate epithelial cells to produce transforming growth factor (TGF-βα) leading to mucosal epithelial metaplasia and fibroblast proliferation. Pro-inflammatory mediators include tumor necrosis factor (TNF) and granulocyte macrophage colony stimulating factor (GM-CSF), which stimulate the expression of vascular adhesion molecules on endothelial cells and cause inflammatory leukocytes to enter the bronchial tree. This study evaluated the therapeutic potential of dried fruit products grapes on ovalbumin-induced allergic asthma in rat models.

　　Method: Animals: Male Wistar rats (180-220g; 8-10 weeks old), animals are raised under controlled conditions (room temperature 25-2°C, humidity 45-5%, light for 12 hours: dark cycle). All animals can eat and drink freely.

　　 Experimental design: Male wistar rats were randomly divided into 5 groups (6 in each group). Group 1, non-sensitized control, received vehicle (0.4 mL/kg); group 2, ovalbumin (OVA) sensitized or asthma control, received vehicle only; group 3, (OVA + dexamethasone) , The reference standard is OVA sensitization to give dexamethasone (2.5 mg/kg body weight); groups 4 and 5, the experimental group (OVA + vvhe 1 and OVA + vvhe 2) were sensitized to OVA and obtained grape ethanol extract (vvhe) ) (31 and 42.5 mg/kg/body weight). Grape fruit is an important component (API) mentioned in the Indian Ayurvedic Pharmacopoeia. Oral drugs or excipients every morning for 28 consecutive days. All animals (except group 1) were sensitized by intraperitoneal injection of allergen suspension (ovalbumin, 40 mg/mouse + aluminum hydroxide, 2 mg/mouse) on day 1. After 15 days of sensitization, the animals were exposed to physiological saline containing 1% ovalbumin for 20 minutes. Animals in the non-sensitized group were exposed to saline in the same environment. The nebulized inhalation solution is inhaled once a day in a closed room (size 40×32×32 cm) for 8 consecutive days, that is, from 15 to 22 days, and then inhaled on 25 and 28 days.

　　 Pulmonary function and bronchoconstriction test: On the 28th day, after exposure to OVA, rats were anesthetized by intraperitoneal injection of sodium pentobarbital (105 mg/kg). A 12G cannula (2 mm diameter) was used for tracheal intubation. The cannula is connected to a respiratory rate recorder and a differential pressure sensor to measure the respiratory rate (f, times/min). The lung tidal volume (VT, ml) is obtained through the electronic integration of the airflow signal. Femoral vein intubation records (1) before and after the use of excipients, 2) before and after methacholine treatment, an average of one data point, changes in F and VT were obtained for 10-12 respiratory cycles. Vecuronium (0.2 mg/kg) is injected intravenously to avoid spontaneous breathing. Use a small polyethylene tube to discharge excess bronchial secretions without disturbing the trachea.

　　 Bronchoalveolar lavage fluid (BAL) collection: After measuring the pulmonary function parameters, lavage the lungs 3 times with 5 ml of 0.9% sterile saline solution (5 ml × 3) through a hollow catheter. The BAL liquid collected from each mouse was combined and centrifuged (1500 rpm, 4°C, 10 minutes). The supernatant was separated and stored at −80°C until analysis of IgE and cytokines. The cell pellet was resuspended in 1 ml of normal saline, and the total number of cells and white blood cell count were determined by the method described by Jung et al.

　　 Serum preparation and cell counting: After collecting BAL fluid, blood was collected by puncture at two different parts of the heart. The first part (2.5–3 ml) is collected in a non-heparin anticoagulant tube; centrifuged (3000 rpm, 10 minutes) and the serum is stored at −80°C to evaluate IgE and cytokines. (0.5ml) is a heparin anticoagulation tube collected and stored at 4°C until the total number of cells and the classification count are determined. Within 30 minutes after heparinized blood is collected, the leukocytes in the blood are automatically analyzed and counted separately. Cell counts and smears were stained with Leishman (1.5% methanol for 6 minutes). IgE, LTD4, cytokines, nitric oxide, serum and nitrite content in BAL fluid: use enzyme-linked immunosorbent assay (ELISA) reagent to determine serum (500μL) and bronchoalveolar lavage fluid (5ml) IgE, LTD4 And cytokine (TNF, IL-4, IL-5 and IL-1β) levels. The samples are analyzed on an automatic elisa plate reader. The concentration of total NO and nitrite in serum and BAL fluid was measured with a nitric oxide calorimeter.

　　 Lung lavage fluid histamine analysis: After collecting BAL fluid, the lung tissue lobes of each animal were dissected. One of the lung lobes was homogenized with 2.5 ml of normal saline, and histamine was analyzed with a 650 nm spectrophotometer according to the method described by Shore et al.

　　 Histological examination: The dissected lung tissue was washed with saline and fixed in a 10% neutral formaldehyde solution at 4°C for 24 hours. Embed in paraffin and sectioned. Observe after HE staining.

　　Result: The effect of vvhe on lung function parameters: Compared with the non-sensitized group, the OVA sensitized group had a significant increase in respiratory rate after the injection of methacholine. Compared with the OVA-sensitized control group, the respiratory rate of animals receiving vvhe 1 (31 mg/kg) or vvhe 2 (42.5 mg/kg) decreased by 13 or 21%, respectively. Compared with the control group, the tidal volume of OVA-sensitized animals was significantly reduced. Compared with the OVA sensitized control group, the tidal volume of rats treated with vvhe 2 (42.5 mg/kg/body weight) was significantly increased by 11%. The respiratory rate and tidal volume of the dexamethasone control group were increased by 37 and 28%, respectively. There are differences Significance. The effect of vvhe on the cell count in the circulating blood: The total number of circulating white blood cells, eosinophils and neutrophils in the blood of the control group was significantly higher than that of the non-sensitized control group. In contrast, the lymph in the blood of sensitized rats The number of cells is less than that of the normal group. Compared with the asthma control group, the number of eosinophils and neutrophils increased significantly after treatment with dexamethasone and vvhe 2. The two drug treatments also normalized the lymphocyte count in the animal's blood. The effect of

　　vvhe on the number of inflammatory cells in BAL fluid: Compared with the non-sensitized control group, the total number and cell count of the BAL fluid group increased significantly in the OVA sensitized control group (p<0.001). Compared with the sensitized control group, eosinophils, lymphocytes, macrophages and neutrophils in the dexamethasone and VVHE experimental groups were significantly reduced. Low-dose VVHE can also reduce the number of inflammatory cells in the lavage fluid, which is statistically significant for lymphocytes and macrophages. The effect of vvhe on the levels of LTD4 and cytokines in serum: Compared with the non-sensitized group, the OVA-sensitized control group can significantly increase the levels of serum LTD4 and all cytokines IL-4, IL-5, TNF and IL-1β. Compared with the OVA sensitized group, vvhe 1 (31 mg/kg body weight) and vvhe 2 (42.5 mg/kg body weight) reduced serum IL-4 levels by 17 and 24.2%, IL-5 levels by 17.1 and 28.2%, and TNF Level 17 and 30.2%, IL-1β level 5 and 15.3%, LTD4 level 4.3 and 11%. Dexamethasone treatment of OVA sensitized animals significantly reduced the serum LTD4 level by 44.7%, IL-4, 56.6%, IL-5, 47.6%, TNF, 53.3% and IL-1β, 57.11%. The effect of

　　vvhe on IgE levels in serum and bronchoalveolar lavage fluid: the sensitization and challenge of IgE in rat serum and BAL fluid were significantly higher than those in the non-sensitized control group. Compared with the OVA-sensitized control animals, the animals receiving vvhe 1 or vvhe 2 showed a significant reduction of 17% or 31% in serum IgE levels, and a 14% or 26% reduction in IgE levels in BAL fluid. Compared with the OVA-sensitized control group, the serum IgE level of the dexamethasone treatment group was reduced by 47.8%, and the IgE level in the BAL fluid was reduced by 58%. The effect of

　　vvhe on the content of total nitric oxide and nitrite: Compared with the non-sensitized control group, the total NO and nitrite levels of OVA-sensitized animals were significantly increased. Compared with animals in the asthma group, serum nitric oxide and nitrite levels were reduced by 29.7% and 31.4%, respectively. BAL fluid was reduced by 15% and 27%, respectively.

　　 The effect of Vvhe on the histamine level in lung tissue: The histamine content in the lung tissue homogenate of rats in the OVA control group was significantly higher than that in the non-sensitized control group. Compared with the OVA-sensitized control group, Vvhe1 and Vvhe2 decreased by 25 and 41.6%, respectively, and increased histamine levels. In the dexamethasone treatment group, the histamine level of lung tissue homogenate decreased by 66.6%. The effect of

　　 on lung histopathology: The histopathological examination of the lung tissue of the OVA control group showed that the lumen of the bronchioles was reduced, and inflammatory cells infiltrated the tissues around the bronchus and the epithelium fell off. The protection induced by Vvhe and dexamethasone improves lumen size and significantly reduces cell infiltration.

　　 Conclusion: The research results show that vvhe may play an important role in the treatment of bronchial asthma. It is manifested in (1) by inhibiting the release of histamine and the production of cytokines against the continuous inflammatory process of asthma. (2) Improve lung function by antagonizing bronchial hyperresponsiveness caused by allergens; (3) Prevent inflammatory cell infiltration (eosinophils, lymphocytes, neutrophils) from entering the airway. It is recommended to conduct further research to confirm that it is a valuable anti-asthma drug.