动物造模,二甲双胍,呼吸系统 z-bo 2020-09-10 442

　　Introduction: At present, metformin or metformin hydrochloride is considered the first choice for the treatment of type 2 diabetes. It has many effects, including intestinal glucose uptake and insulin-mediated increase in glucose processing, transport of glucose transporters, inhibition of hepatic glucose output, and fatty acid oxidation. The mechanism of metformin may be mediated by the protein kinase AMPK activated by adenosine monophosphate. More importantly, metformin can reduce inflammation and autoimmune encephalomyelitis, especially by protecting blood vessels through the glucocorticoid barrier. It is used for cancer prevention and treatment and other beneficial effects. In contrast, the toxicity of metformin is related to metabolic disorders that cause liver and kidney disease. Its toxicity is mainly related to lactic acidosis, which is caused by acute and chronic exposure. Metformin is gradually recognized as a drug that acts on the central nervous system and may accelerate the onset and/or development of Parkinson's disease. Although metformin is a widely used drug, its effect on the respiratory system has not been fully proven. Vascular endothelial growth factor was originally described as a vascular permeability factor, but it has many other functions. The expression of VEGF-a is usually regulated by hypoxia inducer (HIF-1). In asthma, the activation of AMPK can regulate the HIF/VEGF-a pathway. Vascular endothelial growth factor-a receptors are vascular endothelial growth factor-1 and vascular endothelial growth factor-2, the latter being a kinase domain receptor, which is the cell growth of endothelial cells such as nitric oxide and vascular endothelial growth factor.

　　is mainly responsible for (NO) and prostacyclin production, angiogenesis and vascular permeability. Lung tissue is rich in this protein, which is essential for the development of lung structure and function. It is also highly resistant to eosinophilia, mucosalization, airway remodeling (subcutaneous collagen deposition), smooth muscle hyperplasia and hypersensitivity. important. In addition, in asthmatic mice, metformin reduced the expression of various growth factors. The purpose of our study is the ability of metformin to regulate plasma glucose and insulin levels, as well as the effects of inflammatory cells and bronchoalveolar lavage fluid on nitric oxide concentration, lung tissue collagen content and vascular endothelial growth factor. This is to find out the level. At the same time, in a mouse obesity model caused by overnutrition after birth, the pulmonary power of acetylcholine-induced bronchoconstriction was evaluated over time. The mice were placed at a temperature of 22±2°C and maintained in a light-dark cycle of 12:12 hours. Get food and water for free. Obese Swiss mice are obtained through excessive nutrition after delivery. Animals: On the first day of birth, newborn male rats were randomly divided into 3 (obese group) and 12 (control group) for each animal. After weaning, all male puppies received a normal diet. At 7 weeks of age, the mice were weighed and processed to measure body fluid intake. It is divided into obesity group and normal control group (CC, control group). The obesity group is the obesity control group (OC) and the metformin treatment group (OM). The CC and OC groups received regular drinking water, while the OM group received metformin (mixture; 300 mg/kg/day). Raise each mouse and carefully monitor the water consumption for 2 weeks. Raise the mice separately during the treatment and carefully monitor the water consumption for 2 weeks. Estimate the daily water consumption by weighing a water bottle, and calculate these values at a daily rate (g water consumption/body weight 10g). At the end of the specified time, all animals were weighed and the Lee Obesity Index was calculated. These groups are used for histology (biochemical and histological parameters) or respiratory dynamics analysis. The total number of animals is 63,

　　Biochemical parameters and sample processing: After fasting for 12 hours, use a portable blood glucose meter to puncture the tail blood vessels to obtain blood glucose readings. To measure insulin, mice were treated under inhalation anesthesia (isoflurane) to obtain blood samples. Collect blood samples, centrifuge at 5300pm for 10 minutes at 4°C, and store in the refrigerator. To measure the plasma concentration of insulin, please use the "Insulin Enzyme Combined Immunosorbent Measurement Kit". Fasting blood glucose and serum insulin levels were used to calculate the insulin resistance index (HOMA-IR) using fasting blood glucose (mmol/L) x fasting serum insulin (MU/L/22.5). Bronchoalveolar lavage fluid (BALF) cells and nitrite/nitrate concentration (NOX): BALF and blood samples were from the same mouse. After collecting the blood sample, the mouse trachea was incised to obtain the lavage fluid. During this process, 2.5 ml PBS was injected into the trachea. A hemocytometer was used to detect the concentration of white blood cells (WBC) in BALF, and the survival rate was determined by the tricolor blue exclusion method. The difference in BALF cells was confirmed in the Gimza stained samples. Count at least 200 BALF cells in each sample to assess the difference in cell number (%). Evaluate BALF through the oil reaction to determine the nitrite/nitrate concentration (NOX). Just put 90μL into a 96-well plate, and then mix with 10μL 0.7? U/ml nitrate reductase was incubated for 3 hours. Then 50 μl of 1% sulfonamide and 50 μl of 0.1% naphthalene ethylenediamine were added to each well and incubated at room temperature for 5 minutes. Measure the absorbance of the mixture at 540m with a microplate reader and compare it with the standard nitrite curve. Histological and immunohistochemical analysis: After collecting alveolar lavage fluid, the right lung was collected, fixed with 10% paraformaldehyde and embedded in paraffin. In order to obtain the total collagen fiber content, the fiber content in the alveolar septum (parenchyma) and airway was quantitatively analyzed by picric acid polarization method using slices with a thickness of 4 microns. .. The alveolar septum and airway were measured with a conventional optical microscope at 200x and 400x magnification, respectively. For VEGF-a immunohistochemistry, 7 micron thick sections were used, deparaffinized and washed with PBS. After incubating in 1% hydrogen peroxide and 1% PBS/BSA for 30 minutes, 1:100 anti-VEGF-a was incubated overnight at 4°C. The sections were washed with PBS and incubated with biotinylated anti-rabbit 1:500 and streptavidin Western mustard peroxidase for 1 hour. After washing with PBS, it was incubated with 3.3-diaminobenzidine in 50 mM TBS containing 0.1% hydrogen peroxide. Respiratory dynamics: intraperitoneal anesthesia with pentopental sodium 68 mg/kg and xylazine 12 mg/kg. Small animal respirators are used for tracheal intubation and ventilation (main ventilation volume is 10 ml/kg, 120 breaths/min, and positive expiratory pressure is 3 cmH2O). Inject brominated pan (0.5 ml/kg, i.p.) and tramamol (50 mg/kg, i.m.) and use a heating pad to maintain the animal’s body temperature. A 3 s vibration disturbance was applied to the tracheal intubation tube connected to the airway, and the input impedance (Zrs) of the lung system was measured. By fitting the normal phase model to the obtained data, the mechanical parameters of airway resistance (RAW), tissue attenuation (GTIS) and tissue elasticity (HTIS) can be estimated. In adolescents, an ultrasonic device was used to evaluate the bronchoconstriction of each mouse by spraying saline (vehicle, 0.9% sodium chloride) and 100 mg/ml acetylcholine. After 15 seconds, the respiratory dynamics were evaluated for more than 90 seconds. In order to standardize the lung volume history, the lungs are inflated once before starting to deliver the solution at a pressure of 30 cmH2O. Each mouse obtained respiratory dynamics parameters from the 30s, 60s and 90s, and checked the data with the highest decision factor (≥0.85). At the end of the experiment, the animals were sacrificed under anesthesia. Results: The effectiveness of the model to induce obesity was evaluated by body weight and Lee index. Compared with the CC group, these parameters in the OC and OM groups have been significantly increased (p\u003c0.0001). Metformin treatment did not reduce weight and did not restore Lee index in the OM group. Compared with the CC group, the blood glucose, insulin levels and HOMA of the two obese groups increased significantly. The blood glucose level of OC group was higher than that of CC group (P\u003c0.005), while the blood glucose level of OM group was higher than that of CC group. These results provide evidence for the establishment of metabolic syndrome, and metformin treatment cannot repair metabolic disorders. For BALF inflammatory cells, white blood cells and foam cells in the OC group increased significantly compared with the CC group. Compared with the CC and OC groups, the white blood cells of the OM group recovered to a level similar to that of the CC group, but the foam cells of the OM group decreased. For macrophages, compared with the CC group, OC was found to be significantly lower. However, compared with the CC and OC groups, OM macrophages increased. The ratio of lymphocytes in each group did not change, and the NOX value of each group was similar. Compared with the CC group, the airway total collagen content of the OC group and OM group increased. However, compared with the control group, metformin treatment did not repair the airway. There is no difference in the collagen content of the alveolar compartment. Compared with the CC group, the number of VEGF-a in the OC group increased significantly. The level of vascular endothelial growth factor A in the OM group decreased, but the difference was statistically significant with the CC group. Therefore, the results show that metformin cannot completely restore the basic level of pulmonary vascular properties in the CC group. After spraying saline and acetylcholine in the 30s, 60s and 90s, the mechanical parameters of the lungs were recorded. Compared with the CC group, the obese group (OC and OM) showed significant bronchoconstriction after 30 seconds of inhalation of acetylcholine. Metoformin treatment failed to prevent the early exacerbation of acetylcholine in 30-year-old obese mice. In the 60s and 90s, all groups responded similarly to acetylcholine. At 30, 60 and 90 seconds, the GTI level of acetylcholine sprayed in all groups was higher than that of normal saline spray. No change in HTIS value was observed.

　　Conclusion: Postpartum overnutrition leads to limited improvement in plasma glucose and metformin insulin levels in obese mice, and it has no significant effect on nitrogen oxides. In addition, metformin did not improve BALF macrophages, and obese mice had reduced vascular endothelial growth factor and total collagen levels. Despite these improvements, the use of acetylcholine to induce bronchoconstriction in obese mice to evaluate the efficiency of the drug for repairing lung function has been shown to be very inefficient. This study shows that metformin does not promote overall lung improvement. However, in order to determine the effects of metformin on the lungs, it is necessary to study different animal obesity models and different stages of metabolic syndrome.