Isorhamnetin Alleviates Airway Inflammation Via The Nrf2/Keap1 Pathway In A Mouse Model Of COPD

Background Chronic obstructive pulmonary disease(COPD)is a chronic airway inflammatory disease characterized by limited expiratory airflow,which emphysema and small airway dysfunction and abnormalities are the main pathological manifestations.Patients often present with symptoms,such as chronic cough,expectoration,shortness of breath,wheezing,and incompletely reversible airflow limitation(FEV1/FVC<0.7 after inhalation of bronchodilators)and the progressive aggravation of pulmonary hyperinflation [increases in functional residual capacity(FRC)and total lung capacity(TLC)],which eventually lead to respiratory failure,multiple organ failure and even death.Inflammation is a core hallmark of COPD.Exposure to cigarette smoke(CS)and noxious gases activates inflammatory cells to release multiple pro-inflammatory mediators resulting in progressive lung structural damage and promoting COPD disease progression.Therefore,inhibiting airway inflammation is critical to delay disease progression of COPD.Bronchodilators commonly used in clinical treatment can relieve symptoms and alleviate the decline of lung function.Combination with corticosteroids can improve prognosis and reduce the risk of acute exacerbations,but they rarely delay disease progression.The side effects also limit the clinical application of corticosteroids.Therefore,searching for new drugs with bioactivities that regulate inflammation and reduce side effects may be a promising prospect in the treatment of COPD.Isorhamnetin(Iso)is a natural flavonoid isolated from the fruit of sea buckthorn and ginkgo leaves.It has various benefits that include anti-inflammation,anti-oxidation, and anti-apoptotic properties.Additionally,the activity of Iso plays a role in many pulmonary diseases such as acute lung injury,pulmonary arterial hypertension,and influenza.The mechanism is related to regulating the production of inflammatory mediators,cytokines,and reactive oxygen species(ROS).However,it is unclear whether Iso effectively reduces airway inflammation in COPD.Objectives 1.To construct a mouse model of COPD to evaluate the therapeutic effect of Iso on airway inflammation.2.To preliminarily explore the possible mechanism of regulatory effect of Iso on airway inflammation in COPD.Methods Seventy-five mice were randomly assigned to the following groups: Control group,CS group,CS + Iso(30 mg/kg)group,CS + Iso(60 mg/kg)group and CS + Dex group.A mouse model of COPD was established by CS exposure and lipopolysaccharide(LPS)intratracheal instillation.Except for the control group,mice received LPS by intratracheal instillation on days 1 and 14,and were exposed to CS from day 0 to 90 except for days with LPS administration.Mice from the Iso treatment group were given Iso 30 mg/kg or 60 mg/kg respectively by oral administration from day 61 to 90,to observe the therapeutic effect of on the symptoms and airway inflammation.Meanwhile,Dex was set as a positive control group,and also given by oral administration from day 61 to 90(3 mg/kg/d for the first 15 days of treatment and 0.6 mg/kg/d for the last 15 days of treatment).The weight of each mouse was recorded every week during the experiment.Finally,all mice were sacrificed on day 91.Lung functions were assessed from five mice in each group.Peripheral blood samples of five mice in each group were collected for hematological analysis and peripheral blood samples of four mice in each group were determined by flow cytometry.Lung tissues from five mice in each group were harvested and fixed for H&E and Masson staining.Bronchoalveolar lavage fluid(BALF)from five mice in each group was obtained to leukocytes count and proinflammatory cytokines detection.Lung tissues of three mice in each group were used to detect the expression levels of signaling pathway proteins by Western blotting.Results 1.The effect of Iso on the symptoms in a mice model of COPD.RBCs and HCT were significantly increased in peripheral blood of CS-induced COPD compared with the control group.Iso significantly reduce the above indicators in a dose-dependent manner.The weight of mice in the CS group was significantly lower than that of mice in the control group,which was apparently ameliorated by Iso.Dex treatment appeared to have little effect on the above indicators.Iso(60 mg/kg)resulted in a significant difference in the effect on body weight than treatment with Dex.2.The effect of Iso on lung function in a mice model of COPD.Compared with the control group,CS-exposed mice exhibited significant airflow restriction and pulmonary hyperinflation,which was described by increasing FEV100/FVC and decreasing forced vital capacity,resistance index and chord compliance.Iso significantly reduces the increase in FVC and RI,and descended the deterioration in FEV0.1/FVC and Cchord caused by CS exposure in a dose-dependent manner.Additionally,treatment with Dex obviously improved the CS-induced decline in FVC,Cchord,while not significantly improved the FEV0.1/FVC and RI.There was no significant difference between treatment with Iso and Dex in improving lung function.3.The effect of Iso on pulmonary histopathological changes in a mice model of COPD.H&E: 1)Compared with the control group,histopathological analysis of lungs in CSinduced COPD mice revealed dilated alveoli,an impaired alveolar wall,and alveolar wall fusion,a feature in accordance with the pathology of typical emphysema.Compared with the control group,MLI and MAA were increased in the CS groups.Iso significantly restrained pulmonary structural destruction caused by CS exposure.There was no significant difference between treatment with Iso and Dex in improving above indicators.2)The thickness of the airway wall in CS group mice was significantly increased compared with the control group,which manifested as thickening of the bronchial wall and disorder of epithelial cells.Conversely,the above histological damages were improved in mice treated with Iso in a dosedependent manner.There was no significant difference between treatment with Iso and Dex in improving thickness of the airway wall.3)Compared with the control group,the CS group showed obvious infiltration of inflammatory cells in and around the airway,and the pathological score was significantly increased.Iso significantly improves the infiltration of airway inflammatory cells in CS group mice in a dose-dependent manner.There was no significant difference between treatment with Iso and Dex in improving the infiltration of inflammatory cells in the airway wall.Masson: Extensive collagen around the airway wall and beside the small blood vessels were significantly deposited in CS group mice compared with the control group.Iso significantly reduces collagen deposition around the airway wall in CS group mice in a dose-dependent manner.Additionally,an obvious reduction in collagen deposition was observed around vessels and bronchioles after Dex treatment.There was no significant difference between Iso and Dex in the improvement of pulmonary collagen deposition in COPD mice.4.The effect of Iso on leukocytes count in BALF of CS group mice.The total number of leukocytes in BALF of the CS group was significantly higher than that in the control group.CS also triggers the recruitment of a large number of neutrophils and macrophages.Iso apparently reduces the numbers of leukocytes,neutrophils and macrophages in BALF of the CS group.There was no significant difference between treatment with Iso at a dose of 60 mg/kg and Dex in improving the accumulation of leukocytes,neutrophils and macrophages.5.The effect of Iso on the expression of proinflammatory cytokines in BALF and proinflammatory mediators in lung tissues of CS group mice: Compared with the control group,expressions of interleukin-6(Interleukin-6,IL-6),Monocyte chemoattractant protein-1(MCP-1)and T cell activation secreted regulator(regulated upon activation,normal T-cell expressed and secreted,RANTES)and cyclooxygenase-2(Cyclooxygenase-2,COX-2)were significantly increased.Iso ignificantly reduces the expression of IL-6,MCP-1,RANTES and COX-2 in BALF of CS group mice in a dose-dependent manner.There was no significant difference between Iso and Dex in the improvement of expression of proinflammatory cytokines in BALF and proinflammatory mediators in lung tissues of CS group mice.6.The effect of Iso on the proportion of lymphocytes and T lymphocytes in peripheral blood of COPD mice.Compared with the control group,the proportion of lymphocytes and T lymphocytes(CD3+T)in peripheral blood of CS group mice were significantly increased.Iso significantly reduces the proportion of lymphocytes and T lymphocytes in the peripheral blood of CS group mice in a dosedependent manner.Dex significantly reduces the proportion of lymphocytes in the peripheral blood of CS group mice,but it has little effect on improving the proportion of T lymphocytes.There was no significant difference between Dex and Iso in the proportion of peripheral blood lymphocytes and T lymphocytes in CS group mice.7.The regulatory effect of Iso on Nrf2/Keap1 pathway in lung tissue of CS group mice.The expression levels of Nrf2,Keap1,p62,HO-1,SOD1 and SOD2 were detected by western blotting.Compared with CS group,Iso significantly increases the expression of Nrf2 and promote the expression of downstream cytoprotective factors HO-1,SOD1 and SOD2 by significantly upregulating p62 and downregulating the expression of Keap1.There was no significant difference between Dex and Iso(60 mg/kg)in regulating the expression of the above pathway proteins.Conclusion 1.Iso reduces airway inflammation in COPD mice.2.The effect of Iso on airway inflammation in COPD mice is related to the modulation of the Nrf2/Keap1 pathway.