Protective Effect And Mechanism Of Astaxanthin On Cigarette Smokeinduced Emphysema In Mice

Objective: Chronic Obstructive Pulmonary Disease(COPD)is a common,preventable,and treatable disease caused by airway inflammation and airway remodeling.It is a common and frequently-occurring disease of the respiratory system,and its morbidity and mortality are increasing annually.Studies have found that a variety of factors contribute to the development of COPD,including inflammation,epithelialmesenchymal transition(EMT),protease-antiprotease imbalance,and oxidative stress.On the one hand,after the body is exposed to toxic and harmful gases such as cigarette smoke(Cigarette smoke,CS),inflammatory cells(including monocytes-macrophages,neutrophils,lymphocytes,etc.)and epithelial cells and other structural cells release various factors that amplify the inflammatory process and induce changes in lung tissue architecture.This causes an imbalance between proteases that break down connective tissue components and antiproteases that antagonize this effect in the lung tissue of COPD patients.On the other hand,CS contains a large number of oxidants,which can activate inflammatory cells such as macrophages and neutrophils,and also generate a large number of reactive oxygen species(ROS),resulting in oxidative stress damage to lung tissue cells.CS can antagonize the effect of antioxidants in the body,resulting in the imbalance of oxidative and antioxidant reactions in the body.Oxidative stress can directly damage the airways and lung parenchyma,and finally,long-term exposure to CS in smokers can cause external damage to the airway and lung tissue,resulting in significant changes in the structure of normal epithelial cells,a marked increase in basal cells,decreased epithelial cell connection integrity,epithelial cell reprogramming,and ultimately can lead to EMT.The emergence of EMT further promotes the occurrence and development of pathological changes in the lung tissue of COPD patients and then develops into emphysema and airway remodeling,resulting in airflow limitation.Astaxanthin(AXT),a carotenoid,is a natural antioxidant widely present in marine organisms and microorganisms.It is named after it was first discovered in marine shrimp.Studies have shown that astaxanthin has potent anti-inflammatory and antioxidant effects and can inhibit interleukin-8(Interleukin-8,IL-8),interleukin-6(Interleukin-6,IL-6),tumor necrosis factor α(Tumor necrosis factor α,TNF-α)and other inflammatory factors and these factors are the main factors for the occurrence and development of chronic inflammatory diseases.These factors can promote the continuation of lung inflammation,unbalanced extracellular matrix degradation,collagen deposition,and the formation of oxidative stress.Oxidative stress and inflammatory response are important pathogenic factors of COPD and can be potential targets for COPD therapy.Astaxanthin has potent anti-inflammatory and antioxidant effects,therefore,theoretically,astaxanthin has a potential therapeutic effect on COPD,but there is no related research report.The purpose of this study was to detect the lung tissue structure,collagen deposition,inflammatory factors,and oxidation of astaxanthin before and after treatment in a mouse emphysema model induced by cigarette smoke and bronchial epithelial cells stimulated by cigarette extract(CSE).The expression changes of Oxidative stress and EMT-related proteins were evaluated to determine whether astaxanthin has a protective effect on COPD from the level of cells and animal models and to explore its mechanism of action.Methods:1.Effects of astaxanthin on inflammatory response and oxidative stress in mice with cigarette smoke-induced emphysema in vivo and in vitro(1)Human Bronchial Epithelial Cells(HBE)were cultured in vitro,the activity of HBE was detected by the MTT method,and the appropriate concentration of CSE and the concentration of three AXT intervention doses groups were determined;(2)HBE was divided into three groups.Control group,5%CSE stimulation group,L-AXT group(0.1u M AXT),M-AXT group(1u M AXT),H-AXT group(10u M AXT),5%CSE+ LAXT group,5%CSE+ M-AXT group and 5%CSE+ H-AXT group,Western blot was used to detect the oxidative stress-related protein NF-E2-related factor 2(Nrf2),heme oxygenase 1(HO-1)and quinone oxidoreductase(NADPH quinone dehydrogenase 1,NQO1)expression in each group;(3)60 mice were randomly divided into control group,model group,low-,medium-and high-dose AXT intervention groups,using cigarettes Mice emphysema models were established by smoke exposure and intraperitoneal injection of CSE.Mice in the AXT intervention group were given AXT(5mg/kg,10mg/kg,50mg/kg)by gavage once a day 30 minutes before smoking;(4)One month later,the mice in each group were sacrificed,and the lung tissue was stained with HE to evaluate the histopathological changes.The lung tissue was detected by ELISA to detect malondialdehyde(MDA),glutathione(GSH),IL-6,the levels of TNF-α and Myeloperoxidase(MPO),and the expressions of oxidative stress and inflammatory response-related proteins P-p65,p-IκBα,Nrf-2 and HO-1 in lung tissue were detected by Western blot method.2.Effects of astaxanthin on EMT and airway remodeling in cigarette smoke-induced emphysema mice(1)The changes of Fibronectin,Inducible Nitric Oxide Synthase(INOS),and Vimentin proteins in HBE cells before and after AXT intervention and CSE induction were detected by the immunofluorescence method.The expression changes of AXT on CSEinduced EMT marker molecules α-smooth muscle actin(α-SMA),Vimentin,Fibronectin,and E-cadherin in HBE cells were detected by Western Blot.(2)Immunohistochemical staining of lung tissue of mice in each group to evaluate the effect of AXT on the expression of epithelial marker E-cadherin and mesenchymal marker α-SMA around the airway in emphysema model mice;(3)Each group Masson staining of mouse lung tissue was used to observe the collagen deposition and epithelial cell thickness in the airway and surrounding tissues under a light microscope;(4)Western blot was used to detect EMT-related proteins E-cadherin and α-SMA,Vimentin,Fibronectin,p-smad2/3,Tsmad2/3,Tissue inhibitor of matrix metalloproteinase-1(TIMP-1),Matrix metallopeptidase 9(Matrix metallopeptidase 9,MMP-9)and transforming factor β1(Transforming growth factor-β1,TGF-β1)in mouse lung tissue.Result:1.Effects of AXT on inflammatory response and oxidative stress in mice with cigarette smoke-induced emphysema in vivo and in vitro1.1 Western Blot results showed that AXT could significantly increase the expression of oxidative stress-related proteins Nrf2,NQO1,and HO-1 in CSE-induced HBE cells(P<0.001).1.2 Compared with the control group,the mice in the model group had a reduced diet,with red and yellow coats,weakened activity,increased breathing frequency,and significantly reduced body weight.The above changes in each AXT intervention group were improved.1.3 Compared with the control group,the airway and lung tissue structures of the mice in the model group were disordered,the mean linear intercept(MLI)was significantly increased(P<0.001),the mean alveolar area(MAA)and the alveolar Destructive Index(DI)increased significantly(all P<0.001),which was consistent with the pathological changes of emphysema.The above pathological changes in AXT intervention groups were significantly improved compared with the model group(P<0.001).1.4 Compared with the control group,the levels of MDA,MPO,IL-6,and TNF-α in the lung tissue of the mice in the model group were significantly increased(P<0.001),and the level of GSH was significantly decreased(P<0.001).Compared with the model group,the levels of MDA,MPO,IL-6,and TNF-α in the lung tissue of the mice in each AXT intervention group were decreased,especially in the high-dose group,and the GSH level in the high-dose AXT intervention group was significantly higher than that in the model group.(P<0.001).1.5 Western blot results showed that compared with the control group,the expression levels of p-p65,p-IκBα,Nrf2,and HO-1 molecules in the lung tissue of the model group mice were significantly up-regulated(P<0.01),and the expression of p-p65 and p-IκBαin lung tissue of mice pretreated with AXT decreased significantly compared with model group,while the expression of Nrf2 and HO-1 increased further compared with the model group(P<0.001 in the high-dose group).2.Astaxanthin inhibits EMT and airway remodeling in mice with emphysema induced by cigarette smoke and its extracts2.1 Immunofluorescence results showed that CSE significantly up-regulated Fibronectin,INOS,and Vimentin in HBE cells(P<0.001).Different concentrations of AXT had different degrees of inhibition on the above reactions and showed a certain dose-effect relationship.2.2 Western Blot results showed that the expression of α-SMA,Vimentin,and Fibronectin protein molecules in HBE cells in the CSE stimulation group was significantly higher than that in the control group,and E-cadherin was significantly lower than that in the control group,while the above changes in each AXT intervention group showed a certain reversal effect.2.3 The results of immunohistochemical staining showed that the expression of Ecadherin molecules around the small airways of mice in the model group was lesser than that of the control group,while the expression of the mesenchymal cell marker α-SMA was significantly higher than that of the control group.After AXT intervention,the cigarette smoke-induced downregulation of per airway E-cadherin expression was effectively attenuated(P<0.001 in the high-dose group),and the upregulation of mesenchymal marker α-SMA expression was reversed to some extent(P<0.001 in the middle and high-dose group).2.4 Masson staining results showed that a large amount of collagen deposition was seen in the airway wall and blood vessel wall of the mice in the model group,the structure of the inner airway wall was disordered and irregular,and the airway epithelium was significantly thickened.The amount of collagen deposition in the airway and perivascular in AXT intervention groups were significantly reduced compared with the model group,and the airway structural lesions were also significantly improved.2.5 Western blot results showed that compared with the control group,the expression levels of α-SMA,Vimentin,Fibronectin,P-smad2/3,MMP-9,TGF-β1 molecules in the lung tissue of the model group mice were significantly up-regulated,The expression level of E-cadherin and TIMP-1 were significantly down-regulated,and the above changes were reversed to a certain extent in each group of mice treated with AXT.Conclusion:1.Astaxanthin has the effect of reducing emphysema and airway remodeling in mice induced by cigarette smoke and its extracts;2.Astaxanthin may inhibit oxidative stress injury in mouse lung tissue by activating the Nrf2 pathway,reduce inflammation in mouse lung tissue by inhibiting the NF-κB pathway,and reduce EMT and protease/antiprotease imbalance in mouse lung tissue by inhibiting TGF-β1/Smad pathway.Thus to achieve the protective effect on emphysema and airway remodeling induced by cigarette smoke and its extracts in mice.