The Protective Effect And Its Underlying Mechanism Of Heme Oxygenase-1 On Cigarette Smoke-induced COPD

Objective:Chronic obstructive pulmonary disease（COPD）is a chronic and progressive lung disease with few successful treatments.Since the novel coronavirus has spread worldwide seriously,there is growing concern that patients who have chronic respiratory conditions like COPD can easily be infected and are more prone to having severe illness and even mortality because of lung dysfunction.Cigarette smoke is a major factor in COPD.Cigarette smoke causes chronic inflammation and oxidative stress,which contributes to lung dysfunction in COPD.Therefore,it is particularly important to prevent and reveal the regulatory role of cigarette smoke in inducing the development of COPD.Heme oxygenase-1（HO-1）is an endogenous cytoprotective enzyme that is induced ubiquitously in response to oxidative stress.The main function of HO-1 is to cleave heme to form carbon monoxide,free iron,and biliverdin.The cytoprotective effect of HO-1 may have several distinct underlying mechanisms,including the degradation of heme to the antioxidant bilirubin,the co-ordinate induction of ferritin,which chelates free iron,and the release of CO,which exerts significant anti-inflammatory and anti-apoptotic effects.HO-1 can be induced by numerous oxidizing agents and stimuli,including ultraviolet radiation,cigarette smoke,and heme/hemoglobin.Previous studies have confirmed that HO-1 can provide anti-oxidation and cytoprotection in vitro and in vivo systems.Importantly,HO-1 acts as an antioxidant to protect against cigarette smoke-induced vascular damage and lung injury.The exact mechanism of HO-1 in cigarette smoke-induced COPD is currently unknown.Airway inflammation is one of the main characteristics of COPD and is associated with the increased recruitment of inflammatory cells.Macrophages affect airway inflammation by enhancing the secretion of inflammatory cytokines and chemokines in the pathogenesis of COPD.Meanwhile,the antioxidant defense protein HO-1 plays a protective role against cigarette smoke-induced inflammation in the lungs.Of note,overexpression of HO-1 restricted cigarette smoke-induced airway inflammation and lung epithelial cell inflammatory responses.The above results suggested that HO-1 has a significant negative regulatory role in cigarette smoke-induced inflammatory response in COPD,but its specific mechanism of action needs to be further elaborated.Methods:PartⅠ:In vivo study of COPD in rats caused by cigarette smoke exposureMale SD rats（230±20 g）in a SPF grade were purchased from Jinzhou Medical University Animal Center.All procedures for animal experiments adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals(8^thedition,2011),and this protocol was approved by the First Affiliated Hospital of Jinzhou Medical University.All animals were fed and watered ad libitum at 22±1°C,45%-55%humidity,and a 12 h light/dark cycle.After one week of acclimation,all rats were randomly divided into 2 groups（12 rats per group）:the control group（Con）and the COPD group（CS）.The rats were placed in a homemade glass fumigation box（110 cm×86 cm×72 cm）with a ventilation hole of about 2 cm-3 cm in diameter on each of the four sides of the box,and 40 cigarettes were lit for 30 min each time,and the rats in the COPD group were exposed to room air twice a day for 90 days.The rats in the Con group were exposed to indoor air.At the end of cigarette smoke exposure,all rats in the Anires 2005 Animal Lung Function Analysis System were used to measure the forced expiratory volume at 0.3s(FEV_0.3),forced vital capacity（FVC）,FEV_0.3/FVC,inspiratory resistance（Ri）,expiratory resistance（Re）and peak expiratory flow（PEF）.The differences in alveolar lavage fluid cell classification between the two groups were analyzed.The expression levels of inflammatory cytokines and chemokines in serum and lung tissues were measured by ELISA and qRT-PCR.The histomorphological changes of rat lung pathological sections were observed by HE staining.Part II:Transcriptomic study of cigarette smoke-induced COPD rats’model based on RNA-Seq technologyMacrophages derived from rats in COPD and Con groups were taken and total RNA was extracted for transcriptomic sequencing to screen differentially expressed genes.Meanwhile,the differentially expressed genes were classified into functional and biological pathways according to GO terms and KEGG pathway annotation.In addition,the validation of the Hmox-1（HO-1）expression in lung tissues was performed by qRT-PCR.Part III:HO-1 modulates cigarette smoke extract-induced macrophage inflammatory response and oxidative stress via the HIF-1αpathwayRAW264.7 cells cultured to log phase were transfected with the HO-1overexpression vector（HO-1 vector）or HIF-1αoverexpression vector（HIF-1αvector）or negative vector（vector）,and then 2%cigarette smoke extract（CSE）was used to stimulate the transfected RAW264.7 cells.The RAW264.7 cells were randomly divided into 6 groups:untreated group（Untreated）,vector group（NC）,HO-1 vector group,2%CSE group,2%CSE+NC group,2%CSE+HO-1 vector group,and2%CSE+HO-1+HIF-1αvector group.Subsequently,the MTT assay was used to evaluate the proliferation of RAW264.7 cells.Western blotting was applied to detect the expression of HO-1 protein.qRT-PCR was performed to measure the expression of HO-1,HIF-1α,inflammatory cytokines,and chemokines in RAW264.7 cells.ELISA kits were employed to examine the expression of TNF-α,IL-6,IFN-γ,and IL-1βin RAW264.7 cells.Wright-Giemsa staining was used to evaluate the phagocytic ability of RAW264.7 cells and DCFH-DA probe was performed to measure the level of cellular reactive oxygen species（ROS）.Part IV:In vivo experiments to verify the protective effect of HO-1 on cigarette smoke-induced COPD in ratsThe purchased male SD rats were acclimated and housed for one week,all rats were randomly divided into 3 groups（10 rats per group）:the control group（Con）,COPD group,and Hemin（HO-1 activator）+COPD group.Rats in the Hemin+COPD group were injected with Hemin（20μmol/kg）intraperitoneally 30 min before CS exposure.Subsequently,the protective effect of Hemin on the CS-induced COPD model in rats was analyzed by HE staining and morphological evaluation of emphysema.ELISA kits were used to detect the serum levels of bilirubin and the expression levels of TNF-α,IL-1β,IL-17,and IL-6.qRT-PCR was employed to measure the expression of inflammatory cytokines and chemokines in lung tissues.Commercial kits were performed to detect the levels of oxidative stress indicators（SOD,GSH,and MDA）in lung tissues.Immunohistochemistry and qRT-PCR were performed to examine the expression of HO-1 and HIF-1αin lung tissues.Results:PartⅠ:In vivo study of COPD in rats caused by cigarette smoke exposureCompared with the Con group,the bronchial and alveolar walls of rats’lungs in the CS group were slightly deformed,cilia were disorganized and inverted,epithelial cells were heavily shed,alveoli were deformed and dilated and fused into large cystic cavities,inflammatory cells were significantly infiltrated,and the tracheal walls were significantly thickened and lumen narrowed.Meanwhile,the lung function indexes FEV0.3,FEV0.3/FVC,and PEF in the CS group were significantly lower than those of the Con group.In addition,the total number of macrophages,neutrophils,and leukocytes in alveolar lavage fluid,and the expression of pro-inflammatory cytokines（TNF-α,IL-1β,and IL-17）and chemokines（MCP-1 and MIP-2α）in serum and lung tissues of rats in the CS group were significantly higher than those in the Con group.Part II:Transcriptomic study of cigarette smoke-induced COPD rats’model based on RNA-Seq technology The RNA-seq assay showed a total of 1752 genes differentially expressed（934genes up-regulated and 818 genes down-regulated）.Among them,Homx-1（HO-1）was the most significantly differentially expressed down-regulated gene.Meanwhile,the expression of HO-1 was significantly lower in the lung tissues of the CS group than that of the Con group,suggesting that it may play a regulatory role in COPD induced by cigarette smoke.In addition,HO-1 was mainly involved in mediating the HIF-1αsignaling pathway.Part III:HO-1 modulates cigarette smoke extract-induced macrophage inflammatory response and oxidative stress via the HIF-1αpathwayThe proliferation of RAW264.6 cells treated with 2%CSE for 12 h was significantly lower than that of the Untreated group.Compared with the Untreated group,2%CSE significantly down-regulated the expression of HO-1 in RAW264.7cells.In addition,2%CSE treatment significantly up-regulated the expression of inflammatory cytokines,chemokines,ROS levels,and HIF-1αin RAW264.7 cells.However,overexpression of HO-1 significantly inhibited the damaging effect of CSE on RAW264.7 cells,but overexpression of HIF-1αreversed the expression of HO-1.Part IV:In vivo experiments to verify the protective effect of HO-1 on cigarette smoke-induced COPD in ratsHemin treatment significantly alleviated CS-induced lung injury in rats by increasing the mean alveolar number and decreasing the mean linear intercept.Compared with the COPD group,the levels of total bilirubin（TBil）and indirect bilirubin（IBil）in the serum of rats were significantly increased,and inflammatory cytokines and chemokines in serum and lung tissues of rats were significantly decreased in the Hemin group.Meanwhile,SOD activity and GSH content in lung tissues of the Hemin group were significantly higher than that in the COPD group,and MDA content was significantly reduced.In addition,HO-1 expression was lower in the COPD group than that in the Con group,as well as the expression of HIF-1αwas significantly increased,but Hemin treatment reversed the above results.Conclusion:（1）CS exposure facilitated emphysema and decreased lung function,and accelerated pulmonary inflammatory responses and oxidative stress leading to COPD symptoms in rats.HO-1 was significantly downregulated and the HIF-1αsignaling pathway was activated.（2）CSE promoted macrophage inflammation and oxidative stress by downregulating HO-1,whereas overexpression of HO-1 significantly alleviated CSE-induced macrophage injury by inactivating the HIF-1αsignaling pathway.（3）Hemin can alleviate COPD caused by CS exposure by reducing the inflammatory response and oxidative stress and restoring lung function.