Introduction: Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a recently discovered inhibitor of matrix metalloproteinase (MMP). There is a large number of chronic obstructive pulmonary disease (COPD) patients worldwide; however, the role of RECK on COPD has not been studied. This study explored the expression of RECK in COPD patients and its effect on neutrophil function to provide a new scientific basis for the prevention and treatment of COPD. Method: Fifty patients with acute exacerbation of COPD and fifty healthy controls were enrolled in the study. RECK was detected in lung tissue, sputum, and plasma of subjects as well as in BEAS-2B cells stimulated with cigarette smoke extract (CSE) by immunohistochemistry, ELISA, and qRT-PCR. Meanwhile, lung function (FEV1%pred) and inflammatory cytokines (IL-6 and IL-8) were examined, and correlation analysis was performed with RECK expression. The effect of RECK on proliferation, apoptosis, migration, and inflammatory cytokines and its potential mechanism was further quantified by neutrophil stimulated with recombinant human RECK protein (rhRECK) combined with CSE using CCK8, flow cytometry, Transwell assay, qRT-PCR, ELISA, and Western analysis. Results: RECK was mainly expressed on airway epithelial cells in normal lung tissue and was significantly diminished in COPD patients. The levels of RECK in sputum and plasma were also significantly decreased in COPD patients. Pearson correlation analysis showed that RECK level in plasma was positively correlated with FEV1%pred (r = 0.458, p < 0.001) and negatively correlated with IL-6 and IL-8 (r = −0.386, −0.437; p = 0.006, 0.002) in COPD patients. The expression of RECK was decreased in BEAS-2B stimulated with CSE. The migration, inflammation, and MMP-9 expression of neutrophils were promoted by CSE, while inhibited by rhRECK. Conclusion: RECK is low expressed in COPD patients and negatively correlated with inflammation. It may inhibit the inflammation and migration of neutrophils by downregulating MMP-9.

Chronic obstructive pulmonary disease (COPD), a chronic lung disease characterized by incomplete reversible continuous airway limitation, regards chronic inflammation of airway and destruction of lung function as its main clinical manifestations [1]. Global Burden of Disease (GBD) indicated that COPD was the sixth leading cause of death worldwide in 2019, and the prevalence and mortality of COPD are expected to increase in the coming decades [2, 3]. Smoking is a risk factor for COPD [4].

Neutrophil activation (including inflammatory cytokine secretion and local migration) is significantly increased in COPD and is associated with disease progression and negative prognosis [5, 6]. Lung tissue can be damaged by some proteases, like elastase, myeloperoxidase, and matrix metalloproteinase-9 (MMP-9), which are excessively released by activated neutrophils [7]. MMP-9 can degrade the extracellular matrix, including collagen, laminin, and gelatin, regulate and promote the transepithelial migration and vascular extravasation of neutrophils [8, 9]. Studies found that the level of MMP-9 was related to the increased infiltration of neutrophils in COPD sputum, and the abnormal increase expression of MMP-9 promoted lung parenchymal injury and was related to the decline of lung function [10, 11].

Reversion-inducing cysteine-rich protein with kazal motifs (RECK) is an extracellular protein, which has a protease inhibitor-like domain, and physiologically expresses in multiple organs and tissues [12]. Current studies have shown that RECK is an inhibitor of MMP-2, MMP-8, MMP-9, MMP-13, and other metalloenzymes, which can restrain transcription and the activity of metalloenzymes [13]. RECK has been regarded as a new antioncogene and reduced in malignant tumor, including, non-small cell lung cancer [14, 15], suggesting that reduced expression of RECK may contribute to the invasiveness of tumor. Patients with COPD are at higher risk of developing lung cancer, therefore, RECK is suspected to play a role in the pathogenesis of COPD. One study has found that the expression of RECK mRNA was decreased in alveolar lavage fluid cells of COPD patients [16]. Another study demonstrated that RECK expression on airway epithelial cells was significantly reduced in a COPD rat model [17]. However, these studies only presented the expressional changes of RECK and did not dig deep into the underlying mechanism. This study investigated the expression of RECK in sputum, plasma, and lung tissue of COPD patients as well as in BEAS-2B cells stimulated with cigarette smoke extract (CSE), analyzed its correlations with lung function and inflammation, explored the effect of recombinant human RECK protein (rhRECK) on the inflammation and function of neutrophils, preliminarily clarified the role of RECK in COPD inflammation, and shed new light on finding therapeutic targets for COPD.

Subjects

Fifty patients with acute exacerbation of COPD were recruited in the Department of Respiratory and Critical Care Medicine of the Qingdao Municipal Hospital between November 2019 and December 2020. The inclusion criteria included: (1) diagnosed with COPD according to the criteria of Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2018; (2) hospitalized due to respiratory symptoms rapid aggravation. Exclusion criteria included malignancy, autoimmune diseases, oral/intravenous injection corticosteroid therapy, or other pulmonary diseases (except for COPD). Fifty healthy controls were recruited from the Checkup Center. For confirm the expression of RECK in the lung, lung tissues were collected from 6 successive patients with benign pulmonary nodule during operation, 3 of which were COPD. The study was registered in the Chinese Clinical Trial Registry (ChiCTR2100043767) and approved by the Ethics Committee of Qingdao Municipal Hospital (Approval number: 2020-009). Written informed consent was obtained from all participating patients.

Sputum Processing

Sputum induction in subjects (fifty patients with acute exacerbation and fifty healthy controls) was performed using hypertonic saline (3% NaCl). The collected sputum samples were treated with double volume 0.1% dithiothreitol (DTT) and placed in a constant temperature shaker (120 rpm) for 5 min at 37°C. The supernatant was filtered through a 40 μm filter after treated with PBS and centrifuged. The sputum supernatants were stored at −80°C for subsequent experiments.

Neutrophils Isolation, Cell Culture, and Intervention

Peripheral venous blood of six healthy controls (Healthy individuals undergoing physical examination) with EDTA anticoagulant (10 mL) was centrifuged, and the plasma was collected and stored at −80°C for subsequent experiments. Neutrophils were isolated from the blood cells follow the instruction of human neutrophil isolation solution kit (Beijing Solarbio Science & Technology Co., Ltd., China). The human bronchial epithelial cell line BEAS-2B (ATCC) (presented by the School of Public Health of Qingdao University) and neutrophil were cultivated by RPMI 1640 medium (Procell Life Science &Technology Co., Ltd., China) containing 10% fetal bovine serum (FBS, Invitrogen, USA). BEAS-2B cells were stimulated with 10% CSE for 24 h and 48 h, and cells in the control group were treated with the same dose of PBS, then cell supernatant and cell precipitation were collected and stored at −80°C. Neutrophils were treated with different concentrations of rhRECK and CSE for corresponding tests.

Preparation of CSE

Burn up a 3R4F cigarette (Tar 9.4 mg/cigarette, nicotine 0.73 mg/cigarette, University of Kentucky, USA) in 3 min and cigarette smoke was pushed into a drainage bag containing 25 mL of RIPM 1640 medium. The suspension was adjusted to pH 7.4, filtered through a 0.22 μm filter and then transferred to a centrifuge tube. It is then stored −80°C.

Cell Proliferation Assay

Neutrophil cells were seeded in 6-well plates and treated with different concentrations of CSE for 16 h. Then, 100 μL of the above neutrophils were seeded into 96-well plates (5 multiple holes in each group), and 10 μL of CCK-8 solution (Topscience, USA) was added to each well. After 1.5 h, the absorbance of each cell at 450 nm was detected on a microplate reader (Thermo Fisher Scientific Co., Ltd.,USA).

Cell Apoptosis Assay

Neutrophil cells treated with 3% CSE for 14 h after incubated with different concentrations of rhRECK (Abcam Co., Ltd., UK) for 2 h. Treated cells were collected and washed with ice-cold PBS. Then cells were stained using the FITC-Annexin V/PI detection kit (KeyGEN Bio TECH, Nanjing, China). After 10 min incubation at room temperature in darkness, the proportion of apoptotic cells was measured on a flow cytometer (Becton Dickinson and Company, USA).

Cell Migration Assay

Neutrophil cells were treated with RPMI 1640 medium containing 5% FCS to make the cell density 5 × 105 per milliliter. The corresponding concentration of rhRECK was added and the cells were incubated for 2 h. 600 μL of 5% FCS-RPMI 1640 was added to the lower chamber of transwells, mixed with the CSE stock solution except for the control group. 200 μL cell suspension was added to the upper chamber and incubated for 1 h. The cells in the lower compartment were collected and resuspended with 600 μL PBS. Finally, cell counting plate was used under an optical microscope.

ELISA Assay

The concentrations of RECK in plasma, sputum, and BEAS-2B cell culture supernatant were tested by ELISA kits (Cusabio, Wuhan, China). The levels of inflammatory cytokines (IL-6, IL-8) in plasma and neutrophil culture supernatant were, respectively, tested by ELISA kits (Mlbio, Shanghai, China and Cloud-Clone, Wuhan, China) according to the manufacturer’s instructions. The optical density was measured at 450 nm with a microplate reader (Thermo Fisher Scientific Co., Ltd., USA).

qRT-PCR

Total RNAs were isolated from tissues and cells by Trizol reagent (Takara, Tokyo, Japan). Reverse transcription was conducted with a PrimeScript RT reagent Kit (Takara, Tokyo, Japan). qRT-PCR was performed using a TB Green Premix Ex Taq (Takara, Tokyo, Japan). The primers for RECK were 5-CAT​CAC​ACA​AAC​TGC​CGA​GAA-3′ and 5′-GGC​GCA​ATA​ATT​TTC​CAC​TGC​T-3′. The expression level of β-actin was used as internal reference, and the primers for β-actin were 5ʹ-AGA​AAA​TCT​GGC​ACC​ACA​CCT-3ʹ and 5′-GAT​AGC​ACA​GCC​TGG​ATA​GCA-3ʹ. The 2−ΔΔCt method was used for calculating the relative expression quantity.

Western Analysis

Cellular proteins were harvested and equal amounts of proteins were subjected to SDS–PAGE. Proteins were electrophoretically transferred to PVDF membranes and probed with anti-rabbit primary antibody (Rabbit monoclonal antibody against MMP-9, EP1255Y, Abcam Inc.), followed by incubation with secondary antibodies and detected by ECL. The gray value of each protein was analyzed with Image J software.

Immunohistochemical Staining

The lung tissue sections were placed in a 60°C oven and baked for 25 min. The tissue sections were soaked in xylene for 30 min, and soaked in 100%, 95%, 80%, and 70% ethanol solution for 10 min successively. After incubation with 3% hydrogen peroxide for 10 min and rinsing with PBS three times, the slides were incubated at 4°C with anti-RECK polyclonal antibody diluent (1:350) (rabbit-anti-rat; Proteintech, Wuhan, China). Next, the slides were rinsed with PBS three times and incubated with secondary antibody at room temperature for 60 min. The slides were washed with PBS for five times and mixed with ABC compound at room temperature for 30 min. Finally, the slides were rinsed three times and stained with DAB without light, and then restained with Hematoxylin.

Statistical Analysis

All the statistical analyses were conducted in GraphPad Prism (version: 8.0). The measurement data results were expressed as mean ± standard deviation. T test (normal distribution) or nonparametric Mann-Whitney U test (abnormal distribution) was selected for comparison between two groups. One-way ANOVA followed by Tukey’s post hoc test was used for comparison among three or more groups. χ test was used to compare counting data. Correlation between the parameter was performed by Pearson correlation analysis. p values (two-tailed) smaller than 0.05 were considered statistically significant.

Subject Characteristics

Fifty patients with acute exacerbation of COPD and fifty healthy controls were enrolled in the study. There were no significant differences in sex and age between COPD and healthy controls, despite the distinction in smoking amount, FEV1% predicted, FEV1/FVC% (Table 1).

Table 1.

Clinical characteristics of study subjects

GroupControl (n = 50)COPD (n = 50)p value
Sex (male/female) 40/10 40/10 1.000 
Age, years 67.30±7.15 68.72±10.13 0.420 
Smoking amount 4.19±9.74 27.62±24.05 <0.001 
FEV1% predicted 93.56±14.32 46.50±18.04 <0.001 
FEV1/FVC% 78.24±4.06 53.15±11.47 <0.001 
GroupControl (n = 50)COPD (n = 50)p value
Sex (male/female) 40/10 40/10 1.000 
Age, years 67.30±7.15 68.72±10.13 0.420 
Smoking amount 4.19±9.74 27.62±24.05 <0.001 
FEV1% predicted 93.56±14.32 46.50±18.04 <0.001 
FEV1/FVC% 78.24±4.06 53.15±11.47 <0.001 

Data are shown as mean ± SD or count. Smoking amount = packets of cigarettes per day × smoking years.

FEV1, forced expiratory volume in the first second; FVC, forced vital capacity; COPD, chronic obstructive pulmonary disease; SD, standard deviation.

RECK Was Diminished in COPD

In the present study, the percentage of neutrophils in peripheral venous blood collected from COPD patients was higher than that of health controls (Fig. 1a). Meanwhile, the RECK levels in plasma and sputum supernatant collected from COPD patients and health controls were determined by ELISA. Results showed that the RECK level was significantly decreased in COPD patients than that in health controls in both sputum supernatant and plasma (Fig. 1b, c). Pearson correlation analysis showed a positive correlation between the levels of RECK in sputum and plasma of COPD patients (r = 0.415, p < 0.001, Fig. 1d). In addition, the RECK level in plasma was positively correlated with FEV1%pred in COPD patients (r = 0.458, p < 0.001, Fig. 1e). Subsequently, the expression of RECK in lung tissues collected from patients with benign pulmonary nodules with or without COPD was examined. As demonstrated by immunohistochemistry, RECK was mainly expressed on airway epithelial cells in normal lung tissue. Partial expression on immune cells and vascular endothelial cells was also observed. RECK expression on airway epithelial cells in COPD patients was significantly diminished when compared to the normal lung tissue (Fig. 1f). Altogether, these results suggested that RECK was involved in the development of COPD.

Fig. 1.

RECK was diminished in COPD patients. a Percentage of neutrophils in COPD patients was higher than in health controls. b RECK level in sputum supernatant was significantly decreased in COPD patients than that in health controls. c RECK level in plasma was significantly decreased in COPD patients than that in health controls. d RECK level in sputum supernatant was positively correlated with that in plasma of COPD patients. e RECK level in plasma was positively correlated with FEV1%pred in COPD patients. f RECK expression and localization based on immunohistochemical staining (×100 magnification). Control group: RECK expression was highly abundant on airway epithelial cells. COPD patients: RECK expression on airway epithelial cells was significantly diminished. ***p < 0.001, ****p < 0.0001.

Fig. 1.

RECK was diminished in COPD patients. a Percentage of neutrophils in COPD patients was higher than in health controls. b RECK level in sputum supernatant was significantly decreased in COPD patients than that in health controls. c RECK level in plasma was significantly decreased in COPD patients than that in health controls. d RECK level in sputum supernatant was positively correlated with that in plasma of COPD patients. e RECK level in plasma was positively correlated with FEV1%pred in COPD patients. f RECK expression and localization based on immunohistochemical staining (×100 magnification). Control group: RECK expression was highly abundant on airway epithelial cells. COPD patients: RECK expression on airway epithelial cells was significantly diminished. ***p < 0.001, ****p < 0.0001.

Close modal

RECK Was Associated with Inflammation in COPD

The levels of inflammatory cytokines (IL-6 and IL-8) in plasma were detected by ELISA to explore their relationships with RECK. Results showed that the levels of IL-6 and IL-8 in COPD patients were significantly higher than that in health controls (Fig. 2a, b). Pearson correlation analysis revealed that the RECK level was negatively correlated with IL-6 (r = −0.386, p = 0.006) and IL-8 (r = −0.437, p = 0.002) in the plasma of COPD patients (Fig. 2c, d), indicating that RECK was involved in the inflammation of COPD.

Fig. 2.

RECK was associated with inflammation in COPD. a, b The level of IL-6 and IL-8 in COPD patients were significantly higher than that in health controls. c, d RECK level in plasma was negatively correlated with IL-6 and IL-8 in COPD patients. *p <0.05, ***p < 0.001.

Fig. 2.

RECK was associated with inflammation in COPD. a, b The level of IL-6 and IL-8 in COPD patients were significantly higher than that in health controls. c, d RECK level in plasma was negatively correlated with IL-6 and IL-8 in COPD patients. *p <0.05, ***p < 0.001.

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RECK Was Downregulated by CSE in BEAS-2B Cells

To further verify the relationship between RECK and COPD, BEAS-2B cells were stimulated with 10% CSE, and the expressions of IL-6, IL-8, RECK mRNA in cell and RECK protein in cell culture supernatant were detected by qRT-PCR and ELISA. The expression of IL-6 and IL-8 was significantly increased in CSE-stimulated cells (Fig. 3a, b), suggested that the in vitro cellular COPD model was successfully established. The expression of RECK mRNA in CSE-stimulated cells and protein levels in cell culture supernatant were significantly decreased compared to that in the control group (Fig. 3c, d). These results verified that RECK expression was reduced at both the transcriptional level and the translational level in cellular COPD model.

Fig. 3.

RECK was downregulated by CSE in BEAS-2B cells. a, b IL-6 and IL-8 mRNA were overexpressed in CSE-stimulated cells. c The expression of RECK mRNA was significantly decreased in CSE-stimulated cells. d The level of RECK protein was decreased in cell culture supernatant with CSE stimulation. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3.

RECK was downregulated by CSE in BEAS-2B cells. a, b IL-6 and IL-8 mRNA were overexpressed in CSE-stimulated cells. c The expression of RECK mRNA was significantly decreased in CSE-stimulated cells. d The level of RECK protein was decreased in cell culture supernatant with CSE stimulation. *p < 0.05, **p < 0.01, ***p < 0.001.

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RECK Reduced Neutrophilic Inflammation

As the inflammatory response in COPD is primarily mediated by neutrophils, high purity neutrophils were isolated from the blood cells using a neutrophils isolation kit to explore the effect of RECK on neutrophils. Results of CCK-8 showed no significant decrease in neutrophil survival rate in 1%, 2% CSE, and 2.5% CSE groups, but significant decrease in 3% CSE and 4% CSE groups (both p < 0.001) (Fig. 4a). Therefore, 3% CSE was chosen for the apoptosis assay, and 2% CSE for other tests. Results of flow cytometry revealed that the effects of 3% CSE on neutrophils apoptosis could not be abolished by rhRECK (Fig. 4b, c), indicating that RECK had no effect on the neutrophil apoptosis. Transwell assay revealed that the effects of 2% CSE on neutrophil migration could be abolished by rhRECK (Fig. 4d), suggesting that RECK could inhibit neutrophil migration. Moreover, the effects of RECK on inflammatory cytokines (IL-6 and IL-8) in neutrophils were determined. ELISA results showed that 2% CSE increased the levels of IL-6 and IL-8 in cell culture supernatant, while rhRECK abolished the effects. qRT-PCR revealed the same trend of IL-6 and IL-8 mRNA expression (Fig. 4e–h). These results suggested that RECK could reduce the gene and protein expression of inflammatory cytokines in neutrophils, thereby reducing the inflammation. RECK inhibits neutrophil inflammation by inhibiting the migration of neutrophils.

Fig. 4.

Effect of RECK on neutrophilic inflammation. a Effect of different concentrations of CSE on neutrophil activity. b RECK couldn’t reverse the CSE-induced apoptosis of neutrophils. c Statistical graph of apoptosis rate. d Effects of different RECK on neutrophil migration. e-h RECK inhibited the expression of IL-6 and IL-8 in neutrophils. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4.

Effect of RECK on neutrophilic inflammation. a Effect of different concentrations of CSE on neutrophil activity. b RECK couldn’t reverse the CSE-induced apoptosis of neutrophils. c Statistical graph of apoptosis rate. d Effects of different RECK on neutrophil migration. e-h RECK inhibited the expression of IL-6 and IL-8 in neutrophils. *p < 0.05, **p < 0.01, ***p < 0.001.

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RECK Downregulated MMP-9 Expression in Neutrophils

To explore the underlying mechanism of RECK on neutrophilic inflammation, the expression of MMP-9 in neutrophils stimulated with 2% CSE and rhRECK was detected. qRT-PCR and Western analysis showed that both MMP-9 mRNA and protein expression were significantly increased in CSE-stimulated neutrophils, while rhRECK reduced the gene and protein expression of MMP-9 (Fig. 5a–c), suggesting that RECK inhibited neutrophilic inflammation through downregulating MMP-9.

Fig. 5.

The upregulation of MMP-9 stimulated by CSE could be reversed by rhRECK. a RECK could reverse the MMP-9 mRNA expression of neutrophils stimulated by CSE. b Western analysis image of the MMP-9 protein level. c RECK could reverse the MMP-9 protein expression of neutrophils stimulated by CSE. **p < 0.01, ***p < 0.001.

Fig. 5.

The upregulation of MMP-9 stimulated by CSE could be reversed by rhRECK. a RECK could reverse the MMP-9 mRNA expression of neutrophils stimulated by CSE. b Western analysis image of the MMP-9 protein level. c RECK could reverse the MMP-9 protein expression of neutrophils stimulated by CSE. **p < 0.01, ***p < 0.001.

Close modal

This study first demonstrated the inhibition effect of RECK on neutrophilic inflammation in vitro in COPD. The expression of RECK was robustly decreased in lung tissue, plasma, sputum supernatant of COPD patients, and CSE-stimulated BEAS-2B cells. Moreover, RECK was positively correlated with the lung function and negatively correlated with the inflammatory cytokines. In vitro, CSE promoted migration and inflammation of neutrophils, accompanied by the upregulation of MMP-9, while rhRECK could inhibit the above effects induced by CSE. Our results indicated that RECK may play a vital role in the inflammatory response of neutrophils, which may be enlarged during the development of COPD.

RECK is a new tumor suppressor gene first discovered by Professor Takahashi. It can inhibit tumor invasion and metastasis by downregulating the expression of MMP-9, and widely expressed in human blood, various solid tissues, and undifferentiated cells [12, 18]. Studies have found the significantly reduced expression of RECK in a variety of tumors, including breast cancer, colorectal cancer, cholangiocarcinoma, lung cancer, cervical cancer, pancreatic cancer, hepatocellular carcinoma, and gastric cancer, etc. [18‒25], and it was mostly associated with the prognosis of tumors [15, 23, 26]. RECK also has been demonstrated to be involved in the development of many nonmalignant diseases, including myocardial fibrosis, coronary atherosclerosis, asthma, etc. [27, 28]. Paulissen et al. [28] found that asthmatic patients had a significantly reduced RECK level in induced sputum compared with control patients, and the RECK level was positively correlated with the FEV1. Li et al. [29] found that the expression of RECK in the lung tissues of asthmatic mice were significantly lower than that in the control group. Dexamethasone can increase the expression of RECK and reduce the infiltration of inflammatory cells in the lung tissues. These suggest that RECK plays an important role in the process of inflammation and fibrosis. As chronic airway inflammation and remodeling are the main pathological changes of COPD, we explored the expression of RECK in COPD patients and found that the expression of RECK was decreased in the lung tissue, plasma, sputum supernatant of COPD patients, which was consistent with other studies. For instance, Li et al. [17] found that the expression of RECK was significantly reduced in the lung tissues of COPD rats induced by chronic smoking. Navratilova et al. [16] found that the expression of RECK in alveolar lavage fluid cells of COPD showed a downward trend. The low expression of RECK was also verified in in vitro cellular COPD model in our study. We also demonstrated that RECK level was positively correlated with FEV1%pred in COPD patients, which consistent with the result of asthmatic patients [28]. Moreover, RECK level was negatively correlated with the level of cytokines in the plasma of COPD patients, indicating that RECK could inhibit the inflammation of COPD.

Given the findings above, it is reasonable to hypothesize that the occurrence of COPD may be related to the reduction of RECK induced by smoking; however, the underlying mechanism remains unclear. Chronic airway inflammation in COPD is mainly accomplished by the synergistic action of neutrophils, lymphocytes, airway epithelial cells and related inflammatory mediators, among which the increased of neutrophils, apoptosis resistance and secretion of inflammatory factors play an important role. As a major risk factor for COPD, cigarette smoke promotes and activates neutrophils, releasing pro-inflammatory mediators that lead to airway inflammation and tissue cell damage [30‒32]. In this study, RECK was found to inhibit neutrophilic inflammation and reverse the upregulation of MMP-9 stimulated by CSE, which could also be supported by previous studies. For instance, studies have found that the expression of MMP-9 was higher in COPD patients and model animals, and closely related to lung inflammation, airway remodeling, and decreased lung function [33‒35]. Another study also reported that MMP-9 inhibitors could reduce TNF-α-induced neutrophil transepithelial migration and neutrophil circulation in pancreatitis-associated lung injury [36]. Our study demonstrated that RECK could inhibit neutrophilic inflammation through downregulating MMP-9 It is expected to become a new therapeutic target for COPD.

Several limitations should be considered in the study. First, the clinical samples included in this study were relatively small, which may cause the bias of clinical sample selection. Second, the findings of this study were verified based on cellular model, not animal models. In the future, relevant animal model will be used to confirm the relevant conclusions of this study. Finally, patients we recruited were hospitalized due to acute exacerbation. Measurements have not been done at the normal situation without exacerbation.

In summary, this study demonstrates that the expression of RECK in COPD patients is relative lower than that in normal population, and neutrophilic inflammation induced by CSE can be inhibited by RECK, suggesting one therapeutic strategy to combat COPD by modifying RECK-related pathways.

Thanks for the support of Beijing DAO Research Technology Ltd. (www.lingyankeyan.com) in research design, data processing, statistical analysis, and report.

The study was approved by the Ethics Committee of Qingdao Municipal Hospital (Approval number: 2020-009). Written informed consent was obtained from all participating patients. Written informed consent was obtained from the patient to participate in the study and for publication of the details of their medical case and any accompanying images.

The authors declare that they have no competing interests.

This study was funded by the National Natural Science Foundation of China [Nos. 81973012 and 81900048], TCM science and technology development program of Shandong Province [202103020630] and Medicine and Health science and scientific research plan of Qingdao [2021-WJZD011].

Qinghai Li and Wei Han conceived and designed the study; Jiahui Wang, Yi Su, and Hong Liu performed the experiments; Xinjuan Yu and Wei Han analyzed the data and interpreted the findings; Yongchun Li and Qinghai Li were responsible for the data curation. Jiahui Wang and Yi Su wrote the original draft. Xuejie Fang revised the manuscript. All authors read and approved the final manuscript.

Additional Information

Jiahui Wang and Yi Su contributed equally to this work.Edited by: A. Haczku, Sacramento.

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Data are not publicly available due to ethical reasons. Further inquiries can be directed to the corresponding author.

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