Club cell protein (CC16) is expressed primarily by club cells possesses anti-inflammatory properties and is located in the bronchiolar epithelium. Previous studies have demonstrated that CC16 deficiency is associated with the progression of chronic obstructive pulmonary disease (COPD). In the present study, the therapeutic effects of recombinant rat CC16 protein in mice with COPD were examined and the underlying mechanisms investigated. A total of 30 adult male C57/BL6 mice were randomly divided into three groups (10 mice/group). A mouse COPD model was generated by exposing 20 mice to cigarette smoke (CS) for 24 weeks. A total of 10 mice were treated intranasally with rCC16 (2.5 µg/g body weight) and control mice were exposed to normal room air. Results indicated that rCC16 treatment ameliorated pathological damage in the lungs and reduced the production of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8, which were induced by CS exposure. After rCC16 administration, endogenous CC16 was upregulated and the body weight of COPD mice was increased, whereas the opposite was observed in CS-exposed mice. Additionally, rCC16 treatment inhibited the DNA binding of NF-κB/p65 in lung tissues and reduced nuclear translocation of NF-κB/p65 in BALF and epithelial cells. Moreover, rCC16 treatment lead to a decrease in the total number of BALF cells, including macrophages, which was elevated in COPD mice. In conclusion, the present results demonstrate that rCC16 has therapeutic effects on COPD by downregulating pro-inflammatory factors via the NF-κB pathway.
Chronic obstructive pulmonary disease (COPD), a disabling and life-threatening disease, is associated with chronic inflammatory responses. By 2020, COPD is predicted to be the third leading cause of morbidity and mortality in the world (
The nuclear factor-κB (NF-κB) transcription factor exists mainly as a heterodimer contained subunits of the Rel family p50 and p65 and is trapped in the cytoplasm of unstimulated cells. The inhibitor proteins of NF-κB (IκB) masks the nuclear localization sequence which is contained in the Rel homology domain and retain NF-κB in the cytoplasm (
Club cell secretory protein (CC16) is a secretory protein with anti-inflammatory and immunomodulatory effects (
Adult male C57/BL6 mice (6–8 weeks old; weight, 18–20 g) were purchased from the Laboratory Animal Center of Shanxi Medical University (Shanxi, China) and were maintained under specific pathogen-free conditions in a 12-h light-dark cycle with food and water provided
The COPD mouse model was prepared as previously described (
After anesthetization with sodium pentobarbital (75 mg/kg body weight), blood samples were collected from 5 mice in group via retro-orbital plexus puncture and stored at −20°C for analysis. The chest wall was removed and the left bronchus was ligated with a silk suture (0.5 mm; Bangshan Medtec, Guangzhou, China). The trachea was cannulated using an intravenous catheter (20G Intima; Sanxin Medtec, Jiangxi, China), and the right lung was gently lavaged 1 time via a tracheal cannula with 1 ml ice-cold PBS, followed by 4 times with 1 ml PBS. BALF was collected and centrifuged at 290 × g for 10 min at 4°C. Supernatants of the first fraction were stored at −80°C for subsequent analysis of inflammatory cytokines using ELISA. BALF cells from the five different fractions were combined and resuspended in 500 µl PBS. The total number of cells was counted using a hemocytometer. A differential cell count was performed using Wright-Giemsa (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) staining for cytological determination. Finally, tissues of the left lung were removed and stored at −70°C for mRNA analysis.
After anesthetization, the chest wall was removed from another five mice in each group. BALF cells from the right lung of each animal were collected as described above for further analysis. The right bronchus was ligated and the left lung was perfused with 4% paraformaldehyde through the main left bronchus before it was immersed and maintained in 4% paraformaldehyde for another 24 h. The tissue was embedded in paraffin, and 5-µm thick sections were prepared. The mean linear intercept (MLI) and the mean alveolar number (MAN) were visualized using hematoxylin and eosin (H&E) staining, and this was assessed in five randomly selected fields on each slice by Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). One investigator was responsible for inflating the lung with paraformaldehyde and two blinded investigators analyzed the MLI and MAN in order to avoid observer bias.
Immunohistochemical analysis based on the streptavidin-biotin complex was performed as previously described (
RNA was isolated from mouse lung tissues (whole lung) using the RNApure Tissue & Cell kit (CWBIO, Beijing, China) according to the manufacturer's protocols and was reverse transcribed using a Reverse Transcription kit (CWBIO). For gene expression analysis, RT-qPCR was performed using UltraSYBR mixture (CWBIO) and each reaction was performed in duplicate. The averages of the obtained Ct values were used for further calculations. Gene expression levels were normalized to the expression of the reference gene, β-actin. Gene expression levels were calculated using the comparative Ct method (2−ΔΔCt) (
Serum and BALF levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8 proteins were measured using an ELISA kit (Shanghai Westang Bio-Tech. Co., Ltd., Shanghai, China) according to the manufacturer's instructions. The BALF CC16 level was measured using ELISA (Qiaodu Biotechnology, Shanghai, China) according to the manufacturer's protocols.
An NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to extract nuclear and cytoplasmic proteins from BALF cells and proteins were quantified using the BCA protein assay reagent. The total protein was prepared using SDS lysis buffer containing 50 mM Tris-HCl (pH 6.8), 10% glycerol and 2% SDS. Proteins (20 µg) were loaded onto a 12% SDS-PAGE gel, separated and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skimmed milk, incubated overnight with the indicated primary antibodies at 4°C and then incubated with an anti-rabbit HRP-conjugated IgG antibody for 1 h at room temperature. Protein bands were visualized using the ECL blot detection system (CWBIO). Lamin B and β-actin prime antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA) served as references to detect nuclear proteins, total protein and cytosolic proteins.
Nuclear proteins were extracted from lung tissues and incubated with a biotin-labelled double-stranded oligonucleotide containing the consensus sequence of NF-κB-DNA binding site (5′-AGTTGAGGGGACTTTCCCAGG-3′) as previously described (
Data are presented as the mean ± standard deviation. One-way ANOVA followed by the S-N-K method was used for two-group comparisons. All statistical analyses were conducted using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA). Differences with P<0.05 were considered to be statistically significant.
The amino acid sequences for mouse and rat CC16 were analyzed using DNAMAN software. The alignment showed that mouse and rat CC16 shared a 89.58% homology (
CS exposure for 24 weeks resulted in the typical pathological features of COPD, with major enlargement of alveolar spaces distributed throughout the parenchyma. rCC16 treatment could reduce the degree of alveolar enlargement, and lung structures in the control group were normal. H&E staining also revealed that, compared with exposure to CS alone, rCC16 administration alleviated the influx of inflammatory cells, which was characterized by a large number of neutrophils and macrophages in the alveolar spaces (
CC16 is known to possess anti-inflammatory properties (
Pro-inflammatory cytokines are secreted from the endothelium and immunocytes in response to various stimuli (
Our previous studies showed that rCC16 inhibits an inflammatory mediator via the NF-κB pathway (
The primary characteristic of COPD is restricted pulmonary airflow. This is typically progressive and is associated with an abnormal inflammatory response to the inhalation of noxious particles and gases. CS is a complex mixture of oxidant radicals and different chemical compounds, including reactive aldehydes and semiquinones. CS is widely recognized as a primary risk factor that is associated with the progression of COPD (
CC16 is a secretory protein release by club cells, which are present throughout the respiratory tract epithelium from the nose to the respiratory bronchioles. CC16 possesses anti-inflammatory and immunomodulatory properties (
As CC16 has anti-inflammatory properties (
The administration of anti-inflammatory reagents via a nasal route to treat respiratory inflammation has been reported to be beneficial (
Pro-inflammatory cytokines serve an important role in the development of COPD. TNF-α can trigger the activation of other pro-inflammatory cytokines, such as IL-6 and IL-8 (
Pro-inflammatory cytokines are produced by both epithelial cells and immune cells. The accumulation of inflammatory cells, such as macrophages, neutrophils and lymphocytes, is seen in BALF from CS-exposed mice. Among these cells, the number of macrophages is markedly increased in CS-exposed mice. It is known that an increased number of macrophages in the airway lumen are involved in the inflammatory responses of COPD, and anti-inflammation treatment is a primary strategy for COPD (
Nuclear translocation of NF-κB/p65, which is known to activate the NF-κB signaling pathway (
Our present findings are in agreement with those of previous studies (
In the present study, CS-exposure led to a decrease in body weight in COPD mice, and rCC16 treatment partially reversed the effect. Here, we speculate that this may be because CS exposure leads to dysfunction of respiratory muscles and causes malnutrition in COPD mice. By contrast, rCC16 treatnent inhibits inflammaton in the lungs and improves the strength and endurance of respiratory muscles. The recovery of lung fuction may ameliorate nutritional imbalance in COPD mice (
Not applicable.
The present study was supported by the Research Project Supported by Shanxi Scholarship Council of China (grant no. 2015-101), the Fund Program for the Scientific Activities of Selected Returned Overseas Professionals in Shanxi Province (grant no. 2016-097) and the Startup Foundation for Doctors of Shanxi Medical University (grant no. 03201539).
All data generated or analyzed during this study are included in this published article.
MP conceived and designed the experiments. TL, DW, HYL and XYH performed the experiments. BFY and XRZ analyzed and interpreted the data. RG and HLW contributed in designing the present study and drafted the manuscript. All authors read and approved the final manuscript.
All animal experiments were approved by the Animal Ethics Committee of Shanxi Medical University (Taiyuan, China).
Not applicable.
The authors declare that they have no competing interests.
rCC16 ameliorates CS-induced pathological changes in the lungs of COPD mice. (A) Amino acid sequences of mouse CC16 (NP_035811.1) and rat CC16 (NP_037183.1) were aligned using DNAMAN (Lynnon, Quebec, Canada). The degree of homology is indicated by different colors. Black, 100%; blue, 50%. (B) H&E-stained lung sections from control mice, CS-exposed mice and rCC16-treated mice. Exposure to CS induced the infiltration of inflammatory cells into the lungs, as indicated by arrows (neutrophils, green; macrophages, blue). Treatment with rCC16 reduced the degree of pulmonary inflammation, as indicated by arrows. (C) rCC16 reduced the mean linear intercept, which was increased by CS exposure. (D) rCC16 augmented the mean alveolar number, which was decreased by CS exposure. (E) rCC16 treatment led to an increase in body weight, which was significantly reduced in CS-exposed mice. Data are presented as the mean ± SD (n=5/group). *P<0.05 and **P<0.01 as indicated. Scale bars represent 50 µm. COPD, chronic obstructive pulmonary disease; CS, cigarette smoke; rCC16, recombinant club cell secretory protein; SD, standard deviation.
Effects of rCC16 on TNF-α, IL-6 and IL-8 expression in CS-exposed mice. The expression of TNF-α, IL-6 and IL-8 (A-C) mRNA and (D-F) protein in CS-exposed mice treated with rCC16. mice were exposed to CS for 24 weeks and treated intranasally with rCC16 for the last 4 weeks. For qPCR measurements, the results are expressed as ratios of 2−∆∆Ct values for TNF-α, IL-6 and IL-8 mRNA/β-actin. For ELISA measurements, results are expressed as the absolute protein values of TNF-α, IL-6 and IL-8. Data are presented as the mean ± SD (n=5/group). *P<0.05 and **P<0.01 as indicated. BALF, bronchoalveolar lavage fluid; CS, cigarette smoke; rCC16, recombinant club cell secretory protein; SD, standard deviation.
rCC16 promotes endogenous CC16 expression and inhibits nuclear translocation and DNA binding of NF-κB/p65. (A) Endogenous CC16 expression in lung alveolar epithelial cells from each group was determined using immunohistochemical staining. Arrows indicate positive staining. (B) The intensity of positive staining was evaluated and data are expressed as the mean ± SD (n=5/group). (C) Endogenous CC16 levels in BALF were determined by ELISA. Data are presented as the mean ± SD (n=5/group). (D) Subcellular distribution of NF-κB/p65 in the bronchial epithelium, as detected by immunohistochemical staining. (Blue arrows indicate positive cytoplasmic staining; green arrows indicate positive nuclear staining). (E) The intensity of positive staining was evaluated by the gray value, and data are expressed as the mean ± SD (n=5/group). (F) The distribution of NF-κB/p65 in BALF cells was determined by immunoblotting. Potential contamination of the nuclear fractions by cytoplasmic fractions was checked and excluded. One representative experiment is shown. Lamin B and β-actin were used as internal loading controls for the nuclear fraction and the cytoplasmic fraction, respectively. (G) DNA binding of NF-κB/p65 was assessed by EMSA. One representative experiment is shown. *P<0.05 and **P<0.01 as indicated. Scale bars represent 40 µm. BALF, bronchoalveolar lavage fluid; CS, cigarette smoke; rCC16, recombinant club cell secretory protein; SD, standard deviation.
BALF cells analyzed by Wright-Giemsa staining.
Cells | Control | CS | CS+rCC16 |
---|---|---|---|
Total cells (×105/ml) | 3.60±1.18 | 14.93±4.79 |
10.57±3.89 |
Macrophages (×105/ml) | 3.57±1.09 | 14.53±4.04 |
10.91±4.31 |
Neutrophils (×105/ml) | 0 | 0.38±0.03 |
0.33±0.02 |
Lymphocytes (×105/ml) | 0.02±0.01 | 0.31±0.06 |
0.30±0.01 |
Data are expressed as the mean ± SEM (for n=5/treatment group). Statistical analysis was performed using one-way ANOVA followed by a S-N-K Multiple Comparison test
P<0.01 vs. Control
P<0.05 vs. CS group).