The current study aimed to determine the expression of carnitine palmitoyltransferase 1A (Cpt1a) in the lung tissue of chronic obstructive pulmonary disease (COPD) patients and its correlation with lung function. An increase in Cpt1a expression improved lung function in patients with COPD by inhibiting apoptosis and the inflammatory response of lung endothelial cells. Lung tissues of 20 patients with COPD and 10 control patients were collected, their Cpt1a expression was determined by western blotting and apoptosis and inflammation were assessed by haematoxylin-eosin staining, TUNEL assay and ELISA. Mice with knockout or overexpression of Cpt1a were constructed by lentivirus
Chronic obstructive pulmonary disease (COPD) is a common and frequently occurring respiratory disease with high morbidity and mortality. COPD has become an important public health problem due to the economic burden it imposes on society (
Carnitine palmitoyltransferase 1A (Cpt1a) is a key enzyme located to the inner mitochondrial membrane and involved in the regulation of fatty acid oxidation. Cpt1a transports fatty acids from the cytoplasm to the mitochondria for subsequent fatty acid oxidation (
Therefore, the hypothesis of the current study is that Cpt1a alleviates cigarette smoke-induced chronic obstructive pulmonary disease by promoting fatty acid oxidation to inhibit the inflammatory response and cell apoptosis. The main contents of the present study were as follows: i) clarifying the correlation between Cpt1a and lung apoptosis, inflammation and lung function in patients with COPD at the clinical level; ii) confirming whether Cpt1a can treat cigarette-induced COPD in an animal model; and iii) clarifying the mechanism underlying Cpt1a protection of COPD lungs.
All animals were housed in accordance with the Guide for the Care and Use of Laboratory Animals. All procedures of the present study were approved by the Ethics Committee of the Second Hospital of Shanxi Medical University (CMTT number 2013012) and in accordance with international standards. Prior to the clinical study, the subjects (n=20) were informed of the nature, purpose, possible benefits and risks of the trial and the subjects voluntarily confirmed their consent to participate. The inclusion criteria were as follows: i) Patients who met the Global Initiative for Chronic Obstructive Lung Disease (GOLD) in 2015 with severe COPD, ii) no bronchiectasis, iii) no bronchial asthma, iv) no heart failure and v) patients who underwent pulmonary resection. The exclusion criteria were as follows: patients i) with severe hepatic and renal dysfunction, ii) hematologic diseases, iii) usage of immunosuppressants in recent 3 months, or iv) severe immune system diseases. All patients were detected for lung function which was diagnosed as different degrees of dyspnoea, shortness of breath, cough, chronic cough and other symptoms. In addition, patients (n=10) without COPD who underwent pulmonary resection in The Second Hospital of Shanxi Medical University during the same period were selected as the control group. Postoperatively, the lung tissues of all selected patients were collected and frozen in a -80˚C refrigerator.
The tails of healthy adult C57BL/6 mice (5 groups, 10 mice/group, 50% male and 50% female, Age: 8~10 weeks, weight 18~22 g, 12 h light/12 h dark, Temperature is 18~22˚C, humidity, 50-60%, all mice purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., China)were injected with pGLVU6/GFP-short hairpin (sh)RNA control, pGLVU6/GFP-shRNA Cpt1a, pGLVU6/GFP control and pGLVU6/GFP-Cpt1a lentivirus to establish mouse models with knockdown of Cpt1a or overexpression of Cpt1a. At two weeks after the injection, the above four groups of mice were placed in a self-made tobacco smoke inhalation exposure device (30x40x90 cm plexiglass cuboid with nine evenly distributed circular exhaust holes, 2 cm in diameter, on the cover) and a wooden rectangular box containing cigarettes (Furong brand, China Tobacco Hubei Industrial LLC) was placed in it. The principal combustion products of cigarette were as follows: nicotine content in smoke, 1.2 mg/cigarette; carbon monoxide content in smoke, 14 mg/cigarette; and tar content, 15 mg/cigarette. The control group were exposed to smoke-free air and raised normally. To establish the model of COPD in mice, the mice in the model group were exposed to cigarette smoke four times a day in the device. A total of six cigarettes were lit each time and left burning for 1 h, with the smoke concentration in the closed box reaching 100-120 mg/m3. The mice were allowed to breathe smoke-free air for 30 min between two times of smoke exposure. The procedures were conducted six days per week and lasted for four weeks. The experiment included five groups (n=10): control group; pGLVU6/GFP-shRNA control + COPD group; pGLVU6/GFP-shRNA Cpt1a + COPD group; pGLVU6/GFP control + COPD group; and pGLVU6/GFP-Cpt1a + COPD group.
The lung function of mice was tested by using Buxco Fine Pointe Series Whole Body Plethysmography (Buxco Research Systems). The test indexes were as follows: Tidal volume (TV), peak expiratory flow (PEF), 50% expiratory flow (EP50), forced expiratory volume in 0.3 seconds (FEV0.3) and forced vital capacity (FVC).
PMVECs from different experimental groups were serum-starved in 12-well plates, then washed with warm PBS. The cells were incubated with 14C-labeled FAO (Fatty acid oxidation (FAO) medium consisting of DMEM-low glucose (Invitrogen; Thermo Fisher Scientific, Inc.), 0.25 µCi/ml (1-14C) palmitate, 0.25 µCi/ml (1-14C) oleate, 50 µM palmitate, 50 µM oleate, 0.5% BSA, 1 mM carnitine and 12.5 mM HEPES (pH-7.4) at 37˚C for 3 h. The procedures were thrice repeated for each group. After 3 h, the culture medium was collected from each well and an equal portion of the culture medium was distributed to a sealed trapping device. 14CO2 was removed from the medium fraction by adding perchloric acid and captured in NaOH, which was collected and analyzed by liquid scintillation counting to determine the complete FAO ratio of CO2. The acidified medium was collected, refrigerated and centrifuged 5 min at 16,000 x g and 4˚C. The acid soluble metabolite (ASM) of FAO was determined by liquid scintillation counting analysis of the equal samples. Cells were thrice rinsed with cold Hank's balanced salt solution (HBSS) and lysed with SDS lysis buffer. The protein concentration of the lysate was determined by the BCA assay. The results of FAO were expressed as a percentage of CO2.
The lung tissue homogenate sample from mice was placed into the centrifuge tube, then the internal standard mix was added. After vortex oscillation blending, the sample was centrifuged (1,000 x g, 4-8˚C, 5 min) and the supernatant was collected for later detection. Ceramide was analysed by LC-MS. Agilent 6530 Q-TOF was used to identify and analyse levels of ceramide, which was confirmed by comparing retention times and tandem mass spectrometry data with standard compounds. The results were corrected for naturally occurring 13C impurity of the tracers. MassHunter Quantitative Analysis software (Agilent Technologies, Inc.) was used to quantify the ceramide content.
Protein was extracted from RIPA lysate (Huaxingbio) containing protease inhibitor (Thermo Fisher Scientific, Inc.). The tissues or cells were ultrasonically disrupted, then centrifuged at 14,000 x g and 4˚C for 15 min. The supernatant was collected and the protein concentration was determined using the BCA assay. Proteins (30 µg) were separated by SDS-PAGE (10%) together with a pre-stained protein ladder (Thermo Fisher Scientific, Inc.), then transferred to nitrocellulose membranes (MilliporeSigma, blocked with 5% non-fat milk in Tris-Buffered saline and Tween-20 (TBST; 20 mmol/l Tris-Cl, 150 mmol/l NaCl, 0.05% Tween 20, pH 7.4) at 4~8˚C for 2 h and incubated overnight with primary antibody (cat. no. #97361; 1:1,000, CST) at 4˚C. After being washed with buffer, the membranes were incubated with secondary antibodies(Anti-rabbit IgG, HRP-linked Antibody, cat. no. #7074, 1:1,000, CST, USA) at 4˚C for 40 min. The relative protein content was determined by Bio-Rad laser imaging system (Bio-Rad Laboratories, Inc.) and Image Lab software v6.1 (Bio-Rad Laboratories, Inc.) after the DyLight 800-labeled secondary antibody (1:10,000 dilution; KPL, Inc. ) was incubated at room temperature for 1 h the next day(Visualization reagent kit, Cat. No. P0020, Beyotime Institute of Biotechnology). Primary antibody Cpt1a (cat. no. 97361, 1:1,000) secondary Antibodies (Cat. No. #7074,1:1,000) and GAPDH (cat. no. 5174, 1:1,000) were purchased from Cell Signaling Technology, Inc.
Total RNA in tissues(cells number: 2x106) was extracted using TRIzol® reagent (Thermo Fisher Scientific, Inc.). The first-stand cDNA was synthesized using First Strand cDNA Synthesis kit with gDNA Eraser according to the manufacturer's protocol. PCR was performed using cycling conditions: Denaturation 95˚C for 30 sec, annealing 60˚C for 40 sec and extension at 72˚C for 60 sec; 35 cycles) (Takara, Osaka, Japan). RT-qPCR was performed with SYBR Green Master Mix to examine the relative mRNA levels of indicated genes with an AJ qTOWER 2.2 Real-Time PCR system (Analytik Jena AG) by using a quantitative real-time PCR kit (Takara Bio, Inc.). Sequences for RT-qPCR primers were: Mouse Cpt1a, 5'-CTCCGCCTGAGCCATGAAG-3', mouse GAPDH: 5'-AGGTCGGTGTGAACGGATTTG3'. GAPDH was used as an internal control. Relative gene expression level was calculated by 2-ΔΔCq method (
Lung samples were dissected and fixed at 18~25˚C in 4% paraformaldehyde solution (Sangon Biotech Co., Ltd.) for 72 h. Tissues were embedded in paraffin (Sangon Biotech Co., Ltd.). Sections of lung were cut at 3-4 µm and prepared for haematoxylin and eosin staining by standard procedures. Samples were immersed in xylene and alcohol, stained with hematoxylin for 5 min, stained with eosin for 3 min and re-immersed in alcohol and xylene. The sections were counterstained with Harris haematoxylin (Sangon Biotech Co., Ltd.) and normal IgG (Merck Millipore Sigma Aldrich) as a negative control. Images were captured with a brightfield DM4B microscope (Leica Microsystems GmbH).
Lung samples were collected at enrolment and immediately stored at -80˚C in a single biologic resource centre. Lung tissues inflammatory factors (TGFβ, IL-6, IL-β and TNF-α) were determined by commercial ELISA kits (cat. nos. 70-EK981-96, 70-EK206/3-96, 70-EK201B/3-96 and 70-EK282/4-96) purchased from Multisciences (Lianke, Hangzhou, China) Biotech Co., Ltd., according to the manufacturer's instructions. The detection threshold was 0.156 and 1.56 ng/ml. Samples, reagents and buffers were prepared strictly in accordance with the manufacturer's guideline.
Primary PMVECs were isolated by following these steps. C57BL/6J mice (8-10 weeks old, both male and female) were anesthetized by abdominal injection with pentobarbitone sodium (100 mg/kg body weight). and lungs were removed. The lung samples were enzymatically digested by a mouse lung dissociation kit (Miltenyi Biotec GmbH). Following removal of CD45+ cells, CD45-cells were collected, washed and incubated with CD31-conjugated beads (Invitrogen; Thermo Fisher Scientific, Inc.). CD31+ cells were enriched using a MACS column and magnetic field by protocol of reagent kits. For magnetic separation, MACS ART MS Columns were placed into a MiniMACS Separator, rinsed once with 1 ml of MACS ART Binding Buffer (discarded after flow-through), and the CD45- cells suspension was then placed in the column. The PMVECs, which were bound to CD31-conjugated magnetic microbeads, were then retained in the column. That was because the column was placed in MiniMACS Separator, which is basically a magnet forming magnetic field, which causes the retention of magnetically labeled cells. This CD31+ cells was then washed by adding 4 ml of medium and centrifuged 10 min at 1,000 rpm in 4~8˚C. The freshly isolated cells were considered as passage 0, which was cultured in dish coated with human fibronectin (30 µg/ml). Cells within 5 passages were used for experiments.
Haematoxylin-eosin staining is a basic method of histology and pathological examination as the haematoxylin produces crisp, intense blue nuclei providing optimal contrast to the eosin-stained cytoplasm. TUNEL staining detects the DNA breaks formed when DNA fragmentation occurs in the last phase of apoptosis. Lung samples were dissected and fixed at 18~25˚C in 4% paraformaldehyde solution (Sangon Biotech Co., Ltd.) for 72 h. Tissue was embedded in paraffin (Sangon Biotech Co., Ltd.). Sections of lung were cut at ~3-4 µm and prepared for haematoxylin and eosin staining by standard procedures. Then the tissue wax was cut into serial 5 µm sections. After being reconstructed by protease K, the sections were stained by haematoxylin and eosin. The cell samples were collected, washed, fixed and then stained by TUNEL and DAPI. Histological sections were observed in six randomly selected fields for analysis) under a Flirorescent microscope (Nikon Eclipse 80i; Nikon Corporation).
Flow cytometry was used to measure Annexin V-positive cells that are considered as apoptotic cells by Flowjo7.6.1 (Treestar Inc., Ashland, OR). Briefly, cells were collected using the TrypLE (Thermo Fisher Scientific, Inc.). After washed with cold phosphate buffered saline, Annexin V binding buffer (300 µl) was added to resuspend cells. A total 1x105 cells were collected and incubated with 100 µl Annexin V binding buffer along with 5 µl of fluorescein isothiocyanate conjugated Annexin V (Thermo Fisher Scientific, Inc.) and 5 µl of propidium iodide (Life Technology) for 20 min at room temperature. Finally, Annexin V binding buffer (500 µl) was added and mixed gently. FC-500 (Beckman Coulter, Inc.) was used to detect the Annexin V-positive cells with a total of 20,000 events analyzed. The apoptotic rate was calculated by percentage of early and late apoptotic cells.
Experiments of cell cultivation were performed with six biological replicates with a total of five times of technical repetitions for measurements. Data are expressed as mean ± standard error of the mean. One-way analysis of variance with the post Tukey's test was used to determine whether there is any statistical significance between the means of groups. The Student-Newman-Keuls test and Pearson's correlation analysis were used to examine which specific groups of means were statistically different. P<0.05 was considered to indicate a statistically significant difference.
To determine the correlation between Cpt1a expression and lung function in patients with COPD, clinical data were analysed and lung tissues from patients with COPD were collected for the following tests. In this study, 30 samples were collected. Clinical baselines and data are presented in
Lung inflammation and apoptosis are the most basic pathologic features of patients with COPD. Previous studies have shown that Cpt1a is involved in apoptosis of pulmonary microvascular endothelial cells (
To identify the role of Cpt1a in patients with COPD, lentivirus mediated Cpt1a knockout and overexpression models were established and the following tests were carried out. Transfection of pGLVU6/GFP shRNA Cpt1a and pGLVU6/GFP Cpt1a significantly reduced or elevated the levels of Cpt1a protein and mRNA in lung tissues, respectively (
The results of previous studies in our laboratory showed that Cpt1a is mainly expressed in pulmonary microvascular endothelial cells and participates in anti-apoptosis. However, whether overexpression of Cpt1a reduces apoptosis of PMVECs in COPD mice is unknown. Therefore, lung microvascular endothelial cells were isolated from COPD mice. Annexin V-PI flow analysis showed that compared with COPD model mice, knockdown of Cpt1a significantly aggravated the apoptosis of PMVECs induced by COPD, while overexpression of Cpt1a alleviated the apoptosis induced by COPD (
The results of our previous experiments showed that there is a correlation between the difference in Cpt1a expression in clinical patients and the lung function. To identify whether Cpt1a served a role in the treatment of patients with COPD, the present study examined the lung function of the two murine models
The prevalence of COPD has increased in recent years, especially among young individuals. COPD has become one of the commonest diseases affecting the health and quality of life of individuals worldwide (
The CPT system consists of two separate proteins located in the outer (Cpt1) and inner (Cpt2) mitochondrial membranes (
Tobacco smoke is deadly and has more than 7,000 chemicals, 69 of which are verified as carcinogens (
Pulmonary vascular endothelial injury (apoptosis and an inflammatory response) is a common pathologic manifestation in the progression of COPD (
Ceramide is an important lipid molecule that regulates cell differentiation, proliferation, apoptosis, aging and other life activities (
Fatty acid is an important component of blood lipids and the main product of lipid digestion. Fatty acid oxidation plays an essential role in regulating triglyceride metabolism (
In conclusion, the present study showed for the first time that overexpression of Cpt1a could alleviate lung dysfunction and reduce inflammatory response and apoptosis of lung tissues in COPD mice and protect cigarette-induced COPD by promoting the oxidation rate of substrate fatty acids, thus inhibiting the production of ceramide to suppress apoptosis of endothelial cells and inflammatory responses. The data suggested that Cpt1a may be a potential new target for the treatment of patients with COPD.
Not applicable.
All data generated or analyzed during this study are included in this published article.
HZ and LL performed the conception and design of the study, and drafted and revised the manuscript. LL, YZ and JG analyzed data and prepared figures. LL, GY, SZ and DR performed the experiments and manuscript review. HZ, GY and LL confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
The study was approved by the Ethics Committee of the Second Hospital of Shanxi Medical University (CMTT number 2013012). All patients were informed in detail about the objective and methods of this study and signed a consent form. All procedures of the present study, including animals and patients, were approved by the Ethics Committee of the Second Hospital of Shanxi Medical University (CMTT number: 2013012) and in accordance with international standards.
Not applicable.
The authors declare that they have no competing interests.
Cpt1a levels and ceramide in lung tissues from patients with COPD and control group. (A) Western blots and (B) quantification of protein expression for Cpt1a in lung tissues from control and patients with COPD. (C) Ceramide were measured in lung tissues from control and patients with COPD by mass spectrometry. Data are expressed as mean ± standard error of the mean; n=10; **P<0.01 vs. Control; ##P<0.01 vs. COPD with low expression of Cpt1a. Cpt1a, carnitine palmitoyltransferase 1A; COPD, chronic obstructive pulmonary disease.
Cpt1a levels are associated with apoptosis and inflammation in lung tissues from patients with COPD. (A) Haematoxylin and eosin-stained sections showing the lung injury in patients with COPD (Bar=100 µm, n=10; **P<0.01 vs. COPD with low expression of Cpt1a). (B) Apoptosis were assessed by TUNEL (Bar=200 µm, n=10; *P<0.05 vs. COPD with low expression of Cpt1a). (C) ELISA demonstrating the levels of TGF-β, IL-6, TNF-α and IL-1β in lung tissues from patients with COPD and control group. Data are expressed as mean ± standard error of the mean; n=10, **P<0.01 vs. Control; ##P<0.01 vs. COPD with low expression of Cpt1a. Cpt1a, Carnitine Palmitoyl Transferase 1A; COPD, Chronic Obstructive Pulmonary Disease.
Enhancing Cpt1a attenuating CSE-induced apoptosis and inflammation
Enhancing Cpt1a increased FAO attenuating CSE-induced apoptosis
Enhancing Cpt1a inhibited ceramide attenuating CSE-induced lung dysfunction. (A) pulmonary function indicators. (B) Ceramide was measured in lung tissues from model mice by mass spectrometry. Data are expressed as mean ± standard error of the mean; n=10; **P<0.01 vs. Control; #P<0.05, ##P<0.01 vs. pGLVU/GFP-shRNA control; &&P<0.01 vs. pGLVU/GFP control. Cpt1a, carnitine palmitoyltransferase 1A; CSE, cigarette smoke extract; COPD, chronic obstructive pulmonary disease; FAO, fatty acid oxidation.
A proposed model showing how Cpt1a suppresses vascular endothelial apoptosis and inflammation induced by COPD. Cpt1a, carnitine palmitoyltransferase 1A; COPD, chronic obstructive pulmonary disease; ECs, endothelial cells.
Comparison of lung function Indices between patients with COPD with differential expression of Cpt1a and the control group.
Indicator | Control (n=10) | COPD (n=10) Cpt1a low expression | COPD (n=10) Cpt1a high expression | P-value |
---|---|---|---|---|
Tidal volume (ml) | 2.73±0.32 | 1.47±0.25a | 1.87±0.31b | 0.001 |
Peak expiratory flow (ml/s) | 37.15±0.18 | 16.38±2.13a | 20.48±1.56b | 0.003 |
50% expiratory flow (ml/s) | 1.79±0.20 | 1.35±0.22a | 1.56±1.19b | 0.021 |
Forced expiratory volume in 0.3 seconds (ml) | 4.42±0.33 | 2.33±0.32a | 3.43±0.12b | 0.013 |
Forced expiratory volume in 0.3 sec/forced vital capacity (%) | 87.61±4.41 | 64.49±4.30a | 74.73±5.30b | 0.001 |
Results are expressed as mean ± standard deviation. Differences between groups were analyzed by the independent Student t test, χ2 text, or Wilcoxon test. P-values a vs. b. COPD, chronic obstructive pulmonary disease; Cpt1a, carnitine palmitoyltransferase 1A.
Detection of lung function indices of model mice.
Group | Tidal volume (ml) | Peak expiratory flow (ml/s) | 50% expiratory flow (ml/s) | Forced expiratory volume in 0.3 sec (ml) | Forced vital capacity (ml) | Forced expiratory volume in 0.3 sec/forced vital capacity (%) |
---|---|---|---|---|---|---|
Ctrl | 2.76±0.16 | 37.21±1.66 | 1.79±0.13 | 14.69±0.49 | 20.03±0.69 | 0.73±0.04 |
pGLVU6/GFP-shRNA Control | 1.47±0.20 | 15.60±2.17 | 1.29±0.16 | 10.62±0.58 | 17.13±0.55 | 0.62±0.03 |
pGLVU6/GFP-shRNA Cpt1a | 1.20±0.19 | 13.28±2.19 | 1.10±0.13 | 7.75±0.48 | 14.68±0.61 | 0.53±0.03 |
pGLVU6/GFP-Control | 1.41±0.24 | 17.07±1.85 | 1.38±0.11 | 11.04±0.55 | 16.96±0.75 | 0.65±0.06 |
pGLVU6/GFP-Cpt1a | 1.91±0.21 | 21.76±1.68 | 1.69±0.12 | 13.80±0.48 | 19.78±0.41 | 0.70±0.03 |
P |
0.001 | 0.0123 | 0.024 | 0.001 | 0.0342 | 0.0453 |
P |
0.001 | 0.001 | 0.001 | 0.002 | 0.001 | 0.0234 |
Results are expressed as mean ± standard deviation. Differences between groups were analyzed by the independent Student t test, χ2 text, or Wilcoxon test.
Pa, pGLVU6/GFP-shRNA Cpt1a vs. pGLVU6/GFP-shRNA Control;
Pb, pGLVU6/GFP-control vs. pGLVU6/GFP-Cpt1a.