Small airway remodeling in diabetic and smoking chronic obstructive pulmonary disease patients

Baseline data of COPD patients

All the clinical data from 347 COPD patients, including their baseline characteristics (gender, age, smoking status, body mass index, fasting blood glucose, etc.) and pulmonary ventilation test results (residual volume [RV]/total lung capacity [TLC], FEV1/forced vital capacity [FEV1/FVC], FEV1%pred [the patient’s FEV1% divided by the average FEV1% in the population], peak expiratory flow [PEF], the forced expiratory flow when 25%, 50% or 75% of the FVC had been exhaled [FEF25, FEF50 and FEF75], maximal mid-expiratory flow [MMEF] and so on), were collected from hospital record databases. We divided the COPD patients into four subgroups – simple (non-smoking/non-diabetic COPD patients), smoking, diabetic, and smoking diabetic – and calculated the mean or median of each indicator in the different groups (Table 1).

The degree of airway obstruction and airflow limitation differed among the COPD subgroups

The most important and widely accepted indicators used to detect persistent airflow limitations in COPD patients are FEV1%pred, FEV/FVC (both measured after bronchodilator inhalation) and RV/TLC. RV/TLC reflects the proportion of the residual volume of the lungs, which is relevant to airway obstruction. RV/TLV tended to be lower in the simple COPD group than in the other groups, though the differences were not significant (Table 2). These results suggested that the co-existence of hyperglycemia-induced lesions worsened the RV of COPD patients (Figure 1A). FEV1 was used to assess the severity of airflow limitations in accordance with the Global Initiative for Chronic Obstructive Lung Disease (GOLD) system. The airflow limitations were more serious in smoking COPD patients and diabetic COPD patients than in simple COPD patients, and were the worst in smoking diabetic COPD patients (Figure 1B).

Figure 1. (A) The distribution of the RV/TLC% in the different COPD subgroups. (B) The distribution of the FEV1%pred in the different COPD subgroups. (C) Small airway disorders identified using the pulmonary ventilation test in the different COPD patients. * indicates a significant difference between two groups; NS indicates no significant difference between two groups.

Pulmonary ventilation tests revealed small airway disorders in smoking diabetic COPD patients

We used the FEF25, FEF50, FEF75 and MMEF values from the pulmonary ventilation test to assess the presence of small airway disorders. The respiratory community has deemed that a patient has a small airway disorder if any two of the above indicators are less than 60%. All 347 of our included patients had small airway disorders, but some of them exhibited a more serious condition. The smoking COPD patients and diabetic COPD patients displayed a significantly greater degree of damage in their small airways than the simple COPD patients. The worst small airway function was observed in diabetic COPD patients who were former or current smokers (Table 2 and Figure 1C).

Pulmonary ventilation test results were worse in diabetic than in non-diabetic rats

We then assessed the lung ventilation function of control rats, COPD rats (induced by CS), diabetic rats and COPD diabetic rats (Table 3). The tidal volume [box] (TVb) and minute volume [box] (MVb) did not differ among the groups (Table 3). However, the peak expiratory flow [box] (PEFb) and the expiratory flow when 50% of the TV had been expired (EF50) were reduced and the expiratory time (Te) was prolonged in COPD rats, diabetic rats and COPD diabetic rats compared with control rats. COPD diabetic rats had worse pulmonary ventilation test results than COPD or diabetic rats alone. These results implied that diabetes can impair lung ventilation. However, the above indicators were improved in COPD diabetic rats injected with SB431542 (an inhibitor of the activin receptor-like kinase [ALK] receptors ALK5, ALK4 and ALK7).

Hematoxylin and eosin staining revealed that the airway lumen diameters were lower in diabetic rats and COPD rats than in control rats, and were the lowest in rats with both diabetes and COPD (Figure 2). These results indicated that diabetes can impair pulmonary ventilation in both healthy and COPD rats, and may be associated with airway remodeling.

Figure 2. CS-induced COPD and streptozotocin-induced diabetes damaged the lungs of rats. Hematoxylin and eosin staining of bronchioles from control, COPD, diabetic and COPD diabetic rats. →: Bronchiole with smooth muscle hyperplasia, globe cell metaplasia and inflammatory cell infiltration; ★: Hyperplastic parenchyma with inflammatory cell infiltration.

High glucose levels and CS exposure activated EMT in AECs

EMT in AECs is a key contributor to airway remodeling. To investigate the mechanism(s) whereby diabetes and COPD induced airway remodeling, we measured markers of the EMT both in vivo and in vitro. Immunohistochemical staining of the airways indicated that Zo1 (an epithelial biomarker) was downregulated and vimentin (a mesenchymal biomarker) was upregulated in rats with diabetes or COPD, and the combination of both diseases amplified these effects (Figure 3A).

Figure 3. High glucose levels and CS exposure activated EMT in AECs. (A) Immunohistochemical results and positive ratios of Zo1 and vimentin in control, COPD, diabetic and COPD diabetic rats. (B–E) AECs were treated with 25 mM glucose, 1% CSE or both for 24 hours. Western blots of Zo1 and vimentin in BEAS-2B (B) and HSAEpiC (C) cells. qRT-PCR analyses of Zo1 and vimentin in BEAS-2B (D) and HSAEpiC (E) cells. * p<0.05, ** p<0.01 compared with the control group.

We then performed an in vitro experiment by treating AECs (BEAS-2B and HSAEpiC cells) with 25 mM glucose and/or 1% CS extract (CSE). These treatments reduced Zo1 and increased vimentin protein levels in both BEAS-2B and HSAEpiC cells (Figure 3B, 3C), indicating that high glucose levels and CS exposure induced EMT. These conclusions were supported by quantitative real-time PCR (qRT-PCR) analyses of the corresponding mRNA levels (Figure 3D, 3E).

High glucose levels and CS exposure activated the TGF-β signaling pathway

To determine the molecular pathway through which diabetes and COPD activated EMT in AECs, we measured the expression of TGF-β signaling molecules in rat airways and AECs. As shown in Figure 4A–4C, high glucose levels and/or CS exposure clearly induced TGF-β expression both in vivo and in vitro. Smad4 was also activated in the airways of the COPD, diabetic and combination rat models (Figure 4D). In AECs treated with 25 mM glucose and/or 1% CSE, Smad2/3/4 mRNA and protein levels and phosphorylated (p)-Smad2/3 levels were greater than those in control AECs (Figure 4E–4H), further indicating that the TGF-β signaling pathway was activated in vitro.

Figure 4A–E. High glucose levels and CS exposure activated the TGF-β signaling pathway. (A) Immunohistochemical results and positive ratios of TGF-β1 in control, COPD, diabetic and COPD diabetic rats. (B, C) AECs were treated with 25 mM glucose, 1% CSE or both for 24 hours. qRT-PCR results of TGF-β1 in BEAS-2B (B) and HSAEpiC (C) cells. (D) Immunohistochemical results and positive ratios of Smad4 in CS-induced COPD, diabetic and COPD diabetic rats. (E–H) AECs were treated with 25 mM glucose, 1% CSE or both for 24 hours.

Figure 4F–H. High glucose levels and CS exposure activated the TGF-β signaling pathway. Western blot results of Smad2, Smad3, Smad4, p-Smad2 and p-Smad3 in BEAS-2B (F) and HSAEpiC (E) cells. qRT-PCR results of Smad2, Smad3 and Smad4 in BEAS-2B (H) and HSAEpiC (G) cells. * p<0.05, ** p<0.01 compared with the control group.

TGF-β signaling pathway activation/inhibition determined the EMT level in AECs

The TGF-β signaling pathway has different functions in different cell types and conditions. To determine the function of this pathway in AECs, we treated cells with a TGF-β signaling pathway activator (TGF-β, 230 pg/mL, 2 hours) or inhibitor (SB431542, 10 μg/mL, 30 minutes). Western blotting and qRT-PCR analyses indicated that activating the TGF-β signaling pathway with TGF-β induced EMT in AECs with or without 1% CSE, as evidenced by reduced Zo1 expression and increased vimentin expression (Figure 5A–5D). Inhibiting the TGF-β pathway with SB431542 had the opposite effects (Figure 5E–5H).

Figure 5A–D. The TGF-β signaling pathway determined the EMT level. (A–D) AECs were treated with 230 pg/mL TGF-β1 for 2 hours. Western blots of Zo1 and vimentin in BEAS-2B (A) and HSAEpiC (B) cells. qRT-PCR analyses of Zo1 and vimentin in BEAS-2B (C) and HSAEpiC (D) cells.

Figure 5E–H. The TGF-β signaling pathway determined the EMT level. (E–H) AECs were treated with 10 μg/mL SB431542 for 30 minutes. Western blots of Zo1 and vimentin in BEAS-2B (E) and HSAEpiC (F) cells. qRT-PCR analyses of Zo1 and vimentin in BEAS-2B (G) and HSAEpiC (H) cells. * p<0.05, ** p<0.01 compared with the control group; # p<0.05, ## p<0.01 compared with the CSE group SB: SB431542.

High glucose levels and CS exposure induced EMT through the TGF-β signaling pathway

The above results separately demonstrated that high glucose levels and CS exposure could activate EMT and the TGF-β signaling pathway in AECs, and that the TGF-β signaling pathway could activate EMT in AECs. Thus, we further investigated whether the TGF-β signaling pathway induced EMT in AECs upon high glucose and CS exposure. When COPD diabetic rats were injected with the TGF-β inhibitor SB431542, their epithelia were less injured than those of rats injected with the buffer (Figure 6A). Immunohistochemical analyses of the airways indicated that EMT was suppressed in SB431542-injected rats compared with buffer-injected rats, as evidenced by lower vimentin expression and higher Zo1 expression (Figure 6B). In vitro experiments in AECs treated with 25 mM glucose and 1% CSE supported these conclusions, as Zo1 expression increased and vimentin expression decreased after SB431542 treatment (Figure 6C–6F), while TGF-β treatment had the opposite effects (Figure 6G–6J).

Figure 6A–E. High glucose levels and CS exposure induced EMT through the TGF-β signaling pathway. (A) Hematoxylin and eosin staining of bronchioles from control, COPD, COPD diabetic and SB431542-injected COPD diabetic rats. (B) Immunohistochemical results and positive ratios of Zo1 and vimentin in control, COPD, COPD diabetic and SB431542-injected COPD diabetic rats. (C–F) AECs were treated with 25 mM glucose and 1% CSE for 24 hours, and SB431542 was added 30 minutes before further treatment. Western blots of Zo1 and vimentin in BEAS-2B (C) and HSAEpiC (D) cells. qRT-PCR analyses of Zo1 and vimentin in BEAS-2B (E) cells.

Figure 6F–J. High glucose levels and CS exposure induced EMT through the TGF-β signaling pathway. qRT-PCR analyses of Zo1 and vimentin in HSAEpiC (F) cells. (G–J) AECs were treated with 25 mM glucose and 1% CSE for 24 hours, and TGF-β was added 2 hours before the cells were harvested. Western blots of Zo1 and vimentin from BEAS-2B (G) and HSAEpiC (H) cells. qRT-PCR analyses of Zo1 and vimentin in BEAS-2B (I) and HSAEpiC (J) cells. * p<0.05, ** p<0.01 compared with the control group; # p<0.05, ## p<0.01 compared with the CSE group.

Study population

In this retrospective study, data from 347 patients with new discharge diagnoses of COPD based on the GOLD 2014 criteria were obtained from the hospital record database of Shandong Provincial Hospital from January 1^st, 2014 to December 31^st, 2017. Patients were only included if they were over 40 years old, had records of pulmonary ventilation test results and were alive when discharged from the hospital.

The 347 patients were divided into four subgroups: a) the simple COPD group: COPD patients who were non-smokers without diabetes, b) the smoking COPD group: COPD patients who were current smokers or ex-smokers but did not have diabetes, c) the diabetic COPD group: COPD patients who were non-smokers but had diabetes, and d) the smoking diabetic COPD group: COPD patients who were current smokers or ex-smokers and had diabetes.

As this was a retrospective study, there was no randomization, no exploration of new treatments and no potential harm to the patients.

Animals

Adult male Wistar rats weighing around 200 g were used for all the experiments. The rats were raised in a room with a 12-hour/12-hour dark/light cycle, controlled temperature (22 ± 2 °C) and humidity (55 ± 5 %), and free access to food and water. The rats were randomly divided into four groups: control (n=5), COPD (n=5), diabetes (n=5) and COPD combined with diabetes (n=6). The combination group was further divided into the SB431542-injected group (n=3) and the buffer-injected group (n=3). The models and treatments are described in greater detail below.

Type 2 diabetes model

Streptozotocin, an antineoplastic agent that is particularly toxic to the mammalian insulin-producing pancreatic beta cells, is widely used to induce diabetes in animals [33, 34]. In our experiments, rats were fed a high-fat, high-glucose diet for one month before being intraperitoneally injected with streptozotocin (35 mg/kg), and kept feeding such diet till they were sacrificed [35]. One week after the injection, blood glucose was measured through the tail vein, and diabetes was established if the blood glucose level was greater than 11.1 mmol/L and the rat exhibited typical diabetes symptoms. Blood was drawn every two weeks, and streptozotocin was reinjected (35 mg/kg) if the blood glucose level dropped below 11.1 mmol/L. The non-diabetic group was intraperitoneally injected with buffer only.

COPD model

We established the COPD rat model by exposing rats to CS for 30 minutes twice a day, with an interval of at least 6 hours between exposures. The exposure procedure was performed five times per week for 12 weeks. The non-COPD groups were placed in a cage with free access to fresh air. After the exposure period, all the rats were fed in the room previously described, with free access to fresh air.

We used a pulmonary ventilation test instrument for small animals (Fine Pointe Buxco WBP 601-1400-001, Data Sciences International, MN, USA) to measure the TVb, MVb, PEFb, Te and EF50 values of the rats. TVb is the tidal volume (box); MVb is the minute volume (box), the rate of ventilation; PEFb is the peak expiratory flow (box), the estimated maximum expiratory flow that occurs in one breath; Te is the expiratory time, the time spent exhaling during each breath, from the start to the end of expiration; and EF50 is the expiratory flow at the point when 50% of the tidal volume has been expired. The rats were sacrificed after the pulmonary ventilation test was performed, and their lung tissues were obtained for further experiments.

SB431542 injection

Ten days before the rats were sacrificed, the SB431542 group was intraperitoneally injected with 2.5 mg/kg SB431542 dissolved in 5% dimethyl sulfoxide, and the buffer group was intraperitoneally injected with 5% dimethyl sulfoxide only [36–38]. The injections were performed every 48 hours for a total of five times.

Histology and morphometric analyses

The lung tissues were fixed in 4% paraformaldehyde for more than 24 hours, and were embedded in paraffin before being cut into 4.5-μm-thick sections. The sections were stained with hematoxylin and eosin.

Immunohistochemical staining

The paraffin-embedded lung tissue sections were deparaffinized and dyed overnight. Antigen retrieval was performed with 5 minutes of pressure at a temperature greater than 95 °C in a pressure cooker containing citrate antigen retrieval solution. Then, the sample was cooled down on ice. Nonspecific reactions were blocked with goat serum for 15 minutes at 37 °C. Then, the sections were incubated with primary antibodies against vimentin (Proteintech, 10366-1-AP, Chicago, IL, USA, 1:2000), Zo1 (Proteintech, 21773-1-AP, 1:200), TGF-β (Proteintech, 21898-1-AP, 1:200) or Smad4 (Proteintech, 10231-1-AP, 1:50) for 1 hour at 37 °C. The sections were incubated with the components of a two-step secondary antibody kit (ZSGB-BIO, PV9003, Beijing, China) for 20 minutes each at 37 °C. The samples were viewed under a confocal FV 1000 SPD laser scanning microscope (Olympus, Japan), and positive ratio analyses were performed with ImageJ.

Cell culture, exposure and treatment

Human bronchial epithelial cells (BEAS-2B) were obtained from Genechem Co. Ltd. (Shanghai, China), and human small airway epithelial cells (HSAEpiC) were obtained from Xinmin Biotechnology Co. Ltd. (Shanghai, China). BEAS-2B cells were cultured in Dulbecco’s modified Eagle’s medium (containing 1 g/L glucose) supplemented with 20% fetal bovine serum (Gibco, 10099-141, Grand Island, NY, USA), 100 U/mL penicillin and 0.1 mg/mL streptomycin (Beyotime, C0222, Shanghai, China). HSAEpiC cells were cultured in RPMI-1640 medium (Gibco, 8118073), supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 0.1 mg/mL streptomycin. The cells were incubated at 37 °C in 5% CO₂ and 95% air.

The cells were treated with 25 mM glucose for 24 hours, with or without simultaneous exposure to 1% CSE. The CSE was prepared from Daqianmen Brand cigarettes. Each cigarette was bubbled slowly in 10 mL of RPMI-1640 medium until a 100% CSE solution was obtained. The solution was sterile-filtered through a 0.22-μm filter, segregated, sealed and stored at -80 °C. Repeated freezing and thawing was avoided.

To investigate the function of the TGF-β signaling pathway, we treated cells with 230 pg/mL TGF-β1 (Proteintech, HZ-1011) for 2 hours or 10 μg/mL SB431542 (Selleck, S1067, Houston, TX, USA) for 30 minutes before performing further treatments. The cells were starved for 16 hours before being provided complete culture medium for further treatment.

Western blot

Radioimmunoprecipitation assay buffer supplemented with 1% phenylmethanesulfonyl fluoride was used to lyse the cells. Then, an 8% sodium dodecyl sulfate polyacrylamide gel was used to electrophoretically separate 25 μg of protein from each sample, and the proteins were transferred onto a 0.45-μm polyvinylidene difluoride membrane. The membrane was blocked with 5% fat-free milk (or 5% bovine serum albumin for phosphorylated protein detection) for 1 hour at room temperature, and then was washed with Tris-buffered saline with 0.05% Tween-20 (Solarbio, T8220, China) three times. The membrane was then incubated with primary antibodies against glyceraldehyde-3-phosphate dehydrogenase (Proteintech, 10494-1-AP, 1:10000), vimentin (CST, 5741, Boston, MA, USA, 1:1000), Zo1 (Proteintech, 21773-1-AP, 1:1000), Smad2/3/4 or p-Smad2/3 (CST, 12747, 1:1000) for 16-20 hours at 4 °C. Then, the membrane was washed three times with Tris-buffered saline with Tween-20 before and after being incubated with the secondary antibody (ZSGB-BIO, ZB-2301, 1:5000) for 1 hour at room temperature. Proteins were detected with an ECL Plus detection system (Thermo Scientific, Pittsburgh, PA, USA). Grayscale was detected with ImageJ.

RNA extraction and qRT-PCR

TRIzol (Invitrogen, USA) was used to extract total RNA from BEAS-2B and HSAEpiC cells, and 2.5 μg of total RNA was reverse-transcribed into cDNA. Then, qRT-PCR was performed with an Ultra SYBR Mixture kit (Cwbio, CW2601M). The sequences of the primers were as follows: Forward-vimentin: 5’-AATGGCTCGTCACCTTCGTG-3’, Reverse-vimentin: 5’-CAGAGAAATCCTGCTCTCCTCG-3’; Forward-Zo1: 5’-GAAATACCTGACGGTGCTGC-3’, Reverse-Zo1: 5’-GAGGATGGCGTTACCCACAG-3’; Forward-TGF-β: 5’-CTGCAAGTGGACATCAACGG-3’, Reverse-TGF-β: 5’-TCCGTGGAGCTGAAGCAATA-3’; Forward-Smad2: 5’-GAGAGCAGAATGGGCAGGAA-3’, Reverse-Smad2: 5’-AGAGCAAGTGCTTGGTATGG-3’; Forward-Smad3: 5’-AGCTGTGTGAGTTCGCCTTC-3’, Reverse-Smad3: 5’-CACAGGAGGTAGAACTGGTGTC-3’; Forward-Smad4: 5’-AAACCATCCAGCATCCACCA-3’, Reverse-Smad4: 5’-TATACTGGCAGGCTGACTTGTG-3’.

Statistics

All the data represent the results of three independent experiments. EpiData (Version 3.1) was used to collect and record data. Statistical analyses were performed with SPSS Version 23.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 7 (GraphPad, San Diego, CA, USA). Parametric data are presented as the mean ± the standard error of the mean or range. Non-parametric data are presented as the median (interquartile range). The Kruskal-Wallis test was used for multi-group comparisons, and then the Mann-Whitney U test was used to compare pairs of groups. The chi-squared test (χ2) was used to compare proportions among groups. T tests were used to analyze parametric data. P<0.05 was considered as the threshold of significance for all statistical analyses.

Study approval

The clinical study was approved by the Medical Ethics Committee of Shandong Provincial Hospital affiliated to Shandong University (Ethical Review of Medical Research on Human Beings No. 2019-001). The animal research protocol was approved by the Medical Experimental Animal Care Commission of Shandong Provincial Hospital affiliated to Shandong University (Ethical Review of Medical Research on Animals No. 2019-019).