A new mouse model of biomass smoke-related chronic obstructive pulmonary disease combining porcine pancreatic elastase nebulization

Introduction

Background

Chronic obstructive pulmonary disease (COPD) is a prevalent chronic respiratory disorder and a leading cause of global morbidity and mortality (1). Most studies have focused on cigarette smoking which is a major risk factor of COPD, while biomass smoke has been neglected which requires further attention (2,3).

Long-term biomass smoke exposure (BME) has recognized as a critical independent risk factor for COPD, particularly in rural populations (4-6). According to the World Health Organization, household air pollution contributes to COPD-related mortality among approximately 3.8 billion people worldwide. Women and children, who are typically responsible for household chores such as cooking or collecting firewood, bear the greatest health burden (7).

Rationale and knowledge gap

Few studies have employed animal models to investigate the effects of BME. Although we have numerous methods to build up a tobacco smoke-related COPD animal models, accumulating evidence highlights significant phenotypic differences between biomass smoke-related COPD and tobacco smoke-related COPD (8,9). Our previous clinical studies identified a distinct COPD phenotype associated with BME, characterized by early-stage small airway disease. Compared with those with tobacco smoke exposure induced COPD, patients with BME-induced COPD exhibited more severe emphysema, airway remodeling, and reductions in pectoralis major muscle area but with slow decline in pulmonary function (10,11). Additionally, Previous animal models of biofuel-related air pollution have focused primarily on PM2.5, overlooking other harmful components of biofuel, such as nitrous oxide, sulfur dioxide, and other pollutant gases on animals modeling (12,13). Therefore, it is necessary to establish a characteristic type of COPD animal model for BME, which plays a vital role in exploring the mechanism of COPD disease and testing treatment strategies.

Objective

To address these gaps, we propose a novel murine model of COPD by combining BME with elastase nebulization. Wood chips were selected as the biomass source due to their low cost, wide availability, and ecological validity. In our previous study, rats exposed to biomass smoke for 6 months developed a phenotype consistent with COPD (14). We also conducted a preliminary exploration of mouse models involving inducing the condition solely through biofuel exposure. We found that at least 6 months are required to obtain animal models exhibiting a clearly defined phenotype (15). Owing to the complexity of COPD pathophysiology, it is challenging to simultaneously model chronic bronchitis and emphysema in a single system (16). Therefore, in this experiment, we employed a hybrid modeling approach that reduced the induction period to 4 months while yielding a broader range of COPD-related pathophysiological features.

In addition, we proposed that elastase was administered by nebulization instead of airway instillation for the first time, which greatly reduced the physiological and psychological adverse effect on the mice, reducing the mortality of the mice, which fully embodied the 3R principle of experimental animal ethics (replacement, reduction, refinement). Many researchers preferred to administer drugs by airway instillation. Tracheotomy is required during the process of invasive intratracheal titration, leading to heavy trauma and infection, which is difficult to recover (17). Non-invasive airway instillation cannot guarantee uniform distribution of elastase throughout the lungs. The action of elastase on the large airways rather than the small airways is inconsistent with the pathological characteristics of COPD.

Given that existing biomass smoke-related COPD models are severely constrained by their long preparation period, invasive procedures, and incomplete phenotypic representation, there is an urgent need to develop a faster, more clinically relevant model that recapitulates all key pathological features. We present this article in accordance with the ARRIVE reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1502/rc).

Methods

Animals and COPD modeling

Twelve female C57BL/6 mice (6 weeks old, 18–20 g) were obtained from Guangdong Pharmachem Biotechnology Co. and housed in the laboratory animal centre of Guangzhou Medical University under barrier conditions. Food and water were provided ad libitum. After a 7-day acclimatization period, the mice were prepared for subsequent experiments. The mice were randomly assigned to either a BME group (n=6) or a clean air control group (CON group; n=6) using a computer-generated random number sequence. Mice in the BME group were exposed to biomass smoke for 3 hours twice daily, 6 days per week, for 4 months. Control group was exposed to filtered air. Porcine pancreatic elastase was administered via nebulization once weekly. After pulmonary function testing, mice were then euthanized. Lung tissues and blood were collected for further analysis. All animal experiments were performed under a project license (No. GY2023-474) granted by the Guangzhou Medical University Animal Research Ethics Board, in compliance with institutional guidelines for the care and use of animals. A protocol was prepared before the study without registration.

BME system

A whole-body inhalation exposure system (TSE, Bad Homburg, Germany) was used to model. Biomass smoke was generated via controlled combustion of wood chips (10 g per tray) using a piston pump system. The harmful particles and gas were transported into a smoke exposure box where the rodent cage was placed.

Elastase administration

A compressed-air nebulizer (PARI BOY; PARI GmbH, Starnberg, Germany) was used to deliver porcine pancreatic elastase (LS002292; Worthington Biochemical Corporation, Lakewood, NJ, USA). The dose was 6 U per mouse, nebulized for 15–20 minutes each time, once a week for 4 months.

Pulmonary function measurement

Mice were anesthetized with 0.3% pentobarbital sodium (50 mg/kg, intraperitoneal). Forced Pulmonary Function Test System (Buxco^® Research Systems, Data Sciences International, Willmar, MN, USA) were connected by tracheal intubation for detection. After calibration of the system, the pulmonary function parameters were measured by a computer-controlled ventilator, including Boyle’s Law functional residual capacity (FRC), fast flow volume, quasi-static pressure volume, resistance and compliance. Methods were as described previously for invasive pulmonary function testing (12).

Blood sampling and bronchoalveolar lavage sampling

After pulmonary function testing, 500–700 µL of blood was collected from the cardiac apex into Eppendorf tubes containing sodium heparin (1:10 v/v) as an anticoagulant. The sample was mixed and left to be measured. The right lung was lavaged by phosphate-buffered saline while 3 mL bronchoalveolar lavage fluid (BALF) sample was collected by Eppendorf tube.

Lung tissue sampling and staining

After blood sampling, the right lung was snap-frozen in a 2 mL cryovial. Then left lung was removed and fixed in 4% paraformaldehyde (BL539A, biosharp, Hefei, Anhui, China) at 4 ℃ overnight. Lung tissue was subjected to gradient dehydration and paraffin-embedded for pathological analysis; 4 µm paraffin sections were generated and used for hematoxylin & eosin (H&E) staining.

Pathologic detection of lung tissue

For quantifying the injury of mouse lung tissue, we examined the mean linear intercept (MLI) and basement membrane perimeter speciation indices (WAt/Pbm) in mice. To ensure the objectivity of the pathological assessment, all lung tissue sections were labeled with unique, non-identifiable codes that concealed group allocation (BME or CON) from the pathologists.

MLI quantification: emphysema was quantified based on the measurement of the MLI (18). Leica Aperio CS2 (Leica, Wetzlar, Germany) was used to scan the H&E stained lung tissue sections (4 µm thickness). Semi-quantitative analyses were performed with the aid of Image-Pro Plus 6.0. Briefly, the MLI was measured by dividing the length of a line (L) drawn across the lung section by a total number of intercepts counted within this line (NS). According to the formula: MLI = L/NS to express the mean alveolar intervals.

Basement membrane perimeter speciation indices (WAt/Pbm) quantification: the WAt/Pbm was designed to quantitatively assess airway inflammation. Basement membrane circumference of the airway wall <1,500 µm were defined as small airways on H&E-stained sections. The thickness of small airway walls was measured. A brief description was as follows: ImageScope software was used to measure basement membrane circumference of the airway wall (perimeter basement membrane, Pbm), and the area (Abm), followed by the area of the outer membrane of the tube wall (Ao). According to the formula Wat = Ao − Abm, the airway wall thickness was expressed by WAt/Pbm.

RNA extraction and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)

To explore pulmonary inflammation, we performed a RT-PCR assay. Total RNA was extracted using Trizol reagent (15596018CN, Invitrogen, Carlsbad, California, USA). Hifair III 1^st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (11141ES60, Yeasen, Shanghai, China) was applied to reverse transcribe total RNA into cDNA, then Hieff^® qPCR SYBR^® Green Master Mix (Low Rox Plus) (11202ES08, Yeasen) was used for qRT-PCR under the following conditions: denaturation at 95 ℃ for 30 s, followed by 40 cycles of 95 ℃ for 10 s and 60 ℃ for 30 s. Primer sequences were synthesized as Table S1. At least three biological replicates were included per group per experiment. RT-PCR was performed on a LightCycler96 System (172-5201AP, Bio-rad, Hercules, California, USA). The results were analyzed using the 2^−ΔΔCT method.

Multiplexed liquid chip

Plasma levels of inflammatory factors interleukin-1β (IL-1β), interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α), interferon-gamma (IFN-γ), interleukin-6 (IL-6), interleukin-5 (IL-5), interleukin-4 (IL-4), interleukin-10 (IL-10) (pg/mL) were measured using a commercialized corresponding cytokine liquid microarray kit (Mouse Inflammation 10 Factor Panel, LXRLBM10-1) (Univision Bio-technology, Suzhou, Jiangsu, China) according to the manufacturer’s instructions.

Enzyme-linked immunosorbent assay (ELISA)

Levels of IL-10, CXCL2/MIP-2, and CCL2/MCP-1 in BALF were determined by ELISA using commercial kits (70-EK210, 70-EK2142, and 70-EK287; MultiSciences, China) following the manufacturer’s protocols.

Statistical analyses:

Analyses were performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). Normality was assessed using the Shapiro-Wilk test. Normally distributed data were presented as mean ± standard deviation and non-parametric data were expressed as the median (25th percentile, 75th percentile) from at least three independent experiments. Differences between two groups were analyzed by Student’s t-test. The Mann-Whitney U test was used for the non-parametric data. Values of P<0.05 were considered statistically significant.

Results

BME significantly impaired pulmonary function

To evaluate the effects of combined BME and elastase nebulization on respiratory function, pulmonary function tests were performed. BME significantly increased FRC (CON vs. BME =0.3617±0.0354 vs. 0.4956±0.0296 mL, P<0.0001), quasi-static compliance (Cchord, CON vs. BME =0.0024±0.0002 vs. 0.0029±0.0002 mL/cm H₂O, P<0.01), static compliance (Cfvc50, CON vs. BME =0.0800±0.0044 vs. 0.1021±0.0104 mL/cm H₂O, P<0.001) and forced vital capacity (FVC; CON vs. BME =0.8077±0.0522 vs. 1.033±0.0747 mL, P=0.0001), while reducing dynamic pulmonary compliance (Cdyn, CON vs. BME =0.0653±0.0316 vs. 0.0295±0.0097 mL/cm H₂O, P<0.05) compared with CON group as Figure 1 shown.

Figure 1 BME combined with elastase nebulized inhalation impairs pulmonary function in mice after 4 months. Each group n=6. All data were expressed as mean ± standard deviation. All analyzed by Student’s t-test. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. BME, biomass smoke exposure; Cchord, quasi-static compliance; Cdyn, dynamic lung compliance; Cfvc50, compliance at 50% vital capacity; CON, control; FEV100/FVC, forced expiratory volume in 100 ms to forced vital capacity ratio; FEV200/FVC, forced expiratory volume in 200 ms to forced vital capacity ratio; FEV20, forced expiratory volume in the first 20 ms of expiration; FEV300/FVC, forced expiratory volume in 300 ms to forced vital capacity ratio; FEV400/FVC, forced expiratory volume in 400 ms to forced vital capacity ratio; FRC, functional residual capacity; FVC, forced vital capacity.

Consistently, we observed persistent airway obstruction throughout the entire forced expiration from the very early phase to the late phase compared to controls. Forced expiratory volume in the first 20 ms of expiration was significantly reduced in BME group (FEV20; CON vs. BME =0.0025±0.0003 vs. 0.0020±0.0003, P<0.05) with a ratio of forced expiratory volume at 20 to 400 ms and forced vital capacity significantly reduced in BME group compared to control group (FEV20/FVC; CON vs. BME =0.0031±0.0004 vs. 0.0022±0.0007, P<0.05; FEV100/FVC, CON vs. BME =0.1731±0.0101 vs. 0.1374±0.0108, P<0.001; FEV200/FVC, CON vs. BME =0.4106±0.0733 vs. 0.3258±0.0314, P<0.05; FEV300/FVC, CON vs. BME =0.6206±0.0328 vs. 0.4932±0.0367, P<0.0001; FEV400/FVC, CON vs. BME =0.8345±0.0424 vs. 0.6671±0.0495, P<0.0001). These data suggested that BME induced a significant damage to pulmonary function which shown the characteristics of emphysema and airway obstruction.

BME induced emphysema and bronchiectasis

To further determine the effects of BME on alveolar structure, we performed pathological examination of mouse lung tissue sections with H&E staining. A significant reduction in the number of small alveoli was observed with alveolar septal destruction and fusion leading to large airspaces (Figure 2A). Emphysema severity was significantly greater in BME group, with an increase in the mean alveolar linear intercept (MLI; CON vs. BME =11.21±0.8613 vs. 14.94±0.6444 µm, P<0.0001) (Figure 2B).

Figure 2 BME induced emphysema and bronchiectasis in physiology. Representative pictures of paraffin sections (×150, scale bar =200 μm, A; ×200, scale bar =50 μm, C; ×200, scale bar =200 μm, D) stained with H&E. Histological structures of lungs, the status of airway epithelial layer and alveolar septa were observed in sections of lung specimens. (A) The alveolar structure of the CON group was normal while the alveolar was destructed and alveolar cavity was enlarged in BME group. (B) MLI. (C) Airway epithelial layer was normal in CON group and injured in BME group. The arrowhead refers to normal cilia and cilia missing lodging, respectively. (D) Small airway wall thickness was normal in CON group and enlarged in BME group. The arrow refers to the small airway wall. (E) WAt/Pbm expressed the average small airway wall thickness. (B,E) Morphometric measurements of MLI and WAt/Pbm in lung tissues stained with H&E as described in the text. Each group n=6. All data were expressed as mean ± standard deviation analyzed by Student’s t-test. *, P<0.05; ****, P<0.0001. BME, biomass smoke exposure; CON, control; H&E, haemotoxylin & eosin; MLI, mean linear intercept; WAt/Pbm, basement membrane perimeter speciation indices.

In addition, bronchitis was observed. H&E-stained lung tissue sections showed damage of the bronchial airway epithelial cells in BME group, with cilia shedding and inverting, thickening of the walls of the small airways (Figure 2C). Quantitative analysis confirmed significant increase in small-airway wall thickness (WAt/Pbm) in BME group (WAt/Pbm; CON vs. BME =8.11±2.8500 vs. 11.87±0.5721 µm, P<0.05) (Figure 2D,2E).

BME increased the expression of inflammatory cytokines

To quantify lung inflammation, we performed qRT-PCR to observed the changes of inflammatory cytokines expression. Increasing of IL-1β, IL-10 (IL-10, CON vs. BME =0.8399±0.3122 vs. 3.908±1.720, P<0.01) and TNF-α (TNF-α, CON vs. BME =0.8025±0.2361 vs. 5.051±2.166, P<0.001) was found in BME group while IL-10 and TNF-α had significant difference (Figure 3A).

Figure 3 BME increased the expression of inflammatory cytokines. (A) mRNA expression of IL-1β, IL-10 and TNF-α in lung tissue. IL-10, TNF was higher in BME group than CON group significantly. (B) IL-10, CXCL2, CCL2 (pg/mL) expression in BALF measured with ELISA. IL-10 was significant increased in BME group while CXCL2 and CCL2 decreased. Each group n=6. Data were analyzed by Student’s t-test and expressed as mean ± standard deviation. ns, no significant difference; **, P<0.01; ***, P<0.001. BALF, bronchoalveolar lavage fluid; BME, biomass smoke exposure; CCL2, chemokine ligand 2; CON, control; CXCL2, C-X-C motif chemokine ligand 2; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; TNF-α, tumor necrosis factor-α.

We also observed a significant elevation of IL-10 (IL-10, CON vs. BME =186.7±5.097 vs. 201.8±8.610 pg/mL, P<0.01) whereas a marked decrease in CXCL2 (CXCL2, CON vs. BME =10.25±0.8394 vs. 8.480±0.3730 pg/mL, P<0.001) and CCL2 (CCL2, CON vs. BME =313.7±1.480 vs. 308.8±2.326 pg/mL, P<0.01) in BALF (Figure 3B).

BME induced an imbalance in pro-inflammatory/anti-inflammatory pathways

To investigate BME related alterations in COPD inflammatory pathways, we examined classical inflammatory signalling pathways associated with COPD. We observed increased expression of mitogen-activated protein kinase 1 (MAPK1; CON vs. BME =0.9824±0.1314 vs. 1.679±0.2294, P<0.0001) and vascular endothelial growth factor (VEGF) in lung tissue from the BME group, alongside a significant reduction in matrix metalloproteinase-9 (MMP9; CON vs. BME =1.086±0.1528 vs. 0.5265±0.0539, P<0.0001). Additionally, expression levels of nuclear factor-kappaB (NF-κB), signal transducer and activator of transcription 3 (STAT3; CON vs. BME =1.209±0.2176 vs. 0.8128±0.1550, P<0.01), and signal transducer and activator of transcription 6 (STAT6) were diminished (Figure 4).

Figure 4 BME induces an imbalance in pro-inflammatory/anti-inflammatory pathways. qRT-PCR assessing relative MAPK1, VEGF, NF-κB, MMP9, STAT3 and STAT6 expression level in lung tissue. Each group n=6. Data were presented as mean ± standard deviation. All analyzed by Student’s t-test. ns, no significant difference; **, P<0.01; ****, P<0.0001. BME, biomass smoke exposure; CON, control; MAPK1, mitogen-activated protein kinase 1; MMP9, matrix metalloproteinase-9; NF-κB, nuclear factor-kappaB; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; STAT3, signal transducer and activator of transcription 3; STAT6, signal transducer and activator of transcription 6; VEGF, vascular endothelial growth factor.

BME mediated systemic effects

COPD is not only a lung disease, but also a systemic inflammatory process (19,20). Having established pulmonary pathology, we next assessed systemic inflammation. To verify the severity of bronchitis in mice, we used multiple liquid chip technology to detect multiple inflammatory factors in mice plasma. It was found that IL-2 [IL-2, CON vs. BME =0.7800 (0.7800, 0.8650) vs. 1.120 (1.120, 1.953) pg/mL, P<0.05] was significantly higher in BME group compared with CON group (Figure 5). Other indicators including TNF-α, IFN-γ, IL-5 although increased but no statistical difference.

Figure 5 BME mediated the increase of plasma IL-2 in mice. Levels of inflammatory factors IL-1β, IL-2, TNF-α, IFN-γ, IL-6, IL-5, IL-4, IL-10 (pg/mL) in plasma were measured with multiplexed liquid chip technology. IL-2 was higher in BME group than CON group significantly. Others were no significant statistical difference. Each group n=6. IL-2, IL-1β, IFN-γ, IL-6, IL-4, IL-10 were used Mann-Whitney U test to analysis, data were expressed as the median [25th percentile, 75th percentile]. TNF-α, IL-5 were analyzed by Student’s t-test, data were expressed as mean ± standard deviation. ns, no significant difference; *, P<0.05. BME, biomass smoke exposure; CON, control; IFN-γ, interferon-gamma; IL, interleukin; TNF-α, tumor necrosis factor-α.

Discussion

Key findings

This study firstly proposes a novel biomass-related COPD mouse model by BME combined with elastase nebulization for 16 weeks. The model successfully replicated key pathophysiological features of COPD, including emphysema, bronchitis, lung inflammation and systemic inflammation. Elastase nebulization shortened model induction and enhanced phenotypic stability, providing a robust platform for mechanistic and therapeutic studies of biomass-related COPD. Results of this research advance our current understanding of the pathophysiological change through which biomass smoke induced COPD and may inspire to the new modeling method.

Strengths and limitations

However, there are several limitations in this study. Firstly, this was a small sample experiment and there may be bias in the results. Future studies with larger cohorts are warranted to dissect the mechanisms underlying BME-induced COPD. Secondly, we selected female mice for this experiment. It has been shown that estrogen is associated with an increased risk of small airway diseases in female mice (21). This sex choice is justified by the higher prevalence of BME-induced COPD among women, who are traditionally exposed to cooking fumes. To clarify whether gender contributed to the differences in biomass smoke-related COPD animal model phenotype, we need further research. Third, the current work provides only a snapshot of BME-induced inflammation in COPD and signaling pathway alterations. Further investigation is required into other COPD-inducing mechanisms, such as oxidative stress and protease/antiprotease imbalance. Moving forward, we shall expand our sample size and integrate BME-induced human COPD cohorts to further investigate biomarkers and therapeutic targets for BME-induced COPD. Finally, if we detected the situation after several days or 1 months will be better to explore the changes in the early stage of BME. Meanwhile we will include additional groups exposed to (I) biomass smoke only; (II) elastase only; (III) vehicle only; and (IV) biomass smoke for 6 months, to further refine the experimental design.

Explanations of findings and comparison with similar research

Emphysema and small airway obstruction are the two major pathophysiological processes underlying COPD (22). Pulmonary function tests revealed increased static compliance and FRC, alongside reduced dynamic compliance and FEV100/FVC (Figure 1). It suggested the pulmonary function impairment, airway obstruction and emphysema meeting with pathophysiological characteristics of COPD. These findings align with previous reports of decreased dynamic compliance and FEV100/FVC and increased static compliance in COPD patients and animal models (11,23,24). To further assess lung damage, our pathological examination of lung tissue sections showed an apparent reduction in the number of alveoli, severe destruction of alveolar walls and airway epithelial cell injury with an increase of MLI and small airway wall thickness (Figure 2). Moreover, the level of inflammatory cytokines IL-10 and TNF-α mRNA were increased in lung tissue while BALF IL-10 concentrations was increased. Previous article showed that the level of TNF-α increased after 1 month but had no significant different after 6 months of biofuel smoke exposure (14). Our finding suggested that inflammatory cytokines remained elevated from the early phase through to 4 months of BME. These results reflected emphysema, bronchitis and airway remodeling, which is consistent with results of our previous studies. We previously showed that, compared with BME-exposed controls, patients with BME-induced COPD present more severe emphysema and small airway remodeling, display an early, slowly progressive decline in pulmonary function (10), and exhibit feature of small airway disease from an early stage (11).

Notably, when the entire forced expiratory curve was examined, FEV20 was already significantly lower in the BME group, and every ratio from FEV20/FVC to FEV400/FVC declined progressively (all P<0.05–0.0001). The results suggested that airflow limitation is detectable from the first 20 ms and persists throughout the entire forced expiratory curve. BME-induced COPD may be considered as another phenotype of COPD, a pattern seldom reported in existing COPD models. This coexistence of markedly enlarged lung volumes and severely impaired small-airway function mirrors the clinical signature we previously observed in biomass exposed COPD patients (11).

To further investigate the inflammatory mechanisms underlying BME-induced COPD, we propose IL-10 as a potential key mediator. Elevated IL-10 levels were observed in BALF, coinciding with reduced expression of CXCL2 and CCL2. Within lung tissue, expression of NF-κB mRNA decreased, while MAPK1 expression increased significantly. We found a significant drop in STAT3 mRNA and no change in STAT6. These findings indicate that the anti-inflammatory effect of IL-10 in biomass-related COPD do not relay on the activation of STAT3 and STAT6-related pathways, which is different in lung cancer (25). Rather, we hypothesize that IL-10 may appears to associate to the inhibition of NF-κB pathway activated by TNF-α and IL-1β (26), thereby further inhibiting the chemotaxis and activation of inflammatory cells such as CXCL2 and CCL2. This is consistent with previous research (27,28). Additionally, IL-10 did not inhibit MAPK1-related pathways, which usually we considered activated by TNF and IL-1β (29), to exert its anti-inflammatory effects in BME-induced COPD. This suggests that IL-10 exerts a selective anti-inflammatory program in BME-induced COPD.

MMP9 expression is markedly elevated in COPD patients, particularly during acute exacerbations (30), where it participates in airway remodeling by degrading the extracellular matrix. We found that IL-10 may exert anti-inflammatory effects by suppressing MMP9 expression, consisting with previous research (31). Furthermore, we observed a modest elevation in VEGF levels in biomass-related COPD, consistent with previous research that demonstrated a positive correlation between IL-10 and VEGF levels (32). Consequently, IL-10 may serve as a biomarker for biomass-related COPD, but its mechanistic role requires further elucidation.

In addition, we found an increasing of IL-2 in mice plasma presenting a systemic inflammatory response. Previous studies showed that systemic inflammation may also initiate or worsen comorbid diseases, such as ischaemic heart disease, heart failure, osteoporosis, normocytic anaemia, lung cancer, depression and diabetes (20,33). Comorbid diseases potentiate the morbidity of COPD, leading to increased hospitalizations, mortality and healthcare costs (19). Several previous clinical cohort studies have shown BME lead to systemic inflammatory responses and increased oxidative stress with increased serum levels of IL-6, IL-8, TNF-α, CRP, and ROS generation (34,35). IL-2 can activate T cells and macrophages, promote the proliferation of B cells. Biomass users showed an increase in the number of neutrophils, eosinophils, lymphocytes, and alveolar macrophages in the sputum (36-38). The elevation of plasma IL-2, a cytokine linked to T-cell activation and systemic inflammation, represents a novel finding in biomass-related COPD. This suggests a unique inflammatory profile distinct from tobacco-induced COPD, warranting further investigation. The effect of IL-2 in biomass smoke-related COPD remained to be investigated. Further studies on the inflammatory response in BME-induced COPD are needed, as inflammatory features may be distinct from this COPD phenotype.

Implications and actions needed

In summary, although there are still some limitations in our BME combined with elastase nebulization for 4-month COPD mouse model, its advantages over the existing biomass-related COPD modeling methods include shorter cycle time, inexpensive materials, simple manipulation, relevance to human biology and well-defined pathologic phenotypes. This has important implications for our study of the pathogenesis and treatment on BME-induced COPD.

In the future, we will enlarge the cohort and establish four sequential time-points (weeks 4, 8, 12 and months 6) to longitudinally track disease progression. Bronchoalveolar lavage fluid and serum will be collected at each interval to profile local and systemic inflammation, enabling direct comparison between sexes. On this basis, we will extend our investigation into the underlying mechanisms of biomass smoke-related COPD and test potential therapeutic strategies. We also encourage that researchers will develop animal models that are more consistent with the phenotype of BME-induced COPD and have a shorter modeling period.

Conclusions

The results of this study indicate that exposure to biomass smoke significantly induced pulmonary function impairment, leading to pathological changes such as emphysema and bronchiectasis, accompanied by both lung and systemic inflammation. IL-10 may play a significant role in the pro-inflammatory/anti-inflammatory mechanisms of COPD by regulating the expression of key signal pathway proteins. A novel COPD modeling method incorporating elastase was developed for the first time, which significantly reduced the modeling time and enabled the observation of diverse phenotypes.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1502/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1502/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1502/prf

Funding: This work was supported by National Natural Science Foundation of China (No. 8217010337).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1502/coif). All authors report funding support from National Natural Science Foundation of China (No. 8217010337). The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal experiments were performed under a project license (No. GY2023-474) granted by the Guangzhou Medical University Animal Research Ethics Board, in compliance with institutional guidelines for the care and use of animals. A protocol was prepared before the study without registration.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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