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Effects of particulate matter from straw burning on lung fibrosis in mice
Accepted Manuscript Title: Effects of particulate matter from straw burning on lung fibrosis in mice Authors: Yang Hu, Liu-Sheng Wang, Yan Li, Qiu-Hong Li, Chun-Lin Li, Jian-Min Chen, Dong Weng, Hui-Ping Li PII: DOI: Reference:
S1382-6689(17)30287-9 https://doi.org/10.1016/j.etap.2017.10.001 ENVTOX 2893
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
27-6-2017 4-10-2017 6-10-2017
Please cite this article as: Hu, Yang, Wang, Liu-Sheng, Li, Yan, Li, Qiu-Hong, Li, ChunLin, Chen, Jian-Min, Weng, Dong, Li, Hui-Ping, Effects of particulate matter from straw burning on lung fibrosis in mice.Environmental Toxicology and Pharmacology https://doi.org/10.1016/j.etap.2017.10.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effects of particulate matter from straw burning on lung fibrosis in mice Yang Hu1*, Liu-Sheng Wang1*, Yan Li1*, Qiu-Hong Li1*, Chun-Lin Li2, Jian-Min Chen2, Dong Weng1§, Hui-Ping Li1#
1
Department of Respiratory Medicine, Shanghai Pulmonary Hospital, Tongji
University, School of Medicine, Shanghai 200433, China 2
Shanghai Key Laboratory of Atmospheric Particle Pollution and Prevention (LAP3),
Department of Environmental Science and Engineering, Fudan University, Shanghai 200433, China
*Contributed equally to this work #
Corresponding author: Prof. Dr. Hui-Ping Li, PhD & MD, 507 Zheng Min Road,
Shanghai 200433, China. Email: [email protected]; Fax: 86-21-65111298; Phone: 86-21-65115006-2103 §
Co-corresponding author: Prof. Dong Weng, PhD, 507 Zheng Min Road, Shanghai,
200433, China, Email: [email protected]
Authors' information (optional) Yang Hu Department of Respiratory Medicine, Shanghai Pulmonary Hospital Tongji University, School of Medicine 507 Zheng Min Road, Shanghai 200433, China Email: [email protected]
Liu-Sheng Wang Department of Respiratory Medicine, Shanghai Pulmonary Hospital Tongji University, School of Medicine 507 Zheng Min Road, Shanghai 200433, China Email: [email protected]
Yan-Li Department of Respiratory Medicine, Shanghai Pulmonary Hospital Tongji University, School of Medicine 507 Zheng Min Road, Shanghai 200433, China Email: [email protected]
Qiu-Hong Li Department of Respiratory Medicine, Shanghai Pulmonary Hospital Tongji University, School of Medicine 507 Zheng Min Road, Shanghai 200433, China Email: [email protected]
Chun-Lin Li Shanghai Key Laboratory of Atmospheric Particle Pollution and Prevention (LAP3), Department of Environmental Science and Engineering, Fudan University, 220 Han Dan Road, Shanghai 200433, China Email: [email protected]
Jian-Min Chen Shanghai Key Laboratory of Atmospheric Particle Pollution and Prevention (LAP3), Department of Environmental Science and Engineering, Fudan University, 220 Han Dan Road, Shanghai 200433, China Email: [email protected]
#Corresponding author: Hui-Ping Li, PhD & MD
Department of Respiratory Medicine, Shanghai Pulmonary Hospital Tongji University, School of Medicine 507 Zheng Min Road, Shanghai 200433, China Email:[email protected] Fax: 86-21-65111298 Phone: 86-21-65115006-2103
§
Co-corresponding author:
Dong Weng, PhD, 507 Zheng Min Road, Shanghai, 200433, China, Email: [email protected]
Highlights
Exposure of Particulate matter 2.5(PM2.5) can aggravate lung injury based on bleomycin-induced lung fibrosis of mice;
The mechanism related to inflammatory factors induced by PM2.5 exposure;
N-acetylcysteine can exert protective role for lung fibrosis mice exposed to PM2.5
Abstract Objective: To investigate the impacts of particulate matter 2.5 (PM2.5) from straw burning on the acute exacerbation of lung fibrosis in mice and the preventive effects of N-acetylcysteine (NAC). Methods: The composition, particle size, and 30-minute concentration change in an exposure system of the PM2.5 from straw-burning were determined. Forty C57BL male mice were equally randomized to two groups: bleomycin (BLM)-induced lung fibrosis with an exposure to air (BLM+air) and BLM+PM2.5 groups. On day 7 after
receiving intratracheal injection of BLM, mice were exposed to air or PM2.5 in an exposure system for 30 minutes twice daily and then sacrificed after one-week or four-week exposure (10 mice/group). Mouse survival, lung histopathology, macrophage accumulation in the lung, and pro-inflammatory cytokine levels in alveolar lavage fluid (ALF) were determined. Results: PM2.5 from straw burning were mainly composed of organic matter (74.1%); 10.92% of the inorganic matter of the PM2.5 were chloride ion; 4.64% were potassium ion; other components were sulfate, nitrate, and nitrite. Particle size was 10nm-2μm. Histopathology revealed a greater extent of inflammatory cell infiltration in the lung, widened alveolar septum, and lung fibrosis in the BLM+PM2.5 group than in the BLM+air group and a greater extent of those adverse effects after fourweek than after one-week exposure to PM2.5. The BLM+PM2.5 group also showed macrophages containing particular matter and increased pulmonary collagen deposition as the exposure to PM2.5 increased. Interleukin (IL)-6 and TNF-α levels in ALF were significantly higher in the BLM+PM2.5 group than in the BLM+air group (P<0.05) and significantly higher after four-week exposure than after one-week exposure to PM2.5 (P<0.05). TGF-β levels in ALF after four-week exposure were significantly higher in the BLM+PM2.5 group than in the BLM+air group (P<0.05). The levels of IL-6, TNF-α, and TGF-β in peripheral serum were not significantly different in the BLM+PM2.5 and BLM+air groups. Lung hydroxyproline contents increased as the exposure to PM2.5 increased and were significantly higher after fourweek than after one-week exposure (P=0.019). Exposure to PM2.5 did not affect the survival of normal mice (100%) but reduced the survival of mice with BLM-induced IPF (30%), whereas NAC extended the survival (70%, vs. BLM+PM2.5, P=0.032). Conclusion: Exposure of mice with BLM-induced IPF to PM2.5 from straw burning exacerbated lung inflammation and fibrosis and increased mortality; NAC increased the mouse survival, indicating protective effects.
Abbreviations PM2.5: Particulate matter less than 2.5 micrometers in diameter BLM: Bleomycin NAC: Acetylcysteine AM: Alveolar macrophages
ALF: Alveolar lavage fluid IL-6: Interleukin-6 TNF-α: Tumor necrosis factor-α TGF-β: Transforming growth factor-β IPF: Interstitial pulmonary fibrosis COPD: Chronic obstructive pulmonary disease
Key words: particulate matter 2.5; lung fibrosis; straw burning
Background Particulate matter 2.5 (PM2.5), which are fine particles with an aerodynamic diameter smaller than 2.5 µm, are the main component of atmospheric pollutants (smog). Because of their small diameter, large specific surface area, complex chemical composition, and their capability to easily enrich toxic and harmful substances, PM2.5 can reach the respiratory fine bronchi and alveolar cavity after entering the respiratory track, harming human healthy at substantially greater extent than particulate matter with a diameter bigger than 2.5 µm.
One of the key sources of air pollutants in China is the smoke (containing large amounts of PM2.5 (Li et al., 2016, Shi et al., 2016, Chen et al., 2015)) from straw burning in rural area. A previous study has demonstrated that long-term inhalation of biofuel smoke contributes predominantly to the high prevalence of chronic obstructive pulmonary disease (COPD) in rural area (Guan et al., 2016). PM2.5 are also found to correlate closely with the increased incidences of common chronic airway diseases, such as COPD and bronchial asthma (Kelly and Fussell, 2011). Our previous study has shown that exposure to PM2.5 exacerbates the pulmonary injury and increases the mortality of mice with emphysema (Wang et al., 2015). However, the association between PM2.5 and the development and progression of idiopathic pulmonary fibrosis (IPF) has not been studied. Whether PM2.5 can exacerbate IPF remains unclear. In the current study, to simulate the exposure of patients with IPF to severe air pollution, we exposed mice with bleomycin (BLM)-induced IPF to PM2.5 generated from straw burning and investigated the effects of air pollution on IPF deterioration and the underlying mechanisms. Our study also explored potential preventive approaches.
Materials and methods Preparation of PM2.5 pollutants and the exposure system Smoke generated from rice straw burning was used as the source of PM2.5 pollutants. Rice straw was cut into 5 cm-length small pieces, washed, and dried at 80°C in an oven for 4 hours. The dried rice straw was aliquoted into 5g per sealed bag. The whole body inhalation exposure system (Tianjin Hope Industry & Trade Co., Ltd, HOPE-MED8050 exposure controlling system) has a volume of 0.3m3 and allows to
expose a maximum of 20 mice simultaneously. The exposure temperature was 25°C; the relative humidity of exposure was 40%. After exposure, the pollutants in the exposure system were replaced with air (≥ 30 minutes). The exposure system was cleaned daily.
Monitoring PM2.5 in the exposure system Rice straw (5g) was burned inside the exposure system; the mini fan inside the system was switched on to allow the PM2.5 distribute evenly. The sampling port was connected to two aerosol monitors (Model: AM510, Product No.: #32 and #27). The mass concentrations of PM2.5 and PM 1.0 inside the exposure system were monitored for a 30-minute period and the monitoring was repeated 4 times. The time course of the mass concentration of PM2.5 and PM 1.0 of the aerosol in the exposure system was then determined. The particle size of particulate matter was analyzed by widerange particle (WPS) Sizer (Model: 1000XP, TSI Inc, USA). The distributions of concentration, surface area, and volume of particles with a size between 10 nm and 10 μm were also determined. Particles were collected by a particulate matter sampler, and then the size and composition of the particles were analyzed by a transmission electron microscope (TEM, Model: JEOL-2100F).
Animals The protocol for handling mice has been approved by the Institutional Animal Care and Use Committee of Shanghai Pulmonary Hospital of Tongji University School of Medicine (Approval No. SYXK [SH] 2012-0031). A total of C57BL male 40 mice aged 8-week and weighted 16g-18g were randomized into two groups (20 mice/group): bleomycin-induced IPF + exposure to air (BLM + air) group and bleomycin-induced IPF + exposure to PM2.5 (BLM + PM2.5) group. Mice in both groups received intratracheal injection of bleomycin (4mg/mL, dissolved in 50 µL saline) at experiment day 0 according to our previous description (Wei et al., 2016). Mice in the control group were injected with 50 µL saline intratracheally. On day 7 after bleomycin injection, the mice were exposed to air or PM2.5. Each group was further divided into one-week and four-week groups, which were sacrificed after oneweek and four-week exposure (10 mice/group), respectively, and mouse specimens were collected (Figure 1).
To determine mouse survival, 1) mice in the control groups (Control + PM2.5, 15 mice) were exposed to PM2.5 twice daily (30 minutes every exposure) for 28 days; 2) mice in the BLM + PM2.5 group (20 mice) were exposed to PM2.5 after BLMinduced IPF was established, and the exposure was twice daily (30 minutes every exposure) for 28 days; 3) mice in the BLM + air group (12 mice) were observed for survival for 28 days; 4) mice in the N- Acetylcysteine (NAC) intervention group (BLM+PM2.5+NAC, 20 mice) were exposed to PM2.5 twice daily (30 minutes every exposure) for 28 days after BLM-induced IPF was established, and NAC (150 mg/kg, dissolved in 2mL saline) was injected intraperitoneally daily before exposure to PM2.5 (Demiralay et al., 2013).
Exposure to PM2.5 Mice were housed in standard cages before being exposed to PM2.5. Rice straw (5g) was burned completely in the exposure system, and then the cages containing mice were placed inside the exposure system. The temperature and humidity of the exposure system was 25°C and 40%, respectively. Each exposure lasted 30 minutes, and 1L air was supplied to the exposure system every 15 minutes. The exposure was twice daily and the time interval between daily exposures was less than 4 hours. Mice in the BLM + air group were exposed to air in the similar manner. Mice not being exposed to PM2.5 or air were housed and fed normally.
Collecting and testing mouse specimens Mouse blood samples were collected from the eye socket after mice were sacrificed and the eye balls were removed. The blood samples were centrifuged at 3000 rpm for 15 minutes, and the serum was collected. Serum levels of IL-6, TNF-α, and TGF-β were measured by ELISA.
To collect alveolar lavage fluid (ALF), the lung and trachea were dissected. A perfusion needle was placed inside the trachea, and a suture was used to ligate the trachea to prevent leakage. The perfusion needle was secured inside the trachea. A total of 1 mL saline was injected with a 1 mL syringe to expand the lung, and the saline was collected 15 seconds after the injection. A minimum of 0.75 mL saline was
collected. Perfusion was repeated twice for each mouse. IL-6, TNF-α, and TGF-β levels in the collected ALF were determined by ELISA.
To collect lung tissue specimens, a perfusion needle was placed at the left trachea, which was ligated by suture. The perfusion needle was secured inside the left trachea. A total of 0.75 mL formalin was injected into the left lung. The perfusion needle was then removed. The left lung was isolated and fixed in 10% formalin. Paraffin tissue sections were prepared. H&E staining and Masson staining were performed. Hydroxyproline contents were determined. Histopathological characteristics and lung tissue collagen contents were observed.
Results Components of the smoke from straw burning and particle size determination PM2.5 from straw burning were mainly composed of organic matter (74.1%); 30% of the organic matter were polycyclic aromatic hydrocarbons; 10% were L-glucose and mannose-oligosaccharides. Figure 2A shows that the orange area (1.59%) represents carbon (Inorganic particles contained small amounts of hydrogen and sulfur) and the green area represents inorganic matter. Inorganic matter were mainly chloride ion. Other components are displayed in Figure 2B. TEM revealed that the particle size was smaller than 2.5 µm and the particles contained potassium, calcium, magnesium, and organic matter (Figure 3). The distributions of particle concentration, surface area, and mass concentration also showed that the majority of particles in the smoke had a size smaller than 2.5 µm (Figure 4). The concentrations of PM2.5 and PM1.0 in the smoke were at effective levels for 30 minutes inside the exposure system (PM2.5 > 75mg/m3 according to the WHO 2005 Air Quality Guidelines, http://www.euro.who.int/data/assets/pdf_file/0005/78638/E90038.pdf?ua=1, Figure 5).
Mouse survival Inhalation of PM2.5 for 28 days did not adversely affect normal mouse survival (survival rate: 100%, Figure 6). However, compared to exposure to air, inhalation of PM2.5 for 28 days increased the mortality of mice with BLM-induced IPF although the difference was not statistically significant (Survival rate of the BLM + air group:
58.3% versus survival rate of the BLM + PM2.5 group: 30%, P = 0.214, Figure 6). NAC intervention extended the survival of mice with BLM + PM2.5 (Survival rate of the BLM + PM2.5 group: 30% versus survival rate of BLM + PM2.5 + NAC group: 70%, P = 0.032, Figure 6).
Histopathological characteristics After BLM-induced IPF was established, the lung showed widened alveolar septum, damaged alveolar wall, obvious damaged alveolar fusion structure, and locally formed lesion-like nodules (Figure 7). These histopathological damages deteriorated as the exposure to PM2.5 increased (Figure 7). In addition, macrophage accumulation increased in the BLM + PM2.5 group and black particles were found inside the macrophages, whereas the BLM + air group did not showed such types of macrophages (Figure 8).
Compared with the BLM + air group, lung collagen contents in the BLM + PM2.5 group increased substantially after one-week exposure to PM2.5, and the difference was further enhanced after four-week exposure (Figure 9). These results suggest that exposure to PM2.5 may increase collagen deposition in the lung and thus exacerbate lung fibrosis. Compared with one-week exposure to PM2.5, four-week exposure to PM2.5 further increased collagen deposition in the lung, indicating that extended exposure to PM2.5 may aggravate lung fibrosis.
Cytokine levels in serum and alveolar lavage fluid ELISA revealed that serum levels of IL-6, TNF-α, and TGF-β were low and similar in different groups, suggesting that exposure to PM2.5 may not cause obvious systemic inflammation. IL-6 levels in ALF were significantly higher in the BLM+ PM2.5 group than in the BLM + air group after one-week (P = 0.027) and four-week exposure to PM2.5 (P = 0.010) (Figure 10). Additionally, IL-6 levels in ALF were significantly higher after four-week exposure than after one-week exposure to PM2.5 (P =0.036, Figure 10). Similar to IL-6 levels, TNF-α levels in ALF were significantly higher in the BLM+ PM2.5 group than in the BLM + air group after one-week (P = 0.035) and four-week exposure to PM2.5 (P = 0.001), and the TNF-α levels after fourweek exposure were significantly higher than those after one-week exposure (P =
0.048) (Figure 11). These data indicate that exposure to PM2.5 may induce lung inflammation. Figure 12 shows that TGF-β levels in ALF were significantly higher in the BLM+ PM2.5 group than in the BLM + air group after four-week exposure to PM2.5 (P =0.044), whereas were not statistically significantly different in the two groups after one-week exposure. These findings suggest that extended exposure to PM2.5 may exacerbate pulmonary fibrosis.
Lung hydroxyproline contents Lung hydroxyproline contents in the BLM+PM2.5 group were significantly higher than those in the BLM + air group after 4-week exposure (P = 0.032), and lung hydroxyproline contents after four-week exposure were significantly higher than those after one-week exposure to PM2.5 (P = 0.019, Figure 13), indicating that exposure to PM2.5 may exacerbate BLM-induced lung fibrosis.
Discussion A previous epidemiological study has demonstrated that environmental pollutants, in particular particulate matter (PM) in the air, can lead to acute and chronic lung diseases in China (Qiu et al., 2012). The current study exposed mice with BLMinduced IPF (Wei et al., 2016) to PM2.5 generated from the smoke of straw burning to investigate the effects of exposure to air PM2.5 on IPF progression in patients.
PM generated from straw burning are the key source of atmosphere PM. The PM are mainly composed of organic matter and some inorganic ions, including sodium, potassium, calcium, and sulfate ion, and nitrate ion. The particle size of most PM is smaller than 2.5 µm. Thus, the PM can enter alveoli so to seriously and adversely affect human respiratory system (Nie et al., 2016). The current study used smoke from straw burning to generate air pollutants PM2.5 and simulate the air pollutants in China.
Alveolar macrophages (AM) are the effector cells to phagocytose foreign bodies inside the alveoli. AM can rapidly phagocytose PM when they are exposed to air PM in vitro or in vivo (van Eeden et al., 2001, Mukae et al., 2000). Consistently, the current study also found that the accumulation of PM-containing macrophages in
mouse lung tissue increased considerably after the mice were exposure to PM2.5 for one to four weeks. AM can secret pro-inflammatory factors, such as IL-6 and TNF-α (Sijan et al., 2015). Injection of the conditional culture media from PM-stimulated AM into rabbit trachea causes substantial inflammatory reaction, which is similar to the inflammation caused by a direct injection of PM into rabbit trachea (Mukae et al., 2001). Xu et al (Xu et al., 2013) have found that on day 5 after exposure to PM2.5, inflammatory cells, mainly including macrophages and neutrophils, started to accumulate in mouse lung tissue and ALF. Pirela et al (Pirela et al., 2013) have also shown that an injection of PM into mouse trachea induces inflammatory reactions in mouse lung tissue and increases pro-inflammatory cytokine levels in ALF. These studies suggest that AM and the pro-inflammatory cytokines secreted by AM may play an important pro-inflammatory role in PM-induced lung inflammation. However, the effects of PM2.5 on lung fibrosis disease have been reported rarely.
The current study investigated the effects of PM2.5 on lung fibrosis progression by exposing mice with BLM-induced IPF to PM2.5 from straw burning. We found that exposure of mice with IPF to PM2.5 elevated the levels of the acute pro-inflammatory factors, IL-6 and TNF-α, in the ALF, and the pro-inflammatory factor levels were further increased as the exposure duration was extended. These data indicate that PM2.5 may promote local inflammatory reaction in mice with IPF. Anti-inflammatory factors, such as IL-10, were not detected in ALF. Thus, phagocytosis of PM2.5 by AM may promote inflammation, which is consistent with a previous study (Yin et al., 2004). H & E staining of lung tissue also showed that exposure to PM2.5 exacerbated lung inflammatory reaction in mice with IPF. The levels of pro-fibrosis cytokine, TGF-β, were also increased by extended exposure to PM2.5. Masson staining of lung tissue and hydroxyproline content measurement revealed that four-week exposure to PM2.5 increased collagen deposition in lung tissue greatly, suggesting that exposure to PM2.5 may not only exacerbate lung fibrosis but also induce acute inflammatory reaction. These observations resemble the disease course of acute exacerbation of IPF in patients.
Recent studies have found that PM2.5 are associated with multiple organ fibrosis and thus affect organ function. For example, Tan et al (Cucoranu et al., 2005) have shown
that PM2.5 in blood circulation were accumulated in the liver and were phagocytosed by Kupffer cells; they have also found that PM2.5 stimulated Kupffer cells to secret cytokines and induced hepatic stellate cells to secret collagen so to exacerbate nonalcoholic liver disease-mediated fibrosis. Goss et al (Goss et al., 2004) have demonstrated that increased air PM2.5 was associated with acute exacerbation of cystic lung fibrosis and lung dysfunction. Gorr et al (Gorr et al., 2014) have found that long-term exposure to PM2.5 led to cardiovascular tissue remodeling, collagen deposition in myocardial tissue, and cardiac fibrosis, which ultimately exacerbated primary disease and caused heart failure. These studies consistently indicate that longterm exposure to PM2.5 may correlate with the deterioration of multiple organ fibrosis. The current study first showed that exposure of mice with BLM-induced IPF to PM2.5 exacerbated IPF, induced additional inflammatory reaction, and thus increased mouse mortality. The intervention with NAC reduced the mortality of IPF mice exposed to PM2.5, suggesting the preventive effects of NAC. A previous study has shown that air PM2.5 induce strong oxidative stress of mouse macrophages, which can be inhibited by NAC (Bekki et al., 2016). The NAC-mediated therapeutic benefits in IPF mice exposed to PM2.5 may be associated with the antioxidant effects of NAC. However, the underlying mechanism remains to be further investigated.
Conclusion Exposure of mice with BLM-induced IPF to PM2.5 from straw burning exacerbated lung inflammation and fibrosis and increased mouse mortality. NAC extended the survival of IPF mice exposed to PM2.5 and showed some protective effects.
Ethics approval and consent to participate The protocol for handling mice has been approved by the Institutional Animal Care and Use Committee of Shanghai Pulmonary Hospital of Tongji University School of Medicine (Approval No. SYXK [SH] 2012-0031). Consent for publication Not applicable Availability of data and material Please contact author for data requests. Competing interests
The authors declared that they have no competing interests. Authors' contributions Experimental design: H.P.L., Y.H., L.S.W., Y.L. Q.H.L and D.W.; Data acquisition and analysis: Y.H., L.S.W., Y.L., Q.H.L. C.L.L., J.M.C., D.W., and H.P.L.; Writing the manuscript: Y.H., L.S.W., Y.L., Q.H.L., D.W., and H.P.L. All authors read and approved the final manuscript.
Funding Supported by grants from the National Science Foundation of China (No: 81730002，81670055, 81670056, 91442103, 81500052 and 81570057), Ministry of Science and Technology of the People’s Republic of China (2016YFC1100200, 2016YFC1100204), the Health Bureau Program of Shanghai Municipality (SHDC12014120），Grant from Shanghai Hospital Development Center （16CR3054A), Shanghai Municipal Commission of Health and Familiy Planning (201640157) and Tongji University (1511219020).
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Figure 1 Animal experiment protocol
Mice were randomized into bleomycin-induced IPF + exposure to air (BLM + air) group and bleomycin-induced IPF + exposure to PM2.5 (BLM + PM2.5) group (20 mice per group). Mice in both groups received intratracheal injection of bleomycin (4mg/mL, dissolved in 50 µL saline) at experiment day 0 to establish lung fibrosis. On day 7 after the bleomycin injection, the mice were exposed to air or PM2.5. Each group was further divided into oneweek and four-week groups, which were sacrificed after one-week and four-week exposure, respectively, and mouse specimens were collected.
Figure 2 Composition of PM emission from straw burning
The main component of PM2.5 from straw burning was organic matter (74.1%); 30% of the organic matter was polycyclic aromatic hydrocarbons and 10% was L-glucose. A: The orange area (1.59%) represents carbon (Inorganic carbon particles contain small amounts of hydrogen and sulfur). The green area represents inorganic matter. The main component of the inorganic matter is chloride ion (the grey area in the right panel).
Figure 3 Examination of the particle size distribution of PM from straw burning by scanning electron microscope
Single particle scanning electron microscopy revealed that the particle size was smaller than 2.5 µm. Electron microscopic ion chromatography found that PM contained potassium, calcium, magnesium, and organic matter.
Figure 4 Particle size distribution of PM2.5 from straw burning in the exposure system
Particle size of most PM in the exposure system was smaller than 2.5μm. (a) Particle concentration distribution shows the distribution of the number of particles with difference size per cm3. The number of particles with a size of 100 nm per m3 is the highest. (b) Surface area distribution shows the distribution of the surface area of particles with difference size per cm3. The surface area of particles with a size of 500 nm per m3 is the largest. (c) Mass concentration distribution shows the distribution of total mass of particles with different size per m3. The total mass of particles with a size of 400 nm per m3 is the highest. The majority of particles in the exposure system has a size smaller than 2.5 μm.
Figure 5 Time course of the concentrations of PM2.5 and PM 1.0 from straw burning in the exposure system
In the exposure system, PM2.5 concentration reduced gradually within 30 minutes. The average concentration was maintained at > 100mg/m3, which meets the concentration criterion for animal experiments.
Figure 6 Effects of exposure to PM2.5 on the survival of mice with BLM-induced IPF
Inhalation of PM2.5 did not adversely affect the survival of normal mice (survival: 100%). However, compared to exposure to air (BLM+ air group: 58.3%), inhalation of PM2.5 reduced the survival of mice with BLM-induced IPF (BLM+PM2.5 group: 30%, P = 0.214). NAC extended the survival of mice with BLM+PM2.5 (BLM+PM2.5+NAC group: 70% vs. BLM+PM2.5 group: 30%, P = 0.032).
Figure 7 H&E staining images of lung tissue-histopathology
A & a: H&E staining images of the BLM+ air group (one week). B & b: H&E staining images of the BLM+ air group (four weeks). C & c: H&E staining images of the BLM+ PM2.5 group (one week). D & d: H&E staining images of the BLM+ PM2.5 group (four weeks). After BLM-induced IPF was established, the lung showed widened alveolar septum, damaged alveolar wall, obvious damaged alveolar fusion structure, and inflammatory cell infiltration. Lung tissue damage in B was greater than that in A, showing worse alveolar fusion and bronchiectasis caused by local lesion contraction. Inflammatory cell infiltration in lung tissue increased and alveolar septum is wider in C compared with A. Inflammatory cell infiltration in lung tissue increased; lung tissue damage is worse; fibrosis lesions increased in D compared with C. A, B, C, and D represent 100 x magnification. a, b, c, and d represent 400 x magnification.
Figure 8 lung tissue histopathology- macrophages containing PM
A. Lung tissue in the BLM + air group (one week). B. Lung tissue in the BLM + air group (four weeks). C. Lung tissue in the BLM + PM2.5 group (one week). D. Lung tissue in the BLM + PM2.5 group (four weeks). Macrophages in the lung tissue contain black particles (pointing by the black arrows). Such macrophages are the most in the BLM + PM2.5 group exposed for four weeks, but are absent in the BLM + air group (H & E, 400× magnification).
Figure 9 Lung tissue histopathology-Masson staining
A & a: H&E staining images of the BLM+ air group (one week). B & b: H&E staining images of the BLM+ air group (four weeks). C & c: H&E staining images of the BLM+ PM2.5 group (one week). D & d: H&E staining images of the BLM+ PM2.5 group (four weeks). Blue areas represent collagen fibers. Collage deposition increased in C than in A and increased in D than in B, indicating exposure to PM2.5 increased collagen deposition. Collage deposition increased in D than in C, suggesting that extended exposure to PM2.5 increased collage deposition in lung tissue. A, B, C, and D represent 100 x magnification. a, b, c, and d represent 400 x magnification.
Figure 10 IL-6 levels in mouse alveolar lavage fluid
IL-6 levels in mouse ALF after exposure for one week (A) and four weeks (B) increased in the BLM + PM2.5 group than in the BLM + air group (P = 0.027 and P = 0.001, respectively). IL-6 levels increased in the BLM + PM2.5 four-week group compared with one-week group (C, P = 0.036). IL-6 levels reduced in the BLM + air four-week group compared with one-week group (D, P = 0.001). These data suggest that exposure to PM2.5 may increase IL-6 levels in mouse ALF.
Figure 11 TNF-α levels in mouse alveolar lavage fluid
TNF-α levels in mouse ALF after exposure for one week (A) and four weeks (B) increased in the BLM + PM2.5 group than in the BLM + air group (P = 0.035 and P = 0.001, respectively). TNF-α levels increased in the BLM + PM2.5 four-week group compared with one-week group (C, P = 0.048). TNF-α levels reduced in the BLM + air four-week group compared with one-week group (D, P = 0.543). These data suggest that exposure to PM2.5 may increase TNF-α levels in mouse ALF.
Figure 12 TGF-β levels in mouse alveolar lavage fluid
TGF-β levels in mouse ALF after exposure for one week (A) and four weeks (B) increased in the BLM + PM2.5 group than in the BLM + air group (P = 0.724 and P = 0.044, respectively). TGF-β levels increased in the BLM + PM2.5 four-week group compared with one-week group (C, P = 0.103). TGF-β levels reduced in the BLM + air four-week group compared with one-week group (D, P = 0.054).
Figure 13 Hydroxyproline contents in lung tissue
Lung hydroxyproline contents in the BLM + PM2.5 group were significantly higher than those in the BLM + air group, indicating collagen contents increased. The P values of the comparison are: BLM + air one-week vs. BLM + air four-week, P = 0.537; BLM + air one-week vs. BLM + PM2.5 one-week, P = 0.162; BLM + air oneweek vs. BLM + PM2.5 four-week, P < 0.0001; BLM + air four-week vs. BLM + PM2.5 one-week, P = 0.694; BLM + air four-week vs. BLM + PM2.5 four-week, P = 0.032; BLM + PM2.5 one-week vs. BLM + PM2.5 four-week, P = 0.019. These data suggest that extended exposure to PM2.5 may exacerbate fibrosis.
S1382-6689(17)30287-9 https://doi.org/10.1016/j.etap.2017.10.001 ENVTOX 2893
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
27-6-2017 4-10-2017 6-10-2017
Please cite this article as: Hu, Yang, Wang, Liu-Sheng, Li, Yan, Li, Qiu-Hong, Li, ChunLin, Chen, Jian-Min, Weng, Dong, Li, Hui-Ping, Effects of particulate matter from straw burning on lung fibrosis in mice.Environmental Toxicology and Pharmacology https://doi.org/10.1016/j.etap.2017.10.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effects of particulate matter from straw burning on lung fibrosis in mice Yang Hu1*, Liu-Sheng Wang1*, Yan Li1*, Qiu-Hong Li1*, Chun-Lin Li2, Jian-Min Chen2, Dong Weng1§, Hui-Ping Li1#
1
Department of Respiratory Medicine, Shanghai Pulmonary Hospital, Tongji
University, School of Medicine, Shanghai 200433, China 2
Shanghai Key Laboratory of Atmospheric Particle Pollution and Prevention (LAP3),
Department of Environmental Science and Engineering, Fudan University, Shanghai 200433, China
*Contributed equally to this work #
Corresponding author: Prof. Dr. Hui-Ping Li, PhD & MD, 507 Zheng Min Road,
Shanghai 200433, China. Email: [email protected]; Fax: 86-21-65111298; Phone: 86-21-65115006-2103 §
Co-corresponding author: Prof. Dong Weng, PhD, 507 Zheng Min Road, Shanghai,
200433, China, Email: [email protected]
Authors' information (optional) Yang Hu Department of Respiratory Medicine, Shanghai Pulmonary Hospital Tongji University, School of Medicine 507 Zheng Min Road, Shanghai 200433, China Email: [email protected]
Liu-Sheng Wang Department of Respiratory Medicine, Shanghai Pulmonary Hospital Tongji University, School of Medicine 507 Zheng Min Road, Shanghai 200433, China Email: [email protected]
Yan-Li Department of Respiratory Medicine, Shanghai Pulmonary Hospital Tongji University, School of Medicine 507 Zheng Min Road, Shanghai 200433, China Email: [email protected]
Qiu-Hong Li Department of Respiratory Medicine, Shanghai Pulmonary Hospital Tongji University, School of Medicine 507 Zheng Min Road, Shanghai 200433, China Email: [email protected]
Chun-Lin Li Shanghai Key Laboratory of Atmospheric Particle Pollution and Prevention (LAP3), Department of Environmental Science and Engineering, Fudan University, 220 Han Dan Road, Shanghai 200433, China Email: [email protected]
Jian-Min Chen Shanghai Key Laboratory of Atmospheric Particle Pollution and Prevention (LAP3), Department of Environmental Science and Engineering, Fudan University, 220 Han Dan Road, Shanghai 200433, China Email: [email protected]
#Corresponding author: Hui-Ping Li, PhD & MD
Department of Respiratory Medicine, Shanghai Pulmonary Hospital Tongji University, School of Medicine 507 Zheng Min Road, Shanghai 200433, China Email:[email protected] Fax: 86-21-65111298 Phone: 86-21-65115006-2103
§
Co-corresponding author:
Dong Weng, PhD, 507 Zheng Min Road, Shanghai, 200433, China, Email: [email protected]
Highlights
Exposure of Particulate matter 2.5(PM2.5) can aggravate lung injury based on bleomycin-induced lung fibrosis of mice;
The mechanism related to inflammatory factors induced by PM2.5 exposure;
N-acetylcysteine can exert protective role for lung fibrosis mice exposed to PM2.5
Abstract Objective: To investigate the impacts of particulate matter 2.5 (PM2.5) from straw burning on the acute exacerbation of lung fibrosis in mice and the preventive effects of N-acetylcysteine (NAC). Methods: The composition, particle size, and 30-minute concentration change in an exposure system of the PM2.5 from straw-burning were determined. Forty C57BL male mice were equally randomized to two groups: bleomycin (BLM)-induced lung fibrosis with an exposure to air (BLM+air) and BLM+PM2.5 groups. On day 7 after
receiving intratracheal injection of BLM, mice were exposed to air or PM2.5 in an exposure system for 30 minutes twice daily and then sacrificed after one-week or four-week exposure (10 mice/group). Mouse survival, lung histopathology, macrophage accumulation in the lung, and pro-inflammatory cytokine levels in alveolar lavage fluid (ALF) were determined. Results: PM2.5 from straw burning were mainly composed of organic matter (74.1%); 10.92% of the inorganic matter of the PM2.5 were chloride ion; 4.64% were potassium ion; other components were sulfate, nitrate, and nitrite. Particle size was 10nm-2μm. Histopathology revealed a greater extent of inflammatory cell infiltration in the lung, widened alveolar septum, and lung fibrosis in the BLM+PM2.5 group than in the BLM+air group and a greater extent of those adverse effects after fourweek than after one-week exposure to PM2.5. The BLM+PM2.5 group also showed macrophages containing particular matter and increased pulmonary collagen deposition as the exposure to PM2.5 increased. Interleukin (IL)-6 and TNF-α levels in ALF were significantly higher in the BLM+PM2.5 group than in the BLM+air group (P<0.05) and significantly higher after four-week exposure than after one-week exposure to PM2.5 (P<0.05). TGF-β levels in ALF after four-week exposure were significantly higher in the BLM+PM2.5 group than in the BLM+air group (P<0.05). The levels of IL-6, TNF-α, and TGF-β in peripheral serum were not significantly different in the BLM+PM2.5 and BLM+air groups. Lung hydroxyproline contents increased as the exposure to PM2.5 increased and were significantly higher after fourweek than after one-week exposure (P=0.019). Exposure to PM2.5 did not affect the survival of normal mice (100%) but reduced the survival of mice with BLM-induced IPF (30%), whereas NAC extended the survival (70%, vs. BLM+PM2.5, P=0.032). Conclusion: Exposure of mice with BLM-induced IPF to PM2.5 from straw burning exacerbated lung inflammation and fibrosis and increased mortality; NAC increased the mouse survival, indicating protective effects.
Abbreviations PM2.5: Particulate matter less than 2.5 micrometers in diameter BLM: Bleomycin NAC: Acetylcysteine AM: Alveolar macrophages
ALF: Alveolar lavage fluid IL-6: Interleukin-6 TNF-α: Tumor necrosis factor-α TGF-β: Transforming growth factor-β IPF: Interstitial pulmonary fibrosis COPD: Chronic obstructive pulmonary disease
Key words: particulate matter 2.5; lung fibrosis; straw burning
Background Particulate matter 2.5 (PM2.5), which are fine particles with an aerodynamic diameter smaller than 2.5 µm, are the main component of atmospheric pollutants (smog). Because of their small diameter, large specific surface area, complex chemical composition, and their capability to easily enrich toxic and harmful substances, PM2.5 can reach the respiratory fine bronchi and alveolar cavity after entering the respiratory track, harming human healthy at substantially greater extent than particulate matter with a diameter bigger than 2.5 µm.
One of the key sources of air pollutants in China is the smoke (containing large amounts of PM2.5 (Li et al., 2016, Shi et al., 2016, Chen et al., 2015)) from straw burning in rural area. A previous study has demonstrated that long-term inhalation of biofuel smoke contributes predominantly to the high prevalence of chronic obstructive pulmonary disease (COPD) in rural area (Guan et al., 2016). PM2.5 are also found to correlate closely with the increased incidences of common chronic airway diseases, such as COPD and bronchial asthma (Kelly and Fussell, 2011). Our previous study has shown that exposure to PM2.5 exacerbates the pulmonary injury and increases the mortality of mice with emphysema (Wang et al., 2015). However, the association between PM2.5 and the development and progression of idiopathic pulmonary fibrosis (IPF) has not been studied. Whether PM2.5 can exacerbate IPF remains unclear. In the current study, to simulate the exposure of patients with IPF to severe air pollution, we exposed mice with bleomycin (BLM)-induced IPF to PM2.5 generated from straw burning and investigated the effects of air pollution on IPF deterioration and the underlying mechanisms. Our study also explored potential preventive approaches.
Materials and methods Preparation of PM2.5 pollutants and the exposure system Smoke generated from rice straw burning was used as the source of PM2.5 pollutants. Rice straw was cut into 5 cm-length small pieces, washed, and dried at 80°C in an oven for 4 hours. The dried rice straw was aliquoted into 5g per sealed bag. The whole body inhalation exposure system (Tianjin Hope Industry & Trade Co., Ltd, HOPE-MED8050 exposure controlling system) has a volume of 0.3m3 and allows to
expose a maximum of 20 mice simultaneously. The exposure temperature was 25°C; the relative humidity of exposure was 40%. After exposure, the pollutants in the exposure system were replaced with air (≥ 30 minutes). The exposure system was cleaned daily.
Monitoring PM2.5 in the exposure system Rice straw (5g) was burned inside the exposure system; the mini fan inside the system was switched on to allow the PM2.5 distribute evenly. The sampling port was connected to two aerosol monitors (Model: AM510, Product No.: #32 and #27). The mass concentrations of PM2.5 and PM 1.0 inside the exposure system were monitored for a 30-minute period and the monitoring was repeated 4 times. The time course of the mass concentration of PM2.5 and PM 1.0 of the aerosol in the exposure system was then determined. The particle size of particulate matter was analyzed by widerange particle (WPS) Sizer (Model: 1000XP, TSI Inc, USA). The distributions of concentration, surface area, and volume of particles with a size between 10 nm and 10 μm were also determined. Particles were collected by a particulate matter sampler, and then the size and composition of the particles were analyzed by a transmission electron microscope (TEM, Model: JEOL-2100F).
Animals The protocol for handling mice has been approved by the Institutional Animal Care and Use Committee of Shanghai Pulmonary Hospital of Tongji University School of Medicine (Approval No. SYXK [SH] 2012-0031). A total of C57BL male 40 mice aged 8-week and weighted 16g-18g were randomized into two groups (20 mice/group): bleomycin-induced IPF + exposure to air (BLM + air) group and bleomycin-induced IPF + exposure to PM2.5 (BLM + PM2.5) group. Mice in both groups received intratracheal injection of bleomycin (4mg/mL, dissolved in 50 µL saline) at experiment day 0 according to our previous description (Wei et al., 2016). Mice in the control group were injected with 50 µL saline intratracheally. On day 7 after bleomycin injection, the mice were exposed to air or PM2.5. Each group was further divided into one-week and four-week groups, which were sacrificed after oneweek and four-week exposure (10 mice/group), respectively, and mouse specimens were collected (Figure 1).
To determine mouse survival, 1) mice in the control groups (Control + PM2.5, 15 mice) were exposed to PM2.5 twice daily (30 minutes every exposure) for 28 days; 2) mice in the BLM + PM2.5 group (20 mice) were exposed to PM2.5 after BLMinduced IPF was established, and the exposure was twice daily (30 minutes every exposure) for 28 days; 3) mice in the BLM + air group (12 mice) were observed for survival for 28 days; 4) mice in the N- Acetylcysteine (NAC) intervention group (BLM+PM2.5+NAC, 20 mice) were exposed to PM2.5 twice daily (30 minutes every exposure) for 28 days after BLM-induced IPF was established, and NAC (150 mg/kg, dissolved in 2mL saline) was injected intraperitoneally daily before exposure to PM2.5 (Demiralay et al., 2013).
Exposure to PM2.5 Mice were housed in standard cages before being exposed to PM2.5. Rice straw (5g) was burned completely in the exposure system, and then the cages containing mice were placed inside the exposure system. The temperature and humidity of the exposure system was 25°C and 40%, respectively. Each exposure lasted 30 minutes, and 1L air was supplied to the exposure system every 15 minutes. The exposure was twice daily and the time interval between daily exposures was less than 4 hours. Mice in the BLM + air group were exposed to air in the similar manner. Mice not being exposed to PM2.5 or air were housed and fed normally.
Collecting and testing mouse specimens Mouse blood samples were collected from the eye socket after mice were sacrificed and the eye balls were removed. The blood samples were centrifuged at 3000 rpm for 15 minutes, and the serum was collected. Serum levels of IL-6, TNF-α, and TGF-β were measured by ELISA.
To collect alveolar lavage fluid (ALF), the lung and trachea were dissected. A perfusion needle was placed inside the trachea, and a suture was used to ligate the trachea to prevent leakage. The perfusion needle was secured inside the trachea. A total of 1 mL saline was injected with a 1 mL syringe to expand the lung, and the saline was collected 15 seconds after the injection. A minimum of 0.75 mL saline was
collected. Perfusion was repeated twice for each mouse. IL-6, TNF-α, and TGF-β levels in the collected ALF were determined by ELISA.
To collect lung tissue specimens, a perfusion needle was placed at the left trachea, which was ligated by suture. The perfusion needle was secured inside the left trachea. A total of 0.75 mL formalin was injected into the left lung. The perfusion needle was then removed. The left lung was isolated and fixed in 10% formalin. Paraffin tissue sections were prepared. H&E staining and Masson staining were performed. Hydroxyproline contents were determined. Histopathological characteristics and lung tissue collagen contents were observed.
Results Components of the smoke from straw burning and particle size determination PM2.5 from straw burning were mainly composed of organic matter (74.1%); 30% of the organic matter were polycyclic aromatic hydrocarbons; 10% were L-glucose and mannose-oligosaccharides. Figure 2A shows that the orange area (1.59%) represents carbon (Inorganic particles contained small amounts of hydrogen and sulfur) and the green area represents inorganic matter. Inorganic matter were mainly chloride ion. Other components are displayed in Figure 2B. TEM revealed that the particle size was smaller than 2.5 µm and the particles contained potassium, calcium, magnesium, and organic matter (Figure 3). The distributions of particle concentration, surface area, and mass concentration also showed that the majority of particles in the smoke had a size smaller than 2.5 µm (Figure 4). The concentrations of PM2.5 and PM1.0 in the smoke were at effective levels for 30 minutes inside the exposure system (PM2.5 > 75mg/m3 according to the WHO 2005 Air Quality Guidelines, http://www.euro.who.int/data/assets/pdf_file/0005/78638/E90038.pdf?ua=1, Figure 5).
Mouse survival Inhalation of PM2.5 for 28 days did not adversely affect normal mouse survival (survival rate: 100%, Figure 6). However, compared to exposure to air, inhalation of PM2.5 for 28 days increased the mortality of mice with BLM-induced IPF although the difference was not statistically significant (Survival rate of the BLM + air group:
58.3% versus survival rate of the BLM + PM2.5 group: 30%, P = 0.214, Figure 6). NAC intervention extended the survival of mice with BLM + PM2.5 (Survival rate of the BLM + PM2.5 group: 30% versus survival rate of BLM + PM2.5 + NAC group: 70%, P = 0.032, Figure 6).
Histopathological characteristics After BLM-induced IPF was established, the lung showed widened alveolar septum, damaged alveolar wall, obvious damaged alveolar fusion structure, and locally formed lesion-like nodules (Figure 7). These histopathological damages deteriorated as the exposure to PM2.5 increased (Figure 7). In addition, macrophage accumulation increased in the BLM + PM2.5 group and black particles were found inside the macrophages, whereas the BLM + air group did not showed such types of macrophages (Figure 8).
Compared with the BLM + air group, lung collagen contents in the BLM + PM2.5 group increased substantially after one-week exposure to PM2.5, and the difference was further enhanced after four-week exposure (Figure 9). These results suggest that exposure to PM2.5 may increase collagen deposition in the lung and thus exacerbate lung fibrosis. Compared with one-week exposure to PM2.5, four-week exposure to PM2.5 further increased collagen deposition in the lung, indicating that extended exposure to PM2.5 may aggravate lung fibrosis.
Cytokine levels in serum and alveolar lavage fluid ELISA revealed that serum levels of IL-6, TNF-α, and TGF-β were low and similar in different groups, suggesting that exposure to PM2.5 may not cause obvious systemic inflammation. IL-6 levels in ALF were significantly higher in the BLM+ PM2.5 group than in the BLM + air group after one-week (P = 0.027) and four-week exposure to PM2.5 (P = 0.010) (Figure 10). Additionally, IL-6 levels in ALF were significantly higher after four-week exposure than after one-week exposure to PM2.5 (P =0.036, Figure 10). Similar to IL-6 levels, TNF-α levels in ALF were significantly higher in the BLM+ PM2.5 group than in the BLM + air group after one-week (P = 0.035) and four-week exposure to PM2.5 (P = 0.001), and the TNF-α levels after fourweek exposure were significantly higher than those after one-week exposure (P =
0.048) (Figure 11). These data indicate that exposure to PM2.5 may induce lung inflammation. Figure 12 shows that TGF-β levels in ALF were significantly higher in the BLM+ PM2.5 group than in the BLM + air group after four-week exposure to PM2.5 (P =0.044), whereas were not statistically significantly different in the two groups after one-week exposure. These findings suggest that extended exposure to PM2.5 may exacerbate pulmonary fibrosis.
Lung hydroxyproline contents Lung hydroxyproline contents in the BLM+PM2.5 group were significantly higher than those in the BLM + air group after 4-week exposure (P = 0.032), and lung hydroxyproline contents after four-week exposure were significantly higher than those after one-week exposure to PM2.5 (P = 0.019, Figure 13), indicating that exposure to PM2.5 may exacerbate BLM-induced lung fibrosis.
Discussion A previous epidemiological study has demonstrated that environmental pollutants, in particular particulate matter (PM) in the air, can lead to acute and chronic lung diseases in China (Qiu et al., 2012). The current study exposed mice with BLMinduced IPF (Wei et al., 2016) to PM2.5 generated from the smoke of straw burning to investigate the effects of exposure to air PM2.5 on IPF progression in patients.
PM generated from straw burning are the key source of atmosphere PM. The PM are mainly composed of organic matter and some inorganic ions, including sodium, potassium, calcium, and sulfate ion, and nitrate ion. The particle size of most PM is smaller than 2.5 µm. Thus, the PM can enter alveoli so to seriously and adversely affect human respiratory system (Nie et al., 2016). The current study used smoke from straw burning to generate air pollutants PM2.5 and simulate the air pollutants in China.
Alveolar macrophages (AM) are the effector cells to phagocytose foreign bodies inside the alveoli. AM can rapidly phagocytose PM when they are exposed to air PM in vitro or in vivo (van Eeden et al., 2001, Mukae et al., 2000). Consistently, the current study also found that the accumulation of PM-containing macrophages in
mouse lung tissue increased considerably after the mice were exposure to PM2.5 for one to four weeks. AM can secret pro-inflammatory factors, such as IL-6 and TNF-α (Sijan et al., 2015). Injection of the conditional culture media from PM-stimulated AM into rabbit trachea causes substantial inflammatory reaction, which is similar to the inflammation caused by a direct injection of PM into rabbit trachea (Mukae et al., 2001). Xu et al (Xu et al., 2013) have found that on day 5 after exposure to PM2.5, inflammatory cells, mainly including macrophages and neutrophils, started to accumulate in mouse lung tissue and ALF. Pirela et al (Pirela et al., 2013) have also shown that an injection of PM into mouse trachea induces inflammatory reactions in mouse lung tissue and increases pro-inflammatory cytokine levels in ALF. These studies suggest that AM and the pro-inflammatory cytokines secreted by AM may play an important pro-inflammatory role in PM-induced lung inflammation. However, the effects of PM2.5 on lung fibrosis disease have been reported rarely.
The current study investigated the effects of PM2.5 on lung fibrosis progression by exposing mice with BLM-induced IPF to PM2.5 from straw burning. We found that exposure of mice with IPF to PM2.5 elevated the levels of the acute pro-inflammatory factors, IL-6 and TNF-α, in the ALF, and the pro-inflammatory factor levels were further increased as the exposure duration was extended. These data indicate that PM2.5 may promote local inflammatory reaction in mice with IPF. Anti-inflammatory factors, such as IL-10, were not detected in ALF. Thus, phagocytosis of PM2.5 by AM may promote inflammation, which is consistent with a previous study (Yin et al., 2004). H & E staining of lung tissue also showed that exposure to PM2.5 exacerbated lung inflammatory reaction in mice with IPF. The levels of pro-fibrosis cytokine, TGF-β, were also increased by extended exposure to PM2.5. Masson staining of lung tissue and hydroxyproline content measurement revealed that four-week exposure to PM2.5 increased collagen deposition in lung tissue greatly, suggesting that exposure to PM2.5 may not only exacerbate lung fibrosis but also induce acute inflammatory reaction. These observations resemble the disease course of acute exacerbation of IPF in patients.
Recent studies have found that PM2.5 are associated with multiple organ fibrosis and thus affect organ function. For example, Tan et al (Cucoranu et al., 2005) have shown
that PM2.5 in blood circulation were accumulated in the liver and were phagocytosed by Kupffer cells; they have also found that PM2.5 stimulated Kupffer cells to secret cytokines and induced hepatic stellate cells to secret collagen so to exacerbate nonalcoholic liver disease-mediated fibrosis. Goss et al (Goss et al., 2004) have demonstrated that increased air PM2.5 was associated with acute exacerbation of cystic lung fibrosis and lung dysfunction. Gorr et al (Gorr et al., 2014) have found that long-term exposure to PM2.5 led to cardiovascular tissue remodeling, collagen deposition in myocardial tissue, and cardiac fibrosis, which ultimately exacerbated primary disease and caused heart failure. These studies consistently indicate that longterm exposure to PM2.5 may correlate with the deterioration of multiple organ fibrosis. The current study first showed that exposure of mice with BLM-induced IPF to PM2.5 exacerbated IPF, induced additional inflammatory reaction, and thus increased mouse mortality. The intervention with NAC reduced the mortality of IPF mice exposed to PM2.5, suggesting the preventive effects of NAC. A previous study has shown that air PM2.5 induce strong oxidative stress of mouse macrophages, which can be inhibited by NAC (Bekki et al., 2016). The NAC-mediated therapeutic benefits in IPF mice exposed to PM2.5 may be associated with the antioxidant effects of NAC. However, the underlying mechanism remains to be further investigated.
Conclusion Exposure of mice with BLM-induced IPF to PM2.5 from straw burning exacerbated lung inflammation and fibrosis and increased mouse mortality. NAC extended the survival of IPF mice exposed to PM2.5 and showed some protective effects.
Ethics approval and consent to participate The protocol for handling mice has been approved by the Institutional Animal Care and Use Committee of Shanghai Pulmonary Hospital of Tongji University School of Medicine (Approval No. SYXK [SH] 2012-0031). Consent for publication Not applicable Availability of data and material Please contact author for data requests. Competing interests
The authors declared that they have no competing interests. Authors' contributions Experimental design: H.P.L., Y.H., L.S.W., Y.L. Q.H.L and D.W.; Data acquisition and analysis: Y.H., L.S.W., Y.L., Q.H.L. C.L.L., J.M.C., D.W., and H.P.L.; Writing the manuscript: Y.H., L.S.W., Y.L., Q.H.L., D.W., and H.P.L. All authors read and approved the final manuscript.
Funding Supported by grants from the National Science Foundation of China (No: 81730002，81670055, 81670056, 91442103, 81500052 and 81570057), Ministry of Science and Technology of the People’s Republic of China (2016YFC1100200, 2016YFC1100204), the Health Bureau Program of Shanghai Municipality (SHDC12014120），Grant from Shanghai Hospital Development Center （16CR3054A), Shanghai Municipal Commission of Health and Familiy Planning (201640157) and Tongji University (1511219020).
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Figure 1 Animal experiment protocol
Mice were randomized into bleomycin-induced IPF + exposure to air (BLM + air) group and bleomycin-induced IPF + exposure to PM2.5 (BLM + PM2.5) group (20 mice per group). Mice in both groups received intratracheal injection of bleomycin (4mg/mL, dissolved in 50 µL saline) at experiment day 0 to establish lung fibrosis. On day 7 after the bleomycin injection, the mice were exposed to air or PM2.5. Each group was further divided into oneweek and four-week groups, which were sacrificed after one-week and four-week exposure, respectively, and mouse specimens were collected.
Figure 2 Composition of PM emission from straw burning
The main component of PM2.5 from straw burning was organic matter (74.1%); 30% of the organic matter was polycyclic aromatic hydrocarbons and 10% was L-glucose. A: The orange area (1.59%) represents carbon (Inorganic carbon particles contain small amounts of hydrogen and sulfur). The green area represents inorganic matter. The main component of the inorganic matter is chloride ion (the grey area in the right panel).
Figure 3 Examination of the particle size distribution of PM from straw burning by scanning electron microscope
Single particle scanning electron microscopy revealed that the particle size was smaller than 2.5 µm. Electron microscopic ion chromatography found that PM contained potassium, calcium, magnesium, and organic matter.
Figure 4 Particle size distribution of PM2.5 from straw burning in the exposure system
Particle size of most PM in the exposure system was smaller than 2.5μm. (a) Particle concentration distribution shows the distribution of the number of particles with difference size per cm3. The number of particles with a size of 100 nm per m3 is the highest. (b) Surface area distribution shows the distribution of the surface area of particles with difference size per cm3. The surface area of particles with a size of 500 nm per m3 is the largest. (c) Mass concentration distribution shows the distribution of total mass of particles with different size per m3. The total mass of particles with a size of 400 nm per m3 is the highest. The majority of particles in the exposure system has a size smaller than 2.5 μm.
Figure 5 Time course of the concentrations of PM2.5 and PM 1.0 from straw burning in the exposure system
In the exposure system, PM2.5 concentration reduced gradually within 30 minutes. The average concentration was maintained at > 100mg/m3, which meets the concentration criterion for animal experiments.
Figure 6 Effects of exposure to PM2.5 on the survival of mice with BLM-induced IPF
Inhalation of PM2.5 did not adversely affect the survival of normal mice (survival: 100%). However, compared to exposure to air (BLM+ air group: 58.3%), inhalation of PM2.5 reduced the survival of mice with BLM-induced IPF (BLM+PM2.5 group: 30%, P = 0.214). NAC extended the survival of mice with BLM+PM2.5 (BLM+PM2.5+NAC group: 70% vs. BLM+PM2.5 group: 30%, P = 0.032).
Figure 7 H&E staining images of lung tissue-histopathology
A & a: H&E staining images of the BLM+ air group (one week). B & b: H&E staining images of the BLM+ air group (four weeks). C & c: H&E staining images of the BLM+ PM2.5 group (one week). D & d: H&E staining images of the BLM+ PM2.5 group (four weeks). After BLM-induced IPF was established, the lung showed widened alveolar septum, damaged alveolar wall, obvious damaged alveolar fusion structure, and inflammatory cell infiltration. Lung tissue damage in B was greater than that in A, showing worse alveolar fusion and bronchiectasis caused by local lesion contraction. Inflammatory cell infiltration in lung tissue increased and alveolar septum is wider in C compared with A. Inflammatory cell infiltration in lung tissue increased; lung tissue damage is worse; fibrosis lesions increased in D compared with C. A, B, C, and D represent 100 x magnification. a, b, c, and d represent 400 x magnification.
Figure 8 lung tissue histopathology- macrophages containing PM
A. Lung tissue in the BLM + air group (one week). B. Lung tissue in the BLM + air group (four weeks). C. Lung tissue in the BLM + PM2.5 group (one week). D. Lung tissue in the BLM + PM2.5 group (four weeks). Macrophages in the lung tissue contain black particles (pointing by the black arrows). Such macrophages are the most in the BLM + PM2.5 group exposed for four weeks, but are absent in the BLM + air group (H & E, 400× magnification).
Figure 9 Lung tissue histopathology-Masson staining
A & a: H&E staining images of the BLM+ air group (one week). B & b: H&E staining images of the BLM+ air group (four weeks). C & c: H&E staining images of the BLM+ PM2.5 group (one week). D & d: H&E staining images of the BLM+ PM2.5 group (four weeks). Blue areas represent collagen fibers. Collage deposition increased in C than in A and increased in D than in B, indicating exposure to PM2.5 increased collagen deposition. Collage deposition increased in D than in C, suggesting that extended exposure to PM2.5 increased collage deposition in lung tissue. A, B, C, and D represent 100 x magnification. a, b, c, and d represent 400 x magnification.
Figure 10 IL-6 levels in mouse alveolar lavage fluid
IL-6 levels in mouse ALF after exposure for one week (A) and four weeks (B) increased in the BLM + PM2.5 group than in the BLM + air group (P = 0.027 and P = 0.001, respectively). IL-6 levels increased in the BLM + PM2.5 four-week group compared with one-week group (C, P = 0.036). IL-6 levels reduced in the BLM + air four-week group compared with one-week group (D, P = 0.001). These data suggest that exposure to PM2.5 may increase IL-6 levels in mouse ALF.
Figure 11 TNF-α levels in mouse alveolar lavage fluid
TNF-α levels in mouse ALF after exposure for one week (A) and four weeks (B) increased in the BLM + PM2.5 group than in the BLM + air group (P = 0.035 and P = 0.001, respectively). TNF-α levels increased in the BLM + PM2.5 four-week group compared with one-week group (C, P = 0.048). TNF-α levels reduced in the BLM + air four-week group compared with one-week group (D, P = 0.543). These data suggest that exposure to PM2.5 may increase TNF-α levels in mouse ALF.
Figure 12 TGF-β levels in mouse alveolar lavage fluid
TGF-β levels in mouse ALF after exposure for one week (A) and four weeks (B) increased in the BLM + PM2.5 group than in the BLM + air group (P = 0.724 and P = 0.044, respectively). TGF-β levels increased in the BLM + PM2.5 four-week group compared with one-week group (C, P = 0.103). TGF-β levels reduced in the BLM + air four-week group compared with one-week group (D, P = 0.054).
Figure 13 Hydroxyproline contents in lung tissue
Lung hydroxyproline contents in the BLM + PM2.5 group were significantly higher than those in the BLM + air group, indicating collagen contents increased. The P values of the comparison are: BLM + air one-week vs. BLM + air four-week, P = 0.537; BLM + air one-week vs. BLM + PM2.5 one-week, P = 0.162; BLM + air oneweek vs. BLM + PM2.5 four-week, P < 0.0001; BLM + air four-week vs. BLM + PM2.5 one-week, P = 0.694; BLM + air four-week vs. BLM + PM2.5 four-week, P = 0.032; BLM + PM2.5 one-week vs. BLM + PM2.5 four-week, P = 0.019. These data suggest that extended exposure to PM2.5 may exacerbate fibrosis.