Mitochondrial damage mediated by ROS incurs bronchial epithelial cell apoptosis upon ambient PM_2.5 exposure

Abstract

Mitochondria can be used as important biomarkers of pollutants on human health, and fine particulate matter (PM_2.5) has been documented to cause respiratory damage. However, current studies about the relationship between PM_2.5 and mitochondria in respiratory tract are limited and warrant further detailed investigations. Hence, the study was aimed to evaluate effects of PM_2.5 on mitochondrial structure, investigate the link between PM_2.5-induced mitochondrial disorder and respiratory damage, and delineate the possible mechanisms using both in vitro and in vivo models. PM_2.5 exposure resulted in damage of mitochondrial structure, including mitochondrial dynamic, DNA biogenesis and morphological alteration 16HBE cells. Furthermore, PM_2.5 elevated ROS formation. However, DPI and NAC (inhibitor of ROS) in supplement restored PM_2.5-induced mitochondrial disorder. PM_2.5 also contributed to the 16HBE cells apoptosis via mitochondrial pathway. Additionally, the results coincided with the in vivo data which were obtained from bronchial tissues of SD rats exposed to PM_2.5 for 30 days. Collectively, this study uncovers that PM_2.5 leads to the disorder of mitochondrial structure via ROS generation, and then results in respiratory damage. It provides further understanding about the detrimental effect of PM_2.5 on respiratory damage, and reveals a mechanistic basis for preventing outcomes in polluted environments.

INTRODUCTION

Greater than three million deaths per year are attributable to ambient air pollution, which is the 9th leading factor contributing to worldwide burden of disease according to the 2010 Global Burden of Disease Study (Lim et al., 2012). Fine particulate matter (PM_2.5; aerodynamic diameter less than 2.5 µm) is well established as a key contributor of ambient air pollution, which can be readily inhaled by the human body and deposited in the respiratory system (Leung et al., 2014). Epidemiological and experimental investigations have linked PM_2.5 exposure to increased respiratory hospital admissions, mortality and morbidity due to the potential bio-accumulation (Stieb et al., 2009; Rückerl et al., 2011; Brunekreef and Holgate, 2002). However, the studies of mechanism on PM_2.5-induced respiratory damage are limited and warrant further detailed investigations.

In most mammalian cells, mitochondria are primarily considered the major supplier of ATP. In which, more than 95% of ATP is synthesized through oxidative reactions (Nunnari and Suomalainen, 2012). During oxidative phosphorylation, electrons are moved thorough the mitochondrial respiratory chain, and a proton gradient is established across the inner mitochondrial membrane as the energy source for ATP production (Prakash and Doublié, 2015). Mitochondria are morphologically dynamic organelles that continuously undergo two opposing events (fission and fusion), which have vital roles in cell metabolism and respiratory (Sheridan and Martin, 2010). Besides, they undergo prominent structural changes including fragmentation and cristae remodelling in response to intense stimulation (Arnoult, 2007). There are emerging data that mitochondrial disorders are closely related to a variety of physiological and pathological processes, including aging, cardiovascular diseases and respiratory illness (Nunnari and Suomalainen, 2012). Therefore, mitochondria can be used as important biomarkers of pollutants on human health and it is imperative to understand the effect of PM_2.5 on mitochondria. However, current studies on the relationship between PM_2.5 and mitochondria are limited and need further assessment. In the present study, we carried out the comprehensive evaluation about the effect of PM_2.5 on structure and function of mitochondria. The structure involves in mitochondrial dynamic, DNA biogenesis and morphological alteration, and the function includes energy metabolism and respiratory function. Studying the effect of mitochondrial structure and function is of great significance for the evaluation of PM_2.5 toxicity on human health.

It has been documented that reactive oxygen species (ROS) captured the attention of biologists and physicians due to their important function in the regulation of multiple cellular and extracellular processes such as activation of stress-induced mitochondrial biogenesis (Murphy et al., 2011). Thus, mitochondria were the target of ROS, which could affect the mitochondrial structure and function in turn (Scherz-Shouval and Elazar, 2007). Besides, mitochondria are also accountable for the generation of ROS, which is generated with the leak of electrons mainly from mitochondrial respiratory chain complexes I and III (Szewczyk et al., 2015). Impairment of mitochondrial dynamics and pathological changes in metabolic properties leading to unnecessarily high ROS formation must result in diverse abnormalities. Notably, PM_2.5-induced excessive formation of ROS could trigger cell damage by oxidizing macromolecular structures and modifying their biological functions, ultimately leading to cell apoptosis, suggesting the pivotal roles of ROS on PM_2.5-mediated adverse health on human body (Kampa and Castanas, 2008).

Due to the extensive vehicle exhaust emissions and coal combustions in residential stoves for cooking and heating, northern Chinese cities still face serious problems of PM_2.5 pollution, especially in winter (Shen et al., 2010). This situation is worsening with the urbanization and industrialization of Taiyuan, a northern city of China and a center of coal-based electricity production and many chemicals industries (Li et al., 2014). Therefore, the current study was focused on winter PM_2.5 from North China and designed to address three goals: (1) to investigate effects of PM_2.5 on mitochondrial structure in respiratory tract; (2) to explore the link between PM_2.5-induced mitochondrial disorder and respiratory damage; (3) to delineate the possible mechanisms using both in vitro and in vivo models.

MATERIALS AND METHODS

PM_2.5 collection and preparation

As described by Li et al. (2014), PM_2.5 samples were collected during winter 2012/2013 at Taiyuan, a site of Shanxi urban background for atmospheric pollution. Briefly, PM_2.5 high volume air sampler (Thermon Anderson, New York, NY, USA) was placed on the rooftop of a building about 25 meters tall. PM_2.5 concentrations were measured using DustTrakTM II Aerosol Monitor (TSI Inc., Knoxville, TN, USA), and daily samples were collected on quartz fiber filters (QFFs) with a pump flow rate of 1.13 m³/min. These filters loading samples were cut and surged in Milli-Q water with sonication. To obtain PM_2.5 suspensions, the above suspensions were freeze-dried in vacuum and weighed. Then the samples were dried and stored at -20°C. The 20 mg PM_2.5 particles supplemented with 1 mL 0.9% physiological saline were blended, treated with ultrasonic oscillation for 30 min. Particles were UV-irradiated overnight to inactivate possible contaminating endotoxin and to be germicidal, as indicated in the paper of Peeters et al. (2014). Then 20 mg/mL PM_2.5 was stored at 4°C, with gentle oscillation before usage. Prior to use, PM_2.5 suspension was diluted with sterilized 0.9% physiological saline, surged for 20 min and mixed fully.

Cells and culture conditions

16HBE cell line (human bronchial epithelial cells), obtained from the Institute of Biochemistry and Cell Biology (SIBS, CAS, Shanghai, China), was maintained in DMEM medium (HyClone, New York, NY, USA) supplemented with 10% FBS (Boster, Wuhan, China) and 1% penicillin/streptomycin (Solarbio, Beijing, China). Cells were kept at 37°C in a 5% CO₂ humidified cell culture incubator. 16HBE cells were treated with different doses of PM_2.5 (0, 10, 50 and 100 μg/mL) for 48 hr, based on the doses used in other in vitro research papers (Gualtieri et al., 2011; Risom et al., 2005). In the experiments involving pharmacological inhibitors, 16HBE cells were pre-treated with fresh media containing DPI (3 μΜ) or NAC (1 mM) for 24 hr and then treated with PM_2.5 suspensions in the presence of inhibitors for 48 hr.

Quantitative real time PCR

Quantitative real time PCR (qRT-PCR) was employed using an Applied Bio-systems platform, according to our previous methods (Jin et al., 2014a). Human or rat primers for targeted or reference genes are shown in Supplementary Table S1 and Table S2, respectively.

Mitochondrial membrane potential (Δψm) assay

Mitochondrial membrane potential (Δψm) was analyzed in 16HBE cells, according to the manufacturer’s protocol for a commercial kit (KeyGEN BioTECH, Nanjing, China). The details were as described for our previous methods (Jin et al., 2014b).

Determination of ROS generation

Generation of ROS was determined by fluorometric analysis, using 2,7-dichlorofluorescin diacetate (DCFH-DA), which was obtained from Beyotime Institute of Biotechnology (Nan tong, China) as described for our previous methods (Jin et al., 2014b).

Flow cytometric analysis of apoptosis

Apoptosis was examined by using the Annexin V-FITC and propidium iodide (PI) staining method. In brief, 5 × 10⁵ cells were collected and incubated in the buffer containing 200 μL Annexin V solutions (10 μL AnnexinV + 200 μL binding buffer) and 300 μL PI (5 μL PI + 300 μL binding buffer) in the dark (15 min at RT). Untreated cells were used as the control. Then the samples were examined using a FAC Sort Flow Cytometer within 45 min after staining.

Trypan blue exclusion counts and LDH release for toxicity estimation

After 16HBE cells were treated by PM_2.5 for 48 hr with or without NAC or DPI pre-treatment for 24 hr, the culture medium was collected and assayed for LDH activity. It was investigated by the linear region of a pyruvate standard graph using regression analysis and expressed as % release, according to a commercial determination kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Total number of viable cells was counted by trypan blue exclusion method using a hemocytometer. Cell viability (% of control) = (Number of control cells – Number of treated cells) / Number of control cells × 100.

Western blotting assay

Proteins extracted from cells and tissues were resolved by 10% SDS-PAGE and transferred onto nitrocellulose membranes for Western blotting, according to our previous methods (Jin et al., 2014a). The antibodies used in this study were as follows: antibodies for Bax, Bcl-2, and Cyt-c were obtained from Bioworld Technology (Minneapolis, MN, USA); antibodies for caspase-3, and caspase-9 were from Beyotime Institute of Biotechnology (Haimen, China); α-tubulin was purchased from Sigma (St. Louis, MO, USA).

Animal experiment

As indicated in our previous study (Li et al., 2015), healthy adult, clean-grade male Sprague-Dawley rats (weighing 200 ± 20 g) were commercially obtained from Experimental Animal Center of the Chinese Military Medical Science Academy (Beijing, China). The animals were kept in standard animal houses under a 12 hr light/dark cycle (lights on at 8:00 a.m.) at a constant temperature of 24 ± 2°C and relative humidity of 50 ± 5%. After 7 days of habituation, the rats were randomly divided into the following five groups (n = 6); (1) control group, (2) 0.3 mg/kg body weight (b.w.) PM_2.5 group, (3) 0.9 mg/kg b.w. PM_2.5 group, (4) 1.8 mg/kg b.w. PM_2.5 group and (5) 2.7 mg/kg b.w. PM_2.5 group. The treatment groups were instilled with a 0.5 mL PM_2.5 suspension, and the final exposure concentrations reached 0.3, 0.9, 1.8, 2.7 mg/kg b.w., while the control group was instilled with physiological saline at the same volume as that was used for the treatment group. The instillation was conducted using a nonsurgical intratracheal instillation method (Bai et al., 2010), and performed one time every 3 days. When not being treated, the rats had free access to food and water. All of these rats were maintained under standard nutritional and environmental conditions throughout the experiment according to the requirement of the National Act on the Use of Experimental Animals (China). Maximal effort was made to minimize animal suffering and the number of animals necessary for the acquisition of reliable data.

As has been reported, the respiratory volume of an adult rat was 200 mL/min, and the respiratory volume for 3 days reached 0.864 m³. According to the China National Ambient Quality Standard (NAAQS, 2012) for PM_2.5 (0.075 mg/m³), the amount of PM_2.5 inhalation over 3 days is 0.0648 mg and the concentration of PM_2.5 exposure for each rat every 3 days is estimated to be 0.324 mg/kg b.w. (rat 200 ± 20 g). PM_2.5 mean mass concentration determined in our samples was 0.161 ± 0.060 mg/m³ on non-haze weather in Taiyuan (Li et al., 2014), and the higher concentrations corresponded to the haze weather reached 0.692 ± 0.272 mg/m³ (Cao et al., 2014). Taken together, the average concentrations of PM_2.5 exposure for each rat every 3 days were estimated in the range from 0.324 to 2.989 mg/kg b.w.. Therefore, we selected 0.3, 0.9, 1.8, 2.7 mg/kg b.w. PM_2.5 as exposure doses in our SD rat experiment.

Statistical analysis

Statistical analysis was carried out using the SPSS 17.0 software program. Data, derived from at least three independent experiments, were presented as the mean ± standard deviation (S.D.). Differences among groups were tested by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. P-values lower than 0.05 were considered statistically significant.

RESULTS

PM_2.5 causes disorders of mitochondrial structure

Since mitochondria are direct stress organelles upon foreign pollutant exposure (Adam-Vizi and Chinopoulos, 2006) and important biomarkers of pollutant on human health, we carried out the comprehensive evaluation about the impact of PM_2.5 on mitochondrial structure and function. The structure of mitochondria is involved in mitochondrial dynamic, DNA biogenesis and morphological alteration. As shown in Fig. 1A and B, expressions of profusion factors (Mfn1 and OPA1) were notably suppressed by 100 μg/mL PM_2.5 exposure, and there was significant alteration in profission factors (Drp1) compared with non-exposed cells. In addition, PM_2.5 induced a significant decrease in mRNA levels of SIRT1 and p53R2, which are related to mitochondrial DNA biogenesis in 16HBE cells (Fig. 1C). Based on Fig. 1D-F, a bright green fluorescence, showing the decrease of mitochondrial membrane potential, was observed in PM_2.5-treated cells, while control cells displayed a broad yellow-green fluorescence. The data indicated that PM_2.5 treatment caused the damage of mitochondrial structure.

Fig. 1

PM_2.5 causes disorders of mitochondrial structure and function in 16HBE cells. Effects of 0, 10, 50 and 100 μg/mL PM_2.5 on the mRNA levels of mitochondrial dynamics markers, including both (A) profission genes and (B) profusion factors, (C) as well as mitochondrial biogenesis markers in 16HBE cells. (D-F) JC-1 was carried out to assess the PM_2.5-induced alteration in mitochondrial structure. The JC-1 green fluorescence intensities were analyzed using the Image J (NIH) software. Data represented were mean ± S.D. of at least three independent experiments. *p < 0.05, **p < 0.01, compared to control.

PM_2.5-induced ROS promotes mitochondrial damage in 16HBE cells

Given that ROS are accountable for mitochondria disorder and are the main target of PM_2.5-caused toxicity on human health (Kampa and Castanas, 2008), their generation was examined. Fluorometric analysis revealed that PM_2.5 induced significant dose-dependent production of ROS (Fig. 2A). In order to further confirm the possible role of ROS on mitochondria disorder, DPI and NAC, inhibitors of ROS, were applied. After pre-treatment with DPI or NAC for 24 hr, ROS levels were reduced compared to PM_2.5 treatment alone, indicating that ROS were induced by PM_2.5 (Fig. 2B). As shown in Fig. 3A and B, DPI or NAC induced a marked increase in mRNA levels of fusion protein Mfn1 and OPA1 decreased by PM_2.5. Additionally, elevated mRNA expressions of mitochondrial biogenesis markers SIRT1 and p53R2 were observed in PM_2.5 + DPI or NAC group compared to PM_2.5 group (Fig. 3C and D). Moreover, based on Fig. 3E and F, the expression of NDUFS2 and UQCRI1 was successfully attenuated by pre-treatment with DPI or NAC for 24 hr. Likewise, mitochondrial membrane potential attenuated by PM_2.5 treatment was alleviated by pre-treatment with DPI or NAC (Fig. 3G and H). The data indicated that enhanced ROS generation promoted the damage of mitochondrial structure and function.

Fig. 2

PM_2.5 elevates the ROS production in 16HBE cells. 16HBE cells were exposed to PM_2.5, and ROS generation was determined. (A) Alterations in the ROS generation of 16HBE cells relative to control. (B) After pre-treated with fresh media containing DPI (3 μΜ) or NAC (1 mM) for 24 hr and then treated with 100 μg/mL PM_2.5 in the presence of inhibitors for 48 hr, the ROS generation was investigated in 16HBE cells. Results were reported as average ± S.D. for at least three independent experiments. Statistically significant different from PM_2.5-treated cells: *p < 0.05, **p < 0.01.

Fig. 3

The effect of PM_2.5-induced ROS on mitochondrial structure and function in 16HBE cells. After pre-treatment with DPI (3 μΜ) or NAC (1 mM) for 24 hr, inhibiting ROS from NOX or total ROS, and 100 μg/mL PM_2.5 exposure for 48 hr, the mitochondrial structure and function were investigated. (A-B) Mfn1 and OPA1. (C-D) SIRT1 and p53R2. (E-F) NDUFS2 and UQCRI1. (G) JC-1 was carried out to assess the alteration in mitochondrial structure. Scale bar is 10 μm. (H) The JC-1 green fluorescence intensities were analyzed using the Image J (NIH) software. The values were showed as means ± S.D. of triplicate independent determinations. *p < 0.05, **p < 0.01, compared to PM_2.5-treated cells.

PM_2.5 contributes to the cell death of 16HBE cells

In order to explore the link between PM_2.5-induced mitochondrial disorder and respiratory damage, we next determined the cell viability of human bronchial epithelial cells upon PM_2.5 exposure. Flow cytometric analysis was performed to determine the effective dose of PM_2.5 on 16HBE cells for 24 hr and 48 hr exposure. As shown in Fig. 4A, PM_2.5 exposure at 10, 50 and 100 μg/mL induced considerable cell death in a dose-dependent manner after 48 hr of exposure. Especially, 100 μg/mL PM_2.5 exposure induced 48.93% apoptosis compared to control cells with 6.15% apoptosis. Trypan Blue Exclusion counts was adopted to confirm the cell damage incurred by PM_2.5. Cells with PM_2.5 exposure presenting shrinkage shape representing apoptotic cells, which were elevated in a PM_2.5 dose-dependent manner (Fig. 4B and C). Similar findings were observed in the LDH assay, displaying a decline of cell viability in response to PM_2.5 (Fig. 4D). These data clarify that PM_2.5 led to the cell death of human bronchial epithelial cells.

Fig. 4

The effect of PM_2.5 on cell viability of 16HBE cells. (A) Apoptosis of 16HBE cells was detected by AnnexinV-FITC/PI double staining using flow cytometric analysis. (B) Trypan Blue Exclusion count assay was used to determine the cell viability of cells with PM_2.5 exposure. (C) The morphological photo of cells is shown. Cells were examined with light microscope (10 ×). (D) LDH assay was applied to measure the cell viability of 16HBE cells in response to 0, 10, 50 and 100 μg/mL PM_2.5. Values were representative of at least three biologically independent experiments with similar results. *p < 0.05, **p < 0.01, compared to controls.

PM_2.5 leads to the cell death of 16HBE cells via mitochondrial pathway mediated by ROS

We further assessed the association between PM_2.5-induced respiratory damage and mitochondrial disorder, as well as the possible mechanism, adopting the DPI or NAC addition. As revealed in Fig. 5A and B, the reduction of cell viability by PM_2.5 was ameliorated by the addition of DPI or NAC, indicating that the cell death of 16HBE cells was mediated by PM_2.5-induced ROS generation. Due to the major role of mitochondrial pathway in cell death, a further step was taken to determine the pathway. The results from Western blotting indicated that the levels of Bax, cytochrome c, active-caspase3 and active-caspase9 were up-regulated in response to PM_2.5 in a dose-dependent manner. In addition, the protein level of anti-apoptotic gene Bcl-2 was down-regulated in PM_2.5-treated cells as compared to control (Fig. 5C). Moreover, these alterations were significantly reversed by the DPI or NAC addition (Fig. 5D). All these results suggested that PM_2.5 possibly caused the induction of permeability transition in mitochondria, which was mediated by ROS generation, and played a key role in cell death of 16HBE cells.

Fig. 5

PM_2.5 contributes to the cell death of 16HBE cells via mitochondrial pathway. (A-B) After pre-treatment with DPI (3 μΜ) or NAC (1 mM) for 24 hr, inhibiting ROS from NOX or total ROS, and 100 μg/mL PM_2.5 exposure for 48 hr, the cell viability of 16HBE cells was investigated using Trypan Blue Exclusion counts and LDH assay. (C) Western blotting was applied to assess the activation of caspase-3 as well as caspase-9 and the expression of cytochrome c, Bax and Bcl2. (D) These proteins were investigated in response to 100 μg/mL PM_2.5 pre-treated with DPI (3 μΜ) or NAC (1 mM) for 24 hr. The above data were representative of at least three independent biological replicates. Statistically significant different from PM_2.5-treated cells: *p < 0.05, **p < 0.01.

PM_2.5 induces mitochondrial damage and apoptosis pathway in SD rats

The above results uncovered that PM_2.5 contributed to mitochondrial disorder and cell apoptosis of human bronchial epithelial cells, which were mediated by ROS. To validate our findings, in vivo SD rat assay was performed to further determine the toxicity induced by PM_2.5. After 0.3, 0.9, 1.8, 2.7 mg/kg b.w. PM_2.5 exposure of SD rats for 30 days with intratracheal instillation, we separated bronchial tissues of SD rats and determined the alteration of mitochondria and relevant apoptosis pathway. The result from qRT-PCR showed that levels of Mfn1, OPA1, SIRT1 and p53R2 in bronchial tissues were dose-dependently reduced in response to PM_2.5 (Fig. 6A and B). A pronounced dose-related attenuation in NDUFS2 and UQCRI1 expressions was also was observed (Fig. 6C). Furthermore, the data from Western blotting indicated that 2.7 mg/kg b.w. PM_2.5 contributed to up-regulations of apoptotic proteins and down-regulations of anti-apoptotic protein Bcl-2 in bronchial tissues (Fig. 6D).

Fig. 6

Mitochondrial alteration and cell death caused by PM_2.5 in bronchial tissues. After 0.3, 0.9, 1.8, 2.7 mg/kg body weight (b.w.) PM_2.5 exposed to SD rats for 30 days with the intratracheal instillation, bronchial tissues of SD rats were separated and homogenized with the liquid nitrogen for qRT-PCR and Western blotting. qRT-PCR assay was applied to analyze the expressions of mitochondrial relevant genes, inducing (A) Mfn1, OPA1, (B) SIRT1, p53R2, (C) NDUFS2 and UQCRI1 in bronchial tissues. (D) Western blotting was performed as mentioned above to examine apoptosis protein expression in control and 2.7 mg/kg b.w. PM_2.5-treated bronchial tissues. Values shown were given as the mean ± S.D. of 6 animals in each group. *p < 0.05, **p < 0.01, compared to control mice.

DISCUSSION

There are emerging data documenting that PM_2.5 exposure is closely associated with the increased respiratory hospital admissions, mortality and morbidity, and mitochondria are important markers of evaluating pollutant’s toxicity. However, current researches about the relationship between PM_2.5 and mitochondria in respiratory tract are limited and warrant further detailed investigations. In this report, we identify the effects of PM_2.5 on respiratory damage and mitochondrial disorder, as well as classify the possible mechanism involved involved the induction of ROS using both in vitro and in vivo models.

The pollutant and chemical characteristics of PM_2.5 in our study have been reported by Li et al. (2014). Briefly, the measured daily average PM_2.5 mass concentration (0.161 ± 0.060 mg/m³) was much higher than those of the Chinese national recommended safety standards (0.075 mg/m³). The levels of 16 polycyclic aromatic hydrocarbons (PAHs) in PM_2.5 were obviously higher than those of the Chinese national standard for PAHs (10 ng/m³). The daily mean levels of SO₄²⁻ and NO₃⁻ ions in the PM_2.5 samples reached 5.87 and 1.71 μg/m³, respectively. These data indicated that PM_2.5 pollution in Taiyuan might be hazardous to human health.

Mitochondria are essential intracellular energy-generating organelles with various cellular functions. Fusion and fission of mitochondria are important for many biological processes, including homeostasis of mitochondrial biogenesis, turnover, subcellular distribution, cell division, and apoptosis. Mitochondrial dynamics play a crucial role in mitochondrial quality control. Through proper fusion/fission dynamics coordinated with contents amplification, new daughter mitochondria are formed (Chan, 2006). Mitochondrial biogenesis is a complex process involving the replication of mitochondrial DNA (mtDNA) and the expression of mitochondrial proteins encoded by both nuclear and mitochondrial genomes (Higashida et al., 2013). In addition, up-regulation of other mitochondrial contents including respiratory chain complexes and their assembly proteins, is important for preventing dilution of the contents for a successful mitochondrial proliferation (Muster et al., 2010). Consequently, mitochondria are considered as a new paradigm for the research of PM_2.5 toxicity. During apoptosis, mitochondria undergo prominent structural changes including fragmentation and cristae remodelling (Arnoult, 2007). This study has demonstrated that PM_2.5 exposure damaged the structure and function of 16HBE cells. Our previous results also implicated that PM_2.5-induced mitochondrial damage was potentially important mechanisms for the heart injury (Li et al., 2015). Consistently, several studies have implicated the aberrant mitochondrial alteration upon PM_2.5 exposure. Li et al. (2003) have reported that ultrafine particulate matter collected in Los Angeles induced mitochondrial structural damage, which was in accordance with the redox cycling potential of the particles. Xu et al. also have documented that long-term ambient PM_2.5 exposure impaired mitochondrial alteration, which was a risk factor for the development of type 2 diabetes (Gualtieri et al., 2011).

The present results indicate PM_2.5 promoted the damage of mitochondrial structure via the ROS generation. Similarly, mitochondria have been regarded as both target and source of ROS, whose generation may induce the oxidative damage to mitochondrial proteins (Scherz-Shouval and Elazar, 2007). Oxidation of the mitochondrial by ROS may contribute to cytochrome c release due to disruption of the mitochondrial membrane potential (Zamzami et al., 1995). ROS are also one of the important mechanisms of cell damage induced by PM_2.5 (Bräuner et al., 2007; Risom et al., 2005). Under normal conditions, the production and elimination of ROS should be in balance, while sustained ROS production is often responsible for oxidative cell damage through lipid peroxidation, DNA breakdown, and protein damage (Finkel and Holbrook, 2000). When mitochondria are damaged by pollutants, ROS can be generated form mitochondrial respiratory chain (Adam-Vizi and Chinopoulos, 2006). The main sources of cellular ROS are mitochondria and NADPH oxidases (NOXs), which are cell membrane-bound proteins (Lambeth, 2004). As shown in Fig. 2B, the reversed effect of NAC (inhibitor of ROS) on ROS levels was more prominent than DPI (inhibitor of ROS from NOXs), suggesting that ROS induced by PM_2.5 could generate from mitochondria and NOXs. The elevated expression of DUOX1 and DUOX1 in bronchial tissues of SD rats exposed to PM_2.5 confirmed our speculation (Fig. S1).

In the present study, we found that PM_2.5 contributed to respiratory damage, which was consistent with some studies at home and abroad. Guo et al. (2014) indicated that PM_2.5 concentration was closely correlated with the increase of patients with bronchitis. Annesi-Maesano et al. (2007) found that the morbidity of asthma and bronchitis induced by exercises was apparently connected with PM_2.5 concentration, which was evidently higher in high-concentration regions than that in low-concentration regions. In our previous study, we observed that exposure to Taiyuan winter PM_2.5 induced cellular oxidative heart damage, and filter-derived quartz debris did not cause bias in our experimental outcomes (Li et al., 2015). PAHs, as an important part of organic compounds toxic substances in PM_2.5, occupy a large proportion. Many studies also have shown that the biological effects of PM_2.5 are directly related to PAHs (Abbas et al., 2013; Billet et al., 2008). Therefore, we speculated that PAHs in PM_2.5 were important for the mitochondrial damage.

To the best of our knowledge, this is the first report to examine the relationship between PM_2.5-induced mitochondrial disorder and respiratory damage. The data implicated that PM_2.5 exposure elevated the ROS generation via binding to cell membrane-bound NOXs, which further provoked the disorder of mitochondrial structure and function in respiratory tract. Moreover, the damaged mitochondria induced the mitochondrial apoptosis pathway, and then resulted in cell apoptosis of respiratory tract. Accordingly, this project contributes to explaining the damage effects of PM_2.5 on human respiratory tract and provides a kind of mechanistic basis for the development of preventive treatments for urban dust-haze.

ACKNOWLEDGMENTS

This work was sponsored by the National Natural Science Foundation of China (No. 21707085, 31770382), the China Postdoctoral Science Foundation (No. 2017M610120), the Shanxi Province Science Foundation for Youths (No. 201701D221244), the Fund for Shanxi “1331 Project” Collaborative Innovation Center (No. 1331 CIC) and the Shanxi Scholarship Council of China (No. 2015-2).

Conflict of interest

The authors declare that there is no conflict of interest.

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