Cytotoxicity induced by fine particulate matter (PM2.5) via mitochondria-mediated apoptosis pathway in rat alveolar macrophages


Although positive associations exist between ambient particulate matter (PM2.5; diameter ≤ 2.5 μm) and the morbidity and mortality rates for respiratory diseases, the biological mechanisms of the reported health effects are unclear. Considering that alveolar macrophages (AM) are the main cells responsible for phagocytic clearance of xenobiotic particles that reach the airspaces of the lungs, the purpose of this study was to investigate whether PM2.5 induced AM apoptosis, and investigate its possible mechanisms. Freshly isolated AM from Wistar rats were treated with extracted PM2.5 at concentrations of 33, 100, or 300 μg/mL for 4 h; thereafter, the cytotoxic effects were evaluated. The results demonstrated that PM2.5 induced cytotoxicity by decreasing cell viability and increasing lactate dehydrogenase (LDH) levels in AMs. The levels of reactive oxygen species (ROS) and intracellular calcium cations (Ca2+) markedly increased in higher PM2.5 concentration groups. Additionally, the apoptotic ratio increased, and the apoptosis-related proteins BCL2-associated X (Bax), caspase-3, and caspase-9 were upregulated, whereas B cell lymphoma-2 (Bcl-2) protein levels were downregulated following PM2.5 exposure. Cumulative findings showed that PM2.5 induced apoptosis in AMs through a mitochondrial-mediated pathway, which indicated that PM2.5 plays a significant role in lung injury diseases.


Hazy-fog episodes in world have recently become a major public health concern because it contains polluting primary or secondary particulate matter (PM) and, possibly, irritable chemicals and can have severe negative effects on the air quality and human health (Pérez-Díaz et al. 2017). PM2.5 is the primary particle of hazy fog, which is related to an elevated respiratory morbidity and mortality (Pope and Dockery 2006; Espitia-Perez et al. 2018; Zhu et al. 2020). According to Lelieveld et al. (2020), the number of premature deaths caused by air pollution in urban and rural areas was approximately 8.4 million in 2020, which was about twice that reported by the World Health Organization in 2016 (WHO 2019). Pozzer et al. (2020) demonstrated that air pollution is an important co-factor increasing the risk of mortality from COVID-19. Inhaled PM2.5 can penetrate deeply into the lungs, deposit in the airways and alveoli, pass into the circulation, and damage not just in the respiratory system but also in the extrapulmonary organs (Gunasekar and Stanek 2011). Since the lung is one of the major targets of PM deposition, PM2.5 can trigger pulmonary inflammatory responses and impair lung function (Pinkerton et al. 2019; Wu et al. 2013). A growing number of publications have demonstrated that PM2.5 causes respiratory diseases including asthma, airway irritation chronic obstructive pulmonary disease (COPD), and lung cancer (Zhao et al. 2020; Tian et al. 2020; Wu et al. 2020;Wei et al. 2020).

Alveolar macrophage (AM) are the most abundant innate immune cells present in all mammalian organs, and play an important role in tissue homeostasis, host defense, clearance of surfactant and cell debris, pathogen recognition, initiation, and resolution of lung inflammation (Joshi et al. 2018; Zhang et al. 2012; Hu and Christman, 2019). In the lung, AMs are the first line of defense responsible for phagocytic clearance of xenobiotic particles that have reached the air spaces of the lungs; this is important as these cells have been considered as a critical participant in allergic lung diseases (Byrne et al. 2016). Bronchoalveolar lavage (BAL) has proven to be useful for detecting an inflammatory response in the lungs of animals exposed to toxicology studies; there is also optimism about the use of BAL analysis as an early predictor of late-occurring pulmonary diseases (Steinberg et al. 1992). Therefore, AM activity analysis is frequently used as an important indicator for evaluating responses in the lungs (Morio et al. 2001).

Reactive oxygen species (ROS) are involved in transmission stress of signaling in physiological and pathological processes; these are recognized widely as playing important roles in PM2.5-mediated cytotoxicity (Huang et al. 2018). PM2.5 exposure induces the elevation of cellular ROS, and then causes an imbalance in cell homeostasis associated with damage to nucleic acids and proteins, lipids, membranes, and organelles, which results in apoptosis (Reyes-Zarate et al. 2016; Yang et al. 2018). Besides, cations (Ca2+) involved in multiple biological responses, they are considered as signaling molecules (Huang et al. 2018). Several studies have shown that in cancer cells, Ca2+ can induce apoptosis by destroying mitochondrial function (Hsieh et al. 2018).

Cellular apoptosis can occur via two major pathways—the death receptor (extrinsic) pathway and the mitochondrial (intrinsic) pathway (Galluzzi et al. 2018; Reed 2000). It has been reported that PM2.5 induces mitochondria ultrastructure damage including mitochondrial swelling, cristae disorder, and even vacuolation (Wei et al. 2019). The present study aimed to investigate whether PM2.5 induced AM apoptosis via the mitochondria-mediated pathway. Primary AMs were collected from lung lavage of healthy male Wistar rats which had been exposed to Taiyuan PM2.5, in doses of either 33, 100 or 300 μg/mL in vitro. Cytotoxicity, ROS levels, intracellular Ca2+ concentrations, and mitochondria-mediated apoptosis pathway were evaluated.

Materials and methods

PM2.5 sampling and suspension preparation

PM2.5 sampling and suspension preparation were carried out as described previously by Wei et al. (2019). The sampling site was located at Shanxi University campus, Taiyuan, China (37° 47′ N, 112° 34′ E), and on the roof of the school of environmental science and resources (about 25 m above the ground level). During the severe haze period from December 28, 2011 to January 1, 2012, a PM2.5 high capacity air sampler (Thermo Anderson, USA) with a pump flow of 1.13 m3/min was used to collect PM2.5 for 24 h/day. The PM2.5 sample was collected on a quartz filter membrane (Whatman QMA, UK; 0.3 μm DOP rejection efficiency > 99.995%; withstand high temperature was 500–900 °C and pore size was 2.2 μm). The quartz filters were preheated at 450 °C for 6 h prior to sampling and the dry weights of the quartz filter membranes were recorded before and after sampling.

After that, the quartz filter was cut into strips, soaked in Milli-Q water (18.2 MΩ-cm, Thermo Anderson, USA), and treated with bath sonication for 1 h. The filtered extract was transferred to a lyophilized bottle using a 0.2 μm syringe filter (Thermo Anderson, USA) and frozen at – 80 °C. Before use, the samples were diluted to 10 mg/mL with normal saline (0.9% w/v NaCl, pH = 7).

Animal treatment and cell isolation

All of the animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the Ministry of Health of the People’s Republic of China. The protocol was approved by the Shanxi University’s Institutional Animal Care and Use Committee (Approved Animal Use Protocol Number: HZ20180503). A total of 15 healthy male Wistar rats weighing between 180 and 220 g were purchased from the Animal Center at Hebei Medical University. Prior to PM2.5 exposure, the animals were fed in the laboratory for a week with a 12-h light-dark cycle, at 24 ± 2 °C with a 50 ± 5% humidity. After that, the rats were anesthetized via a single intraperitoneal injection of pentobarbital sodium (2%, 0.2 mL/100 g body weight, Solarbio, China), and the whole lung was cannulated and lavaged with phosphate-buffered saline (PBS pH = 7.4, without Ca2+, Mg2+, and sterilized). The bronchoalveolar lavage fluid (BALF) obtained was centrifuged at 3000 rpm for 10 min at 4 °C (Geng et al. 2006).

As described by previous study (Wei et al. 2019), prior to resuspension, the cells recovered from the lavage process were measured using 0.04% trypan blue dye (Sigma-Aldrich, USA) exclusion to ensure that the cell viability was greater than 95% (Strober 2015). Then, the cells were plated at 105 cells/35 mm in cell petri dishes and incubated in a CO2 incubator for 2 h at 37 °C in 5% CO2 and greater than 95% humidity conditions to enable adherence of AMs (Wei et al. 2019). Afterward, cells were washed twice with 2 mL sterilized saline, and exposed to PM2.5 suspended in fresh RPMI 1640 medium (Hyclone, USA) at a series of concentrations (final concentrations were 0, 33,100, or 300 μg/mL) for another 4 h. Prior to treatment, the, PM2.5 sample underwent ultrasonic treatment for 5 min. The control group was treated with RPMI 1640 at the same volume as test samples. Five replicate wells were conducted for each group.

Assessment of cytotoxicity

The MTT test

Cell viability, following exposure to PM2.5 at the differing concentrations for 4 h, was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test as described by Geng et al. (2005). The cells were seeded in 96-well plates at a density of approximately 2.0 × 105 cells/well. After 4 h, the cells were exposed to normal saline (0.9% w/v NaCl, pH = 7) containing different concentrations of PM2.5 (33, 100 or 300 μg/mL); Triton-X 100 (25 μg/mL) was used as a positive control (Fuentes-Mattei et al. 2010), and normal saline (0.9% w/v NaCl, pH = 7) served as negative controls. After the removal of the culture medium, cells were washed three times in PBS. Then, 20 μL of MTT solution (Sigma-Aldrich, USA) was applied and the cells were cultured for 4 h. The medium was then removed. Further, 150 μL 0.1% dimethyl sulfoxide (DMSO; Sigma-Aldrich, USA), the purification of DMSO was 99%, was added to each well in order to dissolve any formamide salts which had formed. After 10 min, the absorbance was measured for each well at a wavelength of at 570 nm using a Microplate Reader (Bio-Rad Model 550, USA). This experiment was performed in triplicates. The relative viability of cells was calculated according to the following formula:

$$ \mathrm{Relative}\ \mathrm{viability}\ \mathrm{of}\ \mathrm{cells}\ \left(\%\right)=\left(\mathrm{treated}\ \mathrm{cells}\ \mathrm{OD}/\mathrm{untreated}\ \mathrm{cells}\ \mathrm{OD}\right)\times 100\% $$

The lactate dehydrogenase test

The reduction of intracellular LDH and its release into the extracellular medium is a sensitive indicator of nonreversible cell death due to the damaged caused to the cell membrane caused by cell apoptosis or necrosis (Yang et al. 2018). One hundred microliters of cell culture medium supernatant was collected to determine LDH activity. The test was performed according to the LDH assay kit manufacturer’s instructions (Nanjing Jiancheng, China). As described above, after AMs were treated for 4 h with PM2.5 (at 0, 33, 100, or 300 μg/mL), a 100 μL aliquot of cell culture medium supernatant was collected from each dish (105 cells) in order to test extracellular LDH activity. Briefly, 250 μL of the reconstituted substrate mix and 50 μL Coenzyme I solution (Nanjing Jiancheng LDH assay kit) were added to each sample; following incubation in a 37 °C water bath for 15 min, the enzymatic reaction was stopped with 25 μL of 2,4-dinitrohydrazine (Nanjing Jiancheng LDH assay kit). The mixture was incubated again for 15 min in a 37 °C water bath and 2.5 mL NaOH (0.4 mol/L) was added. The absorbance was then measured at 440 nm using an ultraviolet (UV)-visible spectrophotometer (Beckman DU-640B, USA). All experiments were performed in triplicates.

Intracellular ROS measurement

Intracellular ROS generation induced by PM2.5 was detected via flow cytometry using a DCFH-DA dye (Wang et al. 2013). DCFH-DA is a dye which passively enters cells and reacts with the ROS within the cell, and produces a highly fluorescent compound called dichlorofluorescein (DCF) (Sheikh et al. 2017). The ROS Assay Kit (Jiancheng Biology Engineering Institute, Nanjing, China) was used as previously described (LeBel et al. 1992). Briefly, 2 mL working solution of DCFH-DA was added to the AMs (1 × 105 cells/35 mm petri dish) previously exposed to PM2.5 and incubated at 37 °C for 30 min in dark conditions. Next, the DCFH-DA was removed and the cells were washed three times in PBS (pH = 7.4, without Ca2+, Mg2+, sterilized), then resuspended in order to measure fluorescence intensity which was measured by flow cytometry (Becton-Dickison, USA) at excitation/emission wavelengths of 485/530 nm. ROS inhibitor (NAC) was added to the control and 100 μg/mL treated group to explore whether PM2.5 directly resulted in ROS generation. The results were expressed by calculating the relative fluorescence intensities in comparison to the control group. For each well, more than 1 × 105 cells were counted, and all experiments were performed in triplicates.

Cytosolic Ca2+ measurement

Intracellular ROS may affect intracellular Ca2+ homeostasis, induce lipid peroxidation, and DNA damage (Li et al. 2008). To quantify cytosolic Ca2+ in living cells, a cell-permeant probe, Fluo-4/acetoxymethyl ester (Fluo-4/AM, Beyotime Biotechnology), was used in accordance with previously described methods (Gramdordy et al. 1988). Briefly, AMs treated with PM2.5 were washed with three times with PBS, resuspended in fresh RPMI 1640 culture medium containing 4 μM Fluo-4/AM, and incubated for 45 min at 37 °C in the dark. The Fluo-4 AM was then removed, the AMs were washed three times, and resuspended in 500 μL PBS (pH = 7.4, without Ca2+, Mg2+ and sterilized). Cell fluorescence intensity (F) was measured at excitation/emission wavelengths of 485/530 nm using flow cytometry (Thermo Scientific Varioskan Flash, USA). Then, 0.1% Triton-x 100 was added to get the maximum fluorescence value (FMax) and 0.05 mol/L EDTA was added to get the minimum fluorescence value (FMin). The concentration was calculated according to the following formula:

$$ \left[{\mathrm{Ca}}^{2+}\right]={K}_d\ \left(F-{F}_{\mathrm{Min}}\right)/\left({F}_{\mathrm{Max}}-F\right) $$

Kd represents the dissociation constant, the value of which is 450 nmol/L (Gramdordy et al. 1988). All experiments were performed in triplicates.

AM apoptosis detection

The apoptotic rate of AMs was detected using the Annexin V-FITC/PI (propidium iodide) apoptosis measurement assay kit (Yang et al. 2018). Cells were treated with the different doses of PM2.5 for 4 h, then centrifuged at 1200 rpm for 3 min. Cells were then washed twice in pre-cooled PBS (pH to 7.4, without Ca2+, Mg2+, sterilized), and resuspended in 100 μL annexin-binding buffer (50 mM HEPES, 700 mM NaCl, 12.5 mM CaCl2, pH = 7.4). Then 5 μL of Annexin V-FITC and 1 μL propidium iodide (PI) were added (Sigma-Aldrich, USA), and the solution was incubated at 4 °C in the dark for 15 min. The apoptotic and necrotic cells were detected using flow cytometry at excitation/emission wavelengths of 488/525 nm (Millipore, USA) (Napierska et al. 2009). For each exposure group, more than 1 × 105 cells were counted for analysis. The cytogram was divided into four quadrants: the upper left quadrant represents cell fragments, the lower left quadrant represents live cells, the upper right quadrant represents late apoptotic cells, and the lower right quadrant represents early apoptotic cells.

Western blot analysis

The expression of apoptosis-related proteins, including Bcl-2, Bax, caspase-3, and caspase-9, in AMs were determined by Western blot analysis. The protein were extracted from the AMs, previously exposed to PM2.5, using a protein extraction kit (Sigma, USA). Briefly, the cell culture medium was removed, the cells were washed three times with pre-chilled PBS (pH = 7.4, without Ca2+, Mg2+, sterilized), and lysed in ice-cold Radio Immunoprecipitation. Assay buffer containing 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 1% SDS and sodium orthovanadate, sodium fluoride, EDTA, leupeptin, and PMSF (pH = 7.4). Next, the cells were transferred to EP tubes and centrifuged at 13,000 rpm for 15 min at 4 °C. The concentrations of the proteins were detected using a BCA protein quantification kit (Sigma, USA). A standard curve was prepared according to the Bradford method, using bovine serum albumin (BSA) as the standard protein (Bradford 1976). Each protein was quantified to 30–50 μg.

The protein samples were loaded onto SDS-poly-acrylamide gels (12% separation gels; Sigma, USA) and underwent electrophoresis at 110 V for 90 min. Next, the target protein was transferred to the nitrocellulose membrane for 45 min (350 mA) in an ice bath. Then the membranes were incubated in the blocking solution with PBS containing 3% bovine albumin (Solarbio, China) for 1 h. After blocking, the membranes were incubated in rabbit polyclonal antibodies against Bax, or Bcl-2 (1:200, Biosynthesis, China), or rabbit monoclonal antibodies against caspase-3, caspase-9, or β-actin (1:1000, Cell signaling Technology, USA) overnight at 4 °C. The membranes were then washed three times with PBS for 10 min each time, and incubated with fluorescent labeled secondary antibodies (1:5000, LICOR Biosciences Corporation, USA) at room temperature for 1 h. The nitrocellulose membranes were then washed three times (10 min each) with PBS. The membranes were washed again with PBS and were then analyzed with the LI-COR Odysse (LI-COR, USA) infrared scanning fluorescence detection system (Chen et al. 2017). The results were expressed by the ratio of optical density of target protein to β-actin protein.

Statistical analysis

The data were expressed as mean ± standard deviation (mean ± SD). All cell experiments were repeated three times. One-way analysis of variance (ANOVA) and post hoc Tukey’s test were performed to determine differences due to treatment after confirming homoscedasticity and normality for data. Statistically significant differences were recognized at a level of p < 0.05.


Assessment of cytotoxicity

As shown in Fig. 1a, MTT assay results suggested that compared with the unexposed control AMs, no statistically significant impacts of PM2.5 were detected at the concentration of 33 μg/mL exposure group. However, a dose-dependent decrease of AMs viability was observed in both 100 and 300 μg/mL exposure groups compared to control (Fig. 1a; p < 0.05 and p < 0.01, respectively).

Fig. 1

Cytotoxic effects of PM2.5 extracts in rat alveolar macrophages (AMs). Panel a shows the arithmetic means ± SD of group data from MTT cell viability; panel b shows the arithmetic means ± SD of group data from lactate dehydrogenase activity assays. Both assay types were performed in AMs were exposed for 4 h to PM2.5 extracts at 0, 33, 100, or 300 μg/mL. ap < 0.05, aap < 0.01, and aaap < 0.001 vs. control; bp < 0.05, bbp < 0.01, bbbp < 0.001 vs. PM2.5 33 μg/mL; cp < 0.05, ccp < 0.01 vs. PM2.5 100 μg/mL

The levels of LDH released from the PM2.5 exposure AMs significantly increased within the 100 μg/mL or 300 μg/mL PM2.5 exposure groups when compared to the control AMs (Fig. 1b; p < 0.01 and p < 0.001, respectively). The results indicated that the amount of LDH released from the PM2.5 induced AMs was related to the cell ability.

Intracellular ROS generation detection

In order to confirm whether ROS participated in PM2.5-induced AM apoptosis, intracellular ROS was measured using the cell permeable probe DCFH-DA. Following a 4-h exposure to PM2.5, fluorescence intensity significantly increased in the 33, 100, and 300 μg/mL treated groups (Fig. 2a; p < 0.05, p < 0.001, and p < 0.001, respectively). Fluorescence intensity in 100 μg/mL and 300 μg/mL treated groups which were almost 1.5- and 1.6-folds higher respectively than that of the control group (p < 0.001 and p < 0.001). Results revealed that generation of intracellular ROS was caused by PM2.5 in a dose-dependent way. NAC protected the PM2.5-induced ROS generation effectively in AMs (Fig. 2b).

Fig. 2

Effects of PM2.5 extracts on intracellular levels of reactive oxygen species (ROS) in rat alveolar macrophages (AMs). Panel a shows the arithmetic means ± SD of group data from DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) ROS quantification assays performed after AMs were exposed for 4 h to PM2.5 extracts at 0, 33, 100, or 300 μg/mL; panel b shows NAC effectively protect the PM2.5-induced ROS generation in AMs. ap < 0.05, and aaap < 0.001 vs. Control; bp < 0.05, bbp < 0.01 vs. PM2.5 33 μg/mL

Cytosolic Ca2+ measurement

To determine the correlation between activation of ROS and Ca2+ overload in cytoplasm, the levels of cytosolic Ca2+ were detected using a cell-permeant probe Fluo-4/AM. Results from the flow cytometry assay showed that AM exposure to PM2.5 at concentrations of either 33, 100, or 300 μg/mL induced intracellular Ca2+ accumulation in a concentration-dependent manner, with significant increases ranging from 1.6 to 3.5 times the levels found in controls (Fig. 3; p < 0.05, p < 0.01, and p < 0.001, respectively).

Fig. 3

Effects of PM2.5 extracts on cytosolic Ca2+ concentrations in rat alveolar macrophages (AMs). This figure shows the arithmetic means ± standard deviations of group data from Fluo-3 assays performed after AMs were exposed for 4 h to PM2.5 extracts at 0, 33, 100, or 300 μg/mL. ap < 0.05, aap < 0.01, and aaap < 0.001 vs. control; bbp < 0.01 vs. PM2.5 33 μg/mL; cp < 0.05 vs. PM2.5 100 μg/mL

Apoptosis ratio detection

AMs were exposed to different concentrations of PM2.5 (33, 100, or 300 μg/mL) for 4 h; the apoptotic ratio in AMs was then analyzed by flow cytometric Annexin V-FITC/PI analysis (Fig. 4a). As shown in Fig. 4b, compared to control, the apoptotic rate was obviously elevated at concentrations of 100 and 300 μg/mL, but not at 33 μg/mL (p < 0.05, p < 0.001, and p > 0.05, respectively). The data suggested that PM2.5-induced AM apoptosis increased in severity following rising concentrations of PM2.5.

Fig. 4

Apoptotic effects of PM2.5 extracts on rat alveolar macrophages (AMs). Panel a shows cell apoptosis from Annexin V FITC assays. Panel b shows the arithmetic means ± standard deviations of the ratio of cell apoptotic rate. The assay was performed after AMs were exposed for 4 h to PM2.5 extracts at 0, 33, 100, or 300 μg/mL. ap < 0.05, aaap < 0.001 vs. control; bp < 0.05 vs. PM2.5 33 μg/mL

Changes of mitochondria-mediated apoptosis-related protein expression

The expression of the apoptotic-related proteins, Bax, Bcl-2, caspase-3, and caspase-9, were determined by western blot (Fig. 5a). The data showed that Bcl-2 was downregulated and Bax was upregulated following treatment with PM2.5 at concentrations of 33, 100, or 300 μg/mL. Moreover, caspase-3 and caspase-9 were significantly activated (Fig. 5b; p < 0.05). In the 300 μg/mL exposure group, the expression of caspase-3 and caspase-9 were 1.52- and 1.60-folds higher than the control group (p < 0.001). The ratio of Bcl-2 to Bax was significantly decreased in both the 100 and 300 μg/mL treated groups (Fig. 5c; p < 0.01).

Fig. 5

Effects of PM2.5 on proteins related to the mitochondrial-mediated apoptosis pathway. Panel a shows proteins related to the mitochondrial-mediated apoptosis pathway determined by western blot; panel b shows the ratio of caspase-3, casepase-9, Bcl-2, Bax protein expression; panel c shows the ratio of Bax to Bcl-2. The assay was performed after AMs were exposed for 4 h to PM2.5 extracts at 0, 33, 100, or 300 μg/mL. ap < 0.05, aap < 0.01, and aaap < 0.001 vs. control; bp < 0.05, bbp < 0.01 vs. PM2.5 33 μg/mL; ccp < 0.01 vs. PM2.5 100 μg/mL


Previous studies have shown that PM2.5 pollution produces premature death globally and is the largest environmental cause of diseases (Yang et al. 2018). Results of a large population-based cohort study of approximately 1.1 million people over a period of 10 years showed a causal relationship demonstrating a positive association between PM2.5 and lung injuries (Weichenthal et al. 2017), as well as an increased risk of respiratory morbidity and mortality (Pun et al. 2017; Wang et al. 2020). The PM used in this study was < 2.5 μm in diameter, and able to penetrate into the alveolar regions of the lung to damage the AMs (Deng et al. 2013; Draijer and Peters-Golden 2017). A number of toxic effects in AMs caused by PM2.5 exposure have been reported (Chu et al. 2016; Zhao et al. 2016). However, the cellular biological mechanism of PM2.5-induced AM apoptosis is not completely understood.

To obtain better insights into PM2.5-induced AM cytotoxicity, several AM cytotoxicity factors were determined following PM2.5 exposure. The cell viability assay is an important step in measuring cellular responses to toxicants (Smith et al. 2003). LDH is also a marker for common injuries of cell membrane damage (Aung et al. 2011). Firstly, we examined cell viability and LDH release; the results showed that PM2.5 exposure led to a decrease in AM viability and LDH release in a dose-dependent manner (Fig. 1). Similar research previously reported that PM2.5 exposure increased LDH level in rat AMs in a dose-dependent manner (Geng et al. 2006; Yang et al. 2018).

It has been reported that PM2.5 may induce generation of ROS (Wang et al. 2019), and excessive ROS could regulate cellular redox states (Shang et al. 2013), induce lipid peroxidation and DNA strand breaks, cause severe damage to RNA and proteins, and provoke cell death (Gunasekar and Stanek 2011; Morio et al. 2001; Liu et al. 2020). In the present study, the ROS levels showed that PM2.5 induced high levels of ROS in AMs in a dose-dependent manner (Fig. 2). Our previous study showed that PM2.5 reduced the activities of antioxidant enzymes such as CAT and GSH-PX in AM, and increased the content of MDA (Liu et al. 2018a). Similar results were reported in other cells (Liu et al. 2018b). The oxidative stress induced by PM2.5 has been regarded as a significant cytotoxicity response (Yang et al. 2018; Xu et al. 2020). We have determined the chemical composition of PM2.5 used in this study, and the results showed both organic and inorganic heavy metal elements including PAHs and Cd, Pb, Cr, Zn, etc. (Xia et al. 2010). These chemical components may partially affect oxidative stress responses induced by PM2.5. Increased ROS is reportedly a typical phenomenon during mitochondria-dependent apoptosis (Sheikh et al. 2017; Vaux and Korsmeyer 1999). Therefore, it is also possible that ROS can induce AM cytotoxicity and apoptosis.

Mitochondria are the regulatory centers of apoptosis (Espitia-Perez et al. 2018). Excessive ROS can cause oxidative damage to mitochondria, lipid peroxidation of mitochondrial inner membranes, and damage mitochondrial membrane permeability (Yang et al. 2018). Severe mitochondrial damage will cause mitochondria to dysfunction and lead to apoptosis (Zhou et al. 2017). Previous studies showed that PM2.5 damaged the mitochondrial ultrastructure and caused cristae disorder, mitochondrial swelling, and even vacuolation (Qi et al. 2019; Wei et al. 2019). Therefore, we speculate that the damage caused to mitochondrial function will result in apoptosis. In addition, mitochondrion is the most important Ca2+ pool in cells, which is very important for the balance of Ca2+ concentration in the cytoplasm. Ca2+ has long been known to be critically involved in both the initiation and effectuation of cell death (Orrenius et al. 2015). Mitochondrial membrane lipids are attacked by free radicals, lipid peroxidation occurs, which leads to a decrease in Ca2+ uptake by the mitochondria and therefore a cellular increase of Ca2+. Evidence has shown that the release of Ca2+ from mitochondria is closely related to apoptosis (Huang et al. 2018). On the one hand, intracellular overload of Ca2+ can activate Ca2+-dependence phosphatase and decrease intracellular ATP concentration, which causes release of cytochrome C (Cyt C) in mitochondria. Ca2+ can also activate endonuclease and degrade nuclear DNA. On the other hand, Ca2+ overload within the cytoplasm may cause mitochondrial swelling, outer membrane rupture (Redza-Dutordoir and Averill-Bates 2016; Ha et al. 2019), and even fragmentation under special conditions, which in turn can induce cellular apoptosis (Li et al. 2015; Watanabe et al. 2014). In the present study, intracellular ROS and cytoplasmic Ca2+ in the PM2.5 exposed cells significantly increased (Fig. 2 and Fig. 3). These findings suggested that PM2.5 activated the ROS production initially and then destroyed the ultrastructure of mitochondria, induced the release of Ca2+ from mitochondria into cytoplasm, which lead to AM apoptosis.

In this study, the apoptotic ratio increased in a dose-dependent manner as a reaction to PM2.5 (Fig. 4). The results were in accordance with previous studies which suggested that PM2.5 and PM10 can induce apoptosis in macrophages (Huang et al. 2004; Obot et al. 2002). To understand the mechanism of PM2.5-induced apoptosis in AMs, the mitochondria-mediated apoptosis pathway was investigated. The proteins related to mitochondria-mediated apoptosis including caspase-3, caspase-9, Bax, and Bcl-2 were detected by western blot following 4-h exposure to PM2.5. The results showed that the pro-apoptotic proteins caspase-3, caspase-9, and Bax were upregulated and the anti-apoptotic protein Bcl-2 was downregulated. The ratio of Bcl-2 and Bax also declined. Caspase-3 and caspase-9 are important proteins involved in the mitochondria-mediated pathway (Fig. 5). ROS increase, Ca2+ influx, and severe mitochondrial dysfunction could lead to Cyt C release from mitochondria into the cytoplasm (Liu et al. 2018a; Liu et al. 2019). Then, Cyt C and caspase-9 form apoptotic bodies, activate caspase-3, thereby leading to cellular apoptosis (Scorrano and Korsmeyer 2003). Bcl-2 and Bax are located in the mitochondrial membrane; the ratio of Bcl-2 and Bax in the cell determines the formation of mitochondrial outer membrane pores (Huang et al. 2018; Zhang et al. 2010). The decline in the ratio of Bcl-2 and Bax is a sign that the mitochondrial-mediated apoptosis pathway was activated (Chen et al. 2017; Liu et al. 2019). Consequently, the results suggested that PM2.5 triggered apoptosis in AMs by activating the mitochondrial-mediated apoptosis pathway.


In summary, PM2.5 exposure caused increases in intracellular ROS, Ca2+ expression, and the apoptotic ratio of AMs. In addition, apoptotic-related proteins including caspase-3, caspase-9, Bax, and Bcl-2 were upregulated. Therefore, we concluded from these cumulative results that mitochondrial-mediated apoptosis in AMs was a key pathway in PM2.5-triggered cytotoxicity.

Data availability

Not applicable.


  1. Aung HH, Lame MW, Gohil K, He G, Denison MS, Rutledge JC, Wilson DW (2011) Comparative gene responses to collected ambient particles in vitro: endothelial responses. Physiol Genomics 43:917–929.

    CAS  Article  Google Scholar 

  2. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1-2):248-254.

  3. Byrne AJ, Maher TM, Lloyd CM (2016) Pulmonary macrophages: a new therapeutic pathway fibrosing lung disease? Trends Mol Med 22:303–316.

    CAS  Article  Google Scholar 

  4. Chen M, Li B, Sang N (2017) Particulate matter (PM2.5) exposure season-dependently induces neuronal apoptosis and synaptic injuries. J Environ Sci (China) 54:336-345.

  5. Chu X, Liu XJ, Qiu JM, Zeng XL, Bao HR, Shu J (2016) Effects of Astragalus and Codonopsis pilosula polysaccharides on alveolar macrophage phagocytosis and inflammation in chronic obstructive pulmonary disease mice exposed to PM2.5. Environ Toxicol Pharmacol 48:76-84.

  6. Deng X, Fang Z, Rui W, Long F, Wang L, Feng Z, Chen D, Ding W (2013) PM2.5-induced oxidative stress triggers autophagy in human lung epithelial A549 cells. Toxicol In Vitro 27:1762-1770.

  7. Draijer C, Peters-Golden M (2017) Alveolar macrophages in allergic asthma: the forgotten cell awakes. Curr Allergy Asthma Rep 17:12.

  8. Espitia-Perez L, da Silva J, Espitia-Perez P, Brango H, Salcedo-Arteaga S, Hoyos-Giraldo LS, de Souza CT, Dias JF, Agudelo-Castaneda D, Valdes Toscano A, Gomez-Perez M, Henriques JAP (2018) Cytogenetic instability in populations with residential proximity to open-pit coal mine in Northern Colombia in relation to PM10 and PM2.5 levels. Ecotoxicol Environ Saf 148:453–466.

    CAS  Article  Google Scholar 

  9. Fuentes-Mattei E, Rivera E, Gioda A, Sanchez-Rivera D, Roman-Velazquez FR, Jimenez-Velez BD (2010) Use of human bronchial epithelial cells (BEAS-2B) to study immunological markers resulting from exposure to PM(2.5) organic extract from Puerto Rico. Toxicol Appl Pharmacol 243:381–389.

    CAS  Article  Google Scholar 

  10. Galluzzi L, Vitale I, Aaronson SA, Abrams JM, Adam D et al (2018) Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ 25(3):486-541.

  11. Geng H, Meng Z, Zhang Q (2005) Effects of blowing sand fine particles on plasma membrane permeability and fluidity, and intracellular calcium levels of rat alveolar macrophages. Toxicol Lett 157(2):129-137.

  12. Geng H, Meng Z, Zhang Q (2006) In vitro responses of rat alveolar macrophages to particle suspensions and water-soluble components of dust storm PM(2.5). Toxicol In Vitro 20(5):575-584.

  13. Gramdordy BM, Frossard N, Rhoden KJ et al (1988) Tachykinin induced phosphoinositide breakdown in airway smooth muscle and epthelium: relationship to contraction. Mol Pharmacol 33(5):515- 519.

  14. Gunasekar PG, Stanek LW (2011) Advances in exposure and toxicity assessment of particulate matter: an overview of presentations at the 2009 Toxicology and Risk Assessment Conference. Toxicol Appl Pharmacol 254:141–144.

    CAS  Article  Google Scholar 

  15. Ha J, Lee D, Lee S, Yun C, Kim Y (2019) Ddvelopment of apoptosis-inducing polypeptide via simutaneous mitochondrial membrane disruption and Ca2+ delivery. Biomaterials 197: 51-59.

  16. Hsieh SF, Chou CT, Liang WZ, Kuo CC, Wang JL, Hao LJ, Jan CR (2018) The effect of magnolol on Ca2+ homeostasis and its related physiology in human oral cancer cells. Arch Oral Biol 89:49-54.

  17. Huang YC, Li Z, Harder SD, Soukup JM (2004) Apoptotic and inflammatory effects induced by different particles in human alveolar macrophages. Inhal Toxicol 16(14):863-878.

  18. Huang Z, Liu L, Chen J, Cao M, Wang J (2018) JS-K as a nitric oxide donor induces apoptosis via the ROS/Ca2+/caspase-mediated mitochondrial pathway in HepG2 cells. Biomed Pharmacother 107:1385–1392.

    CAS  Article  Google Scholar 

  19. Hu G, Christman JW (2019) Editorial: alveolar macrophages in lung inflammation and resolution. Front Immunol 10:2275.

  20. Joshi N, Walter JM, Misharin AV (2018) Alveolar macrophages. Cell Immunol 330:86–90.

    CAS  Article  Google Scholar 

  21. LeBel CP, Ischiropoulos H, Bondy SC (1992) Evaluation of the probe 2’,7’-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem Res Toxicol 5(2):227-231.

  22. Lelieveld J, Pozzer A, Pöschl U, Fnais M, Haines A, Münzel T (2020) Loss of life expectancy from air pollution compared to other risk factors: a worldwide perspective. Cardiovasc Res. 116(11):1910-1917.

  23. Li N, Xia T, Nel AE (2008) The role of oxidative stress in ambient particulate matter-induced lung diseases and its implications in the toxicity of engineered nanoparticles. Free Radic Biol Med 44(9):1689-1699.

  24. Li R, Kou X, Geng H, Xie J, Yang Z, Zhang Y, Cai Z, Dong C (2015) Effect of ambient PM(2.5) on lung mitochondrial damage and fusion/fission gene expression in rats. Chem Res Toxicol 28(3):408-418.

  25. Liu CW, Lee TL, Chen YC, Liang CJ, Wang SH, Lue JH, Tsai JS, Lee SW, Chen SH, Yang YF, Chuang TY, Chen YL (2018a) PM2.5-induced oxidative stress increases intercellular adhesion molecule-1 expression in lung epithelial cells through the IL-6/AKT/STAT3/NF-kappaB-dependent pathway. Part Fibre Toxicol 15(1):4.

  26. Liu J, Liang S, Du Z, Zhang J, Sun B, Zhao T, Yang X, Shi Y, Duan J, Sun Z (2019) PM2.5 aggravates the lipid accumulation, mitochondrial damage and apoptosis in macrophage foam cells. Environ Pollut 249:482–490.

    CAS  Article  Google Scholar 

  27. Liu X, Zhao X. Li X, Lv S, Ma R, Qi Y, Abulikemu A, Duan H, Guo C, Li Y, Sun Z (2020) PM2.5 triggered apoptosis in lung epithelial cells through the mitochondrial apoptotic way mediated by ROS-DRP1-mitochondrial fission axis. J Hazard Mater 397:122608.

  28. Liu Y, Sun J, Gou Y, Sun X, Li X, Yuan Z, Kong L, Xue F (2018b) A multicity analysis of the short-term effects of air pollution on the chronic obstructive pulmonary disease hospital admissions in Shandong, China. Int J Environ Res Public Health 15(4):774.

  29. Morio LA, Hooper KA, Brittingham J, Li TH, Gordon RE, Turpin BJ, Laskin DL (2001) Tissue injury following inhalation of fine particulate matter and hydrogen peroxide is associated with altered production of inflammatory mediators and antioxidants by alveolar macrophages. Toxicol Appl Pharmacol 177(3):188–199.

    CAS  Article  Google Scholar 

  30. Napierska D, Thomassen LCJ, Rabolli V, Lison D, Gonzalez L, Kirsch-Volders M, Martens JA, Hoet PH (2009) Size-dependent cytotoxicity of monodisperse silica nanoparticles in human endothelial cells. Small 5:846–853.

    CAS  Article  Google Scholar 

  31. Obot CJ, Morandi MT, Beebe TP, Hamilton RF, Holian A (2002) Surface components of airborne particulate matter induce macrophage apoptosis through scavenger receptors. Toxicology and Applied Pharmacology 184:98–106.

    CAS  Article  Google Scholar 

  32. Orrenius S, Gogvadze V, Zhivotovsky B (2015) Calcium and mitochondria in the regulation of cell death. Biochem Biophys Res Commun 460:72-81.

  33. Pérez-Díaz JL, Ivanov O, Peshev Z, Álvarez-Valenzuela MA, Valiente-Blanco I, Evgenieva T, Dreischuh T, Gueorguiev O, Todorov PV, Vaseashta A (2017) Fogs: physical basis, characteristic properties, and impacts on the environment and human health. Water 9:807–828.

    CAS  Article  Google Scholar 

  34. Pinkerton KE, Chen CY, Mack SM, Upadhyay P, Wu CW, Yuan W (2019) Cardiopulmonary health effects of airborne particulate matter: correlating animal toxicology to human epidemiology. Toxicol Pathol 47:954–961.

    CAS  Article  Google Scholar 

  35. Pope CA, Dockery DW (2006) Health effects of fine particulate air pollution: lines that connect. J Air Waste Manag Assoc 56:709-742.

  36. Pozzer A, Dominici F, Haines A, Witt C, Münzel T, Lelieveld J (2020) Regional and global contributions of air pollution to risk of death from COVID-19. Cardiovasc Res. 116:2247–2253.

    Article  Google Scholar 

  37. Pun VC, Kazemiparkouhi F, Manjourides J, Suh HH (2017) Long-term PM2.5 exposure and respiratory,cancer, and carddiovascular mortality in older US adults. Am J Epidemiol 186:961-969., Long-Term PM2.5 Exposure and Respiratory, Cancer, and Cardiovascular Mortality in Older US Adults

  38. Qi Z, Song Y, Ding Q, Liao X, Li R, Liu G, Tsang S, Cai Z (2019) Water soluble and insoluble components of PM2.5 and their functional cardiotoxicities on neonatal rat cardiomyocytes in vitro. Ecotoxicol Environ Saf 168:378–387.

    CAS  Article  Google Scholar 

  39. Redza-Dutordoir M, Averill-Bates DA (2016) Activation of apoptosis signalling pathways by reactive oxygen species. Biochim Biophys Acta 1863:2977–2992.

    CAS  Article  Google Scholar 

  40. Reed JC (2000) Mechanisms of apoptosis. American Journal of Pathology 157:1415–1430.

    CAS  Article  Google Scholar 

  41. Reyes-Zarate E, Sanchez-Perez Y, Gutierrez-Ruiz MC, Chirino YI, Osornio-Vargas AR, Morales-Barcenas R, Souza-Arroyo V, Garcia-Cuellar CM (2016) Atmospheric particulate matter (PM10) exposure-induced cell cycle arrest and apoptosis evasion through STAT3 activation via PKCzeta and Src kinases in lung cells. Environ Pollut 214:646–656.

    CAS  Article  Google Scholar 

  42. Scorrano L, Korsmeyer SJ (2003) Mechanisms of cytochrome c release by proapoptotic BCL-2 family members. Biochemical and Biophysical Research Communications 304:437-444.

  43. Shang Y, Sun Z, Cao J, Wang X, Zhong L, Bi X, Li H, Liu W, Zhu T, Huang W (2013) Systematic review of Chinese studies of short-term exposure to air pollution and daily mortality. Environ Int 54:100-111.

  44. Sheikh BY, Sarker MMR, Kamarudin MNA, Mohan G (2017) Antiproliferative and apoptosis inducing effects of citral via p53 and ROS-induced mitochondrial-mediated apoptosis in human colorectal HCT116 and HT29 cell lines. Biomed Pharmacother 96:834-846.

  45. Smith KR, Kim S, Recendez JJ, Teague SV, Ménache MG, Grubbs DE, Sioutas C, Pinkerton KE (2003) Airborne particles of the california central valley alter the lungs of healthy adult rats. Environ Health Perspect 111(7):902–908.

    Article  Google Scholar 

  46. Steinberg F, Rehn B, Kraus R, Quabeck K, Bruch J, Beelen DW, Schaefer UW, Streffer C (1992) Activity testing of alveolar macrophages and changes in surfactant phospholipids after irradiation in bronchoalveolar lavage: experimental and clinical data. Environ Health Perspect 97:171-175.

  47. Strober W (2015) Trypan blue exclusion test of cell viability. Curr Protoc immunol 111:A3.B.1-A3.B.3.

  48. Tian H, Shakya A, Wang F, Wu WD, LI W (2020) Comparative ligandomic analysis of human lung epithelial cells exposed to PM2.5 Biomed Environ Sci 33:165-173.

  49. Vaux DL, Korsmeyer SJ (1999) Cell death in development. Cell 96(2):245–254.

    CAS  Article  Google Scholar 

  50. Wang C, Meng X, Meng M, Shi M, Sun W, Li X, Zhang X, Liu R, Fu Y, Song L (2020) Oxidative stress actives the TRPM2-Ca2+-NLRP3 axis to promote PM2.5-induced lung injury of mice. Biomed & Pharmacother 130:110481.

  51. Wang D, Pakbin P, Shafer MM, Antkiewicz D, Schauer JJ, Sioutas C (2013) Macrophage reactive oxygen species activity of water-soluble and water-insoluble fractions of ambient coarse, PM2.5 and ultrafine particulate matter (PM) in Los Angeles. Atmospheric Environ 77:301–310.

    CAS  Article  Google Scholar 

  52. Wang L, Xu J, Liu H, Li J, Hao J (2019) PM2.5 inhibits SOD1 expression by up-regulating microRNA-206 and promotes ROS accumulation and disease progression in asthmatic mice. Int Immunopharmacol 76:105871.

    CAS  Article  Google Scholar 

  53. Watanabe K, Hosono T, Watanabe K, Hosono-Fukao T, Ariga T, Seki T (2014) Diallyl trisulfide induces apoptosis in Jurkat cells by the modification of cysteine residues in thioredoxin. Biosci Biotechnol Biochem 78(8):1418–1420.

    CAS  Article  Google Scholar 

  54. Wei F, Xiong L, Li W, Wang X, Hong X, Chen B (2020) Relationship between short-term exposure to PM2.5 and daily lung cancer mortality in Nanjing. Biomed Environ Sci 33:547–551

    Google Scholar 

  55. Wei H, Zhang Y, Song S, Pinkerton KE, Geng H, Ro CU (2019) Alveolar macrophage reaction to PM2.5 of hazy day in vitro: Evaluation methods and mitochondrial screening to determine mechanisms of biological effect. Ecotoxicol Environ Saf 174:566-573.

  56. Weichenthal S, Bai L, Hatzopoulou M, Van Ryswyk K, Kwong JC, Jerrett M, van Donkelaar A, Martin RV, Burnett RT, Lu H, Chen H (2017) Long-term exposure to ambient ultrafine particles and respiratory disease incidence in in Toronto, Canada: a cohort study. Environ Health 16(1):64.

    CAS  Article  Google Scholar 

  57. WHO (2019): How air pollution is destroying our health,

  58. Wu B, Dong YX, Wang M, Yang WH, Hu LF, Zhou DS, Lv J, Chai TJ (2020) Pathological damage, immune-related protein expression, and oxidative stress in lungs of BALB/c mice induced by haze PM2.5 biological components exposure. Atmos Environ 223:117230.

  59. Wu S, Deng F, Wang X, Wei H, Shima M, Huang J, Lv H, Hao Y, Zheng C, Qin Y, Lu X, Guo X (2013) Association of lung function in a panel of young healthy adults with various chemical components of ambient fine particulate air pollution in Beijing, China. Atmospheric Environ 77:873–884.

    CAS  Article  Google Scholar 

  60. Xia Z, Duan X, Qiu W, Liu D, Wang B, Tao S, Jiang Q, Lu B, Song Y, Hu X (2010) Health risk assessment on dietary exposure to polycyclic aromatic hydrocarbons (PAHs) in Taiyuan, China. Sci Total Environ 408(22):5331–5337.

    CAS  Article  Google Scholar 

  61. Xu F, Shi X,Qiu X, Jiang X, Fang Y, Wang J, Hu D, Zhu T (2020) Investigation of the chemical components of ambient fine particulate matter (PM2.5) associated with in vitro cellular responses to oxidative stress an inflammation. Environ Int 136:105475.

  62. Yang X, Feng L, Zhang Y, Hu H, Shi Y, Liang S, Zhao T, Fu Y, Duan J, Sun Z (2018) Cytotoxicity induced by fine particulate matter (PM2.5) via mitochondria-mediated apoptosis pathway in human cardiomyocytes. Ecotoxicol Environ Saf 161:198-207.

  63. Zhang J, Song W, Guo J, Zhang J, Sun Z, Ding F, Gao M (2012) Toxic effect of different ZnO particles on mouse alveolar macrophages. J Hazard Mater 219-220:148–155.

    CAS  Article  Google Scholar 

  64. Zhang Y, Zhu T, Hu M, Xue L, Huang X, He L (2010) On-line measurement of organic aerosol elemental composition based on high resolution aerosol mass spectrometry. Chinese Science Bulletin 55:3391-3396.

  65. Zhao C, Wang Y, Su Z, Pu W, Niu M, Song S, Wei L, Ding Y, Xu L, Tian M, Wang H (2020) Respiratory exposure to PM2.5 soluble extract disrupts mucosal barrier function adn promotes the development of experimental asthma. Sci Total Environ 730:139145.

  66. Zhao Q, Chen H, Yang T, Rui W, Liu F, Zhang F, Zhao Y, Ding W (2016) Direct effects of airborne PM2.5 exposure on macrophage polarizations. Biochim Biophys Acta 1860:2835-2843.

  67. Zhou W, Tian D, He J, Zhang L, Tang X, Zhang L, Wang Y, Li L, Zhao J, Yuan X, Peng S (2017) Exposure scenario: another important factor determining the toxic effects of PM2.5 and possible mechanisms involved. Environ Pollut 226:412–425.

    CAS  Article  Google Scholar 

  68. Zhu R, Nie X, Chen Y, Chen J, Wu S, Zhao L (2020) Relationship between particulate matter (PM2.5) and hospitalizations and mortality of chronic obstructive pulmonary disease patients: a meta-analysis. Am J Med Sci 359:354–364.

    Article  Google Scholar 

Download references


The authors would like to express their gratitude to EditSprings ( for the expert linguistic services provided.


This work was supported by the National Nature Science Foundation of China (No. 21177078), and the Natural Science Foundation of Shanxi Province (No. 201901D111005).

Author information




The experiment was conceived and supervised by HW. HW and WY wrote the manuscript. WY and HY conducted the animal experiments and other biological experiment analysis. HG analyzed the experimental results. HW and HY contributed to the analysis of the data. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Haiying Wei.

Ethics declarations

Competing interests

The authors declare that they have no competing interests.

Ethics approval and consent to participate

Current study which involves animal subjects has been reviewed and approved by Shanxi University’s Institutional Animal Care and Use Committee (Approved Animal Use Protocol Number: HZ20180503).

Consent for publication

Not applicable.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Responsible Editor: Lotfi Aleya

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wei, H., Yuan, W., Yu, H. et al. Cytotoxicity induced by fine particulate matter (PM2.5) via mitochondria-mediated apoptosis pathway in rat alveolar macrophages. Environ Sci Pollut Res (2021).

Download citation


  • Particulate matter
  • Alveolar macrophage
  • Cytotoxicity
  • Apoptosis
  • Mitochondria-mediated apoptosis pathway
  • Caspase-3
  • Caspase-9