Particulate matter 2.5 triggers airway inflammation and bronchial hyperresponsiveness in mice by activating the SIRT2--p65 pathway

Abstract

Exposure to particulate matter 2.5 (PM2.5) potentially triggers airway inflammation by activating nuclear factor-κB (NF-κB). Sirtuin 2 (SIRT2) is a key modulator in inflammation. However, the function and specific mechanisms of SIRT2 in PM2.5-induced airway inflammation are largely understudied. Therefore, this work investigated the mechanisms of SIRT2 in regulating the phosphorylation and acetylation of p65 influenced by PM2.5-induced airway inflammation and bronchial hyperresponsiveness. Results revealed that PM2.5 exposure lowered the expression and activity of SIRT2 in bronchial tissues. Subsequently, SIRT2 impairment promoted the phosphorylation and acetylation of p65 and activated the NF-κB signaling pathway. The activation of p65 triggered airway inflammation, increment of mucus secretion by goblet cells, and acceleration of tracheal stenosis. Meanwhile, p65 phosphorylation and acetylation, airway inflammation, and bronchial hyperresponsiveness were deteriorated in SIRT2 knockout mice exposed to PM2.5. Triptolide (a specific p65 inhibitor) reversed p65 activation and ameliorated PM2.5-induced airway inflammation and bronchial hyperresponsiveness. Our findings provide novel insights into the molecular mechanisms underlying the toxicity of PM2.5 exposure. Triptolide inhibition of p65 phosphorylation and acetylation could be an effective therapeutic approach in averting PM2.5-induced airway inflammation and bronchial hyperresponsiveness.

Keywords

particulate matter 2.5 / sirtuin 2 / p65 / airway inflammation / bronchial hyperresponsiveness / triptolide

1 Introduction

Air pollutants comprise a complex mixture of airborne particles such as gases, liquids, and particulate matter (PM). In developing countries, the harmful effects of air pollution are a result of rapid industrial growth and economic expansion. Recent reports indicate that PM is the primary cause of cardiovascular and respiratory diseases. Notably, PM has a vast array of potential sources, including motor vehicles, coal, residual oil, and particles derived from the Earth’s crust. Specifically, in industrialized nations, anthropogenic activities represent the primary source of PM. These air contaminants potentially trigger a cascade of detrimental health impacts in the whole body systems via their proinflammatory effects [1]. Particulate matter 2.5 (PM2.5) refers to fine particles with aerodynamic equivalent diameter less than or equal to 2.5 µm in ambient air. It is one of the most harmful air pollutants that can be deposited in bronchi and alveoli through inhalation. Moreover, PM2.5 can even penetrate the air–blood barrier into the blood circulation [2–5]. Epidemiological studies have shown a relationship between PM2.5 exposure and adverse health outcomes, including respiratory diseases, cardiovascular diseases, type 2 diabetes, and autoimmune diseases [5–8]. Nevertheless, the mechanisms underlying PM2.5-induced airway inflammation remain largely unexplored.

Accumulating evidence suggests an essential role of nuclear factor-κB (NF-κB) activation in PM2.5-induced proinflammatory responses [9–11]. The activation of NF-κB is regulated by a variety of post-translational modifications including the phosphorylation and acetylation of RelA/p65 subunit. These modifications determine the duration and strength of NF-κB nuclear activity and transcriptional output [12]. The phosphorylation of p65 at Ser-276 weakens the intramolecular interaction of p65, thereby facilitating the binding of p65 to co-activator CBP/p300. The phosphorylation of p65 Ser-536 also promotes the recruitment of CBP/p300 to p65. Subsequently, the binding of p65 and CBP/p300 induces the upregulation of NF-κB transcription. Furthermore, the acetylation of p65 at Lys-310 is essential for full activation of p65 transcription [13]. Therefore, inhibiting p65 phosphorylation and acetylation can exert NF-κB inhibition and anti-inflammatory effects [14]. However, the underlying mechanism of PM2.5-induced inflammatory injury remains unknown. Here, we hypothesized that inhibiting the acetylation and phosphorylation of p65 potentially mitigates tissue damage and bronchial inflammation following exposure to PM2.5.

Sirtuin 2 (SIRT2), a NAD-dependent class I histone deacetylase, is the primary co-enzyme implicated in the deacetylation of p65 Lys (310) [15]. SIRT2 inhibits NF-κB activation, thereby blocking the release of inflammatory cytokines and alleviating inflammatory responses [16,17]. Moreover, exposure to PM2.5 during postnatal development periods significantly lowers the expression of SIRT2 in the heart tissues of adult mice, resulting in cardiac dysfunction [18]. Thus, we hypothesized that the downregulation of SIRT2 potentially facilitates PM2.5-induced airway inflammation and bronchial injury. Therefore, understanding the molecular mechanisms of the SIRT2–p65 pathway and targeting this pathway to decrease PM2.5-induced airway inflammation might provide novel therapeutic strategies. However, due to the absence of SIRT2 agonists, inhibiting the transcriptional activity of NF-κB might be an effective strategy in controlling PM2.5-induced airway inflammation and bronchial hyperresponsiveness. Triptolide, a small-molecule inhibitor extracted from the natural Chinese herb Tripterygium wilfordii, can block the interaction of p65 to p300/CBP, inhibit the degradation and phosphorylation of IκBα, and reduce the transcription activity of NF-κB [19,20]. Thus, we also explored the inhibition effects of triptolide on p65 phosphorylation and acetylation to alleviate the PM2.5-induced airway inflammation and bronchial hyperresponsiveness. Our study revealed that PM2.5 exposure triggered airway inflammation and bronchial hyperresponsiveness. Mechanistically, PM2.5 exposure lowered the expression and activity of SIRT2 in bronchial tissues. Notably, SIRT2 directly interacted with p65 and regulated the phosphorylation and acetylation activation of p65 to initiate the NF-κB signaling pathway and airway inflammation. Thereafter, the airway inflammation induced thickening in the bronchial smooth muscle layer and basement membrane layer, increased goblet cell proliferation and mucus secretion, tracheal stenosis, and bronchial hyperresponsiveness. Importantly, we found that triptolide, an inhibitor of p65, significantly inhibited PM2.5-induced p65 phosphorylation and acetylation, thereby reducing airway inflammation and bronchial hyperresponsiveness. Our findings provide a novel mechanism underlying PM2.5-induced airway inflammation and bronchial hyperresponsiveness.

2 Methods

2.1 Particle collection and preparation

PM2.5 samples were prepared using protocols from previous research [21,22]. PM2.5 samples were continuously collected between September and December 2018 in Xi’an, China. Fourth Military Medical University Campus was the sampling site, which was not in proximity to large industrial and thermoelectric plants. The sampling site was also far from streets characterized by moderate traffic and commercial activities. Aerosol samplers (Guangzhou Mingye Technology Company, China) were fixed at a platform about 10 m from the ground. PM2.5 samples were collected on quartz filters (8 cm × 10 cm, 2500 QAT-UP, Pallflex Products, Putnam, CT, USA) by the aerosol samplers at a flow rate of 18 m³/h for 12 h (8:00 to 20:00). During the sampling period, a total of 78 samples were collected.

Before sampling, residual organic contaminants were removed by wrapping the quartz filters in aluminum foil and burning in a muffle furnace at 450 °C for 4 h. Subsequently, the quartz filters were equilibrated under stable temperature and humidity (20±1 °C, 45%±5%) for at least 24 h and weighed in a high-precision microbalance. All sampled filters were stored in the dark at –20 °C before sample collection. After sampling, all filters were weighed again using the same analytical balance under the same temperature and humidity conditions to obtain sample mass concentrations. Then, the weighed samples were stored in a refrigerator for follow-up extraction.

The extraction of PM2.5 samples on quartz filters was performed in accordance with previously published protocols [3,23]. Quartz filters were cut into square pieces and immersed in 2 mmol/L sterile phosphate buffered saline (PBS, pH 7.4, prepared with pyrogen-free water) for 30 min, followed by 45 min sonication with an ultrasonic shaker to elute PM. The particles were filtered and concentrated using a vacuum–freeze dry method. The extracted PM2.5 was weighed and stored at –20 °C before being diluted for use in subsequent experiments.

2.2 Scanning electron microscopy, particle size analysis, and component analysis

As previously described [24,25], the size distribution of PM2.5 was determined under a scanning electron microscope (SEM, Model S-4800 Hitachi, Tokyo, Japan) at an accelerating voltage of 3 kV. Then, PM2.5 was suspended and sonicated to disperse its contents using an ultrasonic processor (VCX130, Sonics, Newtown, CT, USA). The suspended PM2.5 was analyzed using a Nano-Zetasizer (1000 HS; Malvern Instrument Ltd., Worcestershire, UK) based on a dynamic light scattering measurement technique to obtain its size distribution. The particle Z-average was reported.

The composition and quantity of the primary chemical components in PM2.5, including organic and elemental carbon (OC and EC), water-soluble inorganic ions, inorganic elements, and polycyclic aromatic hydrocarbons (PAHs), were evaluated. Detailed descriptions of the application of these analytical techniques for the extraction of PM samples were described by Bein and Wexler [26,27]. To suit the experimental concentrations, concentrated PM2.5 samples were diluted with sterile 0.9% saline.

2.3 Animal experiments

Male SIRT2 knockout (SIRT2 KO) mice and their wild-type littermates (WT) on a C57BL/6J background were housed in a standard animal facility maintained at a 12-h dark/light photoperiod and allowed access to adequate food and water. Male SIRT2 KO mice (C57BL/6 background) were provided by Dr. Baohua Liu (Health Science Center, Shenzhen University, China). SIRT2 KO mice were backcrossed into the C57BL/6J background (generation N10) at the animal resource center. All experimental procedures were approved by the Animal Use and Care Committee of the Fourth Military Medical University.

All mice (wild type/WT and SIRT2^−/−) were first allowed to adapt to the environment for a week before the commencement of experiments. After adaptation, mice were exposed to a PM2.5 inhalation exposure system, as previously described [28]. The PM2.5 inhalation system comprised a plexiglass container (20 cm×15 cm×10 cm) connected to a liquid aerosol generator (HRH-WAG6, Hui Ronghe Ltd., Beijing, China) that produced particles with aerodynamic diameters less than 2.5 µm. The particle concentration was measured using a PM2.5 detector (PC-3A, Sujing Instrument Equipment Co., Ltd., Jiangsu, China). The release of PM2.5 to the environment was mitigated by placing a high-efficiency particulate air filter at the outlet of the container designated for inside–outside air exchange.

Mice were placed in the container, and free movement was allowed. The vessel was infused with either PM2.5 (101.5±2.3 µg/m³, flow rate of 75 L/min) or filtered air for 6 h/day for 28 consecutive days. The dose of PM2.5 was adjusted based on previously published experiments [29]. After PM2.5 exposure, mice were harvested at different time points (days 7, 14, 21, or 28) or 4 weeks later for subsequent biochemical analysis. Some mice were subjected to a daily intraperitoneal injection of triptolide (purity≥99%; catalog No. S3604, Selleck, China) at a dose of 0.07 mg/kg body weight, as previously described [30]. This daily dose was administered for 28 consecutive days before the start of the PM2.5 exposure experiments.

2.4 Bronchial hyperresponsiveness

Pulmonary function testing, particularly airway resistance, was evaluated using a FinePoint noninvasive airway mechanics system (Buxco Electronics, Wilmington, NC, USA) as described in previous studies [31,32]. Unrestrained conscious mice were placed in a double chamber plethysmograph, consisting of a nasal (head) chamber and a thoracic (body) chamber. The nasal chamber was a means for delivering aerosolized methacholine (catalog No. A2251, Sigma-Aldrich, St. Louis, MO, USA). Each mouse received nebulized PBS for 1 min, followed by increasing concentrations of methacholine (6.25, 12.5, 25, and 50 mg/mL) to induce bronchoconstriction. Subsequently, increasing doses of aerosolized methacholine were delivered for 1 min, and the response to each treatment was measured for 3 min. Then, airway resistance was recorded as enhanced pause (Penh). Notably, airway resistance is a dimensionless value calculated from the proportion of maximal expiratory to maximal inspiratory box pressure and pause, which indicates the timing of expiration.

2.5 Preparation of bronchoalveolar lavage fluid (BALF) and cell counts

Mice were euthanized to obtain BALF in accordance with a previously described method [33]. Each mouse’s lung was lavaged three times with 1 ml of PBS, and 90% of the total injected PBS volume was consistently recovered. The PBS was pooled and pelleted by centrifugation at 520×g at 4 °C for 20 min, and the supernatant was frozen at –80 °C for subsequent cell counts and cytokine assessment.

Individual lavage fluids were pooled, and the cells were collected by centrifugation (5000 rpm, 4 °C, for 5 min) and resuspended in 200 µL of PBS. Cell viability was determined by staining with trypan blue (catalog No. 15250061, Thermo Fisher Scientific, MA, USA), and the total number of nucleated cells was counted using a hemocytometer. Differential BALF cell counts were determined microscopically after staining with Diff-Quick (catalog No. B4132-1A, Dade Behring, DE, USA) and calculated as percentage of each cell type [34].

2.6 Analysis of cytokine concentrations in BALF

Quantities of cytokines such as interleukin IL-4 (catalog No. PM4000B), IL-5 (catalog No. PM5000), IL-6 (catalog No. PM6000B), and tumor necrosis factor-α (TNF-α, catalog No. PMTA00B) in BALF were determined from BALF supernatant by ELISA kits (R&D Systems, USA) following the manufacturer’s instructions.

2.7 Lung histology

After BALF collection, the lung was inflated using a fixed volume of formalin. Then, the superior lobe of the right lung was fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned. Then, the sections were stained with hematoxylin–eosin (HE) for inflammation assessment, periodic acid–Schiff (PAS) for the analysis of goblet cells of the bronchial mucosa, and Masson’s trichrome for collagen deposition measurement. All staining procedures were performed in accordance with the manufacturer’s protocol. Pathological changes in the lung and bronchial tissues were observed, and images were captured using a light microscope (Nikon, Japan). Scoring was performed by an observer in a blinded manner. Lung inflammation was evaluated by semiquantitative evaluation of bronchial inflammatory cell infiltration (0, none; 1, mild; 2, moderate; 3, marked; 4, severe). The proportion of goblet cells in airway epithelial cells (0, none; 1,<25%; 2, 25%–50%; 3, 51%–75%; 4,>75%) and positively stained areas of collagen in the lung and bronchial tissues were evaluated and analyzed using Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) image analysis software.

2.8 Nuclear–cytoplasmic protein extraction, Western blot, and co-immunoprecipitation (Co-IP) analysis

Lung tissue proteins were extracted and homogenized and quantified using a BCA Protein Assay Kit (catalog No. 23227, Thermo Fisher Scientific, USA). Proteins were resolved via vertical gel electrophoresis and transferred to the PVDF membrane. Subsequently, the PVDF membrane was incubated overnight at 4 °C with antibodies, HRP-conjugated IgG, and enhanced chemiluminescence solution in sequence. The immunoblots were analyzed by a gel imaging system (Bio-Rad, CA, USA). The expression levels of SIRT2, phospho-p65, acetyl-p65, p65, and iNOS were detected. Meanwhile, some lung tissues were harvested, and nuclear–cytoplasmic extraction was performed in accordance with the protocol of the NE–PER Nuclear and Cytoplasmic Extraction Reagents Kit (No. 78835, Thermo Fisher Scientific, USA) as previously described [35]. The levels of p65 in the nucleus or cytoplasm were checked to assess the activity of NF-κB. Some lung tissue proteins were used for Co-IP analysis, which was performed using the Co-Immunoprecipitation Kit (catalog No. 26149, Thermo Fisher Scientific, USA) following the manufacturer’s instructions. Briefly, the SIRT2 antibody was immobilized to the coupling resin. The lung tissue lysates were pre-cleaned and incubated overnight at 4 °C with the immobilized resin. Then, the proteins were eluted and prepared for Western blot analysis. The rabbit anti-SIRT2 (catalog No. ab51023) and rabbit anti-iNOS (catalog No. ab178945) antibodies were purchased from Abcam (Cambridge, UK). The rabbit anti-p65 (catalog No. #8242), rabbit anti-phospho-p65 (Ser536, catalog No. #3033), rabbit anti-acetyl-p65 (Lys310, catalog No. #12629), and rabbit anti-β-actin (catalog No. #8457) were purchased from Cell Signaling Technology (USA).

2.9 SIRT2 activity

The SIRT-Glo^TM Assay (catalog No. G6450, Promega, USA) was used to determine SIRT2 activity. The SIRT2 protein from lung tissue lysate was collected by immunoprecipitation, eluted, quantitated, and diluted with SIRT-Glo^TM Buffer in gradient concentration. The SIRT-Glo^TM substrate and reagents were added to the 96-well plate. The plate was incubated at room temperature for 30 min. Thereafter, the activity of SIRT2 was quantified using a spectrophotometer.

2.10 Measurement of reactive oxygen species (ROS)

Dihydroethidium (DHE) staining (catalog No. D23107, Thermo Fisher Scientific, USA) was used to generate pulmonary ROS. The lower lobes of the right lungs were collected, embedded in optimal cutting temperature compound, frozen in liquid nitrogen, and cryo-sectioned into 14-µm slices. The sections were then incubated with 5 µmol/L of DHE at 37 °C for 10 min protected from light. DHE fluorescence was observed with a confocal microscope (Olympus, Japan) and quantified using Image-Pro 6.0 software.

2.11 Statistical analysis

Data analysis was performed by GraphPad Prism software version 6.0 (GraphPad Software, San Diego, CA, USA). Quantitative data were presented as mean±SD. Differences among treatments were compared through one-way analysis of variance, and means were separated with Turkey’s multiple comparisons test. Statistical significance was considered at P<0.05.

3 Results

3.1 Characterization of PM2.5

The morphological characteristics of PM2.5 observed under a SEM are shown in Fig. 1A. The median diameter of PM2.5 particles was 0.17±0.09 µm, and their sizes ranged between<0.03 µm and 3 µm (Fig. 1B). The composition and quantity of the primary chemical components in PM2.5 were determined. The mass fractions corresponding to different components are summarized in Fig. 1C. OC accounted for 25.72% of the total PM2.5 mass, whereas EC accounted for 4.37% of the total PM2.5 mass. Water-soluble inorganic ions had a mass fraction of 22.56%, whereas inorganic elements accounted for 7.23% of the total PM2.5. PAHs, which were considered as the primary pollutants in PM2.5, had a mass fraction of 34.28%. However, the remaining 5.84% of the total PM2.5 mass could not be identified by the analytical techniques adopted in this study.

3.2 PM2.5 exposure induced airway inflammation and bronchial hyperresponsiveness

To evaluate the impact of PM2.5 exposure on the morphological abnormality and dysfunction of the airway, several parameters related to airway inflammation and bronchial hyperresponsiveness were determined. HE staining of lung tissues is shown in Fig. 2A. The control group exhibited normal lung tissue structure and clear pulmonary alveoli. PM2.5 exposure induced gradual alveoli and bronchial injuries. The inflammation injury was characterized by hemorrhage, edema, thickened alveolar wall, dot and patchy infiltration, smooth muscle layer hypertrophy, narrowed lumen, and gradual muscularization of the bronchus. Following exposure to PM2.5, airway inflammation scores were significantly elevated (Fig. 2C). In addition, PM2.5 exposure triggered the gradual proliferation of PAS-positive goblet cells (Fig. 2B), along with excessive airway secretion of mucus. The PAS-positive cell scores were also increased significantly with the extension of PM2.5 exposure time (Fig. 2D). Moreover, the Penh, an indirect index of airway resistance, gradually increased in mice exposed to PM2.5 (Fig. 2E). The total BALF inflammatory cells in mice exposed to PM2.5 were significantly higher (Fig. 2F) compared with those in the control group. The recorded increase was attributed to a steep rise in the mean percentage of eosinophils and neutrophils in BALF compared with those in the control group (Fig. 2G and 2H).

Of note, cytokines are important inflammatory transmitters and activators in respiratory diseases. Cytokines including IL-4, IL-5, IL-6, and TNF-α were examined in BALF. PM2.5 exposure induced an increase in IL-4, IL-5, IL-6, and TNF-α in BALF. All cytokine concentrations peaked at 14 days after exposure to PM2.5 and remained at a high level (Fig. 2I–2L). These results demonstrate that PM2.5 exposure induced airway inflammation, including bronchial smooth muscle layer hypotrophy, basement membrane thickening, and lumen narrowing. In addition, PM2.5 exposure triggered goblet cell proliferation and mucus secretion, consequently enhancing airway responsiveness.

3.3 PM2.5 exposure inhibited the expression and activity of SIRT2, enhanced the phosphorylation and acetylation of p65, and promoted the expression of iNOS

SIRT2 is a key modulator in the activation of inflammation. The expression and activity of SIRT2 were examined. Western blot analysis showed a gradual downregulation in the expression of SIRT2 after PM2.5 exposure, where it reached the lowest level at 21 days in the observation period and remained at a low level (Fig. 3A and 3B). Notably, p65 is the core subunit in the NF-κB pathway, and its modulation is of great significance in activating its downstream inflammatory signal. PM2.5 exposure did not influence the total expression of p65 but significantly increased the phosphorylation of p65 (Fig. 3A and 3C). In addition, PM2.5 exposure enhanced p65 acetylation (Fig. 3A and 3D). Of note, iNOS is one of the vital p65 downstream inflammatory molecules. The emergence of iNOS plays a vital role in airway inflammation. In this study, iNOS was barely expressed in the control group, but it gradually increased with the increase in PM2.5 exposure (Fig. 3A and 3E). Additionally, the activity of SIRT2 in bronchial tissues was evaluated. As shown in Fig. 3F, SIRT2 activity was dramatically decreased in PM2.5-exposed mice compared with the control group. We checked the levels of nuclear or cytoplasmic p65 to assess the activity of NF-κB. Results showed that cytoplasmic p65 decreased gradually, whereas nuclear p65 increased with the increase in PM2.5 exposure time. These results implied that PM2.5-induced airway inflammation and bronchial hyperresponsiveness are perhaps related to the inhibition of SIRT2. The inhibition of SIRT2 promoted the phosphorylation and acetylation of p65 and enhanced the expression of iNOS.

3.4 SIRT2 KO aggravated PM2.5-induced airway inflammation and bronchial hyperresponsiveness

SIRT2 KO mice were used to validate the key effect of SIRT2 in PM2.5-induced airway inflammation. SIRT2 KO mice were exposed to PM2.5 for 28 days to assess the changes in airway morphology and function. Following PM2.5 exposure, HE staining results showed that the airway inflammation worsened in SIRT2 KO mice compared with WT mice. The airway inflammation score in SIRT2 KO mice was significantly higher compared with that in WT mice (Fig. 4A and 4B). SIRT2 KO mice also exhibited massive infiltration of inflammatory cells around the bronchioles, aggregation of PAS-positive goblet cells, and mucus overproduction in the bronchioles. The scores for PAS-positive cells were significantly increased in SIRT2 KO mice than in WT mice (Fig. 4C and 4D). Similarly, collagen deposition and airway wall thickness were significantly increased in SIRT2 KO mice than in WT mice (Fig. 4E and 4F). Furthermore, SIRT2 KO mice developed typical airway hyperresponsiveness to methacholine, characterized by higher Penh compared with WT mice (Fig. 4G). A steep increase in the number of total inflammatory cells was observed in BALF from PM2.5-exposed SIRT2 KO mice (Fig. 4H). The accumulation of eosinophils and neutrophils in BALF was increased (Fig. 4I and 4J), and the levels of inflammatory cytokines IL-4, IL-5, IL-6, and TNF-α in BALF (Fig. 4K–4N) were promoted in PM2.5-induced SIRT2 KO mice. These findings suggested that SIRT2 KO aggravated the PM2.5-induced airway inflammation and hyperresponsiveness.

3.5 SIRT2 KO promoted the phosphorylation and acetylation of p65 and the expression of iNOS

The key role of SIRT2 in PM2.5 exposure was determined in the above experiments. Subsequently, the regulation of SIRT2 and p65 was examined to verify the underlying mechanism. The binding relationship of SIRT2 and p65 was evaluated through Co-IP experiments. Results showed that p65 and SIRT2 interacted directly; however, PM2.5 exposure weakened the combination (Fig. 5A). The expression levels of SIRT2 and p65 were also examined. As a consequence, PM2.5 significantly lowered the expression and activity of SIRT2 in bronchial tissues of WT mice (Fig. 5B–5D). The phosphorylation and acetylation of p65 in bronchial tissues of SIRT2 KO mice were increased compared with those of WT mice (Fig. 5E and 5F). Similarly, the expression of iNOS in PM2.5-exposed SIRT2 KO mice was enhanced (Fig. 5G) than in WT mice. These results indicated that PM2.5 significantly weakened the modification of SIRT2 to p65, which in turn promoted the phosphorylation and acetylation of p65 and activated the downstream inflammatory factors.

3.6 Triptolide improved PM2.5-induced airway inflammation and bronchial hyperresponsiveness

Owing to the lack of SIRT2 agonists, triptolide was selected to explore whether the inhibition of p65 could alleviate the PM2.5-induced airway inflammation. Triptolide is a small-molecule inhibitor that was first extracted from the natural Chinese herb Tripterygium wilfordii. Triptolide can block the interaction of p65 to p300/CBP and reduce the transcription activity of NF-κB. All mice were subjected to a daily intraperitoneal injection of triptolide at a dose of 0.07 mg/kg body weight for 28 consecutive days. As shown in Fig. 6, triptolide significantly mitigated PM2.5-induced airway inflammation and bronchial hyperresponsiveness in WT and SIRT2 KO mice. The mitigation mainly involved inhibiting the thickening of the bronchial smooth muscle layer and basement membrane and reducing inflammatory cell infiltration around the alveoli and bronchioles. After triptolide treatment, the airway inflammation score was lowered (Fig. 6A and 6D), the number of PAS-positive goblet cells and mucus overproduction in the bronchioles were decreased (Fig. 6B and 6E), and collagen deposition and ROS production were restrained (Fig. 6C, 6F−6H). Additionally, triptolide reduced the high Penh induced by PM2.5 (Fig. 6I). The production and accumulation of eosinophils and neutrophils in BALF were also inhibited (Fig. 6J–6L). Inflammatory cytokines including IL-4, IL-5, IL-6, and TNF-α were significantly attenuated after triptolide treatment (Fig. 6M–6P). These results indicate that triptolide treatment could attenuate PM2.5-induced airway inflammation and alleviate airway resistance in WT and SIRT2 KO mice.

3.7 Triptolide restrained the phosphorylation and acetylation of p65 and the expression of iNOS

The binding relationship of SIRT2 and p65 after triptolide treatment was examined. Results showed that triptolide mildly increased the interaction (Fig. 7A). Moreover, triptolide slightly upregulated the expression of SIRT2 in bronchial tissues in PM2.5-exposed WT mice (Fig. 7C and 7D); however, it did not affect the activity of SIRT2 (Fig. 7B). As an inhibitor of p65, triptolide inhibited the phosphorylation and acetylation of p65 in PM2.5-exposed WT mice. The ratio of phosphorylated p65 to total p65 and the ratio of acetylated p65 to total p65 were significantly reduced following triptolide treatment in the PM2.5-exposed SIRT2 KO group (Fig. 7C, 7E, and 7F). In addition, triptolide treatment reversed the upregulation of iNOS induced by PM2.5 exposure in the WT and SIRT2 KO groups (Fig. 7C and 7G). These results suggested that triptolide potentially enhances the expression of SIRT2 and its binding to p65. Nevertheless, the underlying mechanism merits further investigation. The inhibition of p65 by triptolide effectively mitigated the PM2.5-induced airway inflammation injury.

4 Discussion

This study confirmed that the SIRT2-p65 signaling pathway is implicated in PM2.5-induced airway inflammation and bronchial hyperresponsiveness. Exposure to PM2.5 significantly suppressed SIRT2 interaction with p65. As a result, PM2.5 induced the downregulation of SIRT2, accelerated the phosphorylation and acetylation of p65, and activated the NF-κB signaling pathway. On the other hand, the NF-κB signaling pathway regulated inflammatory cell infiltration around the alveoli and bronchioles, mucus overproduction, and bronchial hyperresponsiveness. Moreover, SIRT2 KO increased the phosphorylation and acetylation of p65 and aggravated subsequent airway inflammation and bronchial hyperresponsiveness induced by PM2.5. More importantly, we showed that the inhibition of NF-κB activation is an effective approach for regulating PM2.5-induced airway inflammation and bronchial hyperresponsiveness. Furthermore, we demonstrated that triptolide inhibited PM2.5-induced p65 phosphorylation and acetylation, thereby attenuating airway inflammation and hyperresponsiveness. Our findings reveal a novel mechanism underlying PM2.5-induced airway inflammation and provide a precise description and possible therapeutic strategies against PM2.5-induced toxicity [36].

Notably, PM2.5 refers to air pollutants whose aerodynamic equivalent diameter in ambient air is less than or equal to 2.5 µm [11,37]. PM2.5 can be easily inhaled and deposited in the respiratory system because it can float in the atmosphere for a long duration. The smaller the diameter of PM2.5, the higher the hazard levels. Particles with a diameter of less than 2.5 µm deposit in bronchi and alveoli and penetrate the air-blood barrier into blood circulation, causing a severe health hazard to humans [38]. Although the chemical composition of PM2.5 varies with climatic conditions and pollution sources, it can be divided into water-soluble, carbon-containing, and inorganic elements [23,39]. In some instances, PM2.5 contains microorganisms including bacteria and fungi [22]. All these substances enriched in PM2.5 aggravate the adverse health effects to respiratory, circulatory, and other human body systems. In our experiment, the median diameter of the collected PM2.5 particles was 0.17±0.09 µm (mean±SD), and the size distribution ranged between<0.03 µm and 3 µm, indicating that nearly all the sampled particles were PM2.5. This study determined the composition and quantity of major chemical components in the sampled PM2.5. As a result, we found that PAHs were the primary pollutant in PM2.5. They exhibited the most significant mass fraction, followed by OC, water-soluble inorganic ions, EC, and other unknown components. These harmful chemical components in PM2.5 are implicated in the toxicological effects of PM2.5 on airway inflammatory damage.

Previous studies reported that toxic and harmful substances enriched in PM2.5 could be easily inhaled and deposited into the bronchi and alveoli. The deposition subsequently induces inflammatory cell infiltration, accumulation of proinflammatory cytokines, oxidative stress, and secretion of airway mucus [40,41]. Extended airway inflammatory injury causes a thickening of the bronchial smooth muscle layer and basement membrane and increases airway resistance and bronchial hyperresponsiveness, ultimately resulting in airway stenosis and remodeling [42–44]. C57BL/6 mice were exposed to PM2.5 for 6 h per day for 28 consecutive days. After 3 days of exposure, significant changes in inflammatory lesions in airway morphology and airway function were observed. The changes were characterized by hemorrhage, edema, thickened alveolar wall, dot and patchy infiltration, wall thickening and hypertrophy of the smooth muscle layer, narrowed lumen, and gradual muscularization of the bronchus. The severity of inflammatory lesions and airway remodeling was increased following an extension of PM2.5 exposure time.

Additionally, PM2.5 induced the production of inflammatory cytokines. The levels of IL-4, IL-5, IL-6, and TNF-α in BALF were significantly upregulated, peaked at 14 days post-exposure to PM2.5, and remained at a high level. Inflammatory cytokines have significant pathophysiological effects on airway inflammation. IL-4 promotes Th2 and B cell proliferation and differentiation and induces the secretion of immune-reactive substances [45]. IL-5 induces eosinophil differentiation, facilitates the production of various inflammatory chemokines, and triggers mucus secretion and contraction of smooth muscles [46]. IL-6 and TNF-α aggravate airway inflammation, and hyperresponsiveness promotes the proliferation of airway fibroblasts and smooth muscle cells [47]. As a consequence, PM2.5-induced inflammatory cell infiltration and inflammatory cytokine production around the alveoli and bronchioles accelerated airway inflammation and bronchial hyperresponsiveness.

The eukaryotic transcription factor NF-κB/Rel family regulates a wide range of host genes that govern inflammatory and immune responses in mammals. In general, the NF-κB/Rel family comprises seven proteins, including p65/RelA, c-Rel, RelB, p100, p52, p105, and p50. Among them, p65 is widely implicated in the regulation of the transcriptional function of NF-κB. The prototypical NF-κB form contains the heterodimer p65/p50, which is primarily sequestered in the cytoplasm by an inhibitor of IκBα in an inactive state [12]. Proinflammatory cytokines or other genotoxic agents activate the phosphorylation of intracellular IκBα at Ser-32 and Ser-36, triggering its poly-ubiquitination and subsequent proteasomal degradation. This is followed by translocation of p65/p50 heterodimer to the nucleus, where it binds to NF-κB promoter/enhancer sites, initiating the transcription of NF-κB target genes by recruiting the transcriptional co-activator CBP/p300 due to activation of the transcriptional domain of p65 [48]. In addition to the IκBα protein, the phosphorylated and acetylated modification of p65 plays a critical role in fine-tuning the transcriptional activity of NF-κB. Evidence has shown that the phosphorylated and acetylated modification of p65 is the embodiment of activation and persistence of the NF-κB signaling pathway [49]. Moreover, the phosphorylation and acetylation of p65 are mutually promotive. For instance, phosphorylation at different serine sites of p65 promotes its binding to CBP/p300 and its stable acetylation facilely [50], which improves the transcriptional activity of NF-κB. Our experiments revealed that the phosphorylated and acetylated p65 induced by PM2.5 profoundly increased along with the PM2.5 exposure time. Therefore, PM2.5-induced p65 phosphorylation and acetylation strengthened the transcriptional activity of NF-κB and enhanced the NF-κB-dependent expression of inflammatory cytokines. This is a key factor contributing to airway inflammation and bronchial hyperresponsiveness. Moreover, NF-κB predominantly regulates the expression of iNOS [51]. Therefore, we found that PM2.5-induced NF-κB activation promoted the expression of iNOS. Conventionally, iNOS is not expressed in the resting state. Upon stimulation by proinflammatory cytokines or other genotoxic agents, iNOS is activated to synthesize large amounts of NO, thereby inducing Th2 cell activation, dilation of blood vessels, and enhanced plasma extravasation and mucus secretion and promoting the development of airway inflammatory injury and responsiveness [52,53]. SIRT2, which is primarily located in the cytoplasm, is vital in inflammation. It is the key coenzyme implicated in the deacetylation of p65. SIRT2 promotes the deacetylation of lysine 310 of p65 and inhibits the activity of NF-κB and NF-κB-dependent expression of inflammatory cytokines, which alleviates inflammation [16,17]. In line with our experimental results, PM2.5 significantly downregulates the expression of SIRT2 [18]. However, the mechanism responsible for the decreased expression of SIRT2 induced by PM2.5 and the relationship between the biological consequences of SIRT2 reduction and PM2.5-induced inflammatory damage remain unknown. Previous studies reported that PAHs (the primary pollutant in the sampled PM2.5) can bind to the aryl hydrocarbon receptor (AHR), a multifunctional ligand-activated transcription factor and environmental sensor, thereby activating AHR-related signaling pathways involved in immunity and inflammation, microbial defense, and stem/progenitor cell homeostasis [54,55]. Additionally, the activation of AHR can affect the NAD metabolism process, including NAD⁺- and sirtuin-mediated deregulation of lipid, glucose, and NAD⁺ homeostasis [56]. Hence, we believe that PAHs in the sampled PM2.5 triggering the activation of AHR and promoting SIRT2 degradation may be a possible mechanism of SIRT2 downregulation. In addition, other compounds in the sampled PM2.5 might affect the activation of p65 phosphorylation and acetylation or the activity of SIRT2 in different ways or different degrees, which all merits more comprehensive investigation in our next experiment. In the present study, we found that PM2.5-induced SIRT2 downregulation accelerated the phosphorylation and acetylation of p65, causing the activation of the NF-κB signaling pathway. Furthermore, SIRT2 interacted with p65 directly. SIRT2 KO further increased the phosphorylation and acetylation of p65 and NF-κB signaling pathway following exposure to PM2.5. Furthermore, it aggravated airway inflammation and bronchial hyperresponsiveness. Thus, the SIRT2-p65 signaling pathway mediated the impacts of PM2.5 on airway inflammation and responsiveness.

Due to the absence of SIRT2 agonists, inhibiting the transcriptional activity of NF-κB might be an effective strategy in controlling PM2.5-induced airway inflammation and bronchial hyperresponsiveness. Notably, triptolide, an inhibitor of NF-κB, inhibits the degradation and phosphorylation of IκBα and reduces the nuclear translocation and activation of NF-κB [19]. Additionally, triptolide inhibits p65 phosphorylation and acetylation and decreases the interaction of p65 and p300/CBP [20], thereby suppressing the transcriptional activity of NF-κB [57]. In our experiment, we observed that triptolide mildly enhanced the combination of SIRT2 and p65. Triptolide also slightly upregulated the expression of SIRT2 in bronchial tissues in PM2.5-exposed WT mice. Nonetheless, it did not influence the activity of SIRT2. In addition, triptolide significantly inhibited PM2.5-induced p65 phosphorylation and acetylation, thus attenuating PM2.5-induced airway inflammation and hyperresponsiveness. We observed similar effects after treatment of SIRT2 KO mice with triptolide. Therefore, these findings present a novel strategy for airway protection after PM2.5 exposure.

5 Conclusions

This study confirmed that PM2.5 significantly downregulated the expression and activity of SIRT2, which increased p65 phosphorylation and acetylation and activated the NF-κB signaling pathway. This induced NF-κB-dependent expression of inflammatory cytokines and ultimately contributed to airway inflammation and bronchial hyperresponsiveness. The SIRT2-p65 signaling pathway was implicated in PM2.5-induced airway inflammation and bronchial hyperresponsiveness. Furthermore, we revealed that triptolide significantly inhibited PM2.5-induced phosphorylation and acetylation of p65, thereby reducing airway inflammation and bronchial hyperresponsiveness. Fig. 8 illustrates the proposed mechanism based on the results of this study. Our findings demonstrate that the SIRT2-p65 signaling pathway is a novel mechanism responsible for PM2.5-induced organ damage, therefore providing a scientific basis and promising therapeutic and prevention strategies against PM2.5-induced toxicity.

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