Inhibitory functions of maslinic acid on particulate matter-induced lung injury through TLR4-mTOR-autophagy pathways

Environmental Research 183 (2020) 109230

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Inhibitory functions of maslinic acid on particulate matter-induced lung injury through TLR4-mTOR-autophagy pathways

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So Yeon Jeonga,1, Jaehong Kimb,1, Eui Kyun Parkc, Moon-Chang Baekd, Jong-Sup Baea,∗ a College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, BK21 Plus KNU Multi-Omics Based Creative Drug Research Team, Kyungpook National University, Daegu, 41566, Republic of Korea b Department of Biochemistry, College of Medicine, Gachon University, Incheon, 21999, Republic of Korea c Department of Pathology and Regenerative Medicine, School of Dentistry, Kyungpook National University, Daegu, 41940, Republic of Korea d Department of Molecular Medicine, CMRI, School of Medicine, Kyungpook National University, Daegu, 41944, Republic of Korea

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A B S T R A C T

Keywords: Maslinic acid Particulate matter Lung injury TLR4-mTOR-autophagy

Particulate matter (PM), the collection of all liquid and solid particles suspended in air, includes both organic and inorganic particles, many of which are health-hazards. PM particles with a diameter equal to or less than 2.5 μm (PM2.5) is a form of air pollutant that causes significant lung damage when inhaled. Maslinic acid (MA) prevents oxidative stress and pro-inflammatory cytokine generation, but there is little information available regarding its role in PM-induced lung injury. Therefore, the purpose of this study was to determine the protective activity of MA against PM2.5-induced lung injury. The mice were divided into seven groups (n = 10 each): a mock control group, an MA control (0.8 mg/kg mouse body weight) group, an opted PM2.5 produced from diesel (10 mg/kg mouse body weight) group, a diesel PM2.5+MA (0.2, 0.4, 0.6, and 0.8 mg/kg mouse body weight) groups. Mice were treated with MA via tail-vein injection 30 min after the intratracheal instillation of a diesel PM2.5. Changes in the wet/dry weight ratio of the lung tissue, total protein/total cell and lymphocyte counts, inflammatory cytokines in the bronchoalveolar lavage fluid (BALF), vascular permeability, and histology were monitored in diesel PM2.5-treated mice. The results showed that MA reduced pathological lung injury, the wet/ dry weight ratio of the lung tissue, and hyperpermeability caused by diesel PM2.5. MA also inhibited diesel PM2.5-induced myeloperoxidase (MPO) activity in the lung tissue, decreased the levels of diesel PM2.5-induced inflammatory cytokines, including tumor necrosis factor (TNF)-α and interleukin (IL)-1β, reduced nitric oxide (NO) and total protein in the BALF, and effectively attenuated diesel PM2.5-induced increases in the number of lymphocytes in the BALF. In addition, MA increased the protein phosphorylation of the mammalian target of rapamycin (mTOR) and dramatically suppressed diesel PM2.5-stimulated expression of toll-like receptor 4 (TLR4), MyD88, and the autophagy-related proteins LC3 II and Beclin 1. In conclusion, these findings indicate that MA has a critical anti-inflammatory effect due to its ability to regulate both the TLR4-MyD88 and mTORautophagy pathways and may thus be a potential therapeutic agent against diesel PM2.5-induced lung injury.

1. Introduction Air pollution due to anthropogenic sources has worsened globally, particularly as a result of the development of heavy industry in recent years (Losacco and Perillo, 2018). Suspended particulate matter (PM) with a diameter equal to or less than 2.5 μm (PM2.5), a well-known index of air pollution, can adversely affect the respiratory and circulatory systems, with approximately 96% of PM2.5 retained in the lungs due to its small size (Xing et al., 2016). PM2.5 is made up of a number of different components that exert toxic effects, including polycyclic

aromatic hydrocarbons, oxygenated volatile organic compounds, and heavy metals (Cho et al., 2018). The relationship between PM2.5 and inflammation has been identified as playing a role in many pulmonary diseases, such as asthma, acute lung injury, and chronic obstructive pulmonary disease, while PM2.5-induced inflammation is associated with the release of numerous cytokines and chemokines, such as interleukins (ILs) and tumor necrosis factor (TNF)-α (Cho et al., 2018; Gent et al., 2003; Gong et al., 2005). Because there is a significant correlation between exposure to PM2.5 and the risk of asthma and also the incidence and mortality of lung cancer (Wang et al., 2018), there is

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Corresponding author. College of Pharmacy, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu, 41566, Republic of Korea. E-mail address: [email protected] (J.-S. Bae). 1 These authors contributed equally. https://doi.org/10.1016/j.envres.2020.109230 Received 15 December 2019; Received in revised form 30 January 2020; Accepted 4 February 2020 Available online 05 February 2020 0013-9351/ © 2020 Elsevier Inc. All rights reserved.

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previously described (Wang et al., 2017a). Twenty-four hours after the injection, the mice were sacrificed and their bronchoalveolar lavage fluid (BALF) and lung tissue were harvested for further studies.

a high-priority need to develop new prevention and treatment strategies for respiratory diseases. The cascade process involved in lung injury is complex. Toll-like receptor 4 (TLR4) is an essential modulator in this respect, triggering innate immune responses and playing a critical role in the regulation of various mediators of inflammation (Iwasaki and Medzhitov, 2004). PM has been reported to upregulate the production of inflammatory mediators through the activation of the TLR4 pathway (Woodward et al., 2017). PM also increases cellular oxidative stress, which causes cellular apoptosis and cellular autophagy, and damages cellular components by inhibiting the activity of the mammalian target of rapamycin (mTOR), which is a sensor of cellular nutritional status (Kim and Guan, 2015; Li et al., 2017). Autophagy is a lysosome-dependent process related to cell homeostasis and adaptation to harmful environments that is associated with protein aggregates, organelle damage, and intracellular pathogen turnover (Choi et al., 2013). Not only is autophagy essential for keeping cell homeostasis, and helpful to protect cells from damage and stress, but autophagy has been reported to be involved in the development of pulmonary diseases, and it plays an important role in the regulation of pulmonary diseases (Hu et al., 2014; Mizumura et al., 2012). Thus, the inhibition of both TLR4 and autophagy may provide therapeutic benefit for pulmonary injury (Chen et al., 2016; Hu et al., 2016). Maslinic acid (MA, 2-α,3-β-dihydroxyolean-12-en-28-oic acid) is a natural triterpenoid found in the olive (Olea europaea) and a variety of medicinal plants (Lozano-Mena et al., 2014; Reyes-Zurita et al., 2009). MA has been shown to have antioxidant (Montilla et al., 2003), antiinflammatory (Huang et al., 2011), antimalarial (Moneriz et al., 2011), and antiprotozoan (De Pablos et al., 2010) activities. However, the effects of MA on pulmonary injury, histology, inflammation, and TLR4autophagy pathways following PM2.5 exposure have yet to be investigated. To address this gap in knowledge, in the present study, we used a mouse model exposed to diesel PM2.5 to demonstrate our hypothesis that MA may inhibit diesel PM2.5-induced proinflammatory responses and autophagy in lung tissue cells and enhance the recovery of tissue damage caused by diesel PM2.5-induced lung injury by inhibiting the TLR4 and autophagy pathways.

2.3. Wet/dry weight ratio of the lung tissue The right lung was weighed to obtain the wet weight. The lung was then dried in an oven at 120 °C for 24 h and weighed again to obtain the dry weight. Lung edema was determined by calculating the lung wet/ dry (W/D) weight ratio. 2.4. Mouse lung microvascular endothelial cell (MLMVEC) culture and siRNA transfection Mouse lung microvascular endothelial cells (MLMVECs) were acquired using a modified version of a previous approach (Kovacs-Kasa et al., 2017; Lee et al., 2019a, 2019d). Briefly, lung tissue was minced and then digested with collagenase A (1 mg/mL) for 45–60 min at 37 °C. Endothelial cells were purified using a separation technique involving the anti-PECAM-1 monoclonal antibody (mAb) coupled to magnetic beads (BD Pharmingen, San Diego, CA, USA) and grown in growth medium for two days. To produce a monolayer culture, the cells were seeded on fibronectin-coated dishes and grown in endothelial cell basal medium supplemented with EGM-2 MV Bulletkit™ medium (Lonza, Walkersville, MD, USA). The mouse mTOR, TLR4, or nonsense control siRNA transfections were performed as previously described (Lee et al., 2015). 2.5. Hematoxylin and eosin (H&E) staining Lung tissue was removed, washed three times with PBS (pH 7.4) to remove the remaining blood, and then fixed with a 4% formaldehyde solution (Junsei, Tokyo, Japan) in PBS for 20 h at 4 °C. The samples were then dehydrated, embedded in paraffin, and processed into 4-μm thick slices. The slides were subsequently deparaffinized, rehydrated, and stained with hematoxylin (Sigma-Aldrich Inc.). An observer blind to the treatments then evaluated the intactness of the pulmonary architecture and the degree of tissue edema under a light microscope using a defined method (Lee et al., 2019d; Ozdulger et al., 2003).

2. Methods 2.1. Reagents

2.6. ELISA analysis of the phosphorylation of p38 MAPK, MPO, NO, IL-1β, and TNF-α

Diesel PM NIST 1650b (Bergvall and Westerholm, 2006), purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA), was mixed with saline and sonicated for 24 h to avoid the agglomeration of suspended diesel PM2.5 particles (Supplementary Fig. 1). MA and dexamethasone (DEX, used as a positive control) were purchased from Sigma-Aldrich Inc. Small interfering RNAs (siRNA) for control, mTOR, and TLR4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Unless otherwise stated, other chemicals and reagents were obtained from Sigma-Aldrich Inc.

The levels of phosphorylated p38 mitogen-activated protein kinase (MAPK) in the MLMVEC lysates were analyzed using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Cell Signaling Technology, Danvers, MA, USA). The concentrations of myeloperoxidase (MPO), nitrous oxide (NO), interleukin (IL)-1β, and tumor necrosis factor (TNF)-α in the BALF were determined using manufacturer-suggested ELISA kits (R&D Systems, Minneapolis, MN, USA). All of the ELISA readings were conducted with an ELISA plate reader (Tecan Austria GmbH, Grödig, Austria).

2.2. Animal care

2.7. Protein concentration and cell count in the BALF

Male Balb/c mice (7 weeks old, approximate body weight of 27 g) were obtained from Orient Bio Co. (Sungnam, Republic of Korea) and used in our study after 12 days of acclimatization. The mice were treated in accordance with the Guidelines for the Care and Use of Laboratory Animals of Kyungpook National University (IRB #: KNU2017-102). The mice were divided into eight groups (n = 10 each): a mock control group, an MA control group, a diesel PM2.5 (10 mg/kg mouse body weight in 50 μL of saline) group, diesel PM2.5 +MA (0.2, 0.4, 0.6, and 0.8 mg/kg mouse body weight) groups, and a DEX group (5 mg/kg mouse body weight). The mice in the control group received an equal volume of PBS. The MA and DEX groups were injected via the tail vein 30 min after being challenged intratracheally with diesel PM2.5 (10 mg/kg mouse body weight in 50 μL of saline) as

After centrifugation at 3000 rpm for 10 min at 4 °C, the BALF supernatant was used to assess the total protein concentration with a QuantiPro™ BCA Assay Kit (Sigma-Aldrich Inc.), and the cytokine levels were measured. Cell pellets were re-suspended in 50 μL of PBS, and the resuspended cells were counted using a hematology analyzer. 2.8. Permeability assays Spontaneous breathing of the mice was allowed during the in-vivo permeability assays. The MA and DEX groups were injected through the tail vein 30 min after the intratracheal administration of PM2.5 (10 mg/ 2

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kg in 50 μL of saline) as described above. For anesthetization, a 2% isoflurane–oxygen mixture (Forane; JW Pharmaceutical, Seoul, Republic of Korea) was delivered using a gas anesthesia machine (RC2 Rodent Circuit Controller; VetEquip, Pleasanton, CA, USA). The mice were anaesthetized first in a breathing chamber and then via a facemask, followed by the intravenous injection of 1% Evans blue dye solution in normal saline. The mice were euthanized via cervical dislocation after 6 h and the BALF was collected. Permeability data were acquired using an ELISA plate reader as described previously (Kim et al., 2019; Lee and Bae, 2019; Lee et al., 2019b).

levels (4.5 folds), total cell (4.3 folds) and lymphocyte (13.8 folds) counts in the mouse BALF were increased in diesel PM2.5-induced group compared to the control group (p < 0.01). However, MA or DEX (5 mg/kg) treatment after the endotracheal instillation of diesel PM2.5 reduced the total cell number and, the number of lymphocytes in the BALF (p < 0.01 compared to PM2.5-treated group). MA treatment also reduced the total protein levels in the BALF in a dose-dependent fashion (23% reduction with 0.4 mg/kg MA, 36% reduction at 0.6 mg/kg, and 45% reduction at 0.8 mg/kg, p < 0.05 each compared to diesel PM2.5treated group) or DEX (42% reduction at 5 mg/kg, p < 0.05 compared to diesel PM2.5-treated group; Fig. 1B). To examine the protective effects of MA against diesel PM2.5-induced lung injury, changes in lung histopathology were investigated using H&E staining. As shown in Fig. 2A, diesel PM2.5 group exhibited inflammatory cell infiltration, with these cells deposited on the alveolar wall. The lung injury score was calculated following treatment with different doses of MA or DEX (37% reduction with 0.6 mg/kg MA and 65% reduction at 0.8 mg/kg, p < 0.05 each compared to diesel PM2.5treated group) or DEX (62% reduction at 5 mg/kg, p < 0.05 compared to diesel PM2.5-treated group; Fig. 2B), revealing that MA mitigated inflammatory cell infiltration and protected the lungs from diesel PM2.5-induced lung injury.

2.9. Western blot analysis Cells were first rinsed with ice-cold phosphate-buffered saline (PBS) and treated with a RIPA lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% sodium dodecyl sulfate (SDS), 1% NP-40, 1% sodium deoxycholate, and protease inhibitors (Zhang and Wang, 2018). Blocking was performed with 5% bovine serum albumin (BSA) for 2 h, and blots were incubated with the following primary antibodies: antilight chain (LC)3 (1:1000), Beclin 1 (1:1000), TLR4 (1:1000), MyD88 (1:1000), mTOR (1:1000), phosphorylated (p)-mTOR (1:1000), Akt (1:1000), p-Akt (1:2000), p-PI3K (1:1000), and PI3K (1:800) (Cell Signaling Technology, Inc., Danvers, MA, USA). Following this, the membrane was washed and incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, 1:10,000).

3.2. Effects of MA on diesel PM2.5-mediated vascular barrier disruption Because PM has been reported to disrupt the integrity of vascular barrier (Wang et al., 2010, 2017c), the effects of MA on diesel PM2.5induced vascular disruptive responses were evaluated. As shown in Fig. 3A, dye leakage in the BALF was higher following diesel PM2.5 treatment (15.5 folds, compared to control group, p < 0.01), which was subsequently suppressed by MA or DEX (22% suppression with 0.4 mg/kg MA, 45% suppression at 0.6 mg/kg, and 64% suppression at 0.8 mg/kg, p < 0.05 each compared to diesel PM2.5-treated group) or DEX (65% suppression at 5 mg/kg, p < 0.05 compared to diesel PM2.5treated group; Fig. 3A). Because the p38 MAPK signaling pathway mediates the vascular damage reaction caused by inflammatory proteins (Qin et al., 2009; Sun et al., 2009), we then determined the effects of MA on diesel PM2.5-induced p38 MAPK activation, finding that diesel PM2.5 upregulated the phosphorylation of p38 MAPK, which MA treatment inhibited (28% inhibition with 0.4 mg/kg MA, 55% inhibition at 0.6 mg/kg, and 69% inhibition at 0.8 mg/kg, p < 0.05 each compared to diesel PM2.5-treated group) or DEX (67% inhibition at 5 mg/kg, p < 0.05 compared to PM2.5-treated group; Fig. 3B).

2.10. Statistical analysis All experiments were independently performed at least three times, and the results are expressed as the mean and standard deviation (SD). Statistical significance was analyzed using one-way ANOVA followed by Dunnett's tests, with a p-value of < 0.05 considered statistically significant. All of the statistical analyses were conducted with SPSS (version 15.0) for Windows (SPSS, Chicago, IL, USA). 3. Results 3.1. Effects of MA on diesel PM2.5-induced lung damage The effect of MA on diesel PM2.5-induced pulmonary edema was measured by calculating the lung W/D weight ratio. As shown in Fig. 1A, the lung W/D weight ratio was increased in diesel PM2.5 treatment group (37% compared to control group, p < 0.01), and this ratio was reduced with the application of MA or DEX (5 mg/kg). Diesel PM2.5-induced inflammatory cell infiltration and total protein levels in the BALF were also measured. As shown in Fig. 1B–D, the total protein

Fig. 1. Effects of MA on diesel PM2.5-induced lung damage. The maslinic acid (MA) and dexamethasone (DEX) groups were injected intravenously 30 min after being challenged intratracheally with diesel PM2.5 (10 mg/kg in 50 μL of saline). The mice were then sacrificed 24 h postdiesel PM2.5-injection and the lung tissue and BALF were harvested. The effects of various concentrations of MA or DEX on (A) the W/D ratio, (B) the total cell numbers in the BALF, (C) the total protein levels in the BALF, and (D) the number of lymphocytes in the BALF were assessed. The data are presented as the mean and standard deviation (SD) from three independent experiments. *p < 0.01 versus diesel PM2.5-challenged group.

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Fig. 2. Effects of MA on diesel PM2.5-induced lung histopathological changes. The MA and DEX groups were injected intravenously 30 min after being challenged intratracheally with diesel PM2.5 (10 mg/kg in 50 μL of saline). The mice were then sacrificed 24 h post-diesel PM2.5-injection, and the lung tissue was harvested. (A) The histology of the lung tissue was examined using H&E staining. Representative images from each group are shown (n = 5). Scale bar: 200 μm. (B) Lung injury score. The data are presented as the mean and SD from three independent experiments. *p < 0.01 versus diesel PM2.5-challenged group.

four groups. To confirm that PM2.5-induced lung injury was dependent on TLR4mTOR signaling pathways, genetic approach was applied to inhibit the expressions of TLR4 and mTOR in purified MLMVECs using TLR4 or mTOR siRNA. As shown in Fig. 6, PM2.5 exposure induced a notable increase of TNF-α and IL-1β expressions in MLMVECs and mTOR knockdown further enhanced the productions of TNF-α and IL-1β, significantly. However, knockdown of TLR4 by siRNA significantly blocked PM2.5-induced production of TNF-α and IL-1β (Fig. 6). These results supported the signaling network between TLR4-mTOR and autophagy in the presence of PM-induced lung injury.

3.3. Effects of MA on diesel PM2.5-induced pulmonary inflammation Because diesel PM2.5-induced vascular barrier disruption was inhibited in vivo by MA (Fig. 3), we next determined the effects of MA against diesel PM2.5-induced pulmonary inflammatory responses. Inflammatory cytokines such as NO, IL-1β, and TNF-α are important indicators of the inflammatory process, and increased lung MPO activity indicates neutrophil tissue infiltration. As shown in Fig. 4, compared with the control group, diesel PM2.5 increased lung tissue MPO (1.6 fold, p < 0.05) activity and NO (7.5 fold, p < 0.05), TNF-α (1.6 fold, p < 0.05), and IL-1β (2.5 fold, p < 0.05) production in the BALF. However, this increase was suppressed by MA or DEX treatment.

4. Discussion 3.4. Effects of MA on diesel PM2.5-induced signaling pathways In the current study, we were interested in the potential application of MA in the treatment of diesel PM2.5-induced lung injury. Previous experimental research has shown that PM increases the inflammatory response of endothelial cells, epithelial cells, and macrophages, leading to local lung inflammation (Liu et al., 2018; Wang et al., 2017b; Xu et al., 2018b). In addition, the overexpression of inflammatory mediators can lead to systemic inflammation and damage other systems (Ling and van Eeden, 2009). Thus, inflammation is considered the primary biological response to PM exposure. Our previous studies have shown that diesel PM2.5 upregulates the expression of molecules associated with inflammation and the disruption of vascular integrity, including p38, reactive oxygen species (ROS), IL-6, and TNF-α (Choi et al., 2019; Lee et al., 2019c, 2019d). The present research demonstrated that MA can inhibit both the infiltration of lung tissue by inflammatory cells and inflammatory cytokine production in our mouse model of diesel PM2.5induced lung injury. The possible mechanisms underlying the antidiesel PM2.5-induced inflammatory effect of MA are the reduction of TLR4 and MyD88 expression, the increase in mTOR phosphorylation, and the prevention of autophagy. Our study employed dexamethasone as a positive control because it is the most frequently used anti-inflammatory agent in the treatment of lung injury (Meng et al., 2018;

This study investigated the regulatory effects of MA on LC3 and Beclin 1 using Western blot analysis. As shown in Fig. 5A, LC3 II and Beclin 1 levels were higher in diesel PM2.5-treated group than in the control group. MA suppressed the increase in LC3 and Beclin 1 levels induced by diesel PM2.5 in mouse lung tissue. This indicates that MA can inhibit diesel PM2.5-induced autophagy. However, these effects were partially abolished following the administration of LY294002. To understand the mechanisms underlying the anti-diesel PM2.5-induced inflammatory and anti-autophagy effects of MA, both the TLR4 and the mTOR-autophagy pathways were investigated using western blotting for TLR4, MyD88, p-mTOR, total mTOR, p-Akt, Akt, p-PI3K, and PI3K in mouse lung tissue. The intratracheal instillation of diesel PM2.5 upregulated the expression of TLR4 and MyD88 in this tissue (Fig. 5B), which was subsequently reduced by treatment with MA (1 mg/kg). Compared with the control group, the levels of p-mTOR, p-Akt, and pPI3K were lower in the PM2.5 group (Fig. 5C). In addition, MA treatment restored the levels of p-mTOR, p-Akt, and p-PI3K, demonstrating that MA can activate the PI3K/Akt/mTOR pathway. However, LY294002 reversed these effects. Additionally, no significant differences in total levels of mTOR, Akt, or PI3K were observed between the

Fig. 3. Effects of MA on diesel PM2.5-induced vascular barrier disruptive responses and p38 MAPK activation. The MA and DEX groups were injected intravenously 30 min after being challenged intratracheally with diesel PM2.5 (10 mg/kg in 50 μL of saline). The effects of MA or DEX on diesel PM2.5induced vascular permeability were examined by (A) measuring the flux of Evans blue in the BALF (expressed as μg/mouse, n = 5) and (B) phosphorylated p38 (p-p38) levels in purified MLMVECs isolated from each mouse using ELISA. *p < 0.01 versus diesel PM2.5-challenged group. 4

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Fig. 4. Effects of MA on diesel PM2.5-induced pulmonary inflammation. The MA and DEX groups were injected intravenously 30 min after being challenged intratracheally with diesel PM2.5 (10 mg/ kg in 50 μL of saline). The mice were then sacrificed 24 h post-diesel PM2.5-injection, and the lung tissue and BALF were harvested. (A) MPO in lung tissue, (B) NO, (C) TNF-α, and (D) IL1-β in the BALF were measured. The data are presented as the mean and SD from three independent experiments. *p < 0.01 versus diesel PM2.5-challenged group.

Rogerio et al., 2008; Yoder et al., 1991). The diesel particulate material used in this study was NIST SRM 1650b, a diesel particulate material generated in 1984 by direct injection four-cycle diesel engines within a dilution tube facility and collected directly from the heat exchangers (NIST, 2013). NIST SRM 1650b is well-characterized, including known polycyclic aromatic hydrocarbons (PAH) (Huggins et al., 2000; NIST, 2013), reactive metal content (Huggins et al., 2000), as well as the relative ratio of organic and elemental carbon content (Tang et al., 2016). The toxicological consequences of NIST SRM 1650b, particularly with regards to pulmonary effects, have been previously explored through in vitro and in vivo studies. Studies have shown NIST SRM diesel materials can recapitulate some rudimentary toxicological effects of modern diesel fuel, including in vitro ROS generation in a human cell line and in vivo pulmonary inflammatory cytokine increase (Choi et al., 2019; Gour et al., 2018; Hemmingsen et al., 2011; Lee et al., 2019c; Lee et al., 2019e; Lee et al., 2019f). However, one of the primary limitations of the NIST SRM 1650b is it does not contain volatile and semi-volatile diesel species generated from combustion and its interactions with particulates cannot be examined with aged material and can only be captured with freshly generated diesel or with real-time ambient exposures (Huggins et al., 2000; NIST, 2013). Although the median particle size was close to the ultrafine size, the substantial presence of larger particles likely comes from aggregation and agglomeration following the original

Fig. 6. Effects of TLR4 and mTOR siRNA on diesel PM2.5-induced production of TNF-α and IL-1β in purified MLMVECs. Isolated MLMVECs were transfected with TLR4, mTOR siRNA or control siRNA. Subsequently, the cells were treated with PM2.5 (0.1 mg/mL) or vehicle control for 6h and the production of TNF-α and IL-1β were measured by ELISA. #p < 0.01.

combustion reaction, as well as the subsequent aerosolization (Huggins et al., 2000; NIST, 2013). Despite these limitations, the advantages of using NIST SRM 1650b are it is a well-characterized diesel material with an exposure method that can be reproduced. Overall NIST SRM 1650b can be considered a general representative of heavy equipment diesel emissions and is still currently used as a reference material for a variety of studies characterizing modern day particulate emissions (Borillo et al., 2018; Nystrom et al., 2016; Sadiktsis et al., 2014). In this study, the administration of diesel PM2.5 in vivo study was 10 mg/kg body weight and was sufficient to induce lung injury and inflammation in mice. Recent studies showed that intratracheal Fig. 5. Effects of MA on diesel PM2.5-induced signaling pathways. MA groups were injected intravenously 30 min after being challenged intratracheally with diesel PM2.5 (10 mg/kg in 50 μL of saline). The mice were then were sacrificed 24 h post-diesel PM2.5-injection, and the lung tissue was harvested. Representative examples from the Western blot analysis demonstrating the expression of (A) LC3 and Beclin 1, (B) TLR4 and MyD88, and (C) p-mTOR, mTOR, p-Akt, Akt, p-PI3K, and PI3K. Representative images from each group are shown (n = 3).

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immunity responses (Cadwell, 2016; Woodward et al., 2017). Because mTOR serves as a major checkpoint for autophagy, which is also involved in PM-induced lung inflammation, it has been suggested that both the mTOR/autophagy and TLR4/yD88 pathways affect lung injury (Hu et al., 2016). The TLR4-MyD88 pathway is known to be involved in upstream signaling for PM-induced inflammation and mediates inflammatory cytokine and oxidant production (Woodward et al., 2017). Oxidizing agents or other cytokines can inhibit mTOR activation, cause tissue cell autophagy, and lead to excessive inflammation and tissue damage (Zeng et al., 2017). Autophagy can also be regulated by multiple signaling pathways including the PI3K/Akt pathway (Shao et al., 2016), which is a key regulator of cell growth and survival that helps to mediate cardiomyocyte survival. Previous studies have reported that the activation of the PI3K/Akt pathway can phosphorylate mTOR, which is an important regulator of autophagy (Shao et al., 2016). Phosphorylated mTOR has been reported to protect against lung injury by reducing autophagy and promoting lung recovery (Herrero et al., 2018; Saxton and Sabatini, 2017). In this study, MA significantly increased the levels of p-mTOR, p-PI3K, and pAkt and decreased the levels of LC3 II and Beclin 1, while LY294002, which is a specific inhibitor of PI3K, strongly reversed the effects of MA, suggesting that MA inhibits excessive autophagy through the activation of the PI3K/Akt/mTOR pathway. Furthermore, our Western blot experiments showed that MA reduced TLR4 and MyD88 expression (Fig. 5B), indicating that MA inhibits diesel PM2.5-induced TLR4 and MyD88 upregulation, thus reducing inflammatory cytokines (e.g., IL-1β and TNF-α) and the production of oxidants (e.g., MPO and NO), which in turn activates mTOR and the autophagy of tissue cells. It is important to note that PM is generated directly from a variety of sources, such as construction sites, smokestacks, fires, and unpaved roads. PM particles have many sizes and morphologies, and PM pollution can be a combination of hundreds of different compounds. Therefore, a limitation of this study is that it does not address whether MA inhibits the pulmonary damage caused by different PM compounds.

instillation of PM2.5 at 10 mg/kg body weight could cause the respiratory diseases and cardiovascular dysfunction by inducing locally and systemically acute inflammations and/or simulating histological and functional changes in lung tissue in mice (Choi et al., 2019; Wang et al., 2012a, 2012b, 2016; Xie et al., 2015; Xu et al., 2018a; Zhang et al., 2016; Zhao et al., 2015). The major methods of PM2.5 exposure in animal models are intratracheal inhalation and intratracheal instillation. Intratracheal instillation is frequently used in mice, rats, and hamsters by inserting a needle into the mouth and throat. In the present study, diesel PM2.5 was administrated by the intratracheal instillation. It has been previously confirmed that the intratracheal instillation of PM2.5 causes pulmonary injury, including pulmonary vascular hyperpermeability, alveolar epithelial dysfunction, and inflammatory responses (Wang et al., 2018; Yan et al., 2017). Although the drawbacks of intratracheal instillation include its invasive and non-physiological nature, the fact that it bypasses the upper respiratory tract, and the confounding effects of the anesthesia and delivery vehicle (Morimoto et al., 2016), intratracheal instillation requires only a single application, thus increasing its accuracy and efficiency and is a convenient and effective method to induce lung injury in mice (Cho et al., 2018). Lung edema, which can be measured using the lung W/D weight ratio (Matsuyama et al., 2008), results from lung injury caused by the infiltration of inflammatory tissue and the secretion of inflammatory cytokines (Herrero et al., 2018). Inflammatory cytokines can stimulate endothelial cells to produce high concentrations of adhesion molecules and can prompt leukocytes to migrate toward lung tissue (Herrero et al., 2018). Impaired epithelial integrity also leads to vascular leakage and protein extravasation, increasing the occurrence of interstitial and alveolar edema (Bae, 2012; Lee et al., 2018). In the present study, the possible protective effects of MA against diesel PM2.5-induced lung injury were studied in mice. The results indicated that MA can lower the diesel PM2.5-induced increase in the lung W/D weight ratio, the total infiltration of inflammatory cells, the total protein levels in the BALF (Fig. 1), and diesel PM2.5-induced hyperpermeability (Fig. 3), thus suggesting that it has a protective function against pulmonary edema (Fig. 2). MPO levels have been employed to determine polymorphonuclear leukocyte activation and oxidative stress in terms of NO production (Blondonnet et al., 2016). The most notable characteristics of PM-induced pulmonary injury are the release of mediators during inflammation, including IL-1β and TNF-α, via the TLR4/MyD88 pathway, the activation of the inflammatory cascade, neutrophil migration to the alveoli, and lung damage (Gu et al., 2017; Zhao et al., 2012). The present study found that the levels of MPO, NO, and inflammatory cytokines such as IL-1β and TNF-α in the BALF were significantly reduced following the treatment of diesel PM2.5-induced lung injury with MA compared to untreated mice (Fig. 4). Autophagy is a lysosome-dependent process that collects damaged organelles, protein aggregates, and degraded cytoplasmic material in autophagic vacuoles (Choi et al., 2013). Autophagy has been shown to be involved in the developmental and regulatory processes of lung injury (Mizumura et al., 2012). In intact lung tissue, mTOR is known to be activated while autophagy-related protein LC3 II is downregulated (Hu et al., 2014) while, following lung injury, the suppression of mTOR is accompanied by the upregulation of LC3 II in human bronchial epithelial cells (Hu et al., 2016). In addition, when TLR4 or MyD88 is knocked down, lipopolysaccharide (LPS)-induced mTOR phosphorylation is downregulated, indicating that mTOR activation is caused by the TLR4 signaling pathway and that autophagy can be inhibited by LPS (Hu et al., 2014). Thus, autophagy may not play an important role in LPS-induced inflammation, though it may be involved in anti-inflammatory effects because rapamycin treatment can improve lung damage after LPS infection through the downregulation of mTOR. As a result, there is a strong possibility of a signaling network between TLR4 and autophagy in the presence of PM-induced lung injury, with autophagy governed by a complex signaling network and TLR4 an important sensor of autophagy that is closely involved in PM-induced

5. Conclusions In conclusion, our results indicate that MA attenuated diesel PM2.5induced pulmonary damage, including reduction of lung W/D weight ratio/total protein levels/the number of lymphocytes, inhibition of inflammatory cells infiltration, inflammatory cytokines expression, and hyperpermeability. Moreover, MA enhanced the recovery of tissue damage caused by diesel PM2.5-induced lung injury by inhibiting the TLR4 and autophagy pathways. The evaluation of MA on diesel PM2.5-induced inflammation and TLR4 and autophagy pathways will enlight the application of MA in diesel PM2.5-medicated adverse health effects. Therefore, this study could contribute the development of new prevention and treatment strategies for PM-induced respiratory diseases, indicating that MA can be used as a potentially efficient therapeutic agent against diesel PM2.5-induced lung injury. Declaration of competing interest The authors declare no conflicts of interest. Acknowledgments This study was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. 2017R1A5A2015391 and 2017M3A9G8083382). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.envres.2020.109230. 6

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