Cirsilineol Treatment Attenuates PM2.5-Induced Lung Injury in Mice

Ultrafine particulate matter with less than 2.5 μm diameter (PM2.5) is an air pollutant that causes severe lung damage. Currently, effective treatment and preventive methods for PM2.5-induced lung damage are limited. Cirsilineol (CSL) is a small natural compound isolated from Artemisia vestita. In this study, the efficacy of CSL on PM2.5-induced lung toxicity was tested, and its mechanism was identified. Lung injury was caused by intratracheal administration of PM2.5 suspension in animal models. Two days after PM2.5 pretreatment, CSL was injected via mouse tail vein for two days. The effects of CSL on PM2.5-induced lung damage, autophagy, apoptosis, and pulmonary inflammation in a mouse model and their mechanisms were investigated. CSL significantly suppressed histological lung damage and lung wet/dry weight proportion. CSL also significantly reduced PM2.5-induced autophagy dysfunction, apoptosis, lymphocyte suppression, and inflammatory cytokine levels in bronchoalveolar fluid (BALF). Furthermore, CSL increased mammalian target of rapamycin (mTOR) phosphorylation and significantly inhibited the expression of Toll-like receptors (TLR) 2 and 4, MyD88, and the autophagy proteins, Beclin1 and LC3II. Thus, CSL exerts protective effects on pulmonary damage by regulating mTOR and TLR2,4–myD88 autophagy pathways. Therefore, CSL can be used as an effective treatment for PM2.5-induced lung damage.


Introduction
Due to the rapid economic development around the world in recent years, air pollutants have become a leading cause of several pulmonary diseases [1,2]. Ultrafine particulate matter with less than 2.5 µm diameter (PM 2.5 ) is the primary air pollutant. Approximately 96% of PM 2.5 accumulates in the lungs owing to its small size and causes various diseases in the respiratory and circulatory systems [3]. High PM 2.5 concentrations promote the production and release of inflammatory mediators, which damage lung tissues, leading to respiratory lung injury and inflammation. Eventually, the respiratory disease leads to severe mortality and morbidity [4,5]. Secretion of cytokines and chemokines by PM 2.5 , such as interleukin (IL) and tumor necrosis factor (TNF)-α, causing inflammation. Furthermore, it results in the development of many respiratory diseases, including chronic obstructive pulmonary disease (COPD), acute pulmonary injury, and asthma [6,7]. Thus, a correlation exists between PM 2.5 exposure and disease-related mortality.
Apoptosis and autophagy are required to maintain lung function. However, uncontrolled autophagy owing to PM 2.5 exposure induces cell death and causes apoptosis, which advances to pathological pulmonary damage [8]. PM 2.5 -induced cellular oxidative stress can induce apoptosis and autophagy. Furthermore, PM 2.5 damages cellular components and inhibits the activation of mammalian target rapamycin (mTOR), a tropic sensor [9,10]. Autophagy, a lysosomal-dependent process, is involved in adaptation, protein aggregation, and organ damage to pathogen rotations within cells [11]. Autophagy is involved not only in the disease itself but also in lung inflammation observed in conditions such as COPD and lung injury [12,13].
Traditional Chinese and African herbal medicines have helped treat various ailments, including stroke, cardiovascular disease, diabetes, inflammatory disorders, and liver diseases; however, these benefits lack scientific validation [14,15]. Research on the application of traditional herbal and botanical medicines are rapidly increasing [15]. Natural products are increasingly preferred owing to their lack of side effects and cost-effectiveness [15]. Several medicinal herbs potently treat lung inflammation [14]. In this study, we evaluated the efficacy of bioactive compounds for treating PM 2.5 -induced pulmonary inflammation. Cirsilineol (CSL), 4,5-dihydroxy-3,6,7-trimethoxyflavone is a flavone bioactive compound present in Artemisia vestita Wall, an herb belonging to the Asteraceae family distributed in China and Tibet. CSL is a potent antioxidant, antibacterial, hypnotic, antitumor, and calmative drug, and exhibits cytotoxicity against many cancer cells [16][17][18][19]. However, the pharmacological role of CSL on PM 2.5 -induced lung injury, inflammatory response, histological changes, and the TLR-autophagy pathway remains unelucidated. Thus, in this study, we investigated the effect of CSL on autophagy and inflammation in cellular and animal models after PM 2.5 exposure and demonstrated that CSL improved the repair of PM 2.5 -induced tissue damage. Figure 1A represents the chemical structure of CSL. The experiments were conducted according to the workflow in Figure 1B. The mice were administrated with PM 2.5 for 48 h, and CSL or DEX was injected for another 48 h. After 24 h, the W/D ratio in the PM 2.5 -treated mice increased substantially and was ameliorated by CSL treatment ( Figure 1C). Furthermore, total cell and neutrophil count in BALF significantly reduced after CSL injection ( Figure 1D,E). Hematoxylin and eosin (H&E) staining confirmed that the lung tissue structure of the control and CSL-treated mice were similar ( Figure 1F). Pathological damages such as inflammatory cell infiltration, alveolar hemorrhage, exudates in the alveolar spaces, and alveolar wall thickening were observed in the PM 2.5 -treated mice lung tissue, and these changes were ameliorated by CSL. CSL administration also substantially decreased the lung tissue injury score ( Figure 1G). These results indicate that CSL administration alleviated PM 2.5 -induced pulmonary damage.

CSL Protects against PM 2.5 -Induced Lung Toxicity
such as COPD and lung injury [12,13].
Traditional Chinese and African herbal medicines have helped treat various ments, including stroke, cardiovascular disease, diabetes, inflammatory disorders, a liver diseases; however, these benefits lack scientific validation [14,15]. Research on application of traditional herbal and botanical medicines are rapidly increasing [15]. N ural products are increasingly preferred owing to their lack of side effects and cost-eff tiveness [15]. Several medicinal herbs potently treat lung inflammation [14]. In this stu we evaluated the efficacy of bioactive compounds for treating PM2.5-induced pulmon inflammation. Cirsilineol (CSL), 4,5-dihydroxy-3,6,7-trimethoxyflavone is a flavone b active compound present in Artemisia vestita Wall, an herb belonging to the Asterac family distributed in China and Tibet. CSL is a potent antioxidant, antibacterial, hypno antitumor, and calmative drug, and exhibits cytotoxicity against many cancer cells [ 19]. However, the pharmacological role of CSL on PM2.5-induced lung injury, inflamm tory response, histological changes, and the TLR-autophagy pathway remains unelu dated. Thus, in this study, we investigated the effect of CSL on autophagy and inflamm tion in cellular and animal models after PM2.5 exposure and demonstrated that CSL proved the repair of PM2.5-induced tissue damage. Figure 1A represents the chemical structure of CSL. The experiments were conduc according to the workflow in Figure 1B. The mice were administrated with PM2.5 for 4 and CSL or DEX was injected for another 48 h. After 24 h, the W/D ratio in the PM treated mice increased substantially and was ameliorated by CSL treatment (Figure 1 Furthermore, total cell and neutrophil count in BALF significantly reduced after CSL jection ( Figure 1D,E). Hematoxylin and eosin (H&E) staining confirmed that the lung sue structure of the control and CSL-treated mice were similar ( Figure 1F). Patholog damages such as inflammatory cell infiltration, alveolar hemorrhage, exudates in the veolar spaces, and alveolar wall thickening were observed in the PM2.5-treated mice lu tissue, and these changes were ameliorated by CSL. CSL administration also substantia decreased the lung tissue injury score ( Figure 1G). These results indicate that CSL adm istration alleviated PM2.5-induced pulmonary damage.  Two days after PM 2.5 exposure (10 mg/kg in 50 µL saline) daily by intratracheal instillation, the mice were intravenously injected with CSL or dexamethasone (DEX) daily for three consecutive days. The control and PM 2.5 groups received an equal amount of vehicle [0.5% dimethyl sulfoxide (DMSO)] at the same time each day. Control mice received the same volume of saline. Mice were euthanized 24 h after the last compound administration, and lung tissue and bronchoalveolar lavage fluid (BALF) were harvested. The effects of different CSL or DEX concentrations on (C) wet/dry (W/D) weight ratio, (D) total cell count in the BALF, and (E) total neutrophils in BALF were assessed. (F) Lung histology was examined using H&E staining. Representative images from each group are shown (n = 5). Scale bar = 160 µm. (G) Lung injury scores. Values represent the mean ± standard deviation (SD) of three independent experiments. * p < 0.01 versus the PM 2.5 -challenged group. # p < 0.01 versus control group.

CSL Prevents PM 2.5 -Induced Autophagy Dysfunction
We tested whether CSL affected autophagy-associated proteins, such as Beclin 1 and LC3, by Western blotting. The expression of these autophagy-associated proteins substantially increased in PM 2.5 -treated mice than that in the control mice. Notably, CSL substantially inhibited these increases in mouse lung tissue ( Figure 2A). These results suggest that CSL inhibits PM 2.5 -induced autophagy. Subsequently, the expression of proteins in the TLR 2,4 and mTOR autophagy pathways were determined to confirm the CSL-mediated anti-autophagy and anti-inflammatory mechanisms. Intratracheally injected PM 2.5 increased TLR2, TLR4, and MyD88 expression levels ( Figure 2B), which were ameliorated by CSL administration. Moreover, phosphorylated mTOR, Akt, and PI3K expression levels in PM 2.5 -treated mice decreased than that in the control mice ( Figure 2C), which were considerably inhibited by CSL treatment. Thus, these results suggest that CSL activates the mTOR/Akt/ PI3K pathway.

CSL Prevents PM2.5-Induced Autophagy Dysfunction
We tested whether CSL affected autophagy-associated proteins, such as Beclin 1 and LC3, by Western blotting. The expression of these autophagy-associated proteins substantially increased in PM2.5-treated mice than that in the control mice. Notably, CSL substantially inhibited these increases in mouse lung tissue ( Figure 2A). These results suggest that CSL inhibits PM2.5-induced autophagy. Subsequently, the expression of proteins in the TLR 2,4 and mTOR autophagy pathways were determined to confirm the CSL-mediated anti-autophagy and anti-inflammatory mechanisms. Intratracheally injected PM2.5 increased TLR2, TLR4, and MyD88 expression levels ( Figure 2B), which were ameliorated by CSL administration. Moreover, phosphorylated mTOR, Akt, and PI3K expression levels in PM2.5-treated mice decreased than that in the control mice ( Figure 2C), which were considerably inhibited by CSL treatment. Thus, these results suggest that CSL activates the mTOR/Akt/ PI3K pathway.

CSL Inhibits PM 2.5 -Induced Apoptosis in Mice Lung Tissues
To determine whether PM 2.5 affects apoptosis in lung tissue, the levels of apoptotic proteins, including Bcl-2, Bax, cleaved caspase 3, and cleaved poly [ADP-ribose] polymerase 1 (PARP1), were evaluated in the lung tissue. The level of Bcl-2, an anti-apoptotic protein, was significantly reduced, whereas those of the pro-apoptotic proteins, Bax, cleaved caspase3, and cleaved PARP1, were substantially elevated ( Figure 3A-D). However, CSL prevented considerable alterations in these apoptotic protein levels ( Figure 3A-D). Consistently, PM 2.5 treatment considerably elevated the apoptosis index, as demonstrated using TUNEL im-

Discussion
Pulmonary toxicity by PM2.5 is closely related to the imbalance between autophagy and inflammation by apoptosis [20]. Therefore, modulating the balance between apoptosis and autophagy may be a therapeutic and preventive strategy for lung diseases. Although CSL can protect the respiratory tract, its effect on PM2.5-induced respiratory diseases has been sparingly investigated. Here, CSL ameliorated autophagic dysfunction and reduced lung inflammation and apoptosis by activating the mTOR signaling pathway in an animal model of PM2.5-induced pulmonary damage. Therefore, CSL treatment may protect the lung from PM2.5-induced damage by controlling the TLR2,4-MyD88 and mTOR-autophagy pathways.

CSL Protected PM 2.5 -Induced Pulmonary Inflammatory Responses in Mice
PM 2.5 treatment substantially increased pro-inflammatory cytokines such as IFNγ, IL-1β, IL-6, IL-18, and TNF-α, which were ameliorated by CSL injection (Figure 4). Conversely, the levels of anti-inflammatory cytokines such as IL-2, IL-4, and IL-10 were significantly decreased after PM 2.5 administration and recovered by CSL injection (Figure 4). These results indicate that CSL can be used to protect mice against PM 2.5 -induced lung inflammation.
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Discussion
Pulmonary toxicity by PM2.5 is closely related to the imbalance between autophagy and inflammation by apoptosis [20]. Therefore, modulating the balance between apoptosis and autophagy may be a therapeutic and preventive strategy for lung diseases. Although CSL can protect the respiratory tract, its effect on PM2.5-induced respiratory diseases has been sparingly investigated. Here, CSL ameliorated autophagic dysfunction and reduced lung inflammation and apoptosis by activating the mTOR signaling pathway in

Discussion
Pulmonary toxicity by PM 2.5 is closely related to the imbalance between autophagy and inflammation by apoptosis [20]. Therefore, modulating the balance between apoptosis and autophagy may be a therapeutic and preventive strategy for lung diseases. Although CSL can protect the respiratory tract, its effect on PM 2.5 -induced respiratory diseases has been sparingly investigated. Here, CSL ameliorated autophagic dysfunction and reduced lung inflammation and apoptosis by activating the mTOR signaling pathway in an animal model of PM 2.5 -induced pulmonary damage. Therefore, CSL treatment may protect the lung from PM 2.5 -induced damage by controlling the TLR2,4-MyD88 and mTOR-autophagy pathways.
PM induces local lung inflammation by increasing the inflammatory responses of epithelial cells, endothelial cells, and macrophages [21][22][23], and systemic inflammation can occur when inflammatory mediators are overexpressed [24]. Therefore, CSL exposure may cause vascular inflammation as a biological response. Previous studies have demonstrated the relationship between PM 2.5 exposure, reduced vascular integrity, and the expression of inflammation-associated molecules, including IL-6, TNF-α, p38, and reactive oxygen species (ROS) [25,26]. Our study established that CSL alleviated PM 2.5 -induced lung injury in a mouse model by suppressing both inflammatory cytokine secretion and lung tissue infiltration. The anti-inflammatory pathway of CSL against PM 2.5 appears to be regulated by decreased TLR2, TLR4, and MyD88 expression, increased mTOR phosphorylation, and autophagy prevention.
An injection of 10 mg/kg PM 2.5 caused pulmonary damages and inflammation in vivo. In previous studies, intraperitoneal PM 2.5 injection caused respiratory and cardiovascular dysfunction by inducing systemic and local acute inflammation and stimulating histopathological and functional changes in mouse lung tissue [25,27,28]. In this study, PM 2.5 was administered by intratracheal instillation, which is one of the leading methods of exposing animal models to PM 2.5 and is usually performed by inserting a needle into the mouth and throat of mice and hamsters. Intratracheal PM 2.5 instillation reportedly causes pulmonary injury by inducing alveolar epithelial dysfunction, inflammatory responses, and high levels of lung vessel permeability [29,30]. Although intratracheal instillation has some drawbacks, such as its non-physiological and invasive nature, the disturbing effect of anesthesia and means of delivery [31], this method is still used effectively and conveniently as only one injection induces pulmonary injury in mice [32].
Autophagy, a lysosomal-dependent process, gathers unnecessary or dysfunctional components in autophagic fluid for destruction [11]. During this process, autophagy is involved in pulmonary injury pathogenesis [33]. The activation of LC3 II, an autophagyassociated protein, in lung tissue is inhibited during mTOR activation [34], and mTOR inhibition is accompanied by upregulated LC3 II in human bronchial epithelial cells [35]. In addition, MyD88 or TLR4 knockdown downregulates lipopolysaccharide (LPS)-induced mTOR phosphorylation. These results suggested that LPS could inhibit autophagy activity, and the TLR4 signaling pathway could trigger mTOR activation [34]. Thus, despite the anti-inflammatory effects of autophagy, mTOR downregulation by rapamycin may not be effective in suppressing pulmonary injury. Therefore, autophagy and TLR4 can interact during PM-induced inflammatory responses, and autophagy can be manipulated by multiple signal transduction pathways. Moreover, TLR4 can function as an autophagy sensor involved in the PM-induced immune response [12,36]. Both TLR4-MyD88 and mTOR-autophagy pathways affect lung injury, and mTOR serves as a critical marker of autophagy in PM-induced pulmonary inflammation [35]. PM-induced inflammatory responses regulate cytokine and oxidant production through the TLP-MyD88 signaling pathway [36]. Some cytokines or oxidizing agents inhibit mTOR activation, induce cellular autophagy, and cause increased levels of tissue damage and inflammation [37]. Other signaling pathways, such as PI3K-Akt pathway [38], which regulates cell growth and survival and reduces cardiomyocyte death [39], may also control autophagy.
Western blot analysis results also indicated that CSL ameliorated the PM 2.5 -induced increase in TLR 2, 4, and MyD88 expression (Figure 2). These results further indicate that CSL activates mTOR expression by reducing inflammatory cytokines and enhancing anti-inflammatory cytokines (Figure 4). Although CSL exhibited no anti-inflammatory effect, it suppressed PM 2.5 -induced lung injury by modulating PM 2.5 -mediated severe inflammatory response and autophagy compared to that in the control group. The pathway analysis suggests that CSL acts as an anti-inflammatory agent because it controls both TLR 2, 4-MyD88, and mTOR autophagy pathways.
In conclusion, CSL protects the lung from PM 2.5 -induced respiratory diseases through autophagy and TLR 2 and 4 pathway modulation. Therefore, we suggest that CSL is a potentially efficient treatment option for PM 2.5 -induced pulmonary damage.

Materials
Diesel PM 2.5 NIST 1650b [42], CSL, and dexamethasone (DEX, positive control) were obtained from Sigma-Aldrich Inc (St. Louis, MO, USA). PM 2.5 was blended in saline and sonicated for 24 h to break up the suspended particle agglomerates.

Animal Experiments
Seven-week-old BALB/c mice were purchased from Orient Bio Co (Seongnam, Korea). After acclimatization for 12 days, five mice were housed per cage and conditioned to a temperature of 20-25 • C. The mice experiments were conducted in accordance with the Care and Use of Laboratory Animals by Kyungpook National University (IRB No. KNU 2017-102). Mice were divided into 7 groups of 10 mice each depending on the treatment conditions: (1) control (dimethyl sulfoxide; DMSO) group, (2) CSL control group (200 µg/kg), (3) PM 2.5 group, (4-6) PM 2.5 + CSL group (50, 100, and 200 µg/kg), (7) PM 2.5 + DEX group (5 mg/kg). An equivalent dose of DMSO was administered to the control group. Briefly, 10 mg/kg PM 2.5 in 50 µL saline was intranasally injected, and CSL and DEX were administered intravenously in the tail 30 min later, as described previously [43,44]. The mice were euthanized 24 h after compound injection. Subsequently, bronchoalveolar lavage fluid (BALF) was collected for analysis. Nasal PM 2.5 injection causes high levels of pulmonary vascular permeability and diseases, including pulmonary inflammation and alveolar epithelial dysfunction [30,45]. Therefore, this method conveniently and effectively induces lung damage with PM 2.5 .

Lung Wet/Dry (W/D) Weight Ratios
W/D ratio was identified to determine pulmonary edema. The weights of the right lung before and after drying in a 120 • C oven for 24 h were measured as wet and dry values, respectively.

Hematoxylin and Eosin (H&E) Staining
To observe phenotypic changes, the mice lungs were isolated and washed thrice with PBS at pH 7.4. Subsequently, it was fixed in 4% formaldehyde in PBS (Junsei, Tokyo, Japan) at 4 • C for 20 h. The fixed samples were embedded, in paraffin, dehydrated, and cut into 4-µm sections. Next, the samples were deparaffinized, rehydrated, and stained with hematoxylin (Sigma-Aldrich, St. Louis, MO, USA). The lung specimens were visualized under a light microscope to monitor the lung structure and tissue edema, as described previously [46,47].

Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay
The mesenchymal of the right lung was used to detect apoptotic cells in lung tissue using the TUNEL assay. The TUNEL reaction mixture (Roche Applied Science; Mannheim, Germany) was prepared and the total apoptotic cells was shown as the % of TUNELpositive cells to the total cells by Hoechst staining.

Statistical Analysis
The experiments were repeated at least thrice and performed independently. The data are expressed as the mean ± standard deviation (SD). SD values were analyzed using a one-way analysis of variance (ANOVA) followed by Dunnett's test. All analyzes were performed using SPSS for Windows version 16.0, and p < 0.05 were considered statistically significant (SPSS, Chicago, IL, USA).