Biomolecules & Therapeutics 2024; 32(1): 162-169
Therapeutic Effects of (+)-Afzelechin on Particulate Matter-Induced Pulmonary Injury
Sanghee Cho, Yun Jin Park and Jong-Sup Bae*
College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 41566, Republic of Korea
Tel: +82-53-950-8570, Fax: +82-53-950-8557
Received: October 30, 2023; Revised: November 14, 2023; Accepted: November 15, 2023; Published online: January 1, 2024.
© The Korean Society of Applied Pharmacology. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Particulate matter (PM) constitutes a hazardous blend of organic and inorganic particles that poses health risks. Inhalation of fine airborne PM with a diameter of ≤ 2.5 μm (PM2.5) can lead to significant lung impairments. (+)-afzelechin (AZC), a natural compound sourced from Bergenia ligulata, boasts a range of attributes, including antioxidant, antimicrobial, anticancer, and cardiovascular effects. However, knowledge about the therapeutic potential of AZC for patients with PM2.5-induced lung injuries remains limited. Thus, in this study, we investigated the protective attributes of AZC against lung damage caused by PM2.5 exposure. AZC was administered to the mice 30 min after intratracheal instillation of PM2.5. Various parameters, such as changes in lung tissue wet/dry (W/D) weight ratio, total protein/total cell ratio, lymphocyte counts, levels of inflammatory cytokines in bronchoalveolar lavage fluid (BALF), vascular permeability, and histology, were evaluated in mice exposed to PM2.5. Data demonstrated that AZC mitigated lung damage, reduced W/D weight ratio, and curbed hyperpermeability induced by PM2.5 exposure. Furthermore, AZC effectively lowered plasma levels of inflammatory cytokines produced by PM2.5 exposure. It reduced the total protein concentration in BALF and successfully alleviated PM2.5-induced lymphocytosis. Additionally, AZC substantially diminished the expression levels of Toll-like receptors 4 (TLR4), MyD88, and autophagy-related proteins LC3 II and Beclin 1. In contrast, it elevated the protein phosphorylation of the mammalian target of rapamycin (mTOR). Consequently, the anti-inflammatory attribute of AZC positions it as a promising therapeutic agent for mitigating PM2.5-induced lung injuries by modulating the TLR4–MyD88 and mTOR–autophagy pathways.
Keywords: (+)-afzelechin, Particulate matter, Lung injury, TLR4–mTOR–autophagy pathway

Due to increased industrial emissions, global anthropogenic-driven air pollution levels have risen in recent times (Losacco and Perillo, 2018). Suspended particulate matter (PM) with a diameter of ≤2.5 μm (PM2.5) poses risks to the respiratory and circulatory systems and serves as a well-recognized indicator of air pollution. Given its minute size, approximately 96% of PM2.5 particles can be retained within the lungs for extended periods (Xing et al., 2016). The detrimental effects of PM2.5 are influenced by heavy metals, polycyclic aromatic hydrocarbons, oxygenated volatile organic compounds, and other factors (Cho et al., 2018). Conditions such as asthma, acute lung injury, and chronic obstructive pulmonary disease have been linked to inflammation triggered by PM2.5 exposure. Furthermore, the inflammation caused by PM2.5 contributes to the release of various cytokines and chemokines, including interleukins (ILs) and tumor necrosis factor (TNF)-α (Cho et al., 2018; Cao et al., 2022; Teng et al., 2022). Consequently, the need for innovative strategies for preventing and treating respiratory diseases, particularly given the strong link between PM2.5 exposure and the increased risk of asthma, as well as the incidence and mortality of lung cancer, is pressing (Wang et al., 2018).

The process of lung damage entails a intricate sequence of molecular events, including the activation of Toll-like receptor 4 (TLR4), a central regulator that triggers innate immune responses and governs inflammatory mediators (Du et al., 2022). It has been observed that particulate matter (PM) can heighten the production of inflammatory mediators by activating the TLR4 pathway (Nagappan et al., 2021). PM obstructs the mammalian target of rapamycin (mTOR), a cellular nutritional status sensor, and intensifies cellular oxidative stress, culminating in cellular apoptosis and autophagy, causing the breakdown of cellular components (Yan et al., 2021). Autophagy, a crucial lysosome-dependent mechanism that contributes to cellular homeostasis, has been connected to protein aggregation, organelle impairment, and the clearance of intracellular pathogens (Gao et al., 2022). Furthermore, autophagy’s implications in lung-related conditions underscore its significance in managing pulmonary disorders (Zhang et al., 2022). Consequently, inhibiting TLR4 activation and autophagy may hold therapeutic promise for addressing pulmonary damage.

Bergenia ligulata, an enduring plant indigenous to the Himalayas and a member of the Saxifragaceae family, is commonly found in rocky regions of Northern India (Saijyo et al., 2008). Recognized as Pashanbheda in India, its rhizomes possess various medicinal attributes encompassing diuretic, tonic, astringent, and laxative effects (Garimella et al., 2001; Saijyo et al., 2008). The plant houses a variety of chemical components, including bergenin, β-sitosterol, β-sitosterol-D-glucoside, and phenolic compounds like (+)-afzelechin (AZC) and catechin, among others (Garimella et al., 2001; Saijyo et al., 2008). Nevertheless, a comprehensive understanding of the mechanisms through which AZC impacts lung damage, histology, inflammation, and the TLR4–autophagy pathways subsequent to PM2.5 exposure is yet to be established. To bridge this knowledge gap, we employed a mouse model of PM2.5 exposure to test our proposition that AZC hinders PM2.5-induced proinflammatory reactions and autophagy in lung tissue cells, thereby ameliorating lung tissue impairment by suppressing the TLR4 and autophagy pathways.



Diesel PM NIST 1650b (Sigma-Aldrich Inc., St. Louis, MO, USA) was combined with saline following the manufacturer’s guidelines and subjected to sonication for 24 h to prevent the aggregation of dispersed PM2.5 particles (Bergvall and Westerholm, 2006). The acquisition of AZC was facilitated through ChemScene (Monmouth Junction, NJ, USA). Dexamethasone (DEX, utilized as a positive control) was procured from Sigma-Aldrich Inc. The negative control, mTOR, and TLR4 small interfering RNAs (siRNAs) were sourced from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Animal care

Male Balb/c mice (approximately 7 weeks old, weighing around 27 g) were procured from Orient Bio Co. (Sungnam, Korea) and utilized in our study after an acclimatization period of 12 days. Adhering to the guidelines of Kyungpook National University concerning the ethical treatment and usage of laboratory animals (IRB #: KNU2019-103), the mice were organized into eight distinct groups (n=10): the mock control group, the AZC control group, four PM2.5+AZC groups (0.25, 0.5, 1, and 2 mg/kg mouse body weight), the DEX group (2 mg/kg mouse body weight), and the PM2.5 group (10 mg/kg mouse body weight in 50 μL of saline). The control group mice were administered an equivalent volume of phosphate-buffered saline (PBS). Subsequently, PM2.5 (10 mg/kg mouse body weight in 50 μL saline) was intratracheally administered to the mice, while the AZC and DEX groups received injections into the tail vein as previously outlined (Kim et al., 2022a). The mice were euthanized 24 h post-injection, and samples of their bronchoalveolar lavage fluid (BALF) and lung tissue were collected for subsequent research endeavors.

Wet/dry weight ratio of the lung tissue

The wet weight of the right lung was measured, and subsequently, the lung was subjected to 24 h of drying at 120°C in an oven before being reweighed (dry weight). The lung wet-to-dry (W/D) weight ratio was then calculated as an indicator of lung edema.

Culture of mouse lung microvascular endothelial cell (MLMVECs) and siRNA transfection

MLMVECs were subjected to the treatment protocol described in earlier studies (Sim et al., 2021; Kim et al., 2022a). In brief, lung tissue was finely minced and subjected to a 45–60 min incubation at 37°C with collagenase A (1 mg/mL). To isolate the endothelial cells, anti-PECAM-1 monoclonal antibody (mAb) coupled with magnetic beads (BD Pharmingen, San Diego, CA, USA) was employed. The separated endothelial cells were cultured in growth media for a duration of 2 days prior to purification. These cells were then seeded onto fibronectin-coated dishes and cultivated using endothelial cell basal media supplemented with EGM-2 MV Bulletkit media (Lonza, Walkersville, MD, USA) to establish a monolayer culture. The introduction of mouse mTOR, TLR4, or irrelevant control siRNAs into the cells was carried out in accordance with previously documented methods (Kim et al., 2022a).

Hematoxylin and eosin staining

The lung cells were fixed using a formaldehyde solution (4%, Junsei, Tokyo, Japan) in PBS. To eliminate residual blood, PBS washing was conducted three times over a span of 20 h, all carried out at a temperature of 4°C. Following this, the materials were embedded within paraffin blocks, subjected to drying, and then sliced into sections measuring 4 micrometers in thickness. These sections were later deparaffinized and rehydrated before being stained using hematoxylin and eosin (Sigma-Aldrich Inc.). To assess the integrity of pulmonary architecture and the extent of tissue edema, a light microscope was used as per standard procedure. The observations were made by an impartial observer who remained unaware of the specific treatments being employed (Kim et al., 2022b; Lee and Bae, 2022).

Enzyme-linked immunosorbent assay of p38, mitogen-activated protein kinase, myeloperoxidase, nitrous oxide, interleukon-1β, and TNF-α phosphorylation

MLMVEC lysates were employed to quantify the levels of phosphorylated p38 mitogen-activated protein kinase (MAPK) through the utilization of an enzyme-linked immunosorbent assay (ELISA) kit from Cell Signaling Technology, Inc. (Danvers, MA, USA). Levels of myeloperoxidase (MPO), nitrous oxide (NO), IL-1β, IL-10, and TNF-α in the bronchoalveolar lavage fluid (BALF) were determined utilizing ELISA kits in accordance with the manufacturer’s guidelines (R&D Systems, Minneapolis, MN, USA). The ELISA readings were conducted using ELISA plate readers (Tecan Austria GmbH, Grödig, Austria).

Protein concentration and cell count in the BALF

After centrifugation of the bronchoalveolar lavage fluid (BALF) supernatant at 3,000 rpm for 10 min at 4°C, the QuantiProTM BCA Assay Kit (Sigma-Aldrich Inc.) was utilized to assess the total protein concentration. Both the total protein concentration and cytokine levels were evaluated using the QuantiProTM BCA Assay Kit. The resuspended cells were counted using a hematology analyzer after being suspended in 50 μL of PBS buffer.

Permeability assays

The administration of injections to the AZC and DEX groups was carried out through the tail vein, 30 min subsequent to the intratracheal administration of PM2.5 (10 mg/kg in 50 μL saline). Anesthesia was induced using a gas anesthetic machine that delivered a combination of 2% isoflurane-oxygen (Forane; JW Pharmaceutical, Seoul, Korea) through the RC2 Rodent Circuit Controller (VetEquip, Pleasanton, CA, USA). Prior to the intravenous infusion of a 1% Evans blue dye solution in regular saline, the mice underwent face mask anesthesia. After 6 h, the mice were euthanized through cervical dislocation, and the collection of bronchoalveolar lavage fluid (BALF) was performed. The measurement of permeability data was conducted using ELISA plate readers, following previously established protocols (Kim et al., 2019; Lee and Bae, 2019; Lee et al., 2019).

Western blot analysis

The cells were initially washed with PBS kept at a cold temperature and subsequently treated with a RIPA lysis solution. This solution consisted of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% sodium dodecyl sulfate, 1% NP-40, 1% sodium deoxycholate, and protease inhibitors (Zhang and Wang, 2018). After blocking with 5% bovine serum albumin for a duration of 2 h, the blots were exposed to primary antibodies, including anti-light chain (LC)3, anti-Beclin 1, anti-TLR4, anti-MyD88, anti-phosphorylated (p)mTOR, anti-Akt, anti-phosphorylated (p)PI3K, and anti-PI3K antibodies. These antibodies were obtained from Cell Signaling Technology, Inc. Following a washing step, the membrane was subjected to secondary antibodies that were conjugated to horseradish peroxidase (Cell Signaling Technology, Inc., at a dilution of 1:10,000). The concentration analyses of the obtained data were performed using the ImageJ Gel Analysis tool provided by NIH, Bethesda, MD, USA.

Statistical analysis

The results are expressed as means along with standard deviations (SD) from a minimum of three distinct trials conducted for each experiment. The statistical significance was evaluated through one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test, where a p-value of equal to or less than 0.05 was considered to indicate statistical significance. Statistical analyses were carried out using SPSS (version 15.0, SPSS Inc., Chicago, IL, USA) for the Windows operating system.


Effects of AZC lung damage by PM2.5

The lung wet-to-dry (W/D) weight ratio was utilized to evaluate the impact of AZC on lung damage induced by PM2.5 exposure. This ratio increased in the PM2.5 group (Fig. 1A), but decreased upon treatment with AZC or DEX (2 mg/kg). Additionally, we assessed the levels of total protein in the bronchoalveolar lavage fluid (BALF) and examined the extent of inflammatory cell infiltration caused by PM2.5. The PM2.5-exposed group displayed elevated total protein levels and an increased count of total cells, lymphocytes, and neutrophils in the mouse BALF compared to the control group (Fig. 1B-1E). Nevertheless, following intratracheal instillation of PM2.5, treatment with AZC or DEX (2 mg/kg) led to a reduction in the total cell and lymphocyte counts in the BALF. Furthermore, the administration of AZC or DEX in a dose-dependent manner resulted in decreased total protein levels in the BALF (Fig. 1B). To examine the protective effects of AZC against PM2.5-induced lung damage, we analyzed alterations in lung histology through hematoxylin and eosin (H&E) staining. In the PM2.5-exposed group, the alveolar wall was infiltrated and covered by inflammatory cells (Fig. 2A). We calculated a lung injury score after treating with varying doses of AZC or DEX (Fig. 2B), revealing that AZC mitigated inflammatory cell infiltration and provided lung protection against PM2.5-induced damage. Moreover, there was no additional efficacy of AZC at more than 2 mg/kg (data not shown). Therefore, these results show that AZC post-treatment attenuated lung damage caused by PM2.5.

Figure 1. The impact of AZC on lung damage induced by PM2.5. The AZC and DEX groups were subjected to intravenous injections 30 min after intratracheal administration of PM2.5 (10 mg/kg in 50 μL saline). Mice were euthanized 24 h post-PM2.5 injection, and their lung tissues and BALF were gathered. The effects of varying AZC and DEX concentrations were assessed for (A) the W/D ratio, (B) total cell count in the BALF, (C) total protein levels in the BALF, (D) lymphocyte and neutrophil counts, and (E) neutrophil countsE. Statistical analysis was performed using ANOVA followed by Tukey’s post hoc test. The presented data represent the means ± SD of three distinct experiments (n=3). #p<0.01 versus control group and *p<0.01 versus PM2.5 group.

Figure 2. Effects of AZC on PM2.5-induced lung injury. The AZC and DEX groups were subjected to intravenous injections 30 min after intratracheal administration of PM2.5 (10 mg/kg in 50 μL saline). Mice were euthanized 24 h post-PM2.5 injection, and their lung tissues and BALF were collected. (A) Histological changes in lung tissue were assessed using H&E staining. Five representative images were analyzed from each group. The scale bar measures 200 μm. Lung injury score (B) was determined. Statistical analysis involved ANOVA followed by Tukey’s post hoc test. The provided data represent the means ± SD of three distinct experiments (n=3). #p<0.01 versus control group and *p<0.01 versus PM2.5 group.

Effects of AZC on vascular barrier disruptive responses by PM2.5

The integrity of the vascular barrier can be compromised by PM, leading to disruptions (Huuskonen et al., 2021). Hence, this parameter was assessed to evaluate how AZC might influence the disruptive vascular reactions triggered by PM2.5. As shown in Fig. 3A, dye leakage in the BALF increased following PM2.5 exposure and was mitigated by AZC or DEX treatment (Fig. 3A). Additionally, we observed that PM2.5 induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK), which was prevented by AZC or DEX treatment. The activation of the p38 MAPK signaling pathway mediates the vascular damage response to inflammatory proteins (Fig. 3B).

Figure 3. Effects of AZC on PM2.5-induced vascular barrier disruptive response and p38 MAPK activation. The AZC and DEX groups were subjected to intravenous injections 30 min after intratracheal administration of PM2.5 (10 mg/kg in 50 μL saline). To evaluate the impact of AZC or DEX on vascular hyperpermeability caused by PM2.5, Evans blue flux in the BALF (expressed as g/mouse, n=5) and the levels of phosphorylated p38 (p-p38) in MLMVECs of mice were assessed using ELISA. Statistical analysis was performed using ANOVA followed by Tukey’s post hoc test. The provided data represent the means ± SD of three distinct experiments (n=3). #p<0.01 versus control group and *p<0.01 versus PM2.5 group.

Effects of AZC on pulmonary inflammatory responses by PM2.5

We investigated the impact of AZC on PM2.5-triggered pulmonary inflammatory reactions and observed that AZC effectively reduced the breakdown of the vascular barrier induced by PM2.5 in vivo (Fig. 3). Elevated lung MPO activity signifies the infiltration of neutrophils into the tissue, while inflammatory indicators like NO, IL-1β, and TNF-α reflect inflammatory processes. As depicted in Fig. 4, PM2.5 exposure led to increased lung tissue MPO activity and elevated production of NO, TNF-α, and IL-1β in the BALF compared to the control group. However, these effects were mitigated by AZC or DEX treatment. To further validate AZC’s anti-inflammatory characteristics, we assessed the levels of the anti-inflammatory cytokine IL-10. Interestingly, our findings indicated that both PM2.5 and AZC alone elevated IL-10 levels in the BALF. Furthermore, when AZC was administered alongside PM2.5, it led to a further increase in IL-10 levels compared to the control group (Fig. 4E). These results strongly suggest that AZC mitigated PM2.5-induced ALI by suppressing inflammatory cytokines and enhancing the expression of anti-inflammatory cytokines.

Figure 4. Effects of AZC on PM2.5-induced pulmonary inflammation. The AZC and DEX groups were administered intravenous injections 30 min after intratracheal delivery of PM2.5 (10 mg/kg in 50 μL saline). Subsequent to euthanizing the mice 24 h following PM2.5 injection, both lung tissues and BALF were collected. The measured parameters encompassed (A) MPO in lung tissue and the levels of (B) NO, (C) TNF-α, (D) IL-1β, and (E) IL-10 in the BALF. The statistical analysis involved ANOVA, followed by Tukey’s post hoc test. The presented data represent the means ± SD from three distinct experiments (n=3). #p<0.01 compared to the control group, and *p<0.01 compared to the PM2.5 group. #p<0.01 versus control group and *p<0.01 versus PM2.5 group.

Effects of AZC on signaling pathways by PM2.5

To assess the regulatory effects of AZC on LC3 and Beclin 1, we conducted Western blot analysis. The PM2.5-exposed group displayed elevated levels of LC3 II and Beclin 1 when compared to the control group (Fig. 5A, 5D). AZC administration effectively mitigated the PM2.5-induced increase in LC3 and Beclin 1 expression levels in the lung tissue, indicating its potential to counteract PM2.5-induced autophagy. However, treatment with LY294002 partially reversed some of these effects. Western blotting was also employed to evaluate the lung tissues of various groups for TLR4, MyD88, p-mTOR, total mTOR, p-Akt, Akt, p-PI3K, and PI3K, aiming to comprehend the connections between the TLR4 and mTOR–autophagy pathways and the anti-inflammatory/anti-autophagy influences of AZC. The instillation of PM2.5 via the intratracheal route led to heightened TLR4 and MyD88 expression levels in lung tissue (Fig. 5B, 5D), which were subsequently reduced by AZC therapy (2 mg/kg). The PM2.5-exposed group displayed lower levels of p-mTOR, p-Akt, and p-PI3K compared to the control group (Fig. 5C, 5D). Our study indicates that AZC potentially activates the PI3K/Akt/mTOR pathway by elevating p-mTOR, p-Akt, and p-PI3K levels. Nonetheless, the effects of LY294002 contrast with these results. Additionally, no significant variations in total Akt, PI3K, or mTOR levels were observed among the four groups. Genetic techniques were employed to silence TLR4 and mTOR expression in isolated MLMVECs using TLR4 or mTOR siRNA to validate the dependence of PM2.5-induced lung damage on the TLR4–mTOR signaling pathways. Exposure to PM2.5 notably increased TNF-α and IL-1β expression levels in MLMVECs, and mTOR knockdown considerably enhanced the production of TNF-α and IL-1β (Fig. 6). Conversely, siRNA-mediated TLR4 knockdown substantially reduced PM2.5-induced TNF-α and IL-1β production (Fig. 6).

Figure 5. Effects of AZC on PM2.5-induced signaling pathways. The AZC groups were administered intravenous injections 30 min after intratracheal instillation of PM2.5 (10 mg/kg in 50 μL saline). The mice were euthanized 24 h after the PM2.5 injection, and their lung tissues were harvested. Western blot analysis was employed to assess the expression levels of (A) LC3 and Beclin 1, (B) TLR4 and MyD88, as well as (C) p-mTOR, mTOR, p-Akt, Akt, p-PI3K, and PI3K. Representative images from each group are presented (n=3). (D) Densitometric intensities of each component in the signaling pathways were normalized to β-actin or total protein. #p<0.01 versus control group, *p<0.01 versus PM2.5 group, and $p<0.01 versus PM2.5 with AZC group.

Figure 6. Effects of TLR4 and mTOR siRNAs on PM2.5-induced TNF-α and IL-1β production in purified MLMVECs. MLMVECs that were isolated underwent transfection with TLR4 and mTOR siRNAs or control siRNA. Following this, the cells were exposed to PM2.5 (0.1 mg/mL) or the vehicle control for a duration of 6 h. Subsequently, the expression levels of (A) TNF-α and (B) IL-1β were assessed using ELISA. The data were subjected to analysis using ANOVA, followed by the Tukey’s post hoc test. The presented data represent the means ± SD of three distinct experiments (n=3). #p<0.01.

The imbalance between autophagy and apoptosis-induced inflammation is closely linked to the lung toxicity resulting from PM2.5 exposure (Li et al., 2017). Therefore, the modulation of this equilibrium between autophagy and apoptosis could serve as a treatment approach to mitigate lung diseases. AZC shares protective attributes with various other bioactive natural compounds for the respiratory system. Nonetheless, few investigations have delved into the safeguarding potential of AZC against respiratory disorders induced by PM2.5. This study primarily focuses on evaluating the potential therapeutic application of AZC for alleviating PM2.5-triggered lung impairment. Earlier research has shown that PM amplifies inflammatory responses in endothelial, epithelial, and macrophage cells, consequently causing localized lung inflammation Liu, Lee, Chen, Liang, Wang, Lue, Tsai, Lee, Chen, Yang, Chuang and Chen, 2018, Wang, Huang, Wang, Chen, Yang, Jin, Bai and Song, 2017, Xu, Qiu, Hu, Shang, Pardo, Fang, Wang, Rudich and Zhu, 2018). Furthermore, the excessive expression of inflammatory mediators could lead to systemic inflammation (Wang et al., 2017; Liu et al., 2018; Xu et al., 2018b). Hence, inflammation is considered the primary physiological response to PM exposure.

We conducted a study to explore the potential protective effects of AZC against lung damage induced by PM2.5 in mice. Our findings indicated that AZC exhibited a protective role by mitigating pulmonary edema, leading to reductions in total protein levels in the BALF, overall infiltration of inflammatory cells, lung wet-to-dry (W/D) weight ratio, and hyperpermeability caused by PM2.5 exposure (Fig. 1-3). Assessments of polymorphonuclear leukocyte activation and oxidative stress were carried out using MPO levels and NO generation (Blondonnet et al., 2016). PM-induced pulmonary damage is characterized by the activation of the TLR4-MyD88 pathway, which triggers the release of inflammatory mediators like IL-1β and TNF-α, initiating an inflammatory cascade that leads to neutrophil migration to alveoli and lung injury (Gu et al., 2017). Our study’s results demonstrated that mice treated with AZC following PM2.5-induced lung damage exhibited significantly reduced levels of MPO, NO, and inflammatory cytokines such as IL-1β and TNF-α compared to untreated mice (Fig. 4). In our mouse model of PM2.5-induced lung injury, AZC effectively curtailed both the infiltration of inflammatory cells into lung tissue and the production of inflammatory cytokines. The underlying mechanisms for this protective effect of AZC against PM2.5-induced lung damage may involve the downregulation of TLR4 and MyD88 expression, the upregulation of mTOR phosphorylation, and the inhibition of autophagy.

In this research, mice were treated with PM2.5 at a dosage of 10 mg/kg body weight, which effectively induced lung inflammation and damage. Recent investigations have indicated that intratracheal instillation of PM2.5 at 10 mg/kg body weight can lead to respiratory illnesses and cardiovascular issues by triggering both localized and systemic acute inflammation, as well as inducing histological and functional modifications in the lung tissue of mice (Wang et al., 2016; Zhang et al., 2016; Xu et al., 2018a; Choi et al., 2019). Animal models employ two primary techniques for PM2.5 exposure: intratracheal inhalation and instillation. Intratracheal instillation involves injecting the substance directly into the animals’ mouth and throat using a needle and has been widely used with mice, rats, and hamsters. In our study, we utilized intratracheal instillation to administer PM2.5 to the mice. While this method has certain drawbacks, such as invasiveness, non-physiological conditions, bypassing the upper respiratory tract, and potential confounding effects from anesthesia and the delivery method (Morimoto et al., 2016), it offers the advantage of being a one-time procedure that enhances accuracy and efficiency. Therefore, despite its limitations, this method remains a straightforward and effective way to establish mouse models of lung damage (Cho et al., 2018).

During the process of lysosome-dependent autophagy, damaged organelles, aggregated proteins, and degraded cytoplasmic debris accumulate within autophagic vacuoles (Gao et al., 2022; Zhang et al., 2022). Autophagy has been recognized to play roles in the regulatory and developmental phases of lung damage. In healthy lung tissue, mTOR activation occurs, leading to the downregulation of LC3 II, an autophagy-related protein. However, in cases of lung damage, human bronchial epithelial cells exhibit upregulated LC3 II expression along with mTOR downregulation (Zhang et al., 2022). Notably, the downregulation of phosphorylated mTOR (p-mTOR) induced by lipopolysaccharide (LPS) is observed when TLR4 or MyD88 is knocked down. This indicates that the TLR4 signaling pathway is involved in mTOR activation and that LPS can inhibit autophagy (Zhang et al., 2021). Due to the intricate nature of the signaling network that governs autophagy and TLR4’s role as a crucial autophagy sensor, which is closely linked to PM-triggered immune responses, the interplay between the TLR4 and autophagy signaling pathways is highly plausible in the context of PM-induced lung damage (Cadwell, 2016; Woodward et al., 2017). Both the mTOR-autophagy and TLR4-MyD88 pathways are proposed to contribute to lung injury, as mTOR serves as a primary suppressor of autophagy, which plays a role in PM-induced lung inflammation (Hu et al., 2016).

The PI3K/Akt pathway has been shown to activate mTOR, a pivotal regulator of autophagy, through phosphorylation (Shao et al., 2016). mTOR, when phosphorylated, can contribute to lung recovery by reducing autophagy and exerting protective effects against lung injury (Saxton and Sabatini, 2017; Herrero et al., 2018). In this study, AZC led to a significant decrease in the expression levels of LC3 II and Beclin 1, coupled with a substantial increase in the levels of phosphorylated mTOR (p-mTOR), phosphorylated PI3K (p-PI3K), and phosphorylated Akt (p-Akt). The effects of AZC were effectively countered by LY294002, a specific inhibitor of PI3K, indicating that AZC hinders excessive autophagy by activating the PI3K/Akt/mTOR pathway. Furthermore, our western blot analyses revealed that AZC downregulated the expression of TLR4 and MyD88 (Fig. 5B), implying that AZC inhibits the PM2.5-induced elevation of TLR4 and MyD88. This inhibition subsequently leads to a reduction in the production of inflammatory cytokines like IL-1β and TNF-α, as well as oxidative substances such as MPO and NO. As a result, the activation of mTOR and autophagy in tissue cells is modulated by AZC.

In summary, our study demonstrated that AZC effectively reduced the lung W/D weight ratio, total protein levels, and lymphocyte counts. It also acted to inhibit inflammatory cell infiltration, the release of inflammatory cytokines, and the hyperpermeability induced by PM2.5. Notably, AZC’s capacity to suppress the TLR4 and autophagy pathways played a significant role in promoting the recovery of tissue damage resulting from PM2.5-induced lung injury. A more comprehensive investigation into the impact of AZC on the TLR4 and autophagy pathways, as well as its influence on PM2.5-induced inflammation, could offer valuable insights into its potential application for addressing health complications arising from exposure to PM2.5. With its potential as an effective therapeutic agent against PM2.5-induced lung damage, the findings from our study have the potential to contribute to the development of innovative preventive and therapeutic strategies for managing respiratory disorders triggered by PM exposure.


This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2020R1A2C1004131).


The authors have no conflicts of interest to declare.

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