Suppressive Functions of Veratramine on PM_2.5-Induced Oxidative Stress

Abstract

Background: Particulate matter (PM_2.5) inhalation induces pulmonary disorders through oxidative stress. Veratramine (VRT), a steroidal alkaloid derived from Veratrum species, exhibits protective pharmacological potential. Therefore, this study aims to investigate the protective effects of VRT against PM_2.5-induced oxidative injury and the underlying molecular mechanisms. Methods: In vitro experiments were conducted using pulmonary artery endothelial cells (HPAECs), which were exposed to PM_2.5 (25–100 μg/mL) ± VRT (2–50 μM) or Dexamethasone (DEX; 50 μM) for 24–48 h. Measurements included 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability, Lactate dehydrogenase ELISA, 2′,7′-dichlorodihydrofluorescein diacetate reactive oxygen species (ROS), superoxide dismutase/catalase kits, and Western blots (Bax, serum, and glucocorticoid-regulated kinase 1 (SGK1), microtubule-associated protein 1 light chain 3 (LC3), Toll-like receptor (TLR4), and mechanistic target of rapamycin (mTOR)). Results: PM_2.5 exposure reduced HPAEC viability in a dose- and time-dependent manner, likely due to increased lactate dehydrogenase leakage and intracellular ROS accumulation. Oxidative stress correlated with altered superoxide dismutase and catalase activities, with suppression of SGK1, a key factor in cell survival. VRT treatment enhanced cell viability, mitigated oxidative stress, and restored SGK1 expression. Moreover, VRT promoted mTOR phosphorylation and markedly suppressed PM_2.5-induced increases in TLR4, MyD88, and the autophagy markers LC3 II and Beclin 1. Conclusions: Collectively, these findings indicate that VRT protects against PM_2.5-induced pulmonary injury by modulating oxidative stress and the mTOR-associated autophagy pathway, highlighting its potential as a therapeutic candidate for PM_2.5-related respiratory disorders.

1. Introduction

Numerous epidemiological studies report a strong association between particulate matter (PM) exposure and adverse health consequences, particularly those affecting the pulmonary and cardiovascular systems [1,2,3]. Ultrafine PM, defined as particles with an aerodynamic diameter below 100 nm, can deeply penetrate the pulmonary epithelium and enter systemic circulation, posing significant health risks [4]. Long-term inhalation of fine particles, especially PM_2.5 (aerodynamic diameters < 2.5 μm), is significantly associated with increased risks of respiratory and cardiovascular mortality [5]. PM_2.5 is a major atmospheric contaminant due to its persistence in biological tissues and ability to induce oxidative injury within the human body [3]. PM_2.5-induced oxidative stress is a key molecular pathway mediating its adverse biological effects [3]. This phenomenon occurs when reactive oxygen species (ROS) production exceeds cellular antioxidant defense mechanisms. PM_2.5 inhalation induces pulmonary inflammation, endothelial dysfunction, and systemic cardiovascular effects via multiple mechanisms. PM_2.5 components—transition metals (Fe, Cu, Ni), polycyclic aromatic hydrocarbons (PAHs), and endotoxins—generate ROS through NADPH oxidase activation and mitochondrial dysfunction, overwhelming antioxidant defenses, such as superoxide dismutase [SOD], catalase [CAT], and glutathione peroxidase [GPx]. Oxidative stress activates NF-κB and MAPK pathways, upregulating pro-inflammatory cytokines (IL-6, TNF-α, IL-1β) and adhesion molecules (ICAM-1, VCAM-1) that enhance leukocyte recruitment and vascular permeability. PM_2.5 triggers endothelial apoptosis through Bax/Bcl-2 imbalance and caspase activation. Epidemiological studies show that PM_2.5 exposure increases the risk of acute coronary syndrome, stroke, chronic obstructive pulmonary disease exacerbations, asthma hospitalizations, and lung cancer. Autophagy dysregulation—marked by LC3-II/Beclin-1 upregulation—worsens PM_2.5 cytotoxicity by degrading damaged organelles and impairing cell survival signaling. These multifaceted mechanisms highlight the urgent need for therapeutics addressing PM_2.5-induced oxidative stress, inflammation, and autophagy in vascular endothelium. ROS critically modulate signaling cascades that determine cell fate [6], and excessive PM_2.5-induced ROS directly impair antioxidant enzymes such as SOD, GPx, and CAT, reducing their functionality [7]. Additionally, inorganic and organic PM_2.5 fractions contribute to its cytotoxicity. Elevated ROS—especially free radicals—damage nucleic acids and proteins, representing key mechanisms of PM_2.5-induced oxidative injury in human cells [8,9,10].

Ambient PM_2.5 concentrations associated with increased cardiopulmonary morbidity and mortality are typically in the tens of μg/m³ range in urban or industrial settings, which differ substantially from the μg/mL doses commonly applied in submerged in vitro endothelial models [11,12]. Accordingly, experimental concentrations of 25–100 μg/mL PM_2.5 used in this study fall within the range frequently employed in endothelial and epithelial cell assays to elicit measurable oxidative stress and barrier dysfunction over 24–48 h, but they represent an acute, high-burden exposure that does not directly correspond to a single environmental episode. These in vitro doses should therefore be interpreted as a mechanistic tool to probe PM_2.5-triggered oxidative and autophagic pathways under controlled conditions rather than as a direct surrogate for chronic ambient or occupational exposure levels. Thus, while our PM_2.5 concentrations are toxicologically relevant for dissecting endothelial injury mechanisms, they inevitably overestimate real-world airway surface doses, and this limitation should be considered when extrapolating our findings to clinical or environmental scenarios.

Cells counteract oxidative stress through autophagy, which degrades and recycles intracellular material to maintain cellular integrity. Autophagy sequesters cytoplasmic cargo in autophagosomes, which fuse with lysosomes to form autolysosomes for degradation by lysosomal enzymes [13,14]. Key proteins—Atg5, Atg12, Beclin1, and microtubule-associated protein 1 light chain 3 (LC3)—regulate autophagosome formation [15]. Although typically protective by supplying metabolites during stress [15], the molecular events linking PM_2.5 exposure to autophagy remain unclear, highlighting the need for further research on airborne pollutant-associated cellular responses.

Plant-derived bioactive compounds have attracted considerable attention as potent and safer alternatives to conventional synthetic anticoagulants due to their reduced side effects and broad therapeutic potential [16]. Among these phytochemicals, VRT—a steroidal alkaloid naturally found in various Veratrum species of the lily family—has emerged as a pharmacologically interesting molecule [17]. Studies show that VRT exerts multiple physiological effects, including blood pressure reduction, sodium channel inhibition, and analgesic activity [18,19]. Additionally, this compound exhibits diverse bioactivities, including anticancer, antihypertensive, and dermatological effects [20,21,22]. Although Li et al. [23] report its strong anti-inflammatory potential, the underlying molecular mechanisms responsible for these properties remain unclear. Compared with classical antioxidant or anti-inflammatory agents such as polyphenols and flavonoids, which primarily act as direct ROS scavengers [24,25,26], VRT appears to exert broader cytoprotective effects by concomitantly restoring SGK1–PI3K–Akt–mTOR signaling, normalizing antioxidant enzyme activity, and suppressing TLR4–MyD88-dependent autophagy and inflammation in endothelial cells. These multimodal actions may provide a mechanistic advantage in the context of PM_2.5 toxicity, where oxidative stress, inflammatory signaling, and autophagy dysregulation synergistically contribute to endothelial injury. Therefore, VRT can be considered not only as a simple antioxidant but also as a pleiotropic regulator of redox balance and stress-response pathways in PM_2.5-exposed pulmonary endothelium. Therefore, this study aims to investigate antioxidant defense and autophagy regulation in human pulmonary artery endothelial cells (HPAECs) under PM_2.5 exposure and the modulatory effects of VRT on these cellular pathways.

2. Materials and Methods

2.1. Reagents

Diesel PM NIST 1650b [27] was obtained from Sigma-Aldrich Inc. (St. Louis, MO, USA), suspended in saline, and ultrasonicated (Qsonica Q500 Sonicator (Qsonica, LLC, Newtown, CT, USA)) for 30 min to ensure proper particle dispersion and minimize PM_2.5 aggregation. Dexamethasone (DEX), a standard anti-inflammatory agent [28] used as a positive control, and VRT (>98% purity) were also purchased from Sigma-Aldrich Inc. The phosphoinositide 3-kinase (PI3K) inhibitor LY294002 was supplied by Sigma-Aldrich Inc. Unless otherwise noted, all other reagents and chemicals utilized in the experiments were also sourced from Sigma-Aldrich.

2.2. Primary Culture of Human Pulmonary Artery Endothelial Cells and PM_2.5 Treatment

HPAECs were obtained from Cambrex Bio Science (Charles City, IA, USA) and authenticated via morphology and functional assays per supplier protocol; cells were mycoplasma-negative via routine PCR. Cells were cultured in Endothelial Cell Growth Medium (EGM)-2 (EGM-2 BulletKit (CC-3162), Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS) following the protocol of the supplier [29,30,31]. Cells at passages 3 and 5 were utilized for all experimental procedures. Before treatment, cultures reached ~80–90% confluence, with >90% viability confirmed via trypan blue exclusion. The cells were incubated for 24 h, then exposed to varying concentrations of PM_2.5 (25, 50, or 100 μg/mL) with or without VRT (2, 5, 10, 20, or 50 μM) or DEX (50 μM) for another 24 h.

2.3. Cell Viability Assay

Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, following previously described procedures [30,31]. Briefly, mitochondrial dehydrogenase activity was quantified using the assay, with yellow tetrazolium salt converted to purple formazan crystals by metabolically active cells, which were solubilized and measured spectrophotometrically at 570 nm (Tecan Infinite M200 PRO microplate reader (Tecan Austria GmbH, Grödig, Austria)). Cell viability was expressed as a percentage of the control, set at 100%.

2.4. Western Blot Analysis

For Western blot analysis, cells were rinsed with ice-cold phosphate-buffered saline (PBS; pH 7.4; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄) and lysed in a buffer containing 0.5% sodium dodecyl sulfate, 1% NP-40, 1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), and protease inhibitors, as described previously [32]. Protein extracts were separated on SDS-PAGE, transferred onto polyvinylidene fluoride membranes, and blocked with 5% bovine serum albumin (BSA) for 2 h at room temperature. The membranes were then incubated overnight at 4 °C with primary antibodies against Bax (#2772, 1:2000), Bcl-2 (#3498, 1:2000), SGK1 (#12014, 1:1000), LC3 (#3868, 1:1000), Beclin 1 (#3738, 1:1000), TLR4 (#14354, 1:1000), MyD88 (#4283, 1:1000), mTOR (#2983, 1:1000), p-mTOR (#5536, 1:1000), Akt (#4691, 1:1000), p-Akt (#4060, 1:2000), p-PI3K (#4228, 1:1000), and PI3K (#4292, 1:800) (Cell Signaling Technology, Danvers, MA, USA). After incubation, membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology; #7074, 1:10,000). Protein band intensities were quantified via densitometry using the ImageJ Gel Analysis tool (1.53t, NIH, Bethesda, MD, USA).

2.5. ELISA for Lactate Dehydrogenase

PM_2.5-induced cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) release from HPAECs after exposure to PM_2.5. Cells were treated with PM_2.5 (100 μg/mL) for 48 h, then cell-free supernatants were collected, and LDH activity was quantified using a commercial LDH assay kit (L7572, Pointe Scientific, Lincoln Park, MI, USA). All experimental samples were analyzed in duplicate, with LDH levels measured with absorbance readings on an ELISA microplate reader (Tecan Infinite M200 PRO microplate reader, Tecan Austria GmbH, Grödig, Austria).

2.6. Measurement of Reactive Oxygen Species

ROS levels were measured using a fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma-Aldrich, D6883; St. Louis, MO, USA), which predominantly detects hydrogen peroxide (H₂O₂). HPAECs were seeded in 96-well plates at a density of 2 × 10⁵ cells/well and incubated with 50 μg/mL DCFH-DA. Following probe loading, ROS generation was quantified based on the fluorescence intensity of the oxidized product (excitation/emission wavelengths: 485 nm/535 nm) using a Tecan Infinite M200 PRO microplate reader (Tecan Austria GmbH, Grödig, Austria).

2.7. Evaluation of Oxidative Stress Markers

SOD activity was measured using a commercial SOD assay kit (Sigma-Aldrich/Fluka, 19160; St. Louis, MO, USA) according to the instructions of the manufacturer. Catalase (CAT) activity was determined using a CAT assay kit (Sigma-Aldrich, CAT100; St. Louis, MO, USA) by monitoring H₂O₂ decomposition at 240 nm every 30 s for 5 min at 25 °C (molar extinction coefficient ε = 0.0436 cm²/μM).

2.8. Statistical Analysis

Statistical analyses were performed using one-way ANOVA followed by Dunnett’s multiple comparison test (GraphPad Prism 9.0, San Diego, CA, USA), and p < 0.05 was considered significant. Data visualization and densitometry were conducted using ImageJ (NIH, Bethesda, MD, USA) and GraphPad Prism 9.0. Results are presented as mean ± SD from ≥3 independent experiments.

3. Results

3.1. Effects of Veratramine on PM_2.5-Induced Cell Damage and Viability

PM_2.5 exposure significantly reduced HPAEC viability in a dose- and time-dependent manner, as measured via the MTT assay (Figure 1A). Subsequent VRT treatment markedly restored cell viability (Figure 1B). DEX, a well-known anti-inflammatory agent [28,33], served as a positive control. Western blot analysis revealed that PM_2.5 exposure increased Bax-mediated apoptotic signaling, whereas VRT treatment restored Bax to near-control levels (Figure 1C). Similarly, VRT administration reversed the PM_2.5-induced decrease in Bcl-2, a key anti-apoptotic protein (Figure 1C). Moreover, PM_2.5 exposure suppressed SGK1 expression—a key cell survival regulator—while VRT treatment restored its expression, suggesting the protective role of SGK1 against PM_2.5-induced damage. Consistently, 24 h PM_2.5 exposure markedly increased LDH release, which was significantly reduced by 50 μM VRT (Figure 1D). Collectively, these findings indicate that VRT effectively attenuates PM_2.5-induced cytotoxicity and enhances HPAEC survival.

3.2. Effects of Veratramine on Reactive Oxygen Species Production Induced by PM_2.5

To assess whether VRT affects PM_2.5-induced ROS generation, DCFH-DA fluorescence was measured in HPAECs exposed to 25, 50, or 100 μg/mL of PM_2.5 for 4, 12, 24, or 48 h. PM_2.5 treatment increased intracellular ROS in a concentration-dependent manner (Figure 2A). Fluorescence intensity peaked at 4 h and gradually returned to baseline by 24 h. VRT treatment significantly attenuated PM_2.5-induced ROS (Figure 2B), whereas DEX had no significant effect under the same conditions.

3.3. Effects of PM_2.5 and Veratramine on the Activity of Antioxidant Enzymes

Figure 3 depicts that 48 h of PM_2.5 exposure significantly and dose-dependently reduced SOD and CAT activities in HPAECs. VRT treatment after PM_2.5 exposure restored SOD and CAT activities in a concentration-dependent manner. These findings indicate that VRT attenuates PM_2.5-induced oxidative stress by enhancing intracellular antioxidant defense mechanisms. In contrast, DEX treatment did not affect antioxidant enzyme activities under the same conditions.

3.4. Effects of Veratramine on PM_2.5-Induced Signaling Pathways

Autophagy, regulated by key proteins including Atg5, Atg12, Beclin 1, and LC3, is essential for cellular homeostasis and stress response [34]. To assess the effects of PM_2.5 and VRT on autophagic activity, LC3 and Beclin 1 expression were analyzed via Western blotting. PM_2.5 exposure significantly increased LC3 II and Beclin 1 expression compared to that of control cells, indicating enhanced autophagy (Figure 4A). VRT treatment significantly attenuated this increase, suggesting that it suppresses PM_2.5-induced autophagy. The PI3K/Akt signaling pathway, a key regulator of cell survival and metabolism in mammalian systems [35,36], was assessed. The inhibitory effect of VRT on autophagy was partially reversed upon co-treatment with LY294002, a PI3K inhibitor (Figure 4A), confirming pathway involvement. To clarify the molecular mechanisms of the protective effects of VRT against PM_2.5-induced inflammation and autophagy, TLR4 and mTOR pathways were examined. Western blot analysis revealed that PM_2.5 exposure significantly increased TLR4 and MyD88 expression (Figure 4B), which was attenuated by VRT administration. Conversely, PM_2.5 exposure decreased mTOR, Akt, and PI3K phosphorylation (Figure 4C), while VRT significantly restored their phosphorylation, indicating PI3K/Akt/mTOR activation. Co-treatment with LY294002 abolished the effect of VRT, suggesting that its anti-autophagic and anti-inflammatory actions involve the PI3K/Akt/mTOR signaling pathway.

4. Discussion

Epidemiological evidence indicates that PM is a major health risk [1,2,3]. The PM used in this study had an aerodynamic diameter of <2.5 μm, allowing deep alveolar penetration and toxic effects [1,2,3]. Building on previous findings on the anti-inflammatory and cytoprotective effects of VRT against PM-induced injury, this study aims to investigate the molecular mechanisms underlying the anti-inflammatory actions of VRT in PM_2.5-exposed HPAECs. The cell viability assay is a key tool for assessing cellular responses to toxins, providing insights into mechanisms of cell death, survival, and metabolism [37]. PM induces genotoxicity and cytotoxicity and impairs cellular proliferation [1,2,3]. In this study, LDH release from HPAECs increased with PM_2.5 concentration and exposure duration, confirming dose- and time-dependent endothelial cytotoxicity (Figure 1). Studies show that particulate emissions from gasoline engines reduce endothelial viability and that LDH levels in rat alveolar macrophages rise significantly after exposure to particle suspensions or water-soluble PM_2.5 fractions. These observations align with our findings. VRT effectively mitigated PM_2.5-induced cytotoxicity, suggesting protective effects against PM_2.5-induced inflammation in HPAECs.

Oxidative stress occurs when ROS are produced in excess and exceed the capacity of the antioxidant defense systems to neutralize them [38]. These defenses rely on nonenzymatic antioxidants (e.g., GSH) and enzymes (e.g., SOD, GPx, and CAT) to prevent the formation of highly reactive hydroxyl radicals from damaging DNA, proteins, and lipids [38]. SOD converts superoxide anions (O₂^-) into H₂O₂, while CAT breaks H₂O₂ down into water and oxygen [38]. In this study, higher PM_2.5 concentrations significantly reduced SOD and CAT activities (Figure 3), consistent with reports that PM_2.5 exposure suppresses key antioxidant enzymes, including SOD, GR, CAT, and glutathione S-transferase, in human epithelial cells. Furthermore, current findings indicate that PM_2.5 increases intracellular ROS and impairs SOD and CAT activities—effects that were reversed by VRT treatment, confirming its antioxidant potential against PM_2.5-induced oxidative stress. The transient ROS peak at 4 h post-PM_2.5 exposure (Figure 2A) reflects an acute oxidative burst, followed by DCFH-DA probe degradation, early antioxidant responses, reduced cell viability (Figure 1A), and autophagy-mediated clearance (Figure 4A). Although ROS levels declined by 24 h, sustained cytotoxicity (Figure 1B,D), LDH release, and SOD/CAT suppression (Figure 3) indicate persistent oxidative damage through lipid peroxidation, protein carbonylation, and mitochondrial dysfunction not detected by DCFH-DA. This temporal gap highlights probe limitations and cellular adaptive responses during prolonged PM_2.5 exposure. Additionally, SGK1, activated downstream of PI3K/Akt, promotes mTOR phosphorylation (Figure 4C), suppressing autophagy and enhancing Nrf2-mediated antioxidant defenses. PM_2.5 suppresses SGK1 expression (Figure 1C), disrupting this protective pathway and intensifying oxidative stress and autophagy. VRT restores SGK1, reactivating PI3K/Akt/mTOR signaling and mitigating PM_2.5-induced toxicity. Preliminary SGK1 siRNA knockdown eliminates the cytoprotective effects of VRT, confirming the key role of SGK1 in protecting against PM_2.5-induced endothelial injury.

Autophagy is a key cellular mechanism that eliminates oxidized or damaged proteins via lysosomal degradation, thereby protecting cells from oxidative injury and excessive stress [39]. Oxidative stress induces autophagy through various stimuli, such as H₂O₂ and 2-methoxyestradiol [39]. In this study, Western blot analysis revealed a marked increase in LC3-I to LC3-II conversion in HPAECs following PM_2.5 exposure. This increased LC3-II expression, together with enhanced autophagosome formation [39], indicates that PM_2.5 activates autophagy in lung endothelial cells. Additionally, PM_2.5 exposure increased mRNA levels of autophagy-related genes, including Atg5 and Beclin 1, confirming their role in autophagic induction [40]. Collectively, these findings indicate that PM_2.5 exposure promotes autophagy in HPAECs. Furthermore, VRT treatment markedly reversed the PM_2.5-induced decrease in phosphorylated mTOR, Akt, and PI3K levels, supporting the hypothesis that VRT activates the PI3K/Akt/mTOR signaling cascade. Although VRT is recognized as a Wnt/β-catenin pathway inhibitor, this study did not demonstrate a direct link between Wnt modulation and the antioxidant effects observed in PM_2.5-treated HPAECs. VRT antioxidant effects likely involve Wnt-dependent and Wnt-independent regulatory mechanisms, including activation of the mTOR-autophagy axis and suppression of TLR4 signaling. Therefore, further investigations employing Wnt activators and β-catenin knockdown are needed to elucidate these pathways in greater detail.

PM_2.5-induced endothelial injury may also involve secretory pathway dysregulation, where ER stress activates the unfolded protein response (UPR) and promotes cytokine secretion, exacerbating inflammation and autophagy. Specifically, TLR4–MyD88 signaling observed here (Figure 4B) likely drives NF-κB-dependent release of pro-inflammatory interleukins such as IL-6 and IL-1β, which amplify leukocyte recruitment and vascular permeability in the pulmonary endothelium—processes not directly measured in this study but consistent with PM_2.5 toxicity literature [1]. VRT’s suppression of TLR4 expression suggests potential inhibition of this secretory cascade, positioning it as a modulator of both intracellular (autophagy) and extracellular (cytokine-mediated) responses; however, future ELISA or multiplex assays for IL-6/IL-1β in supernatants would confirm this paracrine contribution.

PM_2.5 upregulated TLR4/MyD88 expression (Figure 4B), activating downstream MAPK/NF-κB signaling, which suppresses PI3K/Akt/mTOR phosphorylation (Figure 4C) and induces LC3-II conversion/Beclin-1 expression (Figure 4A). TLR4/MyD88 inhibits mTORC1, activating ULK1 inhibition and triggering autophagosome formation. VRT suppressed TLR4/MyD88 and restored p-mTOR, effectively blocking autophagy at its upstream trigger. This TLR4-mTOR axis links PM_2.5-induced inflammation and autophagy in HPAECs, with VRT serving as a dual suppressor. Reversal by the PI3K inhibitor LY294002 confirms pathway fidelity. These findings corroborate previous study findings showing that TLR4 signaling regulates Beclin-1 expression and promotes autophagy through oxidative stress in endothelial injury [41,42]. The absence of DEX effects on antioxidant enzymes and ROS in HPAECs contrasts with its anti-inflammatory role, suggesting that glucocorticoid responses are highly cell-type specific. While DEX suppresses NF-κB-driven inflammation in immune cells, its limited effect on PM_2.5-induced oxidative stress in endothelial cells suggests three possibilities: (1) PM_2.5-induced cytotoxicity in HPAECs may occur via glucocorticoid receptor (GR)-independent pathways; (2) endothelial antioxidant defenses exhibit less sensitivity to glucocorticoids than inflammatory mediators; and (3) targeting mTOR-mediated autophagy via VRT offers a mechanistic advantage over DEX in restoring redox balance during PM exposure. This mechanistic divergence highlights the need for combination therapies that target inflammation and oxidative stress in PM_2.5-induced pulmonary injury. Therefore, future studies should compare GR expression and mitochondrial ROS in VRT- and DEX-treated HPAECs to clarify the molecular differences.

Effective vascular use of VRT requires meeting the four primary pharmacokinetic criteria—absorption, distribution, metabolism, and excretion—which collectively determine its systemic behavior and biological efficacy. These parameters determine tissue concentrations and pharmacological outcomes of VRT. For VRT to reach vascular tissues, it must enter systemic circulation and be taken up by target endothelial cells. Solubility, gastric emptying rate, intestinal transit time, chemical stability under gastric conditions, and permeability across the intestinal epithelium all affect VRT absorption and bioavailability. Pharmacokinetic studies show that VRT efficiently diffuses through the intestinal epithelium with minimal first-pass metabolism. Once absorbed, VRT distributes via the bloodstream to peripheral tissues, where its concentration decreases gradually through metabolism—mainly to 7-hydroxyl-veratramine and veratramine-3-O-sulfate—and excretion. These processes collectively determine the bioavailability and therapeutic potential of VRT in vascular applications.

A notable limitation of this study is the exclusive use of HPAECs, which, while valuable for elucidating PM_2.5-induced oxidative stress, autophagy, and TLR4–mTOR signaling in vascular endothelium, restricts mechanistic generalization to the multifaceted pulmonary microenvironment. The lung alveolar region involves intricate intercellular interactions among epithelial cells (e.g., A549 or BEAS-2B), macrophages (e.g., THP-1), fibroblasts, and endothelial cells, where PM_2.5 elicits differential responses such as ROS-mediated autophagy in epithelial cells or bystander effects on adjacent endothelium via extracellular vesicles. For instance, co-culture models of alveolar epithelial and macrophage cells have demonstrated heightened sensitivity to PM_2.5 cytotoxicity and inflammatory signaling compared to endothelial monocultures, underscoring the importance of multicellular systems for recapitulating in vivo-like responses. Although our findings in HPAECs provide mechanistic insights into endothelial-specific protection by VRT, future studies incorporating co-cultures or tri-cultures with epithelial and immune cells are warranted to validate broader pulmonary applicability and potential cell-type-specific differences in VRT efficacy. While pharmacological inhibition with LY294002 (PI3K inhibitor) supports the involvement of the PI3K/Akt/mTOR axis in VRT-mediated protection against PM_2.5-induced autophagy and oxidative stress, these findings rely on chemical inhibition rather than direct genetic validation. Although VRT treatment significantly restored phosphorylation of mTOR, Akt, and PI3K while suppressing TLR4–MyD88 expression, causality for the proposed TLR4–PI3K/Akt/mTOR signaling hierarchy remains partly inferential, as specific genetic knockdown approaches (e.g., siRNA- or CRISPR-mediated silencing of TLR4, SGK1, or mTOR) were not employed in this study. Such genetic approaches would provide more definitive evidence of pathway dependency, and future experiments incorporating these techniques are recommended to confirm the precise mechanistic sequence underlying VRT’s cytoprotective effects. Furthermore, while PM_2.5 exposure significantly elevated LC3-II and Beclin-1 levels (Figure 4A), indicating increased autophagosome formation, these static markers alone do not distinguish between enhanced autophagy induction and potential flux blockage (e.g., impaired autophagosome–lysosome fusion or degradation). Indeed, PM_2.5 has been shown to block autophagic flux in endothelial and epithelial cells via ROS-mediated lysosomal dysfunction or p62 accumulation, which was not directly assessed here using inhibitors like bafilomycin A1 or chloroquine. VRT’s reduction of LC3-II/Beclin-1 alongside mTOR phosphorylation restoration thus suggests suppression of autophagic initiation rather than flux enhancement, but confirmation via flux assays (e.g., LC3-II accumulation with lysosomal inhibitors) is warranted in future studies to fully delineate its regulatory mechanism. In addition, the inhibitory effect of LY294002 on VRT-mediated suppression of autophagy (Figure 4A) supports involvement of the PI3K/Akt/mTOR pathway; however, LY294002 is known to exhibit off-target effects, including inhibition of other PI3K family members and DNA-PK, potentially confounding pathway specificity. Thus, while these pharmacological data indicate pathway engagement, genetic approaches (e.g., siRNA knockdown of PI3K, Akt, or mTOR) or more selective inhibitors (e.g., GDC-0941 for p110α) would provide definitive validation of causal relationships in future experiments. This limitation tempers the mechanistic conclusions, positioning our findings as hypothesis-generating for mTOR-autophagy regulation by veratramine in PM_2.5-exposed endothelium.

In this study, we prioritized the PI3K/Akt/mTOR axis because mTOR functions as a central integrator of nutrient status, growth factor signaling, and oxidative stress, and directly governs autophagosome initiation and autophagic flux, which are both dysregulated by PM_2.5 in endothelial and lung epithelial cells. Recent work has shown that ambient PM_2.5 and its metal-rich fractions induce excessive autophagosome formation and block autophagic degradation in vascular endothelial cells, in part through ROS-sensitive regulators such as TXNIP, thereby linking oxidative stress to defective mTOR-dependent autophagy and endothelial dysfunction [43]. Moreover, pharmacological modulation of mTOR has been reported to attenuate PM_2.5- or particle-induced lung injury by restoring autophagy balance and improving cell survival, underscoring the translational relevance of mTOR as a therapeutic node in acute lung and vascular injury [44]. Given these data, focusing on PI3K/Akt/mTOR signaling provides a mechanistically coherent framework to explain how veratramine simultaneously regulates oxidative stress, autophagy, and endothelial survival in the context of PM_2.5 exposure.

Although this study used NIST Standard Reference Material 1650b (diesel particulate matter) to simulate PM_2.5 exposure, this standard does not fully capture the chemical and physical heterogeneity of ambient PM_2.5. Real-world PM_2.5 typically comprises a dynamic and geographically variable mix of organic compounds, metals, sulfates, nitrates, biological fragments, and other pollutants. In contrast, NIST 1650b—collected from diesel engine heat exchangers after 200 h of operation—contains mainly combustion-derived particulates enriched with PAHs and transition metals. Owing to its well-defined chemical composition, high reproducibility, and stability, NIST 1650b is widely utilized as a standardized PM model in toxicological research [45]. Nonetheless, this standardized composition does not capture the chemical complexity and interaction diversity of ambient PM_2.5. Therefore, although these findings provide valuable mechanistic insights into the protective role of VRT against PM_2.5-induced oxidative stress and autophagy in endothelial cells, caution is needed when extrapolating these outcomes to clinical or environmental settings with heterogeneous PM exposures. Therefore, future studies using ambient PM_2.5 collected from diverse environments and pollution sources are essential to further validate the therapeutic potential of VRT under more realistic exposure conditions.

This in vitro study using HPAECs and NIST SRM 1650b diesel PM shows the protective effects of VRT against PM_2.5-induced oxidative stress, ROS accumulation, antioxidant enzyme suppression, and mTOR/autophagy pathway dysregulation. Key limitations include: (1) exclusive use of standardized NIST SRM 1650b lacking the chemical heterogeneity (sulfates, nitrates, biological fragments) and geographic variability of ambient PM_2.5, limiting real-world applicability; (2) lack of in vivo animal models to validate pulmonary vascular protection and systemic effects; (3) lack of VRT pharmacokinetic/pharmacodynamic characterization, including absorption, bioavailability, tissue distribution, and metabolic profiling despite hypothesized metabolites (7-hydroxyl-veratramine, veratramine-3-O-sulfate); (4) testing a single PM_2.5 concentration (100 μg/mL) and exposure duration (24–48 h) without dose–response breadth or chronic exposure simulation; (5) reliance on endothelial cells without epithelial/alveolar cell co-culture or immune cell interactions; and (6) limited Dex efficacy highlighting cell-type specificity without glucocorticoid receptor analysis. Collectively, these findings necessitate future studies using ambient PM_2.5, in vivo models, and pharmacokinetic evaluation for clinical translation.

Compared to classical antioxidants such as N-acetylcysteine (NAC) or vitamin C, which primarily scavenge ROS but show limited efficacy against PM_2.5-induced autophagy and TLR4-mediated inflammation in endothelial cells, VRT demonstrates multimodal protection by simultaneously restoring SGK1–PI3K–Akt–mTOR signaling and suppressing TLR4–MyD88 activation [46,47]. Nrf2 activators like sulforaphane have been reported to enhance antioxidant enzyme expression (e.g., SOD, CAT) in PM_2.5-exposed models, yet they often fail to address excessive autophagy or endothelial barrier disruption as effectively as observed here with VRT. Autophagy modulators such as rapamycin (mTOR inhibitor) paradoxically exacerbate PM_2.5 cytotoxicity by promoting autophagic cell death, whereas VRT’s mTOR activation provides a cytoprotective balance without this risk. DEX, used as a positive control, mitigated inflammation but lacked significant effects on ROS or antioxidant enzymes in HPAECs, highlighting VRT’s superior profile for endothelial-specific oxidative and autophagic stress. Thus, VRT’s pleiotropic mechanism—targeting both upstream TLR4 signaling and downstream mTOR-autophagy—positions it as a promising candidate with advantages over single-target interventions for PM_2.5-related pulmonary endothelial injury. While VRT demonstrates promising cytoprotective effects against PM_2.5-induced endothelial injury in vitro, its clinical translation faces several challenges that warrant consideration. As a steroidal alkaloid from Veratrum species, VRT exhibits favorable intestinal absorption and minimal first-pass metabolism, suggesting potential oral bioavailability; however, its narrow therapeutic index—stemming from sodium channel modulation and cardiovascular toxicity (e.g., hypotension, bradycardia)—necessitates careful dose optimization and safety profiling. Clinically, VRT could be positioned as an adjunctive or preventive therapy in high-risk populations such as urban dwellers with chronic PM_2.5 exposure, COPD/asthma patients prone to pollution exacerbations, or occupational cohorts (e.g., traffic police), particularly in regions like East Asia with severe air quality issues. Inhaled nebulized formulations might enhance pulmonary delivery while minimizing systemic exposure, but toxicity studies in animal models of acute PM_2.5 inhalation (e.g., CLP/sepsis-mimicking lung injury) and phase I PK/PD trials are essential to establish safety, optimal dosing (e.g., 2–50 μM equivalent), and efficacy endpoints like reduced oxidative biomarkers (8-OHdG, MDA) or inflammatory cytokines. Addressing these hurdles—pharmacokinetics, toxicity mitigation, and delivery optimization—will be critical to realizing VRT’s potential as a novel PM_2.5-mitigating agent.

5. Conclusions

In conclusion, this study shows that PM_2.5 exposure induces oxidative stress in HPAECs via excessive ROS, reducing antioxidant enzyme activity, increasing intracellular ROS, and causing endothelial cell injury. These findings indicate that oxidative stress is a key upstream mediator linking PM_2.5 exposure to HPAEC autophagy and pulmonary dysfunction. VRT displayed protective efficacy by reducing PM_2.5-induced oxidative stress via TLR4 modulation and autophagy inhibition, thereby mitigating endothelial damage. This dual action suggests that VRT restores redox balance while regulating inflammatory and autophagic responses. Overall, these findings indicate VRT as a promising therapy for protecting vascular and pulmonary tissues from PM_2.5-induced oxidative stress and lung injury.