Cardiac injury elicited by sub-chronic hookah smoke inhalation, and the alleviating effect of procysteine, 2-oxo-(4R)-4-thiazolidinecarboxylic acid, in BALB/c mice
Sumaya Beegam, Suhail Al-Salam, Bilal M. Nemmar, Nur Elena Zaaba, Ozaz Elzaki, Badreldin H. Ali, Abderrahim Nemmar
TL;DR
This study shows that procysteine (OTC) protects the heart from damage caused by hookah smoke by reducing inflammation and oxidative stress in mice.
Contribution
The novel finding is that OTC mitigates hookah smoke-induced cardiac injury through specific molecular mechanisms including Nrf2 activation and NF-κB inhibition.
Findings
OTC reduced oxidative stress, inflammation, DNA damage, and apoptosis in the heart caused by hookah smoke.
OTC suppressed NLRP3 inflammasome and IL-1β activation, inhibited NF-κB, and restored sirtuin-1 expression.
OTC enhanced Nrf2 expression in the heart under hookah smoke exposure with no histological changes observed.
Abstract
Hookah smoke (HS) inhalation is known to induce cardiovascular dysfunction, including oxidative stress and inflammation. The procysteine, 2-oxo-(4R)-4-thiazolidinecarboxylic acid (OTC) is a prodrug of cysteine, a precursor of glutathione, which is a major intracellular antioxidant. This study aimed to evaluate the possible cardioprotective effects of OTC against HS inhalation-induced cardiac injury in mice. The animals were exposed to HS for 30 min per day, five days per week, for one month, while control mice were exposed to normal air. OTC was administered by gavage at a dose of 80 mg/kg 1 h before each exposure session. OTC prevented HS-induced increase in the concentrations of tumor necrosis factor α, interleukin (IL)-6 and galectin-3 in the heart tissue. HS exposure augmented the levels of markers of oxidative stress and adhesion molecules. The latter effects were significantly…
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TopicsGenomics, phytochemicals, and oxidative stress · Heme Oxygenase-1 and Carbon Monoxide · Interstitial Lung Diseases and Idiopathic Pulmonary Fibrosis
Introduction
1
Oxidative stress contributes to cardiovascular pathophysiology, including hypertension, atherosclerosis, and myocardial infarction (Dubois-Deruy et al., 2020; Młynarska et al., 2024; Valaitienė and Laučytė-Cibulskienė, 2024); however, the specific pathways linking oxidative stress to cardiac injury—particularly in response to inhaled toxicants—remain incompletely defined. This gap is especially relevant given the rise of alternative forms of tobacco consumption (Mahfooz et al., 2023; Qasim et al., 2019).
Tobacco smoking in all its forms is estimated to cause about six million deaths annually worldwide (Gallucci et al., 2020; Niemann et al., 2017; (WHO), 2023). The prohibition of smoking in public places has been a significant public health achievement, reducing myocardial infarction rates by 17% (Gallucci et al., 2020; Niemann et al., 2017). However, smoking in its various forms remains widespread, with over one billion smokers globally (Gallucci et al., 2020; Niemann et al., 2017). The cardiovascular effects of smoking result in higher mortality rates compared with deaths from pulmonary conditions (Gallucci et al., 2020; Niemann et al., 2017; Rahman et al., 2025). The mechanistic connections between inhaled toxicants and cardiac injury remain incompletely understood, and oxidative stress has emerged as a central contributor to smoke-induced cardiovascular dysfunction (Niemann et al., 2017).
The procysteine, 2-oxo-(4R)-4-thiazolidinecarboxylic acid (OTC) is a prodrug of cysteine, a precursor of glutathione (Angelovski et al., 2023). OTC is known for its high membrane permeability, good systemic bioavailability and protective effects against inflammation and oxidative stress (Angelovski et al., 2023). Previous studies have shown that OTC treatments exert antithrombotic and cardioprotective effects in endotoxin-induced cardiac dysfunction and isoproterenol-induced myocardial infarction in rats (Angelovski et al., 2022, 2023; Nemmar et al., 2009; Poon et al., 1998). However, its potential protective role against cardiac injury caused by hookah smoke (HS) inhalation has not yet been investigated. The latter smoking method is highly prevalent in Middle Eastern countries and is increasing worldwide, contributing to substantial cardiovascular morbidity (Mahfooz et al., 2023; Nzeako et al., 2025). Despite its widespread use, mechanistic studies addressing HS–induced cardiac injury and potential therapeutic countermeasures remain limited.
Thus, this study investigates whether OTC can protect against cardiac injury induced by HS inhalation and examines associated mechanisms, including oxidative stress, inflammation, DNA damage, and apoptosis. Additionally, we explore regulatory pathways involving the NLRP3 inflammasome, nuclear factor-κB (NF-κB), sirtuin-1, and nuclear factor erythroid-derived 2-like 2 (Nrf2) in cardiac tissue.
Material and methods
2
Animals and hookah smoke inhalation
2.1
The Institutional Ethics Committee of United Arab Emirates University approved this animal study protocol (protocol code ERA- 2019- 5876) on April 9, 2019. The study involved an equal number of male and female BALB/c mice, aged six to eight weeks and weighing between 25 and 30 g. These mice were obtained from the Research Animal Facility at the College of Medicine and Health Sciences, UAEU, in Al Ain, United Arab Emirates.
The mice were housed in rooms with a controlled temperature of 22 ± 1 °C and a 12-h light-dark cycle. They had unrestricted access to commercially available, additive-free laboratory chow from National Feed and Flour and Marketing Co. in Abu Dhabi, UAE, along with filtered water. The animals were kept in cages where food and water were provided ad libitum. After a one-week acclimatization period, the mice were randomly assigned to one of four groups: air (control), HS, OTC + air, and OTC + HS.
Mice were placed in soft restraints and connected to an exposure tower, as described earlier (Nemmar et al., 2017a; Ali et al., 2017). They were exposed to either HS or air using a nose-only exposure system linked to a hookah apparatus (InExpose System, Scireq, Canada). This HS exposure model has been previously described and validated in several studies from our group, demonstrating its reproducibility and physiological relevance (Nemmar et al., 2013a, 2017a, 2021, 2022). The exposure involved commercially available apple-flavored tobacco (Al Fakher Tobacco Trading, Ajman, UAE), whose ingredients include tobacco, glycerin, molasses, and natural flavor with nicotine (0.5%) and tar (Nemmar et al., 2022; Alarabi et al., 2020). The latter was lit with an instant light charcoal disk (Star, 3.5 cm in diameter and 1 cm in width) (Coco Avana, Indonesia). Similar to human use, the smoke from the hookah apparatus first passed through water before being drawn into the exposure tower. The latter step produces the characteristic humid aerosol of HS; however, the system does not permit direct measurement of variability in water content. The exposure regimen was managed by a computerized system (InExpose System, Scireq, Canada), which generated a computer-controlled puff every minute. This resulted in a 2-s puff duration of HS exposure followed by 58 s of fresh air. Each exposure session lasted 30 min per day, as described before (Nemmar et al., 2017a; Ali et al., 2017). Particulate matter (PM) levels within the exposure tower were monitored using the integrated infrared PM transducer of the InExpose system, which provides quasi-quantitative real-time PM measurements based on infrared opacity (Nemmar et al., 2017a). Direct measurement of carbon monoxide (CO) levels within the exposure tower was not performed in the present study. However, in our earlier work using the same setup and identical puffing parameters, we quantified carboxyhemoglobin (COHb) in exposed mice and observed COHb elevations comparable to those reported in human hookah smokers (Nemmar et al., 2017a; Zahran et al., 1982, 1985).
The mice were exposed for one month to either HS or normal air, with or without OTC treatment (Sigma, St. Louis, MO, USA), which was administered by gavage at a dose of 80 mg/kg, 1 h before the exposure to either HS or normal air. The one-month time point was selected because our earlier studies showed that HS at this duration induces respiratory, cardiovascular, and renal complications (Nemmar et al., 2013a, 2013b, 2020a; Beegam et al., 2024a). The OTC dose (80 mg/kg) was selected based on previously published in vivo studies using comparable and effective dosing regimens. Two independent studies administered OTC at 80 mg/kg in mice and demonstrated clear antioxidant and anti-inflammatory protection in models of asthma (Lee et al., 2004) and diesel exhaust particle–induced lung inflammation and microvascular thrombosis (Nemmar et al., 2009). In addition, Choi et al. (2013) reported dose-dependent efficacy of OTC at 25, 50 and 100 mg/kg in acetaminophen-induced hepatotoxicity, establishing a validated therapeutic range. These studies also support our timing (1 h before HS exposure), as OTC was consistently administered 1–2 h before the injurious challenge in all three models (Nemmar et al., 2009; Lee et al., 2004; Choi et al., 2013). No mortality was observed in any experimental group. The sample size was determined based on prior studies employing similar experimental models and endpoints, which indicated that comparable group numbers were sufficient to detect meaningful biological differences (Nemmar et al., 2013a, 2017a, 2022).
Sample collection, biochemical and histological analysis
2.2
Immediately after the last exposure to either air or HS, mice were anesthetized sodium pentobarbital (45 mg/kg given intraperitoneally), and then blood was drawn from the inferior vena cava in EDTA (4%), and spun for 15 min at 4 °C at 900g. The obtained plasma samples were immediately stored at − 80 °C pending further analysis. The hearts were excised, blotted on filter paper, and frozen at − 80 °C. The preparation of the cardiac homogenates for the assessment of various biochemical parameters was achieved as described before (Nemmar et al., 2013a, 2015).
The concentrations of tumor necrosis factor α (TNFα) (R&D Systems, DY410), interleukin-6 (IL-6) (R&D Systems, DY406), interleukin-1β (IL-1β) (R&D Systems, DY401), and galectin-3 (R&D Systems, DY1197) in heart tissue homogenates were determined using ELISA kits from R&D Systems (Minneapolis, MN, USA) (Ali et al., 2013; Nemmar et al., 2017b).
The concentration of lipid peroxidation (LPO) end products, specifically thiobarbituric acid reactive substances, was measured spectrophotometrically, with malondialdehyde from Sigma Aldrich Co. (St. Louis, MO, USA) serving as the standard (Ali et al., 2018; Nemmar et al., 2013c). Superoxide dismutase (SOD) activity was assessed using a kit from Cayman Chemical (706002), and glutathione (GSH) concentration was measured according to protocols from Sigma-Aldrich Fine Chemicals (CS0260). Nitric oxide (NO) activity was quantified using a total NO assay kit from Cayman Chemical (780001), which measures the more stable NO metabolites, nitrite (NO_2_^−^) and nitrate (NO_3_^−^).
The concentrations of adhesion molecules, including P-selectin (R&D Systems, DY737), E-selectin (R&D Systems, DY575), intercellular adhesion molecule-1 (ICAM-1) (R&D Systems, DY796), and vascular cell adhesion molecule-1 (VCAM-1) (R&D Systems, DY643), in heart tissue homogenates were quantified using ELISA kits obtained from R&D Systems (DuoSet, Minneapolis, MN, USA).
Immediately after the animals were euthanized, their hearts were removed. Single-cell suspensions were then generated from the collected hearts using previously established methods (de Souza et al., 2014; Hartmann and Speit, 1997; Nemmar et al., 2016). Each heart was washed in a chilled solution consisting of RPMI-1640, 15% DMSO, and 1.8% (wt/vol) NaCl. The rinsed tissue was transferred into 1.5 mL of this medium and minced into very small pieces in a Petri dish with scissors. After allowing the tissue fragments to settle, the supernatant was transferred to a 15-mL tube. This suspension was centrifuged at 900 g for 5 min at 4 °C. The resulting supernatant was discarded, and the pellet was resuspended in 0.5 mL of the medium. The cell mixtures were then combined with 0.65% low-melting-point agarose and layered onto microscope slides that had already been coated with 1.5% agarose. Five slides were prepared for each treatment group. The slides were incubated in an ice-cold lysis buffer (2.5 M NaCl, 10 mM Tris, 100 mM EDTA, 1% Triton X-100, 10% DMSO) at 4 °C for at least 1 h to remove cell membranes. After lysis, the slides were placed in a horizontal electrophoresis chamber and immersed in an alkaline electrophoresis buffer (0.2 M EDTA, 5 M NaCl, pH 10) for 20 min to allow DNA unwinding and reveal alkali-labile sites. Electrophoresis was then performed for 20 min at 25 V and 300 mA. Following electrophoresis, the slides were neutralized in 0.4 M Tris buffer (pH 7.5) for 5 min and rinsed with methanol. The slides were then stained with propidium iodide as previously described (de Souza et al., 2014; Hartmann and Speit, 1997; Nemmar et al., 2016). All steps were carried out in the dark to prevent additional DNA damage. Fluorescence microscopy was used to evaluate DNA migration, with fifty cells assessed per treatment group, and the results averaged across the five slides. The length of DNA migration (nuclear diameter plus migrated DNA) was quantified using Axiovision 3.1 image-analysis software (Carl Zeiss) (de Souza et al., 2014; Hartmann and Speit, 1997; Nemmar et al., 2016).
The concentrations of NLRP3 (920134, MyBioSource, San Diego, CA, USA), phosphorylated NF-κB (7173C, Cell Signaling Technology, Boston, MA, USA), and sirtuin-1 (2601957, MyBioSource, San Diego, CA, USA) were measured in heart homogenates of mice exposed to HS with or without OTC treatment using ELISA kits according to the protocol provided by the vendors.
To measure the protein expression of Nrf2, Western blotting was employed (Nemmar et al., 2017c; Beegam et al., 2024b). Heart tissues obtained from mice were swiftly frozen in liquid nitrogen and stored at −80 °C. These tissues were then weighed, rinsed with 0.9% NaCl, and homogenized in a lysis buffer (pH 7.4) containing 140 mM NaCl, 300 mM KCl, 10 mM Trizma base, 1 mM EDTA, Triton X-100 (0.5% in distilled water), sodium deoxycholate (0.5% in distilled water), and protease and phosphatase inhibitors. The homogenized heart tissues were centrifuged for 20 min at 4 °C, and the supernatants were collected. Protein concentrations were quantified by means of the Pierce bicinchoninic acid protein assay kit (Thermo Scientific, Waltham, MA, USA). Samples containing 70 μg of protein were segregated by electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and after that transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with a monoclonal rabbit Nrf2 antibody at 1:2000 dilution (Abcam, Hong Kong, China). After washing, the membranes were incubated for 2 h at room temperature with a goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody at 1:5000 dilution (Abcam, Hong Kong, China). The development of signals was achieved by means of Pierce enhanced chemiluminescent Western blotting substrate kit (Thermo Fisher Scientific, MA, USA). Densitometric analysis of the protein bands was achieved using the Typhoon FLA 9500 imaging system (GE Healthcare Bio-Sciences, Uppsala, Sweden). As a loading control, the membranes were re-probed with a mouse monoclonal GAPDH antibody at 1:5000 dilution (Abcam, Hong Kong, China).
For the histological analysis, after removal, hearts were briefly rinsed in chilled saline to eliminate residual blood, gently dried, and weighed. Tissues were then immersed in 10% neutral buffered formalin for fixation. Each heart was cut into four coronal slices, placed in cassettes, and processed through a standard histological protocol involving graded ethanol dehydration, xylene clearing, and paraffin embedding. Sections of 3 μm thickness were obtained from the paraffin blocks and stained with hematoxylin and eosin. A histopathologist affiliated with the project examined all slides under light microscopy while blinded to the experimental groups.
Statistics
2.3
All statistical analyses were performed using GraphPad Prism version 7. Data normality was initially assessed with the Shapiro–Wilk test. Normally distributed datasets were analyzed using one-way ANOVA, followed by Holm–Sidak post hoc multiple comparisons to determine significant group differences. For datasets that did not meet normality criteria, a log10 transformation was applied before analysis. A p-value <0.05 was considered statistically significant.
Results
3
TNFα, IL-6 and Galectin-3 in heart tissue homogenates
3.1
Fig. 1 shows that when compared with the air group, HS inhalation, induced a significant increase in the concentrations of TNFα (p < 0.0001), IL-6 (p < 0.0001) and galectin-3 (p < 0.0001) in heart tissue homogenates. However, when OTC was administered to mice exposed to HS, the concentrations of TNFα, IL-6 and galectin-3 showed significant decrease (p < 0.0001) compared with the group exposed only to HS.Fig. 1. Tumor necrosis factor α (TNFα, A), interleukin (IL)-6 (B) and galectin-3 (C) concentrations in heart homogenates of mice exposed to hookah smoke (HS) with or without procysteine, 2-oxo-(4R)-4-thiazolidinecarboxylic acid (OTC) (80 mg/kg) pretreatment. Data are presented as individual values with the mean shown (n = 8). Statistical analysis by one-way analysis of variance followed by Holm-Sidak's test.Fig. 1
LPO, GSH, SOD and NO in heart tissue homogenates
3.2
Mice exposed to HS displayed a significant increase in the heart levels of markers of oxidative stress LPO (p < 0.0001), GSH (p < 0.0001), SOD (p < 0.0001) and NO (p < 0.0001) compared with the control (air) mice. These augmentations were normalized (p < 0.0001-p<0.01) in mice concomitantly treated with OTC and HS (Fig. 2).Fig. 2. Lipid peroxidation (LPO, A), glutathione (GSH, B), superoxide dismutase (SOD, C) and total nitric oxide (NO, D) levels in heart homogenates of mice exposed to hookah smoke (HS) with or without procysteine, 2-oxo-(4R)-4-thiazolidinecarboxylic acid (OTC) (80 mg/kg) pretreatment. Data are presented as individual values with the mean shown (n = 8). Statistical analysis by one-way analysis of variance followed by Holm-Sidak's test.Fig. 2
VCAM-1, ICAM-1, P-selectin and E-selectin in heart tissue homogenates
3.3
Fig. 3 illustrates that the concentrations of VCAM-1, ICAM-1, P-selectin and E-selectin were significantly increased in mice exposed to HS, compared with the control group (p < 0.0001-p<0.001), and these effects were significantly reduced (p < 0.0001-p<0.05) with the pretreatment with OTC. The concentrations of ICAM-1 (P < 0.01) and P-selectin (P < 0.01) were higher in OTC + HS versus OTC + air. Also, there was a significant increase in the concentration of E-selectin in OTC + air compared with air group (P < 0.0001).Fig. 3. Vascular cell adhesion molecule-1 (VCAM-1, A), intercellular adhesion molecule-1 (ICAM-1, B), P-selectin (C) and E-selectin (D) concentrations in heart homogenates of mice exposed to hookah smoke (HS) with or without procysteine, 2-oxo-(4R)-4-thiazolidinecarboxylic acid (OTC) (80 mg/kg) pretreatment. Data are presented as individual values with the mean shown (n = 7-8). Statistical analysis by one-way analysis of variance followed by Holm-Sidak's test.Fig. 3
DNA injury and cleaved-caspase-3 in heart tissue homogenates
3.4
As shown in Fig. 4, the DNA damage and the level of cleaved-caspase in the hearts of mice with HS inhalation displayed significant increase (p < 0.0001) compared with the air group. However, a significant ameliorative action was seen with the treatment of OTC 1 h before HS inhalation (p < 0.0001).Fig. 4DNA migration (mm) assessed by Comet assay (A) with representative images (magnification 40x), showing the quantification of the DNA migration, under alkaline conditions and cleaved caspase-3 in heart homogenates of mice exposed to hookah smoke (HS) with or without procysteine, 2-oxo-(4R)-4-thiazolidinecarboxylic acid (OTC) (80 mg/kg) pretreatment. Data are presented as individual values with the mean shown (n = 5 for Comet assay and n = 8 for cleaved caspase). Statistical analysis by one-way analysis of variance followed by Holm-Sidak's test.Fig. 4
NLRP3 and IL-1β in heart tissue homogenates
3.5
When compared with the air group, inhalation of HS caused a significantly higher cardiac concentration of the inflammasome NLRP3 and the pro-inflammatory cytokine IL-1β (p < 0.0001, Fig. 5). However, the administration of OTC significantly normalized the concentration of NLRP3 and IL-1β in HS-exposed mice (p < 0.0001).Fig. 5. Nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3 (NLRP3, A) and interleukin (IL)-1β (B) concentrations in heart homogenates of mice exposed to hookah smoke (HS) with or without procysteine, 2-oxo-(4R)-4-thiazolidinecarboxylic acid (OTC) (80 mg/kg) pretreatment. Data are presented as individual values with the mean shown (n = 8). Statistical analysis by one-way analysis of variance followed by Holm-Sidak's test.Fig. 5
Phospho-NF-κB and sirtuin-1 in heart tissue homogenate
3.6
Fig. 6 shows that, compared with air group, the concentration of phospho-NF-кB in heart tissue homogenates was significantly increased (p < 0.05) following HS inhalation, whereas that of sirtuin-1 was significantly decreased (p < 0.0001). These actions were significantly reversed when OTC treatment was given 1 h before HS inhalation (p < 0.01).Fig. 6. Phosphorylated nuclear factor-кB (phosphor-NF-кB, A) and sirtuin-1 (B) expression in heart homogenates of mice exposed to hookah smoke (HS) with or without procysteine, 2-oxo-(4R)-4-thiazolidinecarboxylic acid (OTC) (80 mg/kg) pretreatment. Data are presented as individual values with the mean shown (n = 6-8). Statistical analysis by one-way analysis of variance followed by Holm-Sidak's test.Fig. 6
Nrf2 expression in heart tissue homogenate
3.7
The Western blot analysis of the expression of Nrf2 is illustrated in Fig. 7. Mice exposed to HS showed significantly higher expressions of Nrf2 (p < 0.05) in hearts compared with mice exposed to air. This expression was more substantially increased in OTC + HS group compared with exposed only to HS (p < 0.01).Fig. 7. Nuclear factor erythroid 2-related factor 2 (Nrf2) expression in heart homogenates of mice exposed to hookah smoke (HS) with or without procysteine, 2-oxo-(4R)-4-thiazolidinecarboxylic acid (OTC) (80 mg/kg) pretreatment. Data are presented as individual values with the mean shown (n = 8). Statistical analysis by one-way analysis of variance followed by Holm-Sidak's test.Fig. 7
Cardiac histology
3.8
Fig. 8A–D illustrates the absence of morphological alterations in H&E-stained heart sections from mice across all experimental groups.Fig. 8. Representative light microscopy images of H&E-stained heart tissues from mice after a 1-month exposure to air or hookah smoke (HS), with or without procysteine, 2-oxo-(4R)-4-thiazolidinecarboxylic acid (OTC) pretreatment. (A) Left ventricular sections from air-exposed mice. (B) Left ventricular sections from air-exposed mice treated with OTC. (C) Left ventricular sections from HS-exposed mice. (D) Left ventricular sections from HS-exposed mice treated with OTC.Fig. 8
Discussion
4
In the present study, we demonstrate that OTC administration significantly attenuates HS-induced cardiac injury. This overall protection is reflected in reduced oxidative stress, inflammation, DNA damage, and apoptosis, mediated through inhibition of NLRP3 and NF-κB and activation of sirtuin-1 and Nrf2. These molecular and biochemical alterations occurred without detectable differences in cardiac histology.
In the present study, we show that subchronic exposure to HS induced cardiac inflammation characterized by the increase in the concentration of some important markers of inflammation, including TNFα, IL-6, IL-1β and galectin-3, as well as various markers of oxidative stress including the marker of lipid peroxidation LPO, the antioxidants GSH and SOD and the free radical scavenger NO. Although HS exposure induced increases in GSH, SOD, and NO, this pattern does not contradict the presence of oxidative stress. Such elevations reflect an early compensatory response in which endogenous antioxidant systems are upregulated to counteract excess reactive oxygen species. This adaptive upregulation has been described in models of subacute oxidative challenge, whereas depletion of antioxidants typically occurs only after more prolonged or severe stress once cellular defenses become overwhelmed (Valko et al., 2007; Jones, 2006; Dröge, 2002). Thus, in our 1-month exposure model, the increased antioxidant levels likely represent an attempt to restore redox balance in response to HS-induced oxidative burden. Interestingly, all the eight markers of inflammation and oxidative stress returned to control levels in mice treated with OTC and exposed to HS, supporting the interpretation that OTC may exert anti-inflammatory and antioxidant effects in this model. Our findings are consistent with previous reports that describe the potential of OTC in reducing oxidative stress and inflammation in endotoxin-induced cardiac dysfunction in rabbits and isoproterenol-induced myocardial infarction in rats (Poon et al., 1998; Angelovski et al., 2022).
We have recently reported that the exposure to HS induce a significant augmentation of the concentration of cell adhesion molecules comprising ICAM-1, VCAM-1, E-selectin and P-selectin in aortic tissue homogenates (Nemmar et al., 2023a). These cell surface proteins are indicators of endothelial dysfunction and are recognized as independent risk factors for cardiovascular disease and smoking (Niemann et al., 2017; Angelovski et al., 2022; Mehtap et al., 2021; Nemmar et al., 2019a). Additionally, an increase in the expression of adhesion molecules in cardiac cells in vitro and heart tissue homogenates in vivo have been reported in animal models of myocardial injury or hypertension or following exposure to silver nanoparticles (Kacimi et al., 1998; Kiarash et al., 2001; Nemmar et al., 2023b). Similarly, in the present study, we found that subchronic HS inhalation caused an increase in the expression of ICAM-1, VCAM-1, E-selectin and P-selectin in heart tissue homogenates. These effects can plausibly be ascribed to the cardiac inflammation and oxidative stress caused by HS exposure. Moreover, our data showed that the administration of OTC has effectively alleviated these actions through the inhibition of inflammation and oxidative stress.
Oxidative stress plays a significant role in the development and progression of cardiovascular diseases (Senoner and Dichtl, 2019). This condition disrupts cellular macromolecules such as lipids, proteins and DNA leading to DNA mutations which impair critical cellular functions, and eventually trigger apoptosis (Senoner and Dichtl, 2019). Our data also show the occurrence of cardiac DNA damage and increase in cleaved caspase-3, indicating the presence of apoptosis, and both effects were significantly abated by the treatment with OTC. The manifestation of oxidative stress related DNA damage and apoptosis has been reported both in vitro following the exposure to the pesticide benomyl in H9c2 cardiomyoblast cells, and in vivo in hearts of mice exposed to HS (Mehtap et al., 2021; Nemmar et al., 2019a). The latter in vivo work has reported that the treatment with gum Arabic, a prebiotic with antioxidant, anti-inflammatory properties, abrogated HS-induced cardiac DNA damage and apoptosis in mice (Nemmar et al., 2019a).
The NLRP3 inflammasome has been reported to play a critical role in the pathophysiology of cardiovascular diseases and act as a proinflammatory mediator, and can promote the secretion of IL-1β (Tong et al., 2020). Here, we show that exposure to HS induced an increase in the concentrations of NLRP3 and IL-1β in the heart tissue, and that these effects were prevented in mice given OTC. Earlier work has reported that exposure to cigarette smoke induced the activation NLRP3 which in turn led to the caspase-1 mediated release of proinflammatory cytokines such as IL-1β and IL-18 which was responsible for triggering an inflammatory outburst in the lung of a mouse model of chronic obstructive pulmonary disease (Mahalanobish et al., 2020). Moreover, it has been reported that exposure of human aortic endothelial cell to nicotine promotes atherosclerosis via the stimulation of reactive oxygen species production, activation NLRP3 and caspase-1, and eventually results in pyroptosis of endothelial cells (Wu et al., 2018). The latter effects were prevented by the pretreatment with the antioxidant N-acetyl-cysteine (Wu et al., 2018).
The transcription factor NF-κB is a critical mediator of inflammation involved in the pathophysiology of various cardiovascular diseases (Barnes, 2016; Hall et al., 2006). It has been reported that the activation of NF-κB signaling pathway initiates up-regulations of proinflammatory cytokines, oxidative stress and induce mitochondrial dysfunction in the heart (Barnes, 2016; Hall et al., 2006). On the other hand, it is well established that oxidative stress exerts an inhibitory action on sirtuin-1, and that the inhibition of sirtuin-1 augments the NF-κB signaling and consequently stimulates inflammatory mechanisms (Ministrini et al., 2021; Kauppinen et al., 2013). Here we confirm the antagonistic association between NF-κB and sirtuin-1 as we show that HS exposure induced an increase in the expression of NF-κB and a decrease in that of sirtuin-1. Moreover, we report for the first time that OTC treatment abrogated these effects. A recent report has demonstrated that HS inhalation up regulates the expression of NF-κB and down regulates that of sirtuin-1 in aortic tissue homogenates of mice (Nemmar et al., 2023a). Our data are in line with a previous report that showed that the natural antioxidant catalpol alleviates inflammation and oxidative stress via mechanisms implying sirtuin-1 activation and NF-κB inhibition in a mouse model of chronic kidney disease (Zaaba et al., 2023).
The present data show that HS inhalation caused a significant augmentation in the cardiac expression of Nrf2. Previous studies have demonstrated that exposure to HS led to an upregulation in the expression of cardiac Nrf2 (Nemmar et al., 2019a; Hamadi et al., 2024). The upregulation of Nrf2 expression was associated with the onset of oxidative stress and the activation of an antioxidant response (Vargas-Mendoza et al., 2019). Moreover, here we also show an overexpression of Nrf2 in mice treated with OTC and exposed to HS when compared with HS alone. This pattern suggests that Nrf2 activation may contribute to the protective effects observed with OTC in this model. It has been previously reported that exposure to HS increase the expression of cardiac Nrf2, and that this effect was potentiated in mice simultaneously treated with gum Arabic and HS smoke (Nemmar et al., 2019a). Additionally, Lian et al. (2017) have reported that chrysophanol, an anthraquinone extracted from the Rheum genus plants, exerts its anti-inflammatory, antioxidant and antifibrotic effects via the upregulation of Nrf2 in high fat diet-induced diabetic heart injury in mice.
Despite the marked oxidative stress, inflammation, DNA damage, and apoptosis observed in HS-exposed mice, no overt cardiac morphological alterations were detected at the 1-month time point. Such divergence between molecular injury and preserved tissue structure is plausible, as early biochemical disturbances often precede detectable histopathological changes, and routine H&E staining may not reveal subtle subcellular abnormalities (Aimo et al., 2020; Wang and Kang, 2020). In addition, the heart's intrinsic antioxidant and repair mechanisms can initially counteract or delay structural remodeling (D'Oria et al., 2020). The relatively short duration of exposure may also have been sufficient to induce cellular stress responses but insufficient to produce detectable morphological alterations. These findings are consistent with our previous observations showing lung, but not cardiac, morphological injury following HS exposure, reflecting tissue-specific susceptibility and direct pulmonary deposition of inhaled toxicants (Nemmar et al., 2019a, 2019b, 2020b, 2020c). The ability of OTC to attenuate the molecular indicators of cardiac injury further supports the presence of early pathophysiologic processes despite the absence of overt structural damage.
This study has some limitations. Although both male and female mice were included, the study was not designed to assess sex-specific differences, which will require dedicated future experiments. We also used a single dose of OTC (80 mg/kg), selected based on previous in vivo studies demonstrating protective effects (Nemmar et al., 2009; Lee et al., 2004; Choi et al., 2013). Using standard body surface area–based allometric scaling, this corresponds to an estimated human-equivalent dose of approximately 6.5 mg/kg; however, such extrapolations are approximate, and additional dose-response studies are needed to better define the optimal dosing strategy and its translational relevance. Finally, our work focused on molecular and biochemical endpoints; functional cardiac assessments such as electrocardiogram recordings and echocardiography were beyond the scope of this study but would strengthen future investigations.
Taken together, our findings suggest that administration of OTC mitigates the HS-induced heart injury by reducing oxidative stress, inflammation, DNA damage, and apoptosis through mechanisms involving inhibition of NRLP3 and NF-κB and activation sirtuin-1 and Nrf2. While these results provide insight into potential mechanisms, they remain preclinical, and further studies are required to clarify translational significance. The generated evidence strengthens the justification for future investigations into the potential cardioprotective effects of OTC.
Statement of ethics
The authors have no ethical conflicts to disclose.
Author contributions
Conceptualization, A.N. and B.H.A; Supervision, A.N., Methodology, S.A., B.M.N., N.E.Z., S.B. and O.E.; Investigation, S.A., N.E.Z., S.B. and O.E.; Writing—original draft preparation, A.N.; Writing—review and editing, A.N. and B.H.A., visualization, S.A., N.E.Z., S.B. and O.E, Funding Acquisition, A.N.
Funding
This research was funded by United Arab Emirates University, Zayed Center for Health Sciences (grant number 12R166) and College of Medicine and Health Sciences (grand number 12M112).
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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