Therapeutic Potential of Cannabinoids in Attenuating Amiodarone-Induced Pulmonary Fibrosis: An in vivo Experimental Study in Wistar Rats

Introduction

Drug-induced pulmonary toxicity represents a formidable challenge in contemporary pharmacotherapy, frequently culminating in severe respiratory dysfunction, heightened morbidity, and, in extreme cases, mortality. Among the pharmacological agents associated with pulmonary toxicity, amiodarone (AMIO), a Class III antiarrhythmic agent, remains a cornerstone in the management of atrial fibrillation (AF) and ventricular tachycardia (VT).¹ Despite its well-established efficacy in stabilizing cardiac arrhythmias, AMIO’s long-term therapeutic viability is significantly hindered by its pharmacokinetic properties, including high lipophilicity, extensive tissue accumulation, and an exceptionally prolonged half-life.² These attributes predispose patients to a broad spectrum of adverse effects, with pulmonary toxicity manifesting as interstitial pneumonitis and progressive pulmonary fibrosis recognized as one of its most severe and clinically limiting complications.³ These pulmonary manifestations can lead to substantial impairment of respiratory function, a progressive decline in quality of life, and, in severe cases, treatment discontinuation, thereby presenting a critical therapeutic challenge in cardiology and pulmonology.⁴ While AMIO has been extensively utilized in clinical settings, the precise pathophysiological mechanisms underlying amiodarone-induced pulmonary toxicity (AIPT) remain incompletely understood.⁵ Existing evidence implicates oxidative stress, mitochondrial dysfunction, and chronic inflammation as principal drivers of pulmonary fibrosis progression in AIPT.^6,7 Prolonged AMIO exposure has been linked to an excessive accumulation of reactive oxygen species (ROS), disruptions in mitochondrial homeostasis, and upregulation of mitochondrial dysfunction and increased levels of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β).⁵ These molecular aberrations instigate fibroblast activation, excessive extracellular matrix deposition, and dysregulated alveolar remodeling, ultimately culminating in irreversible fibrosis and progressive pulmonary insufficiency.⁶ Histopathological analyses corroborate these findings, demonstrating substantial alveolar thickening, excessive collagen accumulation, and pronounced architectural disruption of pulmonary parenchyma, underscoring the imperative need for targeted therapeutic interventions that can effectively mitigate fibrosis progression and ameliorate AIPT severity.⁸ Given the dual nature of AMIO, its indispensable role in arrhythmia management juxtaposed with its severe pulmonary toxicity there is a pressing need to develop pharmacological interventions that can counteract fibrosis while preserving its cardioprotective effects.⁹ Various therapeutic strategies, including antioxidants, anti-inflammatory agents, and fibrosis-modulating compounds, have been explored to mitigate AIPT; however, no clinically viable intervention has been definitively established.^10–18 Recent advances in molecular pharmacology have spotlighted cannabidiols as promising candidates for pulmonary fibrosis therapy, attributed to their pleiotropic pharmacological actions, encompassing anti-inflammatory, antioxidant, and antifibrotic properties.¹⁹ Cannabinoids, bioactive constituents derived from Cannabis sativa, exert their effects primarily via modulation of the endocannabinoid system, particularly through CB1 and CB2 receptors, which govern critical immunological and oxidative stress pathways.¹⁹ Among cannabinoids, cannabidiol (CBD), a non-psychoactive Phyto cannabinoid, has garnered considerable attention as a potential therapeutic agent owing to its potent immunomodulatory, antifibrotic, and cytoprotective effects.⁸ Emerging preclinical studies suggest that CBD and its synthetic derivative, cannabidiol (O-2545), exert protective effects against pulmonary fibrosis through multiple mechanisms, including down-regulation of NF-κB signaling, suppression of pro-inflammatory cytokine production, inhibition of fibroblast proliferation, and augmentation of endogenous antioxidant defenses.^{9,12,13,15–18,20} Furthermore, experimental models of pulmonary fibrosis indicate that CBD may mitigate excessive collagen deposition, prevent extracellular matrix dysregulation, and preserve alveolar integrity, positioning it as a promising candidate for ameliorating AIPT.^10,12–18 Thus, the present investigation aims to explore the possible protective benefits of O-2545 in the setting of AMIO-induced lung damage. This research seeks to build an understanding of the mechanisms involved regarding CBD as a novel therapeutic option for the prevention of drug-induced pulmonary fibrosis by studying the compound’s anti-inflammatory, antioxidant, and antifibrotic properties on a molecular level.¹⁹ The increasing clinical burden of AIPT, coupled with the lack of viable antifibrotic medications, may expand the research possibilities aimed at novel pharmacological strategies to improve the safety of AMIO, improve its long-term therapeutic uses, and improve the clinical condition of patients on long-term anti-arrhythmic medications.¹ Accordingly, the primary aim of the present study was to evaluate the potential protective effects of the cannabinoid O-2545 against amiodarone-induced pulmonary fibrosis in male Wistar rats. The secondary aim was to investigate the associated molecular, biochemical, and histopathological alterations in lung tissue, focusing on oxidative stress, inflammation, apoptosis, and fibrotic progression.

Materials and Methods

Material

Amiodarone Injection, USP Isotonic Solution with Preservative is available in vials of 50 mg/mL. Hospira, Inc. manufactures the product. The country of origin is Australia. The cannabis (O-2545) powder was acquired from Hubei Yuncheng Technology Co. Ltd, a Wuhan, China-based company. Zoad International Co., a medical supplier in Jeddah, Saudi Arabia (KSA), supplied dimethyl sulfoxide (DMSO). A medical supplier located in Jeddah, Saudi Arabia (KSA). Zoad International Co. supplied diethyl ether as well as 37% formaldehyde solution for medical use.

Animal

All experimental procedures were approved by Umm Al-Qura University’s Biomedical Research Ethics Committee (Approval No. HAPO-02-K-012-2024-10-2275). All animal experimentation adhered to the guidelines on the care and use of laboratory animals and AVMA Guidelines for the Euthanasia of Animals (2020). Wistar rats were maintained under controlled laboratory conditions and received daily oral administrations for ten consecutive days. The study involved the administration of amiodarone (50 mg/kg, P.O.) and cannabinoid O-2545 (10 mg/kg, P.O). No additional permit number was required according to local regulations.

Twenty-four male Wistar rats weighing between 170 and 190 grams and aged eight weeks were collected from the Mansour Scientific Foundation (MSF) in Jeddah, KSA. The animals were kept for at least a week before the experiment under controlled laboratory settings, which included a cycle of 12 hours of light and darkness, temperatures around 25°C and 26°C, and a humidity ratio between 35% and 75%. Six rats per cage were provided with constant accessibility to food and water.

Study Design

A pulmonary fibrosis model was developed via administration of amiodarone (50 mg/kg) orally via oral gavage (P.O.) for ten days in a row to establish acute pulmonary fibrosis.²¹ The biochemical, inflammatory, and histological alterations associated with pulmonary fibrosis were assessed using this approach. 24 male Wistar rats were divided into four groups at random, with six rats in each group as follows:

• Group I (control group): was administered oral DMSO as the vehicle and subcutaneous injections of 0.9% NaCl (NS) once a day for ten days in a row.
• Group II (AMIO group): was administered oral DMSO as a transport agent and oral gavage of AMIO (50 mg/kg) once daily for 10 days in a row.
• Group III (O-2545 group): received oral O-2545 at 10 mg/kg once daily for 10 days in a row, as well as subcutaneous injections of NS once daily for 10 days in a row.
• Group IV (AMIO + O-2545): received oral O-2545 (10 mg/kg) and AMIO (50 mg/kg) via oral gavage once daily for ten days in a row.

Biochemical Analysis in Serum

On the eleventh day, rats were put under anesthesia and euthanized by decapitation, and blood samples from the carotid arteries were obtained and placed in separate serum gel tubes. Following that, lung samples from rats were collected.^21,22

Blood Samples Preparation

To ensure that the blood and clot activators were mixed properly, the tube was gently inverted five times. To collect serum, the blood was allowed to coagulate for approximately ten minutes at room temperature prior to centrifuging for 15 minutes at 3500 rpm via a SIGMA SM7000 centrifuge (SIGMA, UK). Serum was stored at −80°C over a period of four weeks before biochemical analysis. The biochemical assays were carried out using DSX Best Sells 2000 automated ELISA equipment.

Pulmonary Samples Preparation

Rats were euthanized, and their lungs were immediately extracted. The isolated lung tissue was rinsed with cold normal saline solution and weighed. The left pulmonary lobe has been removed for histological evaluation, while the right lobe was kept at −80°C to test p53, TNF-a and IL-1b, MDA, HDAC, HYDROX and caspase 3, GSH, and adenosine levels in tissue homogenate.

Pulmonary Homogenate Assessment

Pulmonary tissue homogenates were analyzed using commercial ELISA kits (My BioSource, San Diego, USA) according to the manufacturers’ instructions.²³ The biomarkers included glutathione (GSH, Catalog # MBS724319, ng/mL) to assess antioxidant capacity, malondialdehyde (MDA, Catalog # MBS738685, μmol/L) as an indicator of oxidative damage, hydroxyproline (HYDROX, Catalog # MBS017427, μg/mL) for fibrotic progression, TNF-α (Catalog # MBS175904, pg/mL), and IL-1β (Catalog # MBS268833, pg/mL) for inflammatory response, Caspase-3 (Casp3, Catalog # MBS723886, ng/10⁵), and p53 (Catalog # MBS723886, µg/L) for apoptotic activity, adenosine (Catalog # MBS2606939, U/L) for cellular signaling, and HDAC (Catalog # MBS9307760, µM) for epigenetic regulation.²³ Collectively, these assessments provided a comprehensive evaluation of oxidative stress, inflammation, apoptosis, and fibrosis in rat pulmonary tissue, enabling a detailed investigation of amiodarone-induced pulmonary toxicity and the protective potential of cannabinoid O-2545.

Pulmonary Histopathological Assessment

The rat lung tissues were fixed for a week in 10% neutral buffered formalin and then prepared for paraffin embedding. Five-micron thick sections were cut and stained with H&E as per standard protocols.^24,25 After which scoring of lung tissue changes was done based on established criteria.²⁶ The criteria included the following:

(A) Infiltration of inflammatory cells (neutrophils, macrophages, etc)

(B) Alveolar Damage (collapse, edema, etc).

(C) Bronchiolar changes (eg, hyperplasia or dilation)

(D) Mucus production (eg, goblet cell hyperplasia)

(E) Fibrosis (eg, collagen deposition or interstitial changes)

Scoring was as follows: “-” = No changes watched; “+” = Mild changes; “++” = Moderate changes; “+++” = Extreme changes.

Statistical Analysis

All data are presented as mean ± standard deviation (SD) because the variables were normally distributed. Differences among groups were analyzed using one-way ANOVA, followed by post hoc tests (LSD and Tukey’s tests) for multiple comparisons. A p-value < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS version 26 (IBM SPSS Inc., Windows^®), and graphs were generated with GraphPad Prism 9.3.0 (GraphPad Software, USA).

Results

Biochemical Measurements

One-way analysis of variance (ANOVA) revealed a statistically significant overall difference among the four experimental groups for serum oxidative stress and fibrotic markers (p < 0.05). Tukey’s post hoc multiple comparison test was subsequently applied to identify pairwise group differences. The mean serum MDA level was significantly higher (P < 0.05) in the AMIO group (2.080 ± 0.39) than in the Control group (0.358 ± 0.04), with a [5.8-fold increase]. In contrast, the mean level of GSH in the AMIO group was significantly decreased (P < 0.05) (2.067 ± 0.77), (Figure 1). The O-2545 showed a substantial reduction in MDA levels (0.675 ± 0.10) compared to the AMIO group (2.080 ± 0.39), along with increased levels of antioxidant GSH (16.783 ± 0.79). The AMIO + O-2545 group reduced MDA levels (0.873 ± 0.31), an increase in GSH levels (9.617 ± 1.24), (P < 0.05) (Figure 1).

Figure 1 Effect of O-2545 administration on AMIO induced changes of rat’s lung tissue content of MDA, GSH and HYDROX. (A) Serum level of MDA in nmol/mL, (B) GSH levels in ng/mL. (C) HYDROX levels in ug/mL. Values are mean ± standard deviation (SD), n number of rats (6 rats per group). One-way ANOVA, Tukey’s multiple comparison test. *P value <0.05 compared to the control group. #P value <0.05 compared to the AMIO group. σP <0.05 compared to AMIO + O-2545.

The mean serum level of HYDROX in the AMIO group (4.516 ± 0.55) in comparison with the Control group (0.735 ± 0.03), (P < 0.05) with a [6-fold increase]. The O-2545 group shows a mean level of HYDROX (1.266 ± 0.20) closely related to Control group (P < 0.05), AMIO + O-2545 group shows HYDROX mean level were (0.920 ± 0.32) (P < 0.05) (Figure 1).

Lung Homogenate Studies

One-way ANOVA demonstrated a significant overall effect of treatment on inflammatory and apoptotic markers across all experimental groups (p < 0.05). Tukey’s post hoc test was used for multiple comparisons. Similar changes were observed with TNF-α and IL-1 levels, where the level of TNF-α in the AMIO group was (71.167 ± 11.97) in comparison to the control group (12.550 ± 0.62), significant increase (P< 0.05) approximately [6-fold increase]. In addition, the O-2545 group TNF-α level (13.833 ± 1.16) compared to the control group (12.550 ± 0.62), IL-1β level (14.330 ± 0.69) compared to the control group (13.900 ± 0.40) shown in (Figure 2). Notably, in the AMIO + O-2545 group, both TNF-α and IL-1β were significantly reduced in comparison with the AMIO group (P < 0.05). TNF-α level (22.433 ± 6.98), IL-1β level (25.320 ± 6.26). A similar observation on Adens levels, with a significant increase in the AMIO group (P < 0.05) (67.832 ± 8.97) compared to the control group (12.550 ± 0.56). A slight increase in Adens level in the O-2545 group (19 ± 3.57), but a substantial decrease in AMIO + O-2545 group (P < 0.05) levels were (36.500 ± 3.93), shown in (Figure 2).

Figure 2 Effect of O-2545 administration on AMIO induced changes of rat’s lung tissue content of IL-1β, TNF-α and Adens. (A) Serum level of IL-1β in pg/mL, (B) TNF-α levels in pg/mL. (C) Adens levels in U/L. Values are mean ± standard deviation (SD), n number of rats (6 rats per group). One-way ANOVA, Tukey’s multiple comparison test. *P value <0.05 compared to the control group. # P-value <0.05 compared to the AMIO group. σP <0.05 compared to AMIO + O-2545.

On the other hand, the apoptotic markers were significantly changed in the AMIO group (P < 0.05). Casp3 serum levels were highly elevated in AMIO group (14.783 ± 0.40) compared to the control group (1.376 ± 0.05) [approximately 10.5-fold increase]. In addition, p53, was also elevated in the AMIO group (1.905 ± 0.52) compared to the control group (0.172 ± 0.01), as shown in (Figure 3).

Figure 3 Effect of O-2545 administration on AMIO induced changes of rat’s lung tissue content of Casp3, p53 and HDAC. (A) Serum level of Casp3 in ng/10*5, (B) p53 levels in ug/L. (C) HDAC levels in uM. Values are mean ± standard deviation (SD), and the number of rats (6 rats per group). One-way ANOVA, Tukey’s multiple comparison test. *P value <0.05 compared to the control group. #P value <0.05 compared to the AMIO group.

Furthermore, HDAC levels were significantly increased (P < 0.05) in the AMIO group (1.538 ± 0.27) compared with the control group (0.605 ± 0.03), O-2545 group shows a similar result as control group in Casp3, p53 and HDAC, combining O-2545 with AMIO which results as, Casp3 level were (2.213 ± 0.93) and p53 level (0.575 ± 0.03) along with HDAC level (0.571 ± 0.10) markedly decreased levels in comparing to AMIO group (P < 0.05) shown in (Figure 3).

Histopathological Assessment of Lung Tissue

Histopathological examination of lung tissue was performed to evaluate structural changes and to identify and confirm the biochemical data shown in this paper. In (Figure 4) in Figure 4A, G1 (normal control) the lung architecture showed normal patent alveoli with thin lining epithelium, the bronchiole showed also normal lining epithelium and lumen free of any cell debris or inflammatory cells. In contrast, the lung of Figure 4B, G2 (AMIO) showed marked obliteration of alveolar lumina with inflammatory cells, The bronchioles showed deformed shape, disrupted lining epithelium and the lumen is filled by inflammatory exudate and cells.

Figure 4 Sections from rat pulmonary tissue stained by H&E and photographed at x100 scale bar = 200 µm and x400 scale bar = 50 µm to represent: G1: NC, normal control, notice the normal bronchiole and its epithelium (thick black arrow) with its lumen free of any secretion or cell debris (black star). Notice also the patency of most alveoli (thin black arrows). Blood vessel accompanied bronchiole is normal and has thin wall (dotted arrow). G2: AMIO showing loss of alveoli patency. Most are closed by inflammatory exudate (thin black arrows). Most bronchioles are deformed and their lumina are full of inflammatory exudate (red arrows). Notice the marked thickening of blood vessel walls (dotted arrow). G3: O-2545 receiving the treatment alone showing no apparent changes compared to G1 normal control. Alveoli are patent (thin arrows) and bronchioles showed normal epithelium (thick black arrow) and patent arrow with scanty cell debris (star). G4: AMIO + O-2545 group showing marked improvement in histological changes induced by Amiodarone. Most alveoli are patent free of inflammatory cells (Thin black arrows). Bronchiole showed normal lining epithelium but still showed residual secretion within bronchioles (thick black arrow and star). The response seemed to be about 75% compared to untreated lung and to control which could be considered effective.

In Figure 4C, G3 received the O-2545 alone; the treatment did not affect the histology of lung tissue, which appeared normal. In Figure 4D, G4 Received of O-2545 to AMIO treated animals markedly improved changes occurred by Amiodarone administration, except for residual changes in the bronchiolar wall and lumen. Most alveoli are patent free of inflammatory cells. Bronchiole showed normal lining epithelium but still showed residual secretion. The response seemed to be about 75% compared to untreated lung and to control which could be considered effective.

Table 1 presents the histopathological lung alteration scores for each group, including alveolar patency, inflammatory cell infiltration, bronchiolar epithelium integrity, and bronchiolar lumen content.

Table 1 Lung Histopathological Alteration Scoring

Discussion

AMIO, an antiarrhythmic medication used extensively to treat atrial fibrillation disorder, although its use is limited due to side effects such as pulmonary fibrosis by several pathways including oxidative damage, inflammation, apoptosis, and histopathological changes of lung tissues.^{2,4,6,27–29} Our findings in Wistar rats support clinical data showing that AMIO produces oxidative stress by increasing the levels of MDA while decreasing the levels of GSH, as well as increasing HYDROX levels, which indicates severe damage. AMIO increased inflammatory cytokines, including TNF-α and IL-1β, which supports earlier findings associating NF-κB activation with pulmonary fibrosis, along with high adenosine levels, indicating severe inflammation.^30–32 Elevated apoptotic markers P53 and Casp3 confirm the presence of cell death associated with tissue injury and cellular fibrogenesis.^33–36 HDAC activation has been observed, lending epigenetic modification and stimulation of fibrosis.^37–45

The administration of O-2545 greatly reduced these effects. O-2545 restored GSH, decreased MDA and HYDROX, as well as cytokines and apoptotic biomarker levels. Histological examination indicated a reduction in alveolar thickness and inflammatory infiltrates. Activating the CB2 receptor has been shown to reduce NF-κB signaling, boost Nrf2-mediated antioxidant activities, and decrease HDAC activity.^{9,20,31,43–47}

To further contextualize the effects of O 2545, we considered its efficacy alongside other clinically approved therapies for lung fibrosis. Pirfenidone and nintedanib, the only FDA- and EMA-approved anti-fibrotic treatments for idiopathic pulmonary fibrosis (IPF), slow disease progression by reducing fibroblast proliferation, collagen deposition, and inflammatory signaling.^48–50 Corticosteroids, on the other hand, mainly target inflammation and do not directly prevent fibrosis. In our study, O-2545 demonstrated potent antioxidant, anti-inflammatory, and anti-apoptotic effects, effectively reducing oxidative stress, inflammatory cytokines, and apoptotic markers. These results suggest that CB2 receptor activation may provide a complementary or synergistic mechanism alongside conventional anti-fibrotic therapy. Taken together, our findings highlight the therapeutic potential of O-2545 and provide a strong rationale for further studies in chronic models of pulmonary fibrosis.

Our findings are consistent with previous research revealing that selective CB2 agonists attenuate fibrosis in clinical settings, including bleomycin-induced lung damage.⁵¹ Other studies, however, have found that CB1 activation exacerbates fibrosis while CB1 antagonism improves results, emphasizing cannabis’ receptor-specific and context-dependent effects.⁵² These data highlight the need to ensure O-2545 has CB2 selectivity to enhance antifibrotic efficacy.

The study has limitations. The acute 10-day model excludes chronic progression and mortality outcomes, and dose-response associations were not investigated. Further research should focus on prolonged safety, drug kinetics, and translational potential in therapeutically relevant animals.^53–55 In conclusion, O-2545 decreased AMIO-induced oxidative, inflammatory, apoptotic, and fibrotic alterations in rat lungs while enhancing histological architecture. These findings provide preclinical evidence that cannabinoids, via CB2-mediated pathways, may be useful adjuncts for reducing the pulmonary consequences of long-term AMIO treatment.^2,4,46,51

Limitation of the Study

Finally, it is essential to recognize the limitations of the study, which involves that it is an acute study and further investigation of O-2545 on AMIO kinetics, dynamics, or anti-inflammatory effects cannot be explored as a chronic study. These limitations are opportunities for further investigation to fully understand the benefits and findings of the study.

Conclusion

The findings of this study suggest that activation of the CB2 receptor by the synthetic cannabinoid O-2545 may contribute to reducing amiodarone-induced pulmonary fibrosis in an acute experimental model. The observed protective effect appears to be associated with attenuation of several pathological mechanisms, including oxidative stress, inflammation, apoptosis, and fibrotic remodeling, which was reflected in improved lung histological architecture. Although these results provide promising preclinical evidence, they should be interpreted within the limitations of an acute model. Further studies are needed to evaluate long-term effects, optimize dosing, and clarify the potential clinical relevance of O-2545.