Attenuating amiodarone-induced lung toxicity by the vitamin D receptor activator paricalcitol in rats: targeting TLR4/NF-κB/HIF-1α and TGF-β/Smad signaling pathways
Aamal G. El-Waseif, Mahmoud Elshal, Dalia H. El-Kashef, Nashwa M. Abu-Elsaad
TL;DR
Paricalcitol, a vitamin D receptor activator, reduces lung damage caused by amiodarone in rats by targeting inflammation and fibrosis pathways.
Contribution
This study demonstrates that paricalcitol mitigates amiodarone-induced lung toxicity through antioxidant, anti-inflammatory, and antifibrotic mechanisms.
Findings
Paricalcitol reduced lung inflammation and oxidative stress in rats treated with amiodarone.
Paricalcitol suppressed TLR4/NF-κB and TGF-β/Smad signaling pathways, reducing lung fibrosis.
Paricalcitol also decreased HIF-1α expression, contributing to reduced lung damage.
Abstract
Amiodarone, an antiarrhythmic drug, has been reported to precipitate lung injury by various mechanisms. Vitamin D receptor (VDR) is extensively expressed in the lung, and the disrupted vitamin D/VDR axis may underlie various lung disorders. Therefore, the current study intended to explore the beneficial impact of paricalcitol, a VDR activator, on amiodarone-provoked lung injury and elucidate its possible involved molecular mechanisms. Male Wistar rats were intraperitoneally injected with paricalcitol (0.2 µg/kg) and orally administered amiodarone (40 mg/kg) once daily for four weeks. Our findings revealed that paricalcitol diminished BALF leucocyte count and total protein, serum LDH activity, and pulmonary histopathological changes and counteracted pulmonary oxidative stress. Moreover, paricalcitol decreased pulmonary toll-like receptor 4 (TLR4), nuclear factor kappa B (NF-κB) p65,…
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Figure 9- —Mansoura University
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TopicsChronic Obstructive Pulmonary Disease (COPD) Research · Biomarkers in Disease Mechanisms · Vitamin C and Antioxidants Research
Introduction
Drug-induced lung disease (DILD) denotes a range of pulmonary disorders that are directly linked to the administration of medications. About 3 to 5% of patients with lung disease are drug-induced, which represents an incidence of DILD between 4.1 and 12.4 cases/million/year (Spagnolo et al. 2022). DILD is considered a clinical challenge because of its diverse clinical presentations as well as possible severe outcomes. The management of DILD in common practice depends on introducing glucocorticoids (GCs) concurrently with the discontinuation of the insulting medication (Annareddy et al. 2024). After dose lowering, drug discontinuation, and/or simultaneous GCs use, a complete recovery is probable. Conversely, a considerable proportion fails to recover or tracks a progressive clinical course. Primary underlying disease progression, respiratory failure, multiorgan failure, or a GC therapy adverse effect-like infection is the leading causes of DILD mortality (Annareddy et al. 2024).
Amiodarone is an anti-arrhythmic agent that is commonly used; nevertheless, its administration, unfortunately, might be accompanied by numerous adverse effects, including pulmonary injury. Amiodarone-induced pulmonary injury arises in about 4 to 17% of cases and can manifest as interstitial pneumonitis or even progress to pulmonary fibrosis if not treated early (Șorodoc et al. 2024).
Although it was long believed that vitamin D shares in regulating calcium and phosphate homeostasis, as well as musculoskeletal health, lately, a variety of vitamin D’s other actions in regulating several other organs have arisen, involving the lung (Gayan Ramirez and Janssens 2021). What supports that the lung responds to vitamin D actions is that the vitamin D receptor (VDR) is expressed in numerous cell types in the lung and reacts to 1,25-dihydroxy vitamin D (Mathyssen et al. 2020).
The suppression of the vitamin D/VDR axis has been reported to be engaged in lung disorders, including asthma (Cherrie et al. 2018), chronic obstructive pulmonary disease (Ilyas et al. 2019), cystic fibrosis, and idiopathic pulmonary fibrosis (Gaudet et al. 2022). Accordingly, stimulation of the vitamin D/VDR axis might be a promising approach in lung injury management.
Paricalcitol is an analog of calcitriol, the vitamin D active form, which selectively activates VDR. It has been used for secondary hyperparathyroidism prophylaxis and treatment that is accompanied by chronic renal failure (Robinson and Scott 2005). A prior study suggested that it augments trans-alveolar fluid clearance and could potentially be utilized to improve lung edema (Nie et al. 2016). Additionally, paricalcitol administration antagonized the stimulation of the toll-like receptor 4 (TLR4), relieving inflammation, decreasing apoptosis, and maintaining the structure of the lung in hyperoxia-induced pulmonary injury (Yao et al. 2017). Paricalcitol has been revealed to exert anti-inflammatory activities as well on cardiovascular and kidney diseases through modifying the nuclear factor kappa B (NF-κB) signaling (Tan et al. 2008; Lee et al. 2016).
Accordingly, the purpose of the present study was to examine the beneficial impact of paricalcitol on amiodarone-mediated pulmonary injury and uncover the possible signaling pathways involved.
Materials and methods
Drugs and chemicals
Amiodarone hydrochloride (Sunnydarone^®^, 150mg/3ml ampoules) was obtained from Sunny Pharmaceutical (Cairo, Egypt), and the working solution was prepared by dilution with water for injection to 40mg/ml. Zemplar^®^ (AbbVie Ltd., SL6 4UB, UK), paricalcitol, was purchased as 5µg/ml vials, and the working solution was prepared by dilution with water for injection to 0.2µg/ml. All other chemicals and reagents were of the highest analytical grades.
Animals and study design
Adult male Wistar rats weighing 200±20 g were bought from The Egyptian Organization for Biological Products and Vaccines “VACSERA” (Giza, Egypt). Rats were randomly housed with free access to food and water (four rats per cage), adapted for two weeks, and maintained at room temperature 25°C with a 12-hour light/dark cycle throughout the experiment. The study protocol received approval number (RHARM.PhD.23.03.21) from Mansoura University Animal Care and Use Committee (MU-ACUC).
Sixty animals were weighed and randomized into five groups (n=12) as follows: the Control group served as normal animals and received no treatments; the Vehicle group received distilled water orally (1ml/kg) and intraperitoneal (i.p) injection of paricalcitol vehicle (1ml/kg), 1.2% v/v propylene glycol + 0.8% v/v ethanol in water for injection, once daily for four weeks; the Paricalcitol group received 0.2µg/kg paricalcitol (1ml/kg) by i.p. injection once daily for four weeks (Azak et al. 2013; Abood et al. 2021; Xie et al. 2022); the Amiodarone group received amiodarone at a dose of 40mg/kg (1ml/kg) orally (Ibrahim Fouad and Mousa 2021) once daily for four weeks; and the Paricalcitol + amiodarone group rats received both paricalcitol and amiodarone as described. Paricalcitol was administered an hour before amiodarone administration.
The rats were weighed, and blood samples were gathered from the retro-orbital venous plexus under anesthesia with secobarbital (50mg/kg, i.p.) at the end of this experiment. Serum samples were separated by centrifugation for 15 min at 3000 rpm at 4°C to measure lactate dehydrogenase (LDH). For collecting the bronchoalveolar lavage fluid (BALF), six rats from each group were subjected to an incision in the neck skin near the trachea by surgical scissors. The muscle around the trachea was incised to expose the trachea, and then the middle of the exposed trachea between two cartilage rings was punctured carefully with a 16G needle. The catheter was placed into the trachea. Then the catheter was fixed by fastening the trachea around the catheter with a cotton thread. Then a syringe was packed with 1ml of normal saline (0.9% NaCl) and was attached to the catheter, and the solution was gently introduced into the lung. During massaging the chest of the rat, the solution was aspirated gently. The injection and aspiration process was repeated three times, and the aspirated fluid was collected (Leroy et al. 2008). The BALF was then centrifuged at 4°C using a cooling centrifuge for 20 min at 4000 rpm. The supernatant was collected and used for total protein assessment, and the cell pellets were used for the determination of total leucocyte count (TLC) and lymphocyte percentage.
Finally, the lungs of the other six rats in all groups were isolated, and animals were euthanized by cervical dislocation. The left lungs were fixed in 10% v/v buffered formalin for subsequent histopathological examination and immunohistochemistry (IHC). The right lungs were preserved at −80°C for tissue homogenate preparation (10% w/v) in 50mM phosphate buffered saline (PBS) (pH 7.4).
Assessment of serum lactate dehydrogenase (LDH)
LDH activity was determined by kinetic procedure in the serum samples via a commercial kit (Cat. No. LDH 117090, BioMed, Egypt).
Determination of TLC, lymphocyte percentage, and total protein in the BALF
Cell pellets of the BALF were resuspended by using a hemocytometer in 0.5ml normal saline for determination of TLC and lymphocyte percentage (Locke et al. 2007) and auto hematology analyzer (Diagon D-cell 60, Hungary), where the total cell number was counted, and the lymphocytes were identified and counted based on appearance.
Determination of total protein in the BALF samples is based on a colorimetric assay using a commercial kit (Cat. No. 13302, Vitro Scient, Alsharkia, Egypt).
Assessment of lung protein content
Lung tissue protein content using Genei protein determination kit (Cat. No. 2624800021730, Bangalore, India) was assessed based on Bradford method (1976).
Estimation of pulmonary oxidative stress biomarkers
Pulmonary malondialdehyde (MDA) was evaluated along with the formerly documented method (Ohkawa et al. 1979). Pulmonary total antioxidant capacity (TAC) was estimated calorimetrically using a commercial kit (Cat. No. TA 25 13, Biodiagnostic, Giza, Egypt). Finally, pulmonary reduced glutathione (GSH) was assessed using Ellman’s reagent (Ellman 1959).
Determination of pulmonary hydroxyproline, transforming growth factor-beta 1 (TGF-β1), phosphorylated small mothers against decapentaplegic 3 (pSmad 3), toll-like receptor 4 (TLR4), and tumor necrosis factor alpha (TNF-α)
Pulmonary contents of hydroxyproline (Cat. No. EA0040Ra, Shanghai Korain Biotech Co. Ltd, Zhejiang, China), TGF-β1 (Cat. No. ER1378, Wuhan Fine Biotech Co. Ltd., Hubei, China), pSmad 3 (Cat. No. MBS269938, MyBioSource Inc., CA, USA), TLR4 (Cat. No. E-EL-R0990, Elabscience, Houston, Texas, USA), and TNF-α (Cat. No. 438206, BioLegend Inc., CA, USA) were determined using sandwich ELISA kits based on the manufacturers’ instructions.
Determination of hypoxia-inducible factor-1α (HIF-1α) by western blotting in the lung
Proteins extraction from lung tissues was performed and processed as formerly reported (El-Waseif et al. 2025). Briefly, the membrane was incubated with the primary rabbit polyclonal antibody (1:1000) overnight at 4°C against HIF-1α (Cat. No. GTX127309, GeneTex, Inc., Irvine, CA 92606, USA) and β-actin from Sigma Aldrich chemical Co. (Cat. No. SAB5600204, MO, USA) as the protein loading control. Then, the membrane was incubated with the secondary goat polyclonal antibody conjugated with horseradish peroxidase (HRP) from Novus Biologicals LLC (Cat. No. NB7160, Briarwood Avenue, USA) for an hour at room temperature. Image analysis using Fiji ImageJ (version 1.51r; NIH, Maryland, USA) was applied to read the target proteins’ band intensity relative to internal control β-actin.
Pulmonary histopathological and IHC examination
Lung tissues were fixed in paraffin, cut, and stained with hematoxylin–eosin (H&E) or, for staining collagen fibers, with Masson’s trichrome stain. Slides were examined under a microscope (MEIJI MX5200L); magnification ×200, scale bar = 50µm. Scoring of H&E-stained sections for lung edema, inflammation, and hemorrhage was blindly scored consistent with Li et al.’s (2015) scoring system: 0 = normal histological structure, 1 = light alterations, 2 = moderate alterations, 3 = strong alterations, and 4 = intense alterations. The blue positive Masson’s trichrome staining area was analyzed by Fiji ImageJ software on Intel^®^ core I7^®^-based computer (version 1.51r; NIH, Maryland, USA). Five random fields were analyzed from all slides, sized 200×200 µm.
In brief, the expression of nuclear factor kappa B (NF-κB) p65 and alpha smooth muscle actin (α-SMA) was measured via IHC staining using primary rabbit polyclonal antibody (1:200) (Cat. No. bs-0465R, Bioss Inc., Massachusetts, USA) and mouse monoclonal antibody (1:200) (Cat. No. MC0603, Medaysis Company, California, US), respectively; then the biotinylated secondary polyclonal antibody against rabbit and mouse primary antibody (Cat. No. D01-6, GBI Labs, WA, USA) was applied. The slides were evaluated under a light microscope (MEIJI MX5200L) via a ×100 and ×400 objectives. The area percentage of positive brown stain was analyzed using Fiji ImageJ software on an Intel^®^ core I7^®^-based computer (version 1.51r; NIH, Maryland, USA). Five random fields in all sections were analyzed then averaged. Different controls were used so that non-specific binding or background interferences were eliminated.
Statistical analysis
For statistical analysis and graphing, GraphPad Prism software Inc. (version 8, Ca, USA) was used. Using one-way analysis of variance (ANOVA) followed by a post hoc test, and Tukey’s multiple comparison test, a statistical analysis for parametric values among groups was conducted (normality test applied was Shapiro Wilk test). Data were indicated as mean±standard deviation (SD). Non-parametric values of histopathological scoring were indicated as median with interquartile range score and evaluated by Kruskal–Wallis analysis followed by uncorrected Dunn’s test. At p<0.05, differences were presumed significant.
Results
Neither the vehicle nor paricalcitol injection showed any significant changes in the BALF TLC and lymphocyte percentage or oxidative stress biomarkers in the lung in comparison with the control rats (Supplementary Table 1).
Paricalcitol alleviated TLC, lymphocyte percentage, and total protein content in the BALF and serum LDH activity
TLC (Fig. 1a), lymphocyte percentage (Fig. 1b), and total protein content (Fig. 1c) in the amiodarone-administered rats were considerably (p<0.05) increased relative to the control group. The paricalcitol-injected animals exhibited a substantial (p<0.05) decline in the enhanced TLC, lymphocyte percentage, and total protein compared to the amiodarone animals.Fig. 1. Effect of paricalcitol on** a **totall eukocyte count (TLC), **b **lymphocyte percentage, **c **total protein in the bronchoalveolar lavage fluid (BALF), and **d **serum lactate dehydrogenase (LDH) in amiodarone-induced pulmonary injury in rats. Data are shown as mean±standard deviation (SD) (n=6). *p<0.05; #p<0.05, compared to the control and amiodarone groups, respectively, applying one-way analysis of variance (ANOVA) then Tukey’s multiple comparison post hoc test
In addition, amiodarone markedly (p<0.05) elevated serum LDH activity (Fig. 1d) relative to the control animals. Meanwhile, paricalcitol injection significantly (p<0.05) diminished its activity compared to the amiodarone-administered rats.
Paricalcitol ameliorated amiodarone-induced changes in pulmonary oxidative stress markers
Pulmonary MDA level (Fig. 2a) in the amiodarone-administered animals was markedly (p<0.05) increased compared to the control animals. Compared to the amiodarone-administered rats, the paricalcitol + amiodarone group displayed a significant (p<0.05) lowering in pulmonary MDA content.Fig. 2. Effect of paricalcitol on pulmonary** a **malondialdehyde (MDA), **b **total antioxidant capacity (TAC). and **c **reduced glutathione (GSH) in amiodarone-induced pulmonary injury in rats. Data are shown as mean±standard deviation (SD) (n=6). *p<0.05; #p<0.05, compared to the control and amiodarone groups, respectively, applying one-way analysis of variance (ANOVA) then Tukey’s multiple comparison post hoc test
Alternatively, the amiodarone group demonstrated a marked (p<0.05) decrease in pulmonary TAC (Fig. 2b), and GSH (Fig. 2c) concentration compared to the control animals. Meanwhile, paricalcitol treatment remarkably (p<0.05) elevated pulmonary TAC and GSH concentration compared to the amiodarone rats.
Paricalcitol improved amiodarone-induced histopathological changes in the lung
H&E photomicrographs of the lung sections (Fig. 3a) demonstrate normal lung architecture with alveoli that were rounded and separated from each other by thin interalveolar septa, bronchioles, and bronchial arterioles in the control sections. Contrarily, the amiodarone group showed distortion in the pulmonary architecture, thickened interalveolar septa, mononuclear cellular infiltration in septa and around the bronchi, thick congested lung arterioles, and small-sized alveoli. Alternatively, paricalcitol treatment restored normal lung architecture with alveoli, separated by thin interalveolar septa, normal bronchioles with minimal peribronchial cellular infiltration, and bronchial arterioles. In addition, a semi-quantitative scoring demonstrated the difference in pathological changes including lung edema (Fig. 3b), inflammation (Fig. 3c), and hemorrhage (Fig. 3d) among all groups.Fig. 3. Effect of paricalcitol on pulmonary histopathological changes in amiodarone-induced pulmonary injury in rats. a H&E-stained lung section photomicrographs; magnification, ×200; scale bar, 50µm. Green arrow represents alveoli; blue arrow represents interalveolar septa; black arrow represents bronchiole; yellow arrow represents hemorrhage and red arrow represents bronchial arteriole. b–d represent scoring for histopathological changes: b edema score, c inflammation score, and d hemorrhage score. Scores are shown as median with interquartile range. *p<0.05; #p<0.05, significantly different from control and amiodarone groups, respectively, applying Kruskal–Wallis analysis followed by uncorrected Dunn’s test
Paricalcitol suppressed amiodarone-induced fibrotic changes in the lung
Masson’s trichrome photomicrographs of the lung sections are presented in Fig. 4a. The control group showed fine blue stained collagen fibers deposition surrounding the bronchiole. Contrariwise, the amiodarone group displayed elevated collagen fibers deposition around the bronchiole and interalveolar septa. While the paricalcitol + amiodarone group showed less collagen fibers deposition around the bronchiole and interalveolar septa. For more illustration, the fibrosis percentage was calculated and demonstrated in Fig. 4b, where the amiodarone group had dramatically (p<0.05) higher fibrosis percentage compared to the control animals and administration of paricalcitol substantially (p<0.05) repressed this percentage compared to the amiodarone rats.Fig. 4. Effect of paricalcitol on pulmonary fibrosis in amiodarone-induced pulmonary injury in rats.** a** Masson trichrome-stained lung section photomicrographs; magnification, ×200; scale bar, 50µm. b Percentage of fibrotic area. Data are shown as mean±standard deviation (SD) (n=5). *p<0.05; #p<0.05, compared to the control and amiodarone groups, respectively, applying one-way analysis of variance (ANOVA) then Tukey’s multiple comparison post hoc test
Paricalcitol reduced pulmonary TLR4 and TNF-α levels
Amiodarone substantially (p<0.05) increased pulmonary TLR4 level (Fig. 5a) relative to the control group. Meanwhile, paricalcitol administration considerably (p<0.05) declined this level relative to the amiodarone-administered rats.Fig. 5. Effect of paricalcitol on pulmonary** a **toll-like receptor 4 (TLR4) and **b **tumor necrosis factor alpha (TNF-α) levels in amiodarone-induced pulmonary injury in rats. Data are shown as mean±standard deviation (SD) (n=5). *p<0.05; #p<0.05, compared to the control and amiodarone groups, respectively, applying one-way analysis of variance (ANOVA) then Tukey’s multiple comparison post hoc test
Regarding the TNF-α level (Fig. 5b), the amiodarone group revealed a marked (p<0.05) higher level in the lung relative to the control animals. Upon pretreatment with paricalcitol, the pulmonary TNF-α level was significantly (p<0.05) lower relative to amiodarone-administered rats.
Paricalcitol diminished pulmonary NF-κB p65 expression
IHC-stained lung sections against NF-κB p65 are represented in Fig. 6a. Additionally, the NF-κB p65 percentage of expression in the pulmonary tissue (Fig. 6b) was dramatically (p<0.05) raised in the amiodarone rats relative to the control animals. On the contrary, paricalcitol substantially (p<0.05) suppressed the NF-κB p65 percentage of expression compared to the amiodarone-administered rats. Yet, there was a substantial (p<0.05) difference between the paricalcitol pretreated rats compared to the control group.Fig. 6. Effect of paricalcitol on pulmonary** a **nuclear factor kappa B (NF-κB) p65 expression in amiodarone-induced pulmonary injury in rats. Data are shown as mean±standard deviation (SD) (n=5). *p<0.05; #p<0.05, compared to the control and amiodarone groups, respectively, applying one-way analysis of variance (ANOVA) then Tukey’s multiple comparison post hoc test. **b **NF-κB p65 IHC-stained lung section photomicrographs. The upper row original magnification is ×100, with a scale bar 100µm, and the lower row is ×400, with a scale bar 20µm
Paricalcitol reduced pulmonary TGF-β1 and pSmad 3 levels
The amiodarone-administered rats exhibited a significant (p<0.05) elevation in pulmonary TGF-β1 (Fig. 7a) and pSmad 3 (Fig. 7b) levels related to the control rats. Meanwhile, paricalcitol administration significantly (p<0.05) lowered both pulmonary TGF-β1 and pSmad 3 concentrations in comparison with the amiodarone rats.Fig. 7. Effect of paricalcitol on pulmonary** a transforming growth factor-beta 1 (TGF-β1) b **phosphorylated small mothers against decapentaplegic 3 (pSmad 3) levels in amiodarone-induced pulmonary injury in rats. Data are shown as mean±standard deviation (SD) (n=5). *p<0.05; #p<0.05, compared to the control and amiodarone groups, respectively, applying one-way analysis of variance (ANOVA) then Tukey’s multiple comparison post hoc test
Paricalcitol attenuated pulmonary hydroxyproline content and α-SMA expression
As shown in Fig. 8a, amiodarone significantly (p<0.05) boosted pulmonary hydroxyproline concentration compared to the control animals. Meanwhile, compared to the amiodarone group, administration of paricalcitol significantly (p<0.05) repressed hydroxyproline content.Fig. 8. Effect of paricalcitol on pulmonary** a **hydroxyproline content and **b **alpha smooth muscle actin (α-SMA) expression in amiodarone-induced pulmonary injury in rats. Data are shown as mean±standard deviation (SD) (n=5). *p<0.05; #p<0.05, compared to the control and amiodarone groups, respectively, applying one-way analysis of variance (ANOVA) then Tukey’s multiple comparison post hoc test. **c **α-SMA IHC-stained lung section photomicrographs. The upper row original magnification is ×100, with a scale bar of 100µm, and lower row is ×400, with a scale bar 20µm
Regarding α-SMA, the percentage of the expression of α-SMA (Fig. 8b) in the amiodarone-administered animals was substantially (p<0.05) raised in comparison with the control rats. Meanwhile, the paricalcitol + amiodarone group showed a significant (p<0.05) decline in the α-SMA expression percentage compared to the amiodarone-administered rats. Additionally, IHC-stained lung sections against α-SMA are represented in Fig. 8c.
Paricalcitol inhibited pulmonary HIF-1α expression
A western blot trend of the lung HIF-1α protein expression is represented in Fig. 9a. The amiodarone group revealed a significant (p<0.05) higher pulmonary HIF-1α level relative to the control rats. Meanwhile, paricalcitol treatment significantly (p<0.05) suppressed relative expression of pulmonary HIF-1α level relative to the amiodarone group (Fig. 9b).Fig. 9. Effect of paricalcitol on pulmonary hypoxia-inducible factor-1α (HIF-1α) protein expression in amiodarone-induced pulmonary injury in rats.** a** Representative photographs show expression bands of pulmonary HIF-1α and β-actin and b the relative expression of pulmonary expression of HIF-1α. Data are shown as mean±standard deviation (SD) (n=5). *p<0.05; #p<0.05, compared to the control and amiodarone groups, respectively, applying one-way analysis of variance (ANOVA) then Tukey’s multiple comparison post hoc test
Discussion
Amiodarone-induced pulmonary injury is a common form of DILI that still represents a clinical challenge due to limited treatment approaches, which mainly include administration of GCs (Annareddy et al. 2024). The vitamin D/VDR axis has recently been referred to as a significant contributor to lung health (Gayan Ramirez and Janssens 2021). Therefore, this study explored the potential beneficial impact of paricalcitol, a VDR activator, on amiodarone-induced pulmonary injury and elucidated its possible underlying molecular mechanisms.
Herein, amiodarone-induced pulmonary injury in rats was linked to elevated TLC, lymphocyte percentage, and total protein content in the BALF. Increased inflammatory cell count in the lung indicates provoked immune and inflammatory responses (Al-Shammari et al. 2016). Whereas increased total protein may be linked to alveolar and capillary barrier damage (Kulkarni et al. 2022). In addition, amiodarone boosted serum LDH activity, which may indicate alveolar lesions and cell injury (Akdogan et al. 2021).
Treatment with paricalcitol markedly reduced TLC, lymphocyte percentage, and total protein in the BALF, as well as serum LDH activity. This observation is compatible with a prior study by Chang et al. (2021), who manifested that mice placed on a vitamin D-deficient diet revealed aggravated bleomycin-induced pulmonary inflammatory cell infiltration compared to bleomycin-induced pulmonary injury in mice placed on a vitamin D-sufficient diet. Shi et al. (2016) have also reported that in mice lacking VDR, pulmonary permeability was elevated with higher levels of BALF proteins.
In addition, histopathological examination using H&E staining revealed a significant distortion in the normal lung architecture upon amiodarone administration, which confirms the successful modeling of amiodarone-induced pulmonary injury. This distortion was represented by thickened interalveolar septa and mononuclear cellular infiltration in septa and around the bronchi, thick congested lung arterioles, and small-sized alveoli, which are in accordance with preceding studies by Al-Shammari et al. (2016) and Ibrahim Fouad and Mousa (2021). Conversely, treatment with paricalcitol markedly maintained the normal lung architecture, supporting its protective effect on amiodarone-induced pulmonary injury.
Amiodarone is metabolized to an aryl radical that may generate other reactive oxygen species (ROS) (Nicolescu et al. 2007). Gawad et al. (2018) and Ibrahim Fouad and Mousa (2021) reported that excessive ROS production and associated oxidative stress play a role in the amiodarone-induced pulmonary toxicity. This comes in line with the present study, which reports an oxidative stress status in the lung following amiodarone administration in rats. This status is indicated by elevated pulmonary MDA levels, the lipid peroxidation end product, while repressed pulmonary TAC and GSH levels. Redox imbalance, represented by increased damage biomarkers and decreased antioxidant capacity, has been previously reported in amiodarone-induced pulmonary injury in rats (Gawad et al. 2018; Ibrahim Fouad and Mousa 2021). Conversely, treatment with paricalcitol diminished pulmonary MDA while increasing pulmonary TAC and GSH levels, restoring redox balance in the lung. Paricalcitol has been previously reported to diminish oxidative stress and augment the antioxidant enzyme activity in kidney (Wang et al. 2023) and liver (Jia et al. 2021).
The role of inflammation in drug-induced toxicity, including amiodarone-induced lung toxicity development, has been reported (Ibrahim Fouad and Mousa 2021). Toll-like receptors (TLRs) have a crucial role in mediating the inflammatory response mainly via the enhancement of macrophage stimulation and the release of pro-inflammatory cytokines, causing tissue damage (Jha et al. 2021). Among TLRs, TLR4 is widely expressed on the cell surface of different cells, and when activated, it can provoke the transcription factor, NF‐κB. Activated NF-κB mediates the production of different inflammatory cytokines such as TNF-α (Luo et al. 2020).
In the present study, amiodarone-induced pulmonary injury was associated with upregulated TLR4, NF-κB, and TNF-α expression in the lung. These results are in concordance with preceding studies that documented elevated TLR4, NF-κB (Radwan et al. 2020), and TNF-α (Madkour and Ahmed 2013) levels in the lung. Interestingly, treatment with paricalcitol markedly repressed pulmonary TLR4, NF-κB p65, and TNF-α expression. Previously, anti-inflammatory effects of paricalcitol have been reported through suppressing TLR4 (Lee and Choauthor 2015), NF-κB (Deluque et al. 2024), and TNF-α (Tan et al. 2009) levels. Moreover, Kim et al. (2017) showed that paricalcitol counteracts TLR4/NF-κB signaling and so inhibits pro-inflammatory mediators secretion, like TNF-α in hepatic ischemia/reperfusion injury.
Moreover, TLR4 is one of the TLRs that has been reported to have a profibrotic effect in the lung upon stimulation (Bolourani et al. 2021). It was documented that TLR4 boosts TGF-β1 signaling during lung fibrogenesis (Wang et al. 2022). TGF-β1, the most potent profibrotic cytokine, acts via interacting with a transmembrane receptor that phosphorylates Smad 2 and Smad 3 (El-Waseif et al. 2022). These active Smads thereafter move to the nucleus with the binding partner, Smad 4; then this complex modulates a variety of target genes, which are related to tissue fibrosis, including collagen and α-SMA (Shi and Massagué 2003).
Our data revealed that amiodarone-induced upregulated pulmonary TLR4 was accompanied by enhanced TGF-β1 expression and pSmad 3 levels in the lung. Activated TGF-β1/Smad 3 signaling and fibrotic changes were reflected in stimulated collagen deposition, as shown by Masson’s trichrome staining, and elevated hydroxyproline content, as well as upregulated α-SMA expression.
In contrast, administration of paricalcitol effectively reversed these effects by inhibiting the TGF-β1/Smad signaling pathway. Where, in the present study, treatment with paricalcitol reduced pulmonary TGF-β1, pSmad 3, hydroxyproline levels, and α-SMA expression. Additionally, pretreatment with paricalcitol attenuated fibrosis as presented by Masson’s trichrome stained tissue sections. All these fibrotic changes were effectively counteracted by paricalcitol, suggesting another underlying mechanism.
HIF-1α signaling has emerged as an important player in both lung inflammation and fibrosis. HIF-1α signaling represents a crucial linkage between inflammation and hypoxia (Balamurugan 2016). A crosstalk between HIF-1α and NF-κB exists and underlies the pathogenesis of different disorders (Tang et al. 2022). The expression of HIF-1α is raised by non-hypoxic stimuli like ROS and/or the master transcription factor, NF-κB (Korbecki et al. 2021), regulating numerous pro-inflammatory cytokines expression (Suresh et al. 2023). Moreover, TLR4 stimulation has been recognized to enhance not only NF-κB expression, but also HIF-1α activity, which has a positive feedback on the TLR4 expression level (Zhao et al. 2022).
Regarding fibrosis, HIF activation promotes collagen cross-linkage that mediates pulmonary fibrosis (Brereton et al. 2022). It was also reported that TGF-β mediates Smad3-dependent accumulation of HIF-1α via suppression of prolyl-hydroxylase domain expression, which is crucial for HIF-1α degradation (Epstein Shochet et al. 2021; Brereton et al. 2022). Previously, HIF-1α inhibition has attenuated pulmonary fibrosis in mice by decreasing TGF-β1/pSmad 3-induced collagen and α-SMA expression (Xu et al. 2022).
Interestingly, the findings of our study revealed a substantial elevation in relative expression of pulmonary HIF-1α in the amiodarone group. Since HIF-1α triggers the stimulation of TLR4/NF-κB and TGF-β/Smad signaling pathways, and the interaction of these two pathways mediates amiodarone-induced pulmonary injury, these anti-inflammatory and antifibrotic effects of paricalcitol might be due to downregulation of HIF-1α and subsequent dependent genes.
The study limitations include the lack of using pathway-specific inhibitors and apoptosis assessment. Future investigations and pulmonary function tests are warranted in comparison to standard therapy protocols.
Conclusions
The current study showed that paricalcitol protects against amiodarone-induced lung injury in rats via antioxidant, anti-inflammatory, and antifibrotic effects. These beneficial effects are mainly mediated via targeting the interplay between TLR4/NF-κB, TGF-β/Smad 3 signaling pathways, and HIF-1α (Fig. 10).Fig. 10. Schematic representation of the mechanisms underlying the protective effect of paricalcitol against amiodarone-induced pulmonary injury. α-SMA, alpha smooth muscle actin; HIF-1α, hypoxia-inducible factor-1α; LDH, lactate dehydrogenase; NF-κB, nuclear factor kappa B; ROS, reactive oxygen species; Smad, small mothers against decapentaplegic; TGF-β1, transforming growth factor-beta 1; TLC, total leucocyte count; TLR4, toll-like receptor 4; TNF-α, tumor necrosis factor alpha
Supplementary Information
Below is the link to the electronic supplementary material.ESM1(DOCX 7.74 MB)
The reference list from the paper itself. Each links out to its DOI / PubMed record.
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