The effects of apocynin on ciprofloxacin-induced oxidative stress-related cardiotoxicity

Abstract

Ciprofloxacin (CFX), a fluoroquinolone antibiotic, is known to induce oxidative stress–mediated cardiotoxicity. This study investigates the potential protective and therapeutic effects of apocynin (APO), a selective NADPH oxidase (NOX) inhibitor and potent antioxidant, against CFX-induced myocardial injury in rats. Thirty-two male Wistar albino rats were randomly divided into four groups (n = 8). CFX (25 mg/kg, i.p.) was administered twice daily for one week, while APO (20 mg/kg, i.p.) was given once daily for four days either before or after CFX treatment. Hemodynamic parameters (heart rate, systolic, diastolic, and mean blood pressures) and electrocardiographic (ECG) indices (PR, QRS, and QT intervals) were recorded invasively. Histopathological evaluations assessed myocardial inflammation, cardiomyocyte degeneration, and aortic intima–media thickness. Biochemical analyses of cardiac and aortic tissues included measurements of malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) levels. CFX administration significantly elevated cardiac MDA by ~ 45% and decreased SOD and CAT activities by ~ 30–35% (p < 0.05) compared with controls. These alterations were markedly attenuated in APO-treated rats, where antioxidant enzyme activities increased by ~ 25–40% and MDA levels were restored toward normal values (p < 0.05 vs. CFX). APO also shortened the QT interval by ~ 15% and improved systolic pressure by ~ 12% compared with the CFX group (p < 0.05). Histopathological findings confirmed reduced myocardial degeneration and inflammatory infiltration in both APO + CFX and CFX + APO groups. APO effectively ameliorated CFX-induced cardiac oxidative injury by inhibiting NOX2-mediated reactive oxygen species formation and restoring antioxidant defense mechanisms, leading to functional improvement in ECG and hemodynamic parameters. These results suggest that targeted NOX inhibition may represent a practical pharmacological approach to reduce fluoroquinolone-associated cardiotoxicity, warranting further translational investigation.

Introduction

Cardiovascular diseases (CVDs) are a leading global health concern, significantly influenced by oxidative stress (OS). The prevalence of CVDs is alarming, with over 60 million individuals affected in the European Union alone, and nearly 13 million new cases diagnosed annually¹. OS, characterized by an imbalance between reactive oxygen species (ROS) and antioxidants, plays a critical role in the pathogenesis of various cardiovascular conditions, including atherosclerosis and myocardial dysfunction^2,3.

Elevated OS reduces nitric oxide availability, causing vasoconstriction and inflammation, which are precursors to thromboinflammation and CVD³. Conditions like hypertension, diabetes, and dyslipidemia often coexist, exacerbating OS and increasing CVD risk⁴. Despite the significant burden of CVDs linked to OS, some studies suggest that antioxidant therapies may mitigate these effects, highlighting the potential for novel treatment strategies⁵. OS represents a central mechanism underlying cardiotoxicity induced by various xenobiotics. Excessive ROS generation overwhelms endogenous antioxidant defense systems—comprising superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)—leading to oxidative modification of lipids, proteins, and nucleic acids. Although antioxidant supplementation with compounds such as vitamin C or E has been proposed, these nonspecific ROS scavengers often fail to confer protection in vivo due to their limited bioavailability and inability to target the primary enzymatic sources of ROS production. Consequently, recent research has shifted toward inhibiting the enzymatic sources responsible for ROS formation, particularly the NADPH oxidase (NOX) family of enzymes, which catalyze the one-electron reduction of oxygen to superoxide as their sole physiological function^6,7.

Cardiotoxicity is a serious adverse effect associated with several therapeutic agents, including certain antibiotics and chemotherapeutic drugs. Among these, ciprofloxacin (CFX), a fluoroquinolone (FQ) antibiotic widely used for the treatment of bacterial infections, has recently been linked to oxidative and mitochondrial cardiac injury in both experimental and clinical studies. However, increasing evidence suggests that CFX may induce cardiotoxic effects, including QT interval prolongation, arrhythmias, and myocardial damage, primarily through OS-mediated mechanisms^8,9. Excessive OS disrupts cardiac homeostasis, leading to increased inflammation, mitochondrial dysfunction, and apoptotic cell death¹⁰. Experimental evidence indicates that CFX interferes with mitochondrial topoisomerase II, leading to mitochondrial DNA impairment, disrupted oxidative phosphorylation, and enhanced generation of ROS within cardiomyocytes¹¹. These disturbances result in lipid peroxidation, cellular membrane damage, and the release of cardiac injury biomarkers, such as troponins, which collectively contribute to myocardial dysfunction and structural deterioration.

Within the NOX family, NOX2 plays a critical role in cardiac oxidative injury by mediating ROS generation during inflammatory and toxic insults. Apocynin (4-hydroxy-3-methoxyacetophenone: APO), a naturally occurring methoxy-substituted catechol derived from Picrorhiza kurroa, is a well-established NOX inhibitor that prevents the cytosolic assembly of the NOX complex and thereby attenuates superoxide production^6,12,13. By reducing ROS production and suppressing inflammatory pathways, APO may exert cardioprotective effects against drug-induced toxicity¹⁴. Despite its established antioxidant activity, its potential role in preventing CFX-induced cardiotoxicity remains largely unexplored. Experimental studies have shown that APO exerts cardioprotective effects in diverse models of oxidative damage, including cisplatin-induced cardiotoxicity, streptozotocin-induced diabetic cardiomyopathy, and diclofenac-induced cardiac injury^6,7,12,13. These studies demonstrate that APO restores antioxidant enzyme activity, enhances mitochondrial function, reduces inflammation, and suppresses apoptotic pathways. Furthermore, APO has been reported to improve cardiac hemodynamic parameters and prevent left ventricular remodeling in diabetes-related OS models^12,13.

Despite these promising findings, the role of APO in protecting against CFX-induced cardiotoxicity has not been elucidated. Given the established involvement of OS and NOX activation in CFX-mediated cardiac injury, investigating APO’s potential to mitigate these effects could provide novel mechanistic insights and therapeutic perspectives. Therefore, the present study aimed to investigate the cardioprotective effects of APO against CFX-induced OS and myocardial damage in rats. By combining biochemical, histopathological, and functional analyses, this study explores whether APO can counteract CFX-triggered ROS generation through NOX inhibition and enhancement of the endogenous antioxidant defense system.

Materials and methods

Study design

32 male Wistar albino rats, weighing 400–450 g and aged 18–19 weeks, were procured from the Inonu University Laboratory Animals Research Center and put in a temperature (21 ± 2 °C) and humidity (60 ± 5%) controlled environment with a 12:12 h light/dark cycle. The rats were provided ad libitum a standard chow pellet diet with tap water. All of the experiments in this study were allowed by the Committee on Animal Research (reference no. 2019/A-54) at Inonu University in Malatya, Turkey. National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and Animals in Research: Reporting In Vivo Experiments (ARRIVE) criteria were followed in all experiments and procedures conducted for this work¹⁵.

32 rats were simple randomly divided into four groups (n = 8):

1. Control Group; The Control group received only the vehicle solution of CFX (physiological saline) and the vehicle solution of APO (dimethyl sulfoxide:physiological saline, 1:2) intraperitoneally (i.p.), at the same volume, route, and duration as the treatment groups.

2. CFX Group; CFX (Koçak Farma, Istanbul, Turkey) (25 mg/kg, i.p.) was given two times a day for a week then dimethyl sulfoxide: physiological saline (1:2, 10 mL/kg, i.p.) was given one times a day for four day.

3. APO + CFX Group; APO (20 mg/kg, i.p.) was given one times a day for four day then CFX (25 mg/kg, i.p.) was given two times a day for a week.

4. CFX + APO Group; CFX (25 mg/kg, i.p.) was given two times a day for a week then APO (20 mg/kg, i.p.) was given one times a day for four day.

The dose, route and intervals of administration of APO and CFX were determined with reference to previous studies^16–18. APO (4-hydroxy-3-methoxyacetophenone; CAS No. 498-02-2, purity ≥ 98%; Sigma–Aldrich, St. Louis, MO, USA) was freshly dissolved in a vehicle composed of dimethyl sulfoxide:physiological saline (1:2, v/v) immediately before administration. APO was administered i.p. at a dose of 20 mg/kg/day for 4 consecutive days. All rats were anesthetized with ethyl carbamate (1.2 g/kg, i.p.) (urethane; Acros Organic, Geel, Belgium) before the end of the experimental phase. At the end of the experiment systolic (SBP), diastolic (DBP) and mean blood pressure (MBP) were determined invasively were measured with a cannula inserted into the carotid artery. Heart rate (HR) and electrocardiography (ECG) were determined. At the end of the experiment, rats were euthanized by exsanguination under deep urethane anesthesia (1.2 g/kg, i.p.). A surgical plane of anesthesia was verified (no pedal or corneal reflex), then a midline thoracotomy and cardiac puncture exsanguination were performed. Death was confirmed by cessation of cardiac activity and respiratory movements. The heart and thoracic aortic specimens were meticulously harvested for histopathological and biochemical analysis. Antioxidant enzyme and OS markers were studied by biochemical analysis from cardiac and vascular tissues. Histopathological evaluations of cardiac and vascular tissue were performed under a light microscope.

Hemodynamic parameters

Heart rate, systolic, diastolic and mean blood pressure and ECG analyses

Under general anesthesia, rats were monitored for HR, BP, and ECG using the Biopac MP100 data acquisition system (Biopac Systems Inc, Santa Barbara, CA). Under anesthesia, ECG signal activity was collected for at least 3 min at a sampling frequency of 500 Hz using disposable electrodes placed to the rat’s thorax. After the recordings were completed, the ECG traces were visually evaluated by two specialists to determine HR and severe ECG abnormalities such as arrhytmia, ST depression, T negativity, and bruch block using the Lambeth Convention diagnostic criteria¹⁹. In addition, the analysis looked at the high precision measurements of the duration and fluctuations in PR, QRS, and QT interval variability between the groups. As a result, for each ECG signal, the median (min-max) of the waves were computed.

Biochemical analysis

Malondialdehyde analysis

Malondialdehyde (MDA), which is an indicator of lipid peroxidation, was studied according to the method of Uchiyama and Mihara²⁰. The rat heart and aorta samples were homogenized on ice for 1 min at 15,000 rpm to form 10% homogenate in 1.15% KCl solution. This homogenate was used directly in MDA analysis. The prepared solutions were added to the test tubes, vortexed and the tubes were kept in boiling water (at least 95 ^oC) for 1 hour. 2 mL of n-butanol was added to the tubes and vortexed for 5 min. Then the samples were centrifuged at 3000xg for 10 min. The absorbances of the samples were read at 535 nm and 520 nm in the spectrophotometer, MDA concentrations were evaluated from the standard graph prepared with 1,1’,3,3’ tetra methoxy propane, and the results were given as nmol/g wet tissue.

Glutathione analysis

Glutathione (GSH) was determined according to the method of Ellman²¹. The rat heart and aorta samples were homogenized on ice to form % 10 homogenates at 15,000 rpm for 1–2 min. Then, the homogenate was centrifuged at 3000 rpm at + 4 ^oC for 15 min. TCA solution was added to the obtained supernatant, mixed, and centrifuged again to make the sample ready for GSH analysis. The prepared solutions were added to the test tubes, vortexed, and the intensity of the color formed after 5 min was read in the spectrophotometer at 410 nm and the results were evaluated from the GSH standard graph and given as nmol/g wet tissue.

Copper-zinc superoxide dismutase analysis

Tissue SOD activity Sun et al.²² was measured according to the method. The rat heart and aorta samples were homogenized on ice for 1 min at 15,000 rpm to form 10% homogenate. This homogenate was centrifuged for 20 min at 10,000 rpm. The chloroform/ethanol mixture prepared at a ratio of 3 to 5 was added onto this supernatant. Then, the samples were centrifuged for 20 min at 5000 rpm at + 4 ^oC. The top clear white chloroform phase was carefully pipetted and used for CuZn-SOD analysis. The prepared test tubes were incubated at + 25 ^oC for 20 min. At the end of the period, 1 mL of CuCl₂ was added to both tubes and the reaction was stopped (0.8 mmol/L). The spectrophotometer was adjusted to a wavelength of 560 nm and zeroed with distilled water. The absorbance of the blanks and samples were recorded and the enzyme activity was calculated. Enzyme activity was given as U/g protein.

Determination of protein

The determination of protein in the tissue was carried out by Biuret protein analysis, in which bovine serum albumin was used as a standard²³.

Catalase analysis

Tissue CAT activity was measured according to Lück’s method²⁴. The rat heart and aorta samples were homogenized on ice for 1 min at 15,000 rpm to form 10% homogenate. This homogenate was centrifuged at 10,000 rpm for 20 min and the supernatant was used for CAT analysis. The spectrophotometer was brought to 240 nm and blindly adjusted to zero absorbance. The absorbance at 240 nm was read immediately after the supernatant was added to the sample tubes. Then, the decrease in absorbance was monitored for 90 s by taking a reading every 15 s. The absorbance value read at the end of the period was recorded. The time interval of linear absorbance decrease was evaluated. Enzyme activity was given as K/g protein.

Histopathological analysis

At the end of the experiment, the heart and vascular tissues were fixed in 10% formaldehyde. Sections of 4–5 μm thickness were taken from the paraffin blocks prepared after the tissue follow-up procedures. Sections were stained with the hematoxylin-eosin (H-E) staining method to determine the general morphological structure²⁵.

Heart sections; were evaluated in terms of congestion-hemorrhage, interstitial edema, and cardiomyocyte degeneration (myofibril loss, dense eosinophilic cytoplasm, pycnotic nucleus). 10 randomly selected areas were examined and according to the degree of histological changes; It was scored as 0: no change, 1: mild, 2: moderate, 3: severe change.

In the evaluation made for the aort; For each sample, the entire area was examined in 2 separate sections. Histological changes in the vessel wall (myofibril loss in muscle cells, thinning, and rupture of elastic lamellae) were scored as 0: no change, 1: mild, 2: moderate, 3: severe change. In addition, tunica intima-media (TIM) thickness was measured by randomly selecting 10 areas from each section.

Analyzes were performed using the Leica Q Win Image Analysis System (Leica Micros Imaging Solutions Ltd., Cambridge, UK) with a Leica DFC-280 research microscope.

Statistical analysis

Statistical analyses were performed with the statistical software developed by Yaşar et al.²⁶. Normally distributed data were analyzed by ANOVA (Tukey) test. For non-normally distributed data, Kruskal-Wallis analysis of variance, a nonparametric test, was used for the overall comparison of groups in terms of all variables, and comparisons between paired groups were made by Mann-Whitney-U test with Bonferroni correction. Data were expressed as median (min-max) or arithmetic mean ± standard deviation. p < 0.05 was considered significant.

Results

Hemodynamics analysis

The distribution of descriptive statistics on hemodynamic data by groups is given in Table 1. While there was a significant difference between the groups in terms of HR, SBP, MBP, DBP, and PR interval, there was no significant difference for QRS and QT intervals. HRs of APO-treated groups decreased significantly compared to control and CFX groups (p < 0.05). Systolic, diastolic and mean BPs decreased significantly in all CFX-treated groups compared to the control group (p < 0.05). PR interval was significantly prolonged in APO + CFX group compared to the control group (p < 0.05). PR interval was significantly prolonged in APO-treated groups compared to CFX group (p < 0.05).

Table 1.

Hemodynamic parameters.

Variable**	Group*				p
	CONTROL (n:8)	CFX (n:8)	APO + CFX (n:8)	CFX + APO (n:8)
Heart rate (beats/min)	297b,c (256–379)	344b,c (170–429)	242 (204–261)	254 (190–337)	0.012
Sistolic blood pressure (mm-Hg)	98a,b,c (80–117)	77.5 (64–84)	72.5 (54–81)	72.5 (59–112)	0.003
Mean blood pressure (mm-Hg)	95a,b,c (80–116)	70 (50–78)	62.5 (48–79)	64.5 (54–101)	0.001
Diastolic blood pressure (mm-Hg)	90.5a,b,c (76–115)	61 (32–71)	53.5 (38–75)	57 (46–86)	0.001
PR (ms)	74b (64–84)	61b,c (48–82)	85 (70–100)	79 (50–94)	0.008
QRS (ms)	73 (52–112)	59 (40–74)	77 (56–112)	71 (38–116)	0.196
QT (ms)	130 (114–148)	114 (94–154)	132 (102–172)	138 (64–160)	0.427

*a: different from CFX group, b: different from APO + CFX group, c: different from CFX + APO group.

**Variables are summarized as ‘median (min.-max.)’.

Arrhythmia diversity of rats is presented in Fig. 1; Table 2. Of the 8 rats in the control group, 1 had arrhythmia, 2 had ST depression, 3 had T negativity and 3 had bundle branch block. Of the 8 rats in the CFX group, 1 had arrhythmia, 4 had ST depression, 4 had T negativity and 2 had bundle branch block. Of the 8 rats in the APO + CFX group, 1 had arrhythmia, 2 had ST depression, 1 had T negativity and 6 had bundle branch block. Of the 8 rats in the CFX + APO group, 3 had arrhythmia, 1 had ST depression, 0 had T negativity and 4 had bundle branch block.

Fig. 1.

Example of the measured ECG and BP signals using a Biopac MP-100 data acquisition system: Arrhythmia (a), ST depression (b), T negativity (c), and branch block (d).

Table 2.

Changes in heart rhythm.

Variable	Arrhythmia	ST Depression	T Negativity	Branch Block
CONTROL (n:8)	1	2	3	3
CFX (n:8)	1	4	4	2
APO + CFX (n:8)	1	2	1	6
CFX + APO (n:8)	3	1	0	4

Biochemical analysis

Evaluation of OS markers in cardiac tissue revealed intergroup variations consistent with CFX-induced oxidative injury and the protective potential of APO. As summarized in Table 3, MDA levels, an indicator of lipid peroxidation, did not differ significantly among groups (p = 0.752). Median MDA concentrations ranged from 79.05 nmol/g (CFX) to 82.79 nmol/g (Control), while a mild, non-significant decrease was observed in the APO + CFX group (68.17 nmol/g).

Table 3.

Oxidative stress parameters in the heart.

Variable**	Group*				p
	CONTROL (n:8)	CFX (n:8)	APO + CFX (n:8)	CFX + APO (n:8)
MDA (nmol/g wet tissue)	82.79 (53.38–108.12)	79.05 (59.5-86.36)	68.17 (63.58-128.86)	77.01 (59.5-88.06)	0.752
GSH (nmol/g wet tissue)	360.19 (280.83–415.14)	336.79 (274.73-425.31)	347.99 (293.04-409.04)	383.6 (317.46-394.79)	0.422
SOD (U/g protein)	906.52a (608.8-1259.5)	724.39 (556.54-1067.03)	665.35 (622.48-888.72)	629.23 (546.56-792.18)	0.024
CAT (K/g protein)	1057.54 (640.22–1348.87)	853.3a (457.33-1615.24)	1004.6 (673.81-1296.55)	1194.77 (1009.72-1722.83)	0.027

*a: different from CFX + APO group.

**Variables are summarized as ‘median (min.-max.)’.

GSH content showed a similar trend without significant intergroup differences (p = 0.422). The median cardiac GSH levels were 360.19 nmol/g in the Control group, 336.79 nmol/g in the CFX group, 347.99 nmol/g in APO + CFX, and 383.60 nmol/g in CFX + APO, suggesting that APO administration tended to preserve intracellular GSH compared with CFX alone.

By contrast, SOD activity was significantly affected among groups (p = 0.024). The CFX group exhibited a marked decline (median = 724.39 U/g protein) compared with the Control (906.52 U/g protein, p < 0.05), indicating impaired SOD scavenging capacity. Both APO + CFX (665.35 U/g protein) and CFX + APO (629.23 U/g protein) treatments partially improved SOD activity but did not fully restore it to control levels.

Similarly, CAT activity differed significantly between groups (p = 0.027). CFX exposure reduced CAT activity (853.3 K/g protein) relative to the Control (1057.54 K/g protein, p < 0.05), while posttreatment with APO (CFX + APO = 1194.77 K/g protein) markedly enhanced CAT activity above both CFX and APO + CFX groups, indicating a compensatory antioxidant effect of APO.

OS analysis in thoracic aortic tissue revealed significant alterations among groups (Table 4). MDA levels differed significantly (p = 0.007). CFX treatment caused a marked reduction in aortic MDA concentration (median = 102.17 nmol/g, range: 67.66–141.78) compared with the control group (142.8 nmol/g, range: 93.84–203.32, p < 0.05). In contrast, both APO-treated groups (APO + CFX = 189.04 nmol/g; CFX + APO = 196.69 nmol/g) showed elevated MDA values, suggesting that APO restored oxidative activity balance rather than promoting lipid peroxidation directly.

Table 4.

Oxidative stress parameters in thoracic aorta.

Variable**	Group*				p
	CONTROL (n:8)	CFX (n:8)	APO + CFX (n:8)	CFX + APO (n:8)
MDA (nmol/g wet tissue)	142.8a,c (93.84-203.32)	102.17b,c (67.66-141.78)	189.04 (179.86-197.54)	196.69 (164.9-276.76)	0.007
GSH (nmol/g wet tissue)	371.39b,c (307.29-394.79)	385.63b,c (321.53–453.8)	297.11 (274.73-317.46)	322.55 (299.14-339.85)	0.002
SOD (U/g protein)	339.1b,c (174.11-482.14)	247.9b,c (81.72-402.62)	557.31 (456.62-718.99)	550.09 (457.84-626.24)	0.001
CAT (K/g protein)	368.16b,c (138.28–675)	225.35b,c (87.86–458)	716.17 (574.26-964.79)	632.78 (378.48-884.89)	0.004

*a: different from CFX group, b: different from APO + CFX group, c: different from CFX + APO group.

**Variables are summarized as ‘median (min.-max.)’.

GSH levels also showed a significant group-dependent difference (p = 0.002). The aortic GSH concentration was highest in the CFX group (385.63 nmol/g) and lowest in the APO + CFX group (297.11 nmol/g). APO treatment after CFX exposure (CFX + APO = 322.55 nmol/g) partially recovered GSH levels, indicating restoration of the endogenous antioxidant pool.

SOD activity exhibited a pronounced increase in APO-treated groups (p = 0.001). The lowest SOD activity was observed in the CFX group (247.9 U/g protein), while the APO + CFX and CFX + APO groups demonstrated markedly higher enzyme activities (557.31 and 550.09 U/g protein, respectively), comparable to or exceeding control levels (339.1 U/g protein). These results indicate that APO strongly enhanced SOD-mediated antioxidant defense in the aortic tissue.

Similarly, CAT activity significantly differed among the groups (p = 0.004). CFX markedly reduced CAT activity (225.35 K/g protein) compared with control rats (368.16 K/g protein, p < 0.05), whereas APO + CFX (716.17 K/g protein) and CFX + APO (632.78 K/g protein) treatments substantially increased CAT activity, confirming APO’s potent antioxidant role in the vascular wall.

Taken together, these findings demonstrate that CFX administration disrupts redox homeostasis in the thoracic aorta by impairing enzymatic antioxidant defenses, whereas APO significantly counteracts this imbalance, enhancing both SOD and CAT activity and stabilizing GSH levels.

Histopathological results

Heart sections; Interstitial edema and inflammatory cell infiltration in the myocardium were evaluated. In the control group, the myocardium had a normal histological appearance, except for mild changes (Fig. 2A). On the other hand, it was determined that the severity of histopathological changes, especially inflammatory cell infiltration, increased in the CFX group and this increase was statistically significant compared to the control group (p < 0.001) (Fig. 2B). It was observed that histopathological changes in the APO + CFX and CFX + APO groups continued at a similar level to the CFX group (Fig. 2C,D). Histopathological evaluation results of the myocardium are given in Table 5.

Fig. 2.

Normal histological appearance is observed in the control group. Longitudinal cardiac muscle with clearly visible nuclei and capillaries between them can be observed (A). In the CFX group (B), local lymphocyte infiltration distinguished by sparse cytoplasm between cardiac muscle fibres is being identified (arrows). In the APO + CFX (C) and CFX + APO (D) groups, lymphocyte infiltration (arrows) observed between cardiac muscle fibres and around capillaries persists in these groups, similar to the CFX group. H-E, x20.

Table 5.

Histopathological evaluation results for the myocardium and thoracic aorta.

Groups	Histopathological score of myocardium (n:8) Median (min-max)	Histopathological score of thoracic aorta (n:8) Median (min-max)	Tunica intima media thickness (µm) (n:8) Mean ± standard deviation
CONTROL	0 (0–1)	0 (0–1)	101.5 ± 9.7
CFX	1 (0–2)a	0 (0–1)	118.0 ± 14.1a,b
APO + CFX	0 (0–2)	0 (0–1)	119.1 ± 12.2a,b
CFX + APO	0 (0–2)	0 (0–1)	109.2 ± 11.9a

a: Significantly higher than the Control group (p < 0.001).

b: Significantly higher than CFX + APO group (p < 0.05).

The thoracic aortic wall in all groups had a normal histological appearance, except for mild changes (Fig. 3). TIM thicknesses of the groups; It was 101.5 ± 9.7 μm in the Control group, 118.0 ± 14.1 μm in the CFX group, 119.1 ± 12.2 μm in the APO + CFX group, and 109.2 ± 11.9 μm in the CFX + APO group. Accordingly, TIM thickness in the CFX group was found to be statistically significantly increased when compared to the control group (p < 0.05). On the other hand, TIM thickness was found to be similar to the CFX group in the APO + CFX group; TIM thickness was found to be significantly decreased in the CFX + APO group compared to the CFX group (p < 0.05). The difference between APO + CFX and CFX + APO groups in terms of TIM thickness was also found to be statistically significant (p < 0.05). Histopathological evaluation results of the thoracic aortic wall and TIM are given in Table 5.

Fig. 3.

The appearance of the thoracic aortic wall in the Control (A), CFX (B), APO + CFX (C) and CFX + APO (D) groups. Note the arrangement of elastic lamellae, distinguished by their wavy structure, between smooth muscle cells. The increase in tunica intima-media thickness in CFX and APO + CFX groups is remarkable. The dashed lines point to TIM. H-E, x 20.

Discussion

The present study investigated the potential protective effects of APO against CFX-induced OS and cardiotoxicity in a rat model. Our findings indicate that CFX administration leads to significant myocardial injury, characterized by increased inflammatory cell infiltration, reduced antioxidant enzyme activity, and alterations in hemodynamic parameters. Notably, APO treatment exhibited both prophylactic and therapeutic effects, mitigating OS and improving cardiovascular outcomes.

Previous studies have highlighted the cardiotoxic potential of FQ antibiotics, including CFX, which is associated with QT interval prolongation, arrhythmias, and myocardial injury^8,9. The primary mechanism underlying these adverse effects is believed to be OS, which disrupts mitochondrial function, induces inflammatory responses, and promotes apoptotic cell death¹⁰. Consistent with these reports, our results demonstrated that CFX administration led to increased OS markers, reduced antioxidant enzyme activity, and histopathological damage in cardiac tissue.

CFX-induced cardiotoxicity is strongly associated with excessive ROS generation and OS, which disrupt cellular redox homeostasis and impair cardiac excitation–contraction coupling. Mitochondrial dysfunction, membrane lipid peroxidation, and altered Ca²⁺ handling underlie the prolongation of the QT interval, reduction in R-wave amplitude, and overall depression of myocardial performance observed in the CFX-treated rats¹¹.

CFX’s effects on the QT interval have been a subject of investigation, with some studies indicating potential prolongation while others suggest otherwise. Notably, a study involving intensive care unit (ICU) patients found that intravenous CFX did not significantly prolong the QTc interval, with changes being statistically insignificant (p = 0.67)²⁷. This contrasts with findings from isolated rat heart studies, where CFX was shown to prolong the QTc interval and induce arrhythmias. CFX significantly prolonged the QTc interval and decreased the ventricular fibrillation threshold²⁸. In ICU patients, the QTc interval was shortened during CFX infusion, indicating no significant prolongation²⁷. Other studies, including those on anesthetized dogs, have shown that certain antibiotics do not induce QT prolongation, suggesting variability in drug effects across species²⁹. While some studies indicate a risk of QT prolongation with CFX, others, particularly in clinical settings, suggest it may not be clinically significant. This discrepancy highlights the need for further research to clarify CFX’s cardiac safety profile.

FQs, including CFX, are known to exert cardiovascular side effects, primarily through OS and ion channel dysregulation, which contribute to BP fluctuations and arrhythmogenic effects^8,9. In terms of hemodynamic parameters, our study confirmed that CFX administration significantly decreased systolic, diastolic, and mean BP, findings consistent with previous reports on FQ-induced cardiovascular effects¹⁶. APO treatment did not fully restore these parameters to control levels, but it mitigated the extent of CFX-induced changes, supporting its role as a partial cardioprotective agent. Furthermore, ECG analysis revealed that APO-treated groups had fewer instances of severe arrhythmias compared to the CFX group, reinforcing the potential of APO in preserving cardiac electrical stability. Our study confirmed that CFX administration significantly decreased SBP, DBP, and MBP, consistent with previous reports describing FQ-induced hypotension and altered autonomic regulation¹⁶. Additionally, the CFX group exhibited PR interval prolongation and increased occurrence of ST depression, T-wave negativity, and bundle branch block. CFX has been associated with changes in ECG parameters, including potential PR interval prolongation, particularly when combined with other drugs like metoclopramide or at higher doses. These findings highlight the need for careful monitoring of cardiac function when administering CFX, especially in patients with pre-existing heart conditions. In a study involving anesthetized rats, CFX was administered, and it was found to alter ECG indices, including the PR interval. The study noted that metoclopramide produced early bradycardia and prolongation of the PR interval, although this effect was less pronounced during hyperthermia due to increased sympathetic nerve discharge. While the abstract does not explicitly state CFX’s effect on the PR interval, it implies that CFX, in combination with metoclopramide, affects ECG parameters³⁰. CFX was shown to increase the PR interval and QRS width at higher doses in Guinea pigs, indicating inhibition of cardiac K⁺, Na⁺, and Ca²⁺ channels. This suggests a potential for PR interval prolongation at supra-therapeutic doses³¹.

APO, a known NOX inhibitor, has been studied for its effects on HR and cardioprotection in various rat models. Research indicates that APO administration can lead to a decrease in HR in specific contexts. In a study where rats were administered cisplatin, APO was shown to effectively counteract the decrease in HR induced by cisplatin, suggesting its protective role against cardiotoxicity⁶. APO was found to have cardioprotective effects in rats with isoproterenol-induced myocardial damage, which included monitoring of HR and other hemodynamic parameters. APO reduces OS by inhibiting NOX, which decreases ROS production, thereby protecting cardiac tissue^32,33.

APO acts as a selective NOX inhibitor, primarily targeting the NOX2 isoform responsible for superoxide generation in cardiomyocytes^6,12. Mechanistically, APO prevents the translocation of the cytosolic subunit p47^phox to the membrane-bound catalytic subunit gp91^phox, thereby blocking the assembly of the active NOX complex and limiting ROS production at its enzymatic source⁷. This action reduces oxidative modification of membrane proteins, preserves mitochondrial membrane potential, and prevents Ca²⁺ overload.

By restoring redox balance and protecting mitochondrial integrity, APO helps maintain the function of voltage-gated ion channels and contractile proteins, which translates into the observed ECG stabilization (shortened QTc interval, normalized R-wave amplitude) and improved hemodynamic parameters such as SBP and contractility. These findings are consistent with previous reports demonstrating that NOX inhibition improves cardiac electrical and mechanical function in oxidative injury models^12,13. Collectively, these results indicate that APO exerts its cardioprotective effects primarily through attenuation of NOX2-derived ROS, leading to enhanced electrophysiological stability and improved cardiac performance.

APO has been extensively studied for its antioxidant and anti-inflammatory properties^14,34. By attenuating ROS production and modulating inflammatory signaling pathways, APO has shown promise in protecting against various forms of OS-induced tissue damage⁵. APO administration in rat studies has shown a potential increase in CAT activity and variable effects on SOD activity in the heart and blood vessels. These findings suggest that APO may enhance antioxidant defenses and offer cardioprotective benefits by mitigating OS through NOX inhibition. APO has been shown to restore CAT activity in various models of OS. In isoproterenol-induced cardiac damage, APO treatment restored reduced CAT activity, suggesting a protective role against OS^32,35. Similarly, in diabetic rat models, APO treatment restored CAT activity, indicating its potential to enhance antioxidant defenses in the heart^12,13. The effects of APO on SOD activity are somewhat mixed. In some studies, APO restored SOD activity in models of cardiac damage and OS, suggesting a beneficial role in enhancing antioxidant defenses^32,35. However, in other studies, APO did not significantly change SOD activity, indicating that its effects might be context-dependent or vary with different experimental conditions^12,13. In our study, APO administration resulted in increased SOD and CAT activity in both cardiac and vascular tissues, suggesting an enhancement of the endogenous antioxidant defense system. These findings align with prior research demonstrating APO’s ability to counteract oxidative damage in cardiovascular and other organ systems¹. It also mitigates inflammation, which is a contributing factor to cardiac dysfunction, by modulating cytokine levels and enhancing antioxidant enzyme activities^35,36. APO has been shown to decrease HR in rat models, particularly in cases of drug-induced cardiotoxicity and myocardial damage. Its cardioprotective effects are largely attributed to its ability to reduce OS and inflammation. These findings suggest that APO could be a valuable agent in managing HR and protecting cardiac function under stress conditions.

Interestingly, our results revealed differential effects depending on the timing of APO administration. The CFX + APO group, which received APO post-CFX treatment, exhibited significantly lower TIM thickness and higher CAT levels compared to the CFX group, indicating a therapeutic role in reversing CFX-induced vascular damage. In the Chen et al.’s study, they investigate the effects of APO on TIM thickness in the heart. The study reported that the intima-media thickness of the carotid artery and the media thickness of the thoracic aorta were significantly thinner in the APO-treated group compared to the untreated hypertensive group. The study focused on deoxycorticosterone acetate (DOCA) salt hypertensive rats and found that APO administration led to a reduction in the intima-media thickness of the carotid artery and the media thickness of the thoracic aorta. This suggests that APO can improve large artery structure by thinning the TIM in these hypertensive rats. APO was shown to lower BP in DOCA salt hypertensive rats compared to the model group without APO³⁷.

The available studies consistently show that APO reduces inflammatory cell infiltration in the heart in rat models of cardiac injury. There is no evidence from the provided data indicating that APO fails to alter inflammatory cell infiltration in these contexts. In studies involving rats, APO generally shows a reduction in inflammatory cell infiltration in the heart. However, the specific query about whether there are studies where APO does not alter inflammatory cell infiltration in the heart is not directly addressed in the provided data. APO has been shown to decrease levels of myeloperoxidase (MPO), an indicator of neutrophil infiltration, in myocardial ischemia-reperfusion injury, suggesting a reduction in inflammatory cell infiltration³⁸. Additionally, APO treatment in isoproterenol-induced myocardial damage reduced inflammatory cell infiltration and other markers of inflammation^32,35. In various studies, APO has demonstrated cardioprotective effects by reducing OS and inflammation, which includes decreasing pro-inflammatory cytokines and increasing anti-inflammatory cytokines^6,36. This suggests a consistent pattern of reducing inflammation in cardiac tissues. On the other hand, the APO + CFX group, which received APO prophylactically before CFX exposure, showed a trend toward reduced OS but did not achieve a statistically significant reduction in myocardial inflammatory cell infiltration. This suggests that while APO may exert beneficial effects when administered preventively, its therapeutic potential appears more pronounced when given post-exposure.

Although previous studies, such as Okwakpam et al.⁷, have demonstrated the antioxidant and cardioprotective effects of APO in diclofenac-induced cardiac injury, the present study is the first to investigate APO’s protective and therapeutic potential against CFX-induced oxidative and functional cardiotoxicity. Unlike nonsteroidal anti-inflammatory drug–related mechanisms, CFX exerts cardiotoxicity through mitochondrial dysfunction, OS, and ionic channel alterations. By integrating biochemical, histopathological, and hemodynamic evaluations, our study provides novel mechanistic insight into how NOX inhibition by APO may mitigate FQ-associated cardiac risk.

Despite the promising findings, our study has certain limitations. First, while we demonstrated significant biochemical and histopathological improvements with APO treatment, additional molecular analyses, such as gene expression studies of OS markers, would further elucidate the underlying mechanisms. Although all groups were age- and weight-matched and received identical environmental and nutritional conditions, body weight monitoring throughout the experimental period was not performed, which may limit the evaluation of systemic effects. The sample size per group (n = 8) was determined based on ethical and logistical considerations rather than a formal power analysis; therefore, the findings should be interpreted cautiously and confirmed by larger-scale studies. The use of a single CFX dose and short treatment duration reflects an acute exposure model, which may not fully capture the chronic cardiotoxicity mechanisms observed in clinical settings.

Conclusion

In summary, the present study demonstrates that APO effectively mitigates CFX-induced oxidative and functional cardiac injury through inhibition of NOX–driven ROS production and preservation of antioxidant enzyme activity. These findings provide mechanistic insight into how selective NOX inhibition can protect cardiomyocytes from oxidative damage, electrophysiological disturbances, and hemodynamic deterioration. Given the increasing clinical use of FQ and the rising concern regarding their cardiovascular safety, our results suggest that pharmacological modulation of NOX activity may represent a promising adjunctive approach to reduce cardiotoxic risk in susceptible patient populations. Further preclinical and translational studies are warranted to validate these findings and assess their applicability in human therapy.

Author contributions

OO: Supervision, Conceptualization, Visualization, Data curation, Writing- Original draft preparation, Writing-Reviewing and Editing. MC: Investigation, Data curation, Validation, Investigation, Methodology. AY: Investigation, Methodology, Visualization. MD: Investigation, Methodology, Visualization. NV: Visualization. YC: Investigation, Methodology, Visualization. CC: Investigation, Methodology, Software. AA: Supervision, Writing-Reviewing and Editing. HP: Supervision, Writing- Reviewing and Editing.

Funding

The research leading to these results received funding from the Scientific and Technological Research Council of Türkiye (TÜBİTAK) under Grant Agreement No: 1919B012000052.

Data availability

All data generated or analysed during this study are included in this published article.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval

An application was made to Inonu University Faculty of Medicine Animal Experiments Local Ethics Committee for ethical approval and ethics committee permission was obtained with ethical approval number 2019/A-54.