The protective effects of topiramate and spirulina against doxorubicin-induced cardiotoxicity in rats

Abstract

Doxorubicin (DOX) is a widely used chemotherapy drug that can cause significant cardiotoxicity, limiting its clinical application. This study aimed to investigate the potential protective effects of topiramate (TPM) and spirulina (SP), either alone or in combination, in preventing DOX-induced cardiotoxicity. Adult Sprague Dawley rats were divided into five groups, including a normal control group and groups receiving DOX alone, DOX with TPM, DOX with SP, or DOX with a combination of TPM and SP. Cardiotoxicity was induced by administering DOX intraperitoneally at a cumulative dose of 16 mg/kg over 4 weeks. TPM and/or SP administration started 1 week before DOX treatment and continued for 35 days. Body weight, serum markers of cardiac damage, oxidative stress and inflammatory parameters were assessed. Histopathological and immunohistochemical examinations were performed on cardiac tissues. Results showed that TPM and SP monotherapy led to significant improvements in serum levels of cardiac markers, decreased oxidative stress, reduced fibrosis-related growth factor levels, increased antioxidant levels, and improved histopathological features. SP demonstrated more prominent effects in comparison to TPM, and the combination of TPM and SP exhibited even more pronounced effects. In conclusion, TPM and SP, either alone or in combination, hold promise as therapeutic interventions for mitigating DOX-induced cardiotoxicity.

Keywords

Doxorubicin cardiotoxicity topiramate spirulina

Introduction

Doxorubicin (DOX) is a potent chemotherapeutic agent widely used for the treatment of solid tumors and hematological malignancies.^1,2 However, its clinical efficacy is hampered by a high incidence of dose-dependent cardiotoxicity.³ The reported incidence of cardiotoxicity ranges from 5% at a cumulative dose of 400 mg/m² to 48% at 700 mg/m², with even low cumulative doses of anthracyclines posing a significant risk.⁴ DOX-induced cardiotoxicity can manifest as either acute or chronic heart injury. Acute cardiotoxicity occurs following high doses of DOX and is clinically manageable.⁵ However, chronic DOX-induced cardiotoxicity is irreversible, more severe, and can progress to cardiomyopathy.⁶ Both acute and chronic cardiotoxicity can lead to severe heart failure and ultimately death.⁷

Although the exact molecular mechanisms of DOX cardiotoxicity are not fully understood, several mechanisms have been proposed, including increased reactive oxygen species (ROS) and lipid peroxidation, calcium overload, mitochondrial dysfunction, and cardiomyocyte apoptosis.^8,9 Moreover, emerging evidence suggests that ROS overproduction and pro-inflammatory cytokines play a fundamental role in the pathogenesis of DOX cardiotoxicity, contributing to the disruption of normal cardiomyocyte structure and function.^10,11 Currently, the only FDA-approved cardioprotective agent against DOX-induced cardiotoxicity is dexrazoxane.¹² However, dexrazoxane has been associated with various side effects, including the development of secondary malignancies and myelosuppression.^2,13 Therefore, it is warranted to develop effective and safe alternative therapies to prevent DOX-induced cardiotoxicity.¹⁴

Topiramate (TPM) is an antiepileptic medication also used for the treatment of obesity, Lennox Gastaut syndrome, and migraine headaches.^15,16 It exerts its effects by inhibiting voltage-gated calcium and sodium channels, activating GABA, and blocking AMPA/kainate receptors.¹⁷ Furthermore, several studies have suggested that TPM possesses antioxidant, anti-inflammatory, and antiapoptotic properties, making it a potential protective agent against diseases characterized by inflammation and oxidative stress.^18,19

Spirulina (SP), a spiral filamentous blue-green alga,²⁰ is rich in essential amino acids, vitamins, and phytochemicals such as carotenoids, polyphenols, and phycocyanin.²¹ Due to its richness in many bioactive elements, SP exhibits antioxidant, anti-inflammatory, and antiapoptotic activities.^22,23

In this study, we aimed to investigate the potential anti-inflammatory, antiapoptotic, and antioxidant effects of TPM, either alone or in combination with SP, on DOX-induced cardiotoxicity in rats.

Materials and methods

Drugs and chemicals

DOX (Adricin) was purchased from (Hikma Pharmaceuticals, Egypt), TPM (Topamax) was purchased from (Johnson & Johnson, USA), and SP (spirulina 99% green powder) was purchased from (of LANXESS-Germany). Phosphate buffer saline (PBS), potassium phosphate buffer and formalin 10% buffered solution were obtained from (Al-Gomhoria pharmaceutical company, Egypt). Spirulina was dissolved in distilled water to achieve a final concentration of 200 mg/ml. Similarly, topiramate was dissolved in distilled water to attain a final concentration of 28 mg/ml. Consequently, the daily administration volume was adjusted to 0.5–1 ml/dose based on the weight of the animals. All chemicals used in the study were of analytical grade.

Experimental animals

The handling of animals and all experimental procedures were approved by the institutional Research Ethics Committee (REC), Faculty of Medicine, Tanta University, Egypt (Approval no. #34431/01/21). The study included 50 male Sprague Dawley rats, weighing 200–250 g, obtained from a local source. The rats were housed in wire mesh cages in animal laboratory rooms under strict hygienic conditions. They were provided with free access to standard animal diet and water ad libitum throughout the experiment. A period of 2 weeks was allowed for acclimatization before the start of the experiment.

Experimental design and treatment protocol

A schematic presentation of the experimental design is depicted in Figure 1. Rats were divided into five equal groups for the experiment (10 rats in each group). Group 1 (control group) received a weekly intraperitoneal injection of 0.9% sodium chloride solution for 4 weeks. Group 2 (untreated cardiotoxicity) received intraperitoneal injections of DOX at a dose of 4 mg/kg/week for 4 weeks, reaching a cumulative dose of 16 mg/kg. Group 3 (TPM group) received TPM orally at a dose of 35 mg/kg/day. Group 4 (SP group) received SP orally at a dose of 500 mg/kg/day. Group 5 (combination group) received TPM and SP concurrently, following the same dosage regimen as mentioned above. Cardiotoxicity was induced in groups 3, 4, and 5 using the same method as in group 2. The treatment protocol for groups 3, 4, and 5 started 1 week before cardiotoxicity induction and continued for 28 consecutive days after starting the first DOX injection. The initial body weight of each rat was recorded, and subsequent body weight measurements were taken every 7 days until the end of the experiment.

Figure 1.

Summary of the experimental design.

On the 28^th day from the first DOX injection, all rats were anesthetized using pentobarbital (60 mg/kg) via intraperitoneal injection. Blood samples were collected from the retro-orbital plexus of each animal, allowed to clot for 10 min at room temperature, and then centrifuged for 20 min at 2000 r/min to obtain serum for analysis of creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) activity, and N-terminal pro-brain natriuretic peptide (NT-pro BNP) levels. The thoracic cavity was opened, and the heart was extracted. The heart was rinsed three times with PBS solution (pH 7.4) to remove any remaining blood clots. It was then divided into four parts. One part was fixed in 10% formalin for histopathological examination and immunohistochemical expression of caspase-3. The remaining three parts of the heart were homogenized, and the supernatants were stored at -80°C for further analysis of malondialdehyde (MDA), reduced glutathione (GSH) activity, and transforming growth factor beta 1 (TGF-β1) levels.

Biochemical analysis

Determination of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) activity in serum

The serum CK-MB and LDH activity assays were performed using spectrophotometry with kits from Spectrum Diagnostic, Egypt (Catalogue #239 002 and Catalogue #279 002, respectively). The CK-MB assay employed the spectrophotometric method developed by Urdal and Landaas.²⁴ In this procedure, 1 mL of working solution and 40 μL of serum were pipetted into a thermostatized cuvette, mixed, and incubated for 60 s. The initial absorbance (A) of the sample was measured, followed by absorbance readings at 1-min intervals for 5 min. The difference between absorbances and the average absorbance differences per minute (∆A/min) were calculated. The CK-MB concentration was determined using the formula: ∆A/min x 8254 = CK-MB. For the serum LDH assay, the spectrophotometric method developed by Young was utilized.²⁵ In this procedure, 1 mL of working solution and 20 μL of serum were pipetted into a cuvette, mixed, and the initial absorbance was measured after 30 s. Subsequently, absorbance readings were taken at 1, 2, and 3-min intervals. The mean absorbance change per minute (∆A/min) was determined, and the LDH activity was calculated using the formula: U/L = 8095 x ∆A 340 nm/min.

Determination of serum N-terminal pro-brain natriuretic peptide (NT-proBNP) level

Serum levels of NT-proBNP were measured in ng/L using ELISA kits obtained from Sun Red Biotechnology Company, Shanghai, China (Catalogue # 201-11-0068). The procedure was conducted according to the instructions provided in the kit’s manual. Briefly, a double-antibody sandwich ELISA technique was employed to measure the level of rat NT-proBNP in samples. Rat NT-proBNP monoclonal antibody was pre-coated on a microtiter plate. Standards, test samples, and biotin-conjugated reagent are added to the appropriate wells and incubated. Then, avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Unbound conjugates are removed using wash buffer at each step. After incubation and washing steps, chromogen solutions were added, resulting in a color change from blue to yellow. The intensity of the yellow color was directly proportional to the concentration of NT-proBNP in the sample. Standard dilutions were prepared, and the optical density (OD) values were measured to generate a standard curve. The OD values of the samples were then used with the standard curve equation to calculate the corresponding concentrations of NT-proBNP.

Determination of malondialdehyde (MDA) and reduced glutathione (GSH) level in the cardiac tissue

Cardiac tissue MDA levels (nmol/g tissue) and GSH levels (mg/g tissue) were measured spectrophotometrically using kits from Biodiagnostic Company, Egypt (Catalogue # MD 2529 and Catalogue # GR 2511, respectively).

For MDA level measurement, cardiac tissue samples were weighed and homogenized in a 5 mL cold buffer (50 mM potassium phosphate, pH 7.5) per gram of tissue. After centrifugation at 4000 r/min for 15 min, the supernatant was collected and stored at −80°C for. MDA levels were calculated using the equation: Tissue MDA (nmol/g tissue) = $A s a m p l e / A s t a n d e r d x 10 / g t i s s u e u s e d,$ based on the method by Ohkawa et al.²⁶ To measure GSH levels, cardiac tissue samples were weighed and homogenized in 7.5 mL of cold buffer (50 mM potassium phosphate, pH 7.5, 1 mM EDTA) per gram of tissue using a tissue homogenizer. After centrifugation at 4000 r/min for 15 min at 4°C, the supernatant was collected and stored at −80°C. GSH concentration was determined using the equation: $Tissue G S H = A s a m p l e \times 66.66 / g . t i s s u e u s e d = m g / g . tissue,$ following the method by Beutler et al.²⁷

Determination of transforming growth factor beta 1 (TGF-β1) level in the cardiac tissue (pg/ml)

Tissue levels of TGF-β1 were measured using kits obtained from INOVA Company, Beijing, China (Catalogue # In-RA1354). The procedure involved preparing a tissue homogenate by weighing and homogenizing the cardiac tissue in PBS followed by centrifugation. The resulting supernatant was collected and frozen for analysis. The measurement of TGF-β1 was performed using an ELISA technique with a pre-coated Microelisa stripplate. Standards and samples were added to the wells, and a series of incubation and washing steps were carried out. The addition of substrate solution resulted in color development, which was measured spectrophotometrically. The concentration of TGF-β1 in the samples was calculated by comparing the OD to a standard curve. The standard curve was generated using known concentrations of TGF-β1 and their corresponding OD readings.

Histological examination of H&E-stained sections

Specimens from the cardiac tissue sections were collected at the same level of the left ventricular free wall. They were then promptly fixed in 10% neutral formalin, processed, and embedded in paraffin using standard techniques. Subsequently, tissue sections with a thickness of five microns were cut using a microtome. These sections were stained with hematoxylin and eosin (H&E) to facilitate histopathologic evaluation under a light microscope.

Immunohistochemical detection of tissue caspase-3

Multiple 4-μm-thick tissue sections were prepared from paraffin blocks. The sections underwent deparaffinization in xylene and rehydration with descending concentrations of ethanol. For antigen retrieval, the sections were pretreated with 0.01 mol/L citrate-buffered saline (pH 6.0). To inhibit endogenous peroxidase activity, the sections were exposed to 0.3% (v/v) H₂O₂ in phosphate-buffered saline. To prevent nonspecific binding, the sections were incubated with 10% (v/v) normal goat serum for 1 hour. Subsequently, the sections were incubated overnight at 4°C with mouse monoclonal antibodies against caspase 3 obtained from Abcam, United Kingdom. The streptavidin-biotin complex and horseradish peroxidase were employed in the immunohistochemical staining process to visualize the reaction products. Immunohistochemical staining kits were used accordingly. The sections were subjected to a one-minute incubation in diaminobenzidine tetra-hydrochloride to generate the peroxidase labeling. Counterstaining was performed using Mayer’s hematoxylin solution. For quantitative analysis, the area and the percentage of caspase 3 immunoexpression were measured in myocardial sections of both the control and treated groups using ImageJ software.

Statistical analysis

The data were analyzed using one-way ANOVA, followed by post-hoc Tukey’s multiple comparison tests. The parametric values were expressed as mean ± SEM. The significance was considered at values of p < 0.05.

Results

Results of body weight follow up in the different studied groups

Regarding body weight follow up in the different studied groups (Figure 2), there was a significant decrease in the body weight gain in the untreated DOX group, when compared to the normal control group. TPM-treated group showed marked reduction in the body weight gain relative to SP group and combination group. Conversely, treatment with SP in group 4 resulted in a significant increase in body weight gain relative to the untreated DOX, TPM-treated, and combination-treated groups.

Figure 2.

Body weight in different study groups. Cardiotoxicity was induced by administering DOX intraperitoneally at a cumulative dose of 16 mg/kg over 4 weeks starting from day 7. TPM and/or SP administration started 1 week before DOX treatment and continued for 28 days. Data are presented as means ± SEM (n = 10). Abbreviations; DOX: Doxorubicin, TPM: Topiramate, SP: Spirulina. Symbols @, #, $, %, and & indicate statistical significance at p < 0.05 using one-way ANOVA followed by Tukey as a post-hoc test. Specifically, @ is used to compare between the DOX group and the control group. # is used to compare between the SP group and the DOX group, $ is used to compare between the SP group and the topiramate group, % is used to compare between the SP group and the combination group, and & is used to compare between the topiramate group and the combination group.

Effect of topiramate and spirulina on serum CK-MB, LDH and NT-proBNP levels

Serum CK-MB, LDH, and NT-proBNP were assessed to evaluate the cardiotoxicity induced by DOX and the potential protective effects of TPM and SP. DOX-treated group demonstrated a significant increase in serum levels of CK-MB (Figure 3(a)), LDH (Figure 3(b)), and NT-proBNP (Figure 3(c)) compared to the normal control group. However, significant reductions in the serum levels of these cardiac biomarkers were observed in the TPM-treated group, SP-treated group, and the combination group when compared to the untreated DOX group. The combination group showed a more pronounced reduction relative to both the TPM-treated group and SP-treated group. Furthermore, a comparison between the TPM group and SP group revealed that the reduction in serum levels of CK-MB and LDH was more pronounced in the SP group.

Figure 3.

Effect of topiramate and spirulina on serum CK-MB, LDH and NT-proBNP levels. (a) CK-MB, (b) LDH, (c) NT-proBNP. CK-MB & LDH were measured spectrophotometrically, and NT-proBNP was measured by ELISA. Data are presented as means ± SEM (n = 10). Data were analyzed by one-way ANOVA followed by post-hoc Tukey’s multiple comparison test. Abbreviations; DOX: Doxorubicin, TPM: Topiramate, SP: Spirulina.

Effect of topiramate and spirulina on cardiac tissue oxidative stress markers (MDA and GSH)

Assessment of oxidative stress was conducted by measuring the cardiac tissue levels of MDA and GSH in the various study groups. The DOX group demonstrated a significant increase in cardiac tissue levels of MDA (Figure 4(a)), indicating higher oxidative stress, along with a significant decrease in cardiac tissue levels of GSH (Figure 4(b)) compared to the normal control group. However, in each of the TPM-treated group, SP-treated group, and the combination group, there was a significant reduction in MDA levels, indicating decreased oxidative stress, and a significant increase in GSH levels in the cardiac tissues compared to the untreated DOX group. These findings suggest that TPM, SP, and their combination effectively mitigate oxidative stress induced by DOX treatment.

Figure 4.

Effect of topiramate and spirulina on cardiac tissue oxidative stress markers. (a) MDA, (b) GSH. MDA & GSH were measured spectrophotometrically. Data are presented as means ± SEM (n = 10). Data were analyzed by one-way ANOVA followed by post-hoc Tukey’s multiple comparison test. Abbreviations; DOX: Doxorubicin, TPM: Topiramate, SP: Spirulina.

Effect of topiramate and spirulina on cardiac inflammatory marker (TGF-β1)

Cardiac TGF-β1 levels were measured to assess the level of inflammation in the cardiac tissues among the different study groups. The DOX group exhibited a significant increase in cardiac TGF-β1 levels (Figure 5) compared to the normal control group, indicating increased inflammation. However, in each of the TPM-treated group, SP-treated group, and the combination group, there was a significant reduction in cardiac TGF-β1 levels when compared to the untreated DOX group. These findings suggest that TPM, SP, and their combination exert anti-inflammatory effects, leading to a decrease in TGF-β1 levels in the cardiac tissues affected by DOX-induced cardiotoxicity.

Figure 5.

Effect of topiramate and spirulina on cardiac inflammatory marker (TGF-β1). Cardiac tissue was homogenized and TGF-β1 was measured by ELISA. Data are presented as means ± SEM (n = 10). Data were analyzed by one-way ANOVA followed by post-hoc Tukey’s multiple comparison test. Abbreviations; DOX: Doxorubicin, TPM: Topiramate, SP: Spirulina.

Histopathological examination of the cardiac tissues stained with H&E

Figure 6 depicts the histopathological examination of the rat’s myocardium in different experimental groups. Normal architecture of the myocardial tissues was observed in the control group. Massive degeneration of the myocardial cells with severe congestion of blood vessels was observed in the DOX group. However, administration of either TPM or SP to rats resulted in a significant reduction in myocardial degeneration and restoration of the normal architecture of myocardial fibers. These beneficial effects were more pronounced in in the group treated with the combination of TPM and SP surpassing the effects observed in the groups treated with each agent alone.

Figure 6.

Histopathological examination of the cardiac tissues stained with H&E (X100). (a) In the control group: myocardial bundles consisting of normal myocardial cells (blue arrows) with no degeneration; (b) in the DOX group: diffuse severe degeneration of myocardial cells (black arrows) with congested blood vessels (red arrows); (c) DOX + TPM group: moderately degenerated myocardial muscles (black arrows) surrounded by some normal myocardial cells (blue arrow); (d) DOX + SP group: mild degenerated myocardial muscles (black arrow) surrounded by normal myocardial cells (red arrows); (e) DOX+TPM+SP group: normal myocardial bundles (blue arrows) with areas of focal slight degeneration (red arrow) Abbreviations; DOX: Doxorubicin, TPM: Topiramate, SP: Spirulina.

Effect of topiramate and spirulina on immunohistochemical expression of caspase-3

Caspase-3 was used as an indication of apoptosis. Analysis of the percentage of positive tissue caspase-3 staining in the different studied groups is shown in Figure 7(a). Minimal caspase-3 immunostaining of the myocardial tissues was observed in the control group (Figure 7(b)). A marked increase in the percentage of positive tissue caspase-3 staining in the cardiac tissues in of the group treated with DOX alone (Figure 7(c)), indicating occurrence of apoptosis in comparison to the normal control group. In each of the groups with TPM (Figure 7(d)), SP alone (Figure 7(e)), and the combination group (Figure 7(f)), a significant reduction in the percentage of positive tissue caspase-3 staining in the cardiac tissues was observed as compared to DOX treated group. When comparing the TPM-treated group and the SP-treated group, the reduction was more significant in group treated with SP. Furthermore, the reduction in the percentage of positive tissue caspase-3 staining was more pronounced in the combination group relative to other studied groups. This result implicates the superior effectiveness of combination treatment in preventing apoptosis.

Figure 7.

Immunohistochemical expression of caspase-3 in the cardiac tissues (X400). (a) The percentage of positive tissue caspase-3 staining in the different studied groups, Data are presented as means ± SEM (n = 10). Data were analyzed by one-way ANOVA followed by post-hoc Tukey’s multiple comparison test. (b) In the control group: very minimal caspase-3 immunostaining in the myocardial cells (about 0.92% of cells); (c) in the DOX group: strong positive caspase-3 immunostaining in myocardial cells (blue arrows) (about 60.24% of cells); (d) DOX+TPM group: moderate positive caspase-3 immunostaining in myocardial cells (blue arrows) (about 28.44% of cells); (e) DOX+SP group: weak positive caspase-3 immunostaining in myocardial cells (blue arrows) (about 13.27% of cells); (f) DOX+TPM+SP group: very minimal caspase-3 immunostaining in myocardial cells (blue arrow) (about 5.4% of cells). Abbreviations; DOX: Doxorubicin, TPM: Topiramate, SP: Spirulina.

Discussion

DOX is a highly effective chemotherapeutic agent widely used for treating solid and hematological tumors.²⁸ Unfortunately, the development of cardiotoxicity during DOX therapy poses a significant limitation to its clinical use. Although the precise mechanism underlying DOX-induced cardiotoxicity is not fully elucidated, multiple studies have suggested that oxidative stress, lipid peroxidation, inflammation in cardiac tissues, and apoptosis contribute significantly to the development of DOX-induced cardiomyopathy.^29,30 Dexrazoxane, the only FDA-approved medication for preventing DOX cardiotoxicity, is associated with numerous side effects and can potentially reduce the anti-neoplastic activity of DOX.¹³ Therefore, there is still a drastic need for the development of preventive strategies for this cardiotoxicity.

In the present study, it was observed that the administration of DOX resulted in a significant reduction in body weight gain relative to the control group, while the administration of SP led to an increase in body weight gain. Alhowail et al. reported significant reduction of the body weight gain after DOX administration, which was explained by the fact that DOX increases catabolism and decreases food intake.³¹ Another study reported that administration of SP to rats treated with chemotherapeutic agents resulted in significant increase in the body weight gain in comparison to the other groups.³² Furthermore, a study involving HIV-infected children indicated that SP increased the average body weight.³³ This can be attributed to the rich composition of SP, including proteins, essential amino acids, vitamins, carotenoids, minerals, essential fatty acids, polysaccharides, and glycolipids,³⁴ which are readily absorbed and distributed throughout the body, thereby quickly restoring deficient nutritional status to physiological levels.³⁵ TPM, an FDA-approved drug for weight management in combination with phentermine, acts as an appetite-reducing drug, although its exact mechanism of action, possibly involving gamma-aminobutyric acid (GABA), remains poorly understood.³⁶ This explains the effect of TPM on the body weight of groups 3 and 5.

CK-MB is an isoenzyme of creatine kinase predominantly present in cardiac tissues, playing a vital role in the conversion of creatine into phosphocreatine, thus facilitating energy preservation through ATP utilization.³⁷ Oxidative stress caused by DOX administration damages the creatine isoenzyme in the presence of ferrous iron, resulting in the generation of peroxynitrite free radicals.³ DOX-induced oxidative stress leads to lipid peroxidation, accompanied by the release of CK-MB into the bloodstream. Elevated levels of CK-MB are considered indicative of myocardial injury.^38,39 In our study, we observed that DOX administration increased CK-MB levels, whereas both SP and TPM attenuated the DOX-induced elevation of CK-MB. Previous study has shown that SP supplementation effectively restored increased serum levels of CK-MB induced by microcystin toxicity in rats.⁴⁰ Additionally, another study observed a decrease in both serum CK-MB levels and lipid peroxidation biomarkers in cardiac tissues following pre-administration of SP in tilmicosin-induced cardiotoxicity in mice.⁴¹

LDH is expressed in multiple organs and exists as five different isoenzymes, with LDH1 predominantly found in cardiac tissues.⁴² LDH serves as an important marker to assess cardiomyocyte injury.⁴³ Administration of anthracyclines disrupts the cell membranes of cardiac myocytes, leading to the release of intracellular proteins such as LDH into the bloodstream.³⁹ Numerous studies have reported that DOX-induced oxidative stress leads to lipid peroxidation, accompanied by the release of LDH into the bloodstream.^28,44,45 Consistent with our present study, administration of SP was found to reduce serum LDH activity.^40,46 This effect may be attributed to the antagonistic properties of SP against DOX-induced oxidative stress, which include improvements in superoxide dismutase (SOD) and catalase (CAT) activities, reduction of MDA levels, and the potent scavenging activity of SP against free radicals.⁴⁰

Other potential markers for detecting DOX-induced cardiotoxicity include N-terminal pro B-type natriuretic peptide (NT-proBNP), which is released from the cardiac ventricles in response to volume expansion and pressure overload.⁴⁷ Measurement of NT-proBNP levels has demonstrated its early and sensitive utility in the diagnosis, prognosis, and assessment of the severity of heart failure in adults.⁴³ Several experiments have reported an elevation in serum NT-proBNP following DOX chemotherapy administration, which is consistent with the results of our study.^48–50 In line with our findings, El-Haggar et al. observed a significant reduction in serum NT-proBNP levels in children with β-thalassemia following supplementation with SP.⁵¹ This reduction may be attributed to the effect of SP on vascular reactivity, as it enhances the synthesis and release of nitric oxide by the endothelium and acts as a vasodilating cyclooxygenase-dependent metabolite of arachidonic acid. Additionally, SP has been shown to decrease the synthesis and release of vasoconstrictive eicosanoids by the endothelium.⁵² The current study is the first to report the effect of TPM on DOX-induced cardiotoxicity.

Consistent with our study, it was previously reported that TPM exhibit cardioprotective effects in animals with cardiac ischemia.⁵³ Specifically, TPM reduced cardiac damage resulting from coronary artery ligation, decreased the incidence of cardiac rupture, and improved survival rates. These effects may be attributed to a reduction in the proportion of proinflammatory M1/Ly-6C^high macrophages, which are associated with tissue destruction, and an increase in tissue-repairing M2/Ly-6C^low macrophages. Furthermore, another study demonstrated that TPM reduced the accumulation of lipid droplets and the transformation of macrophages into foamy cells in vitro by modulating the intrinsic GABAergic system within the macrophages, when exposed to oxidized LDL derived from human monocytes.⁵⁴

The role of oxidative stress in the development of DOX-induced cardiotoxicity has been well-documented.⁵⁵ According to this theory, DOX is reduced by NADH dehydrogenase in mitochondrial respiratory complex I, leading to the formation of semiquinone radicals. These radicals then react with molecular oxygen, generating superoxide radicals, hydrogen peroxide, and initiating apoptosis through the hydroxyl radical pathway.⁸ Furthermore, the conversion of hydrogen peroxide to hydroxyl radicals results in the production of (ROS), which can react with DNA, proteins, and lipids, leading to DNA damage, depletion of cellular reduced GSH, lipid peroxidation, and an increase in levels of MDA, an end product of lipid peroxidation.⁵⁶ This observation provides an explanation for the elevated levels of oxidative stress markers observed following DOX administration in the current study.

In the present study, both SP group and TPM group demonstrated antioxidant properties characterized by a significant reduction in MDA levels and a significant increase in GSH levels in the cardiac tissues. The robust antioxidant properties of SP have been previously highlighted.⁵⁷ These properties were attributed to the high abundance of phycocyanins, β-carotenes, vitamins, and minerals present in SP species. Another study has explained the antioxidant activity of SP as a result of its phytochemical compounds, which have demonstrated the ability to reduce the generation of ROS.²⁰ This effect may be explain the ability of SP to ameliorate DOX-induced lipid peroxidation, the potent scavenging activity against free radicals, and the enhancement of cellular antioxidant defenses by increasing the concentration of GSH and nicotinamide adenine dinucleotide phosphate (NADP).⁵⁸ Interestingly, it was previously shown that SP increased GSH concentration by protecting against D-galactosamine-induced hepatic damage and improving the antioxidant status.⁵⁹ Furthermore, SP reduced levels of MDA and increased GSH concentration in patients with chronic obstructive pulmonary disease (COPD).⁶⁰

Numerous studies have documented the antioxidant properties of TPM as evidenced by reduction in MDA levels.^61,62 Furthermore, another study specifically demonstrated the significant reduction of kainate-induced lipid peroxidation in both the pyriform cortex and frontal cortex due to TPM administration.⁶³ Furthermore, the inhibition of carbonic anhydrase enzyme by TPM has been proposed as a potential mechanism underlying its effectiveness in mitigating oxidative stress in various cell types.^64,65 Furthermore, TPM was found to increase GSH levels in the brains of epileptic mice⁶⁶ and rats.⁶⁷ Additionally, treatment with TPM resulted in a higher GSH concentration in the hippocampus compared to non-treated rats.⁶⁸

One of the anticancer mechanisms of DOX is the inhibition of mRNA transfer and synthesis of matrix metalloprotease-1 (MMP-1) in tumor cells, leading to reduced cell mobility. However, this inhibition results in the activation of other MMPs, such as MMP-2 and MMP-9, which have been shown to induce cardiac toxicity by promoting collagen formation and increasing TGF-β1 expression, ultimately leading to myocardial fibrosis.⁹ In the present study, both SP group and TPM group demonstrated anti-inflammatory properties characterized by a significant reduction in DOX-elevated TGF-β1 expression. It was previously demonstrated that topical application of SP on cutaneous wounds markedly inhibited TGF-β1 expression, thus preventing scar formation in a rat model.⁶⁹ Similarly, another study in a rat model of pulmonary fibrosis reported a significant decrease in tissue TGF-β1 levels following administration of C-phycocyanin, the main active component of SP.⁷⁰ The potent anti-inflammatory activity of SP has been attributed to its ability to reduce the synthesis of proinflammatory mediators by inhibiting cyclooxygenase-II and lipoxygenase enzymes, attenuating collagen deposition in inflamed tissues, and ameliorating DOX-induced fibrinoid necrosis.⁷¹

In addition, the potent anti-inflammatory effects of TPM and its ability to reduce tissue TGF-β1 levels have been reported.⁷² This study attributed the anti-inflammatory activities of TPM to the activation of GABA-mediated Cl⁻ channels, inhibition of glutamate Ca²⁺ channels, blockage of Na⁺ channels, and changes in adrenergic signaling mediated by TPM. Additionally, in a study conducted on a post-infarction model, TPM-treated mice exhibited significant modulation of inflammation and control of ventricular remodeling, as evidenced by notable decline in TGF-β1 levels compared to non-treated mice. This anti-inflammatory activity was attributed to the ability of TPM to stimulate GABA_A receptors in monocytes/macrophages.⁵³

DOX-induced apoptosis is considered the primary mechanism underlying DOX-induced cardiotoxicity, as DOX triggers both the intrinsic and extrinsic pathways of apoptosis through various mechanisms.⁷³ DOX administration is associated with the upregulation of the mammalian target of rapamycin (mTOR)/Protein kinase B (Akt) signaling, leading to the inhibition of mitogen-activated protein (MAP) kinase and Jun NH2-Terminal Kinase (JNK) activity in cardiac tissues, along with an increase in caspase-3 activity.⁷⁴ In the current study, it was observed that DOX administration significantly increased caspase-3 staining in the heart, indicating an increase in apoptosis signaling, and both SP and TPM mitigated DOX-induced apoptosis. In line with our study, several studies have reported the antiapoptotic activity of SP.^32,75 This activity have been attributed to phycocyanin, which has been shown to induce the expression of heme oxygenase-1 (HO-1), leading to the suppression of caspase-3 activation. Additionally, phycocyanin has been found to increase the nuclear accumulation of nuclear factor erythroid 2 (Nrf-2) and enhance antioxidant response element (ARE)-mediated transcriptional activity.⁷⁶ Furthermore, previous studies conducted on rats have demonstrated the antiapoptotic activity of TPM against methylphenidate-induced neurotoxicity and cerebral ischemia/reperfusion neuronal damage.^18,77 However, the effect of TPM on preventing apoptosis in cardiotoxicity models is still understudied and requires further investigation, including the assessment of other apoptotic and anti-apoptotic markers.

When comparing the TPM group to the SP group, it was observed that the SP group exhibited a marked increase in final body weight compared to the TPM group. Furthermore, the SP-treated group demonstrated a significant reduction in CK-MB and LDH levels, along with a significant increase in anti-apoptotic effects, as evidenced by a significant decrease in caspase-3 expression in cardiac tissues compared to the TPM group. Additionally, the SP group showed notable improvement in histopathological signs of cardiotoxicity. This superior action of SP can be attributed to its natural composition, which possesses powerful anti-inflammatory and antioxidant properties.²²

To the best of our knowledge, this is the first study to investigate the combination of TPM and SP and compare it to monotherapy with either TPM or SP alone. When comparing the combination group to monotherapy with either TPM or SP alone, significant improvements were observed in terms of serum and cardiac tissue parameters, as well as in histopathological and immunohistochemical examinations. These findings suggest that the combination of SP and TPM may have a potential beneficial effect in reducing inflammation, protecting cells from oxidative stress, and preventing cell death.

Conclusion

In conclusion, TPM and SP may represent promising candidates for treatment of DOX-induced cardiotoxicity by their antioxidant, anti-inflammatory, and anti-apoptotic mediated mechanisms. Their combination exhibited more pronounced anti-apoptotic and anti-inflammatory effect together with activation of the antioxidant activities. Further research is vitally needed to explore the exact molecular mechanisms that may underlie these effects and to test the possibility of clinical application of the findings of the present study.

Footnotes

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Ahmed M Kabel

Radwa M Elmorsy

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