Ameliorative effects of betaine on cisplatin-induced cardiotoxicity in rats through anti-inflammatory pathways

Abstract

Cisplatin (CS) is a broad-spectrum chemotherapeutic agent that causes serious adverse effects, such as cardiotoxicity, despite its potent anti-tumor efficacy. This study aimed to evaluate the cardioprotective effects of betaine in rats exposed to repeated low-dose CS administration using histopathological and immunohistochemical methods. Forty female Wistar albino rats were divided into four groups, including control, betaine, CS, and CS + betaine. Betaine (250 mg kg^-1) was administered orally on a daily basis for four weeks, while CS (8.00 mg kg^-1) was administered intraperitoneally once a week for the same duration. Cardiomyocytes were then examined using histopathological and immunohistochemical methods. The data were analyzed using one-way analysis of variance and Tukey tests. Histopathological analysis revealed cardiomyocyte disorganization, myofibril loss, and increased eosinophilia in the CS group. Betaine treatment partially prevented CS-induced histological damage, contributing to the cardiac muscle structure preservation. Immunohistochemical analyses demonstrated a significant increase in transforming growth factor-beta and interferon gamma expressions in the CS group, whereas betaine administration reduced transforming growth factor-beta levels. Interleukin 6 expression was lower in the CS + betaine group compared to the CS group. No significant differences were observed between groups regarding Interleukin-1β expression. These findings suggest that betaine may have protective effects against CS-induced cardiotoxicity. Its anti-inflammatory properties appear to mitigate cardiomyocyte damage.

Introduction

Cisplatin (CS) is a broad-spectrum alkylating chemotherapeutic agent used against various tumor types, including sarcomas and carcinomas.¹ The CS exerts its anti-tumor effect primarily by interacting with guanines in nuclear DNA, thereby arresting cell division.² Unfortunately, the mechanism of action of CS is not specific to cancer cells. While effectively inducing apoptosis in malignant cells, CS is also associated with a range of dose- and time-dependent toxic effects on healthy tissues, including nephrotoxicity, cardiotoxicity, and hepatotoxicity.^3-5 Direct damage to the nuclear and mitochondrial genetic materials, along with the tissue response to the oxidative stress–inflammation–apoptosis cascade induced by CS, ultimately leads to histopathological alterations and functional impairment in the affected tissues.⁶

It is known that homocysteine levels in blood increase in response to CS.⁷ Homocysteine plays a role in a biochemical cascade involving oxidative stress, caspase activation, DNA damage, and dysfunction of the endo-plasmic reticulum and mitochondria.⁸ The formation of methionine from homocysteine can occur through betaine or 5-methyltetrahydrofolate pathways, with betaine playing a crucial role in this mechanism through methylation.⁹

Betaine is a methylated derivative of glycine that is naturally found in various plants and animals.^10,11 Numerous studies have shown that betaine exhibits anti-oxidant properties, providing protective effects in the kidneys, heart, nerves, and liver.^9,12,13

Due to its ability to activate the anti-oxidant defense system, betaine is also referred to as an anti-inflammatory agent.¹⁴ Betaine transfers a methyl group to toxic metabolites, such as homocysteine, through the betaine-homocysteine methyltransferase enzyme, converting it into methionine while itself being transformed into dimethylglycine.^15,16

This study aimed to investigate the cardioprotective properties of betaine against potential side effects induced by CS in healthy rats subjected to the repeated low-dose CS treatment. In this context, the ability of betaine to protect the heart by exhibiting anti-inflammatory properties was examined using histopathological and immuno-histochemical methods.

Materials and Methods

Groups and chemicals.

In the present study, a total of forty female Wistar albino rats, aged 8 - 10 weeks and weighing 200 - 250 g, raised at the Experimental Research Application and Research Center of Erciyes University, Türkiye, were utilized. The rats were housed in plastic cages under standard conditions with a regular pellet diet and water, and maintained at a temperature of 22.00 ± 2.00 ˚C and a 12-hr light/12-hr dark cycle. Four groups were formed from randomly selected rats (n = 10), including control, betaine, CS, and CS + betaine. Betaine hydrochloride (Sigma-Aldrich, St. Louis, USA) at a dose of 250 mg kg^-1 was dissolved in physiological saline solution and administered orally via gavage.¹⁷ Cisplatin (Koçak Farma, Istanbul, Türkiye) at a dose of 8.00 mg kg^-1 was administered intraperitoneally to the corresponding groups.¹⁸ The control group received physiological saline solution for 30 days, while the betaine and CS + betaine groups were treated with betaine dissolved in physiological saline solution via gavage. Cisplatin was administered once a week for four weeks (a total of four CS injections) to the CS and CS + betaine groups. Two weeks after the final dose of betaine, the rats were anesthetized by intraperitoneal administration of ketamine (50.00 mg kg^-1; Pfizer, Istanbul, Türkiye) and xylazine (10.00 mg kg^-1, Bayer, Istanbul, Türkiye), after which they were sacrificed. Ethical approval for conducting the experimental studies was obtained from the Local Ethics Committee for Animal Experiments at Erciyes University, Türkiye (05.12.2024, Approval No: 24/238). All animals were treated humanely in accordance with the Guide for the Care and Use of Laboratory Animals.

Tissue preparation.

For histopathological examination, immediately after euthanasia, heart tissues were rapidly excised and immersed in a 10.00% formaldehyde solution and fixed for 72 hr. The tissues were then processed according to the standard histological tissue processing procedures. Briefly, the tissues were washed in running tap water, dehydrated through increasing concentrations of alcohol, cleared with xylene, and embedded in paraffin blocks. Sections of 5.00 μm thickness were cut from the paraffin blocks and mounted onto poly-L-lysine-coated slides.

Hematoxylin and Eosin (H&E) and immunohistochemical stainings.

The obtained sections were stained with H&E for histopathological evaluation. In addition, immunohistochemical staining was performed to assess inflammation during the histopathological assessment. For this purpose, primary antibodies were used as follows: Transforming growth factor-beta (TGF-β; 1/100 dilution, Bioss, Woburn, USA), interleukin 6 (IL-6; 1/400 dilution, Affinity Biosciences, Cincinnati, USA), IL-1β (1/100 dilution, Bioss), and interferon gamma (IFN-γ; 1/200 dilution, Bioss). Immunohistochemical staining was performed using the avidin-biotin-peroxidase method (Ultravision Polyvalent (Rabbit-Mouse) Horseradish Peroxidase Kit, 125 mL, ThermoFisher Scientific, Waltham, USA). The stained slides were examined under a microscope (Olympus BX51; Olympus, Tokyo, Japan), and images were captured from four randomly selected areas of each preparation. The immunoreactivity intensities of the obtained images were measured using the ImageJ Software (National Institutes of Health, Bethesda, USA).¹⁹

Statistical analysis.

Statistical analyses of the data in this study were performed using GraphPad Prism (version 7.0; GraphPad Software Inc., San Diego, USA). The normality of the data distribution was assessed using the D'Agostino & Pearson normality test. Since all data showed a normal distribution, one-way analysis of variance was used, followed by Tukey post-hoc test for multiple comparisons between groups. A p-value of < 0.05 was considered statistically significant.

Results

Histopathological findings.

The histopathological evaluation was performed based on the findings obtained from H&E staining of myocardial tissue sections, which were examined under a light microscope. In the control and betaine groups, the cardiac muscle fibers exhibited a standard histological architecture with centrally located oval nuclei and homogeneous cytoplasm showing standard acidophilic staining. In the CS group, the muscle fibers were widely separated, showing cardiomyocyte disorganization. The cytoplasm exhibited varying degrees of eosinophilia, mostly observed in necrotic areas with or without visible nuclei. In addition, some cardiomyocytes exhibited loss of myofibrils and minimal cytoplasmic vacuolization, leading to a loss of eosinophilia, thus presenting a disrupted architecture of the heart muscle. In the CS + betaine group, the standard architecture of the heart muscle was partially preserved compared to the CS group. While certain muscle fibers displayed typical cytoplasmic features with centrally positioned nuclei, others demonstrated reduced eosinophilia associated with cytoplasm myofibrils loss. Disorganization, characterized by separation of muscle fibers, was also present (Fig. 1).

Fig. 1.

Photomicrographs of myocardium in A) Control, B) betaine, C) Cisplatin (CS) and D) CS + betaine groups. Asterick: Disorganization of cardiomyocytes with widely separated muscle fibers; black arrow: Loss of myofibrils; green arrow: Minimal cytoplasmic vacuolization; yellow arrow: Intensely eosinophilic cytoplasm, (Hematoxylin and Eosin staining; Bars = 100 µm).

Immunohistochemical findings.

Following histo-pathological evaluation, cardiomyocytes were stained with TGF-β, IL-6, IL-1β, and IFN-γ primary antibodies using immunohistochemical methods (Fig. 2). The obtained density measurements results were statistically analyzed (Table 1). In this context, evaluation of TGF-β expression revealed that the CS group exhibited significantly higher expression levels compared to the control and betaine groups, whereas the CS + betaine group showed a marked reduction in expression relative to the CS group. According to the immunoreactivity assessment for IL-6, no significant difference was observed between the control, betaine, and CS groups, whereas the CS + betaine group showed significantly lower expression than the CS group. As for IL-1β, although there was no significant difference between the groups, the CS group exhibited higher expression. When evaluating IFN-γ, it was found that the CS group had significantly higher levels than the control and betaine groups. At the same time, no significant difference was observed between the CS + betaine and CS groups.

Fig. 2.

Transforming growth factor-beta (TGF-β), interleukin 6 (IL-6), IL-1β, and interferon gamma (IFN-γ) immunohistochemical stainings of the heart tissue in different experimental groups, (Bars = 100 µm). The expressions intensities in the groups are indicated by arrows. CS: Cisplatin

Table 1.

Immunoreactivity intensities of different parameters in the heart tissue of the experimental groups. Data are expressed as mean ± standard deviation of pixels per 305 μm × 446 μm in ImageJ Software.

Parameters	Control	CS	Betaine	CS + Betaine	p-value
Transforming growth factor-beta	73.41 ± 2.37a	75.64 ± 2.48b	71.70 ± 2.14c	71.65 ± 1.63cd	0.0001
Interleukin-6	74.22 ± 3.18ab	75.75 ± 3.60b	74.75 ± 2.60ab	73.79 ± 2.20a	0.0245
Interleukin -1β	72.79 ± 2.05a	73.96 ± 2.33a	72.84 ± 2.33a	72.86 ± 1.71a	0.0441
Interferon gamma	73.35 ± 1.72a	77.74 ± 2.38b	72.60 ± 1.69a	77.25 ± 2.18b	0.0001

CS: Cisplatin.

^a-d Different letters indicate the statistical differences between groups (p < 0.05).

Discussion

In recent years, betaine, which has been used as a dietary supplement in animal nutrition,⁹ has garnered attention in human health research due to the discovery of its anti-oxidant and anti-inflammatory properties.²⁰ This study presented the first data regarding betaine treatment showing anti-inflammatory properties in CS-induced cardiotoxicity. The obtained data demonstrated that betaine consumption suppressed CS-induced inflammation by reducing TGF-β, IL-6, IL-1β, and IFN-γ levels and promoting histopathological improvement in rat cardiomyocytes.

Cisplatin-induced cardiotoxicity, although less common than ototoxicity or gastrointestinal toxicities, is one of the major limiting factors in cancer treatment, and clinical cases have been increasingly reported in recent years.²¹ Clinically, various cardiac events induced by CS have been reported.²² The cause of these symptoms can be attributed to the endothelial damage and various vascular events.²³ The mechanism underlying CS-induced cardiotoxicity is thought to involve the generation of reactive oxygen species (ROS) and consequent oxidative stress, which in turn triggers apoptosis and inflammatory responses.²⁴

As stated, while much of the literature acknowledges that oxidative stress following CS treatment is associated with the resulting damage, there is also evidence indicating that inflammation may be the primary initiator of CS-related toxicity.²⁵ The immune system primarily regulates the inflammatory response through pattern-recognition receptors, which detect pathogen- or damage-associated molecular patterns.²⁶ Activation of various receptors subsequently stimulates key transcription factors, such as nuclear factor kappa B (NF-κB).²⁷ This leads to the release of pro-inflammatory cytokines, such as IL-1β, IL-6, and tumor necrosis factor-alpha (TNF-α), as well as chemokines, like Chemokine (C-X-C motif) ligand 2 and IL-8.²⁸ These mediators coordinate the recruitment of immune cells to the affected area and promote inflammation; however, if this process persists, it may result in tissue damage.²⁵

For this reason, natural or synthetic anti-inflammatory compounds with the potential to significantly alleviate CS-related toxicity have increasingly emerged as promising therapeutic candidates.²⁵ In addition to more commonly used anti-inflammatory agents, such as resveratrol,²⁹ honey,³⁰ and curcumin,³¹ betaine has also been included in studies on CS-induced toxicity, and has been shown to reduce CS-induced nephrotoxicity by inhibiting NF-κB, IL-6, IL-1β, and TNF-α.^32,33

Additionally, it is known that high ROS levels can lead to pathological processes, such as inflammation.³⁴ It has been shown that CS administration produces a variety of inflammatory factors and chemokines in cardiomyocytes, resulting in damage to the cardiomyocytes.³⁵

The formation of ROS and endothelial damage resulting from CS treatment triggers the production of pro-inflammatory mediators.³¹ Following CS-induced intra-cellular damage, the transcription factor NF-κB is activated, leading to the induction of synthesis and expression of cytokines and pro-inflammatory mediators, such as TNF-α, IL-1β, and IL-6, which exacerbate tissue damage.³⁶ The increase in inflammatory cytokines observed in the present study due to the CS treatment was consistent with the results reported in previous studies.³⁶

Therefore, anti-oxidant therapies may provide myocardial protection against CS. For this purpose, many natural anti-oxidants, such as resveratrol, apocynin, and alpha-lipoic acid, can protect cardiomyocytes against ROS.³⁷ Potential cytoprotective strategies against CS include curcumin,³⁸ vitamin E,³⁹ N-acetylcysteine,⁴⁰ and Aloe vera.⁴¹ The general mechanism of action of these agents involves the improvement of mitochondrial dysfunction, attenuation of oxidative damage, and a subsequent reduction in inflammation and cell death.⁴²

In this context, reducing oxidative stress and inflammation may help alleviate CS-induced cardiotoxicity. Therefore, the present study investigated the effects of betaine, a natural anti-oxidant and anti-inflammatory compound, on inflammatory markers in CS-treated rats. Betaine treatment directly reduces homocysteine levels by inducing the remethylation process, which converts homocysteine into methionine.¹² Hence, the beneficial properties of betaine are promising. Studies have shown that hyperhomocysteinemia ultimately induces oxidative stress and apoptosis.^43,44 Betaine exerts its primary anti-oxidant mechanism by acting as a methyl group donor through betaine-homocysteine methyltransferase, converting homocysteine into methionine.²⁰ Additionally, it reduces apoptosis and inflammation markers (NF-κB and IL-1β) levels.¹⁷ The NF-κB pathway regulates many genes involved in inflammation, including pro-inflammatory cytokines, such as TNF-α and IL-1β. Therefore, it is not surprising that chronic NF-κB activation is involved in many inflammatory diseases.⁴⁵ As a result, betaine exerts anti-inflammatory effects by inhibiting the NF-κB signaling pathway.²⁰ In the present study, the anti-inflammatory property of betaine is supported by its ability to reduce specific inflammatory markers. Previously, it has been shown that betaine restores the activity of anti-oxidant enzymes, inhibits TNF-α, NF-κB, and caspase 3, and protects rats from CS-induced nephrotoxicity.³² The CS-induced oxidative stress triggers a series of inflammatory reactions, including the elevation of TNF-α, IL-1β, and IL-6, leading to tissue damage. This damage is being mitigated using various substances, such as taurine, which is known for its anti-inflammatory properties.³⁵ The present study demonstrated that betaine significantly decreased the levels of TGF-β and IL-6, indicating its role in the suppression of inflammatory processes. This finding is consistent with a former study.⁴⁶ However, IL-1β did not show sufficient expression in any of the groups, resulting in no significant outcome. At the same time, IFN-γ was significantly elevated in response to CS, although this could not be adequately reduced by betaine. It has been shown that betaine provides various benefits, including improving heart health by preventing oxidative stress through its anti-oxidant role and reducing inflammation.^20,47 It has been shown that betaine can reverse cardiovascular complications and myocardial infarction.⁴⁷ Therefore, anti-oxidants may offer a promising approach to alleviate cardiac damage in CS chemotherapy patients.⁴⁸

The present study demonstrated significant improvement in cardiac histology at the light microscopic level in the CS + betaine group. Findings, such as partial restoration of the standard histological structure and organization of the heart muscle fibers, were consistent with previous similar studies.⁴⁹ This improvement can be attributed to the previously known anti-oxidant and anti-inflammatory potential of betaine.²⁰ The reduction in inflammatory cytokines observed in the present study following betaine administration is consistent with the findings of earlier studies.⁵⁰

In conclusion, this study provides novel evidence that betaine exerts protective effects against CS-induced cardiotoxicity, primarily through its anti-inflammatory properties. Histopathological analysis revealed that betaine alleviated myocardial structural damage, while immunohistochemical findings demonstrated a significant reduction in TGF-β and IL-6 expressions levels. Although IL-1β and IFN-γ did not show statistically significant improvements, the overall trend supports the regulatory effect of betaine on inflammatory mediators. Considering these results, betaine may represent a promising therapeutic candidate for mitigating CS-related cardiac side effects. Further research is warranted to explore the full therapeutic potential of betaine in cardioprotection.

Conflict of interest

The authors declare no conflict of interest.