Probiotics containing Lactobacillus are promising candidates for cisplatin-induced cardiotoxicity

Abstract

This study examined the effects of the mixture of probiotics Lactobacillus (Lactobacillus rhamnosus, Lactobacillus fermentum, and Lactobacillus brevis) against cardiotoxicity induced by cisplatin (CP) in rats by monitoring markers of oxidative stress, inflammation, and apoptosis in cardiac tissue. For this study, 28 male rats were divided into four groups as follows: Control, CP, Lactobacillus + CP, and Lactobacillus. Experimental cardiotoxicity was induced by CP. The activity of cardiac enzymes in serum and indicators of oxidative stress and nitric oxide (NO) in heart tissues were measured using biochemical methods. Inflammation, apoptosis, and DNA damage were also assessed in cardiac tissue by immunohistochemistry and immunofluorescence. CP administration caused a significant increases (p < 0.01) in malondialdehyde (1.53 ± 0.21), NO (77.83 ± 4.26), and 8-hydroxydeoxyguanosine (8-OHdG) (80.50 ± 1.64) levels as well as marked decreases (p < 0.01) in the activity of superoxide dismutase (0.93 ± 0.15) and the level of glutathione (1.87 ± 0.20) in the CP group compared with those in the control group (0.88 ± 0.19, 53.67 ± 5.32, 21.67 ± 0.81, 1.39 ± 0.29, 2.64 ± 0.28, respectively). This imbalance in the oxidant/antioxidant system was accompanied by increased expression of tumor necrosis factor-alpha (TNF-α), Bcl-2-associated X protein (Bax), and caspase-3. CP also caused degenerative and necrotic changes in cardiac tissues and increased cardiac biomarkers, such as serum creatine kinase, creatine kinase myocardial band, lactate dehydrogenase, and troponin I. In contrast, the Lactobacillus probiotic administration significantly reduced (p < 0.01) malondialdehyde (1.04 ± 0.21) and NO (56.33 ± 5.39) levels in the Lactobacillus + CP group compared with those in the CP group, and significantly increased (p < 0.01) GSH levels (2.33 ± 0.14) in the Lactobacillus + CP group compared with that in the CP group. Lactobacillus probiotic also decreased TNF-α, 8-OHdG, Bax, and caspase-3 expression, and increased the expression of B-cell lymphoma 2. This enhanced the histopathology of the heart and positively affected cardiac biomarkers. In conclusion, the probiotic Lactobacillus reversed cardiotoxicity by suppressing oxidative stress, attenuating the inflammatory process, and improving the proteins that regulate apoptosis. Our findings support the use of probiotics to treat cardiotoxicity resulting from cancer therapy with CP.

Introduction

Most chemotherapeutic drugs used to treat cancer cause severe side effects in patients, such as decreased leukocyte, erythrocyte, and platelet counts, hair loss, discoloration of the skin, nausea, vomiting, and loss of appetite. Some patients get used to these painful side effects and the situation remains under control unless other complications develop, such as organ toxicity. Cisplatin (CP) is a potent chemotherapy drug used for the treatment of various cancers¹. It exhibits its anticancer effects through the induction of apoptosis, which leads to irreparable DNA damage^2,3. The heart is one of the most important organs that is pathologically affected by CP. Cardiotoxicity, which may lead to cardiac ischemia, angina, myocarditis, and cardiomyopathy as well as chronic heart failure, is a serious complication associated with the treatment of cancer patients with CP^4–6. Thus, CP-induced cardiotoxicity has become an important challenge to overcome to improve the treatment of cancer patients. The development of drugs that ameliorate CP-induced cardiotoxicity depends primarily on a broad understanding of the mechanisms underlying cardiotoxicity. Although these specific mechanisms have not been clearly defined, there is evidence that oxidative stress, inflammation, and apoptosis contribute to CP-induced cardiotoxicity⁷. Therefore, the development of agents that exhibit antioxidant, anti-inflammatory, and anti-apoptotic properties, without causing harmful side effects, has attracted attention as a promising option for minimizing CP-induced cardiotoxicity and the resulting complications that debilitate cancer patients and may lead to death. Because of the beneficial effects of probiotics on human health and their ability to prevent and treat many diseases, they have been the subject of many recent studies. In addition, probiotics exert antioxidant and anti-inflammatory properties, which have enhanced their use in combination with chemotherapeutic drugs to treat various types of cancer^8–10. According to The Food and Agricultural Organization, probiotics are “live microorganisms” that confer beneficial health effects on the host following consumption¹¹. Among the probiotics belonging to lactic acid bacteria, Bifidobacterium and Lactobacillus are the most widely used¹². Moreover, recent studies have demonstrated that probiotic supplements potentiate the anticancer effects of chemotherapeutic drugs and reduce some of the side effects associated with these drugs, such as gastrointestinal side effects^10,13,14. In children with acute leukemia, the daily Lactobacillus rhamnosus supplementation reduced gastrointestinal side effects following the use of chemotherapy¹³. In rats with colorectal cancer, probiotic Bifidobacterium infantis supplementation also ameliorated intestinal mucositis following the use of chemotherapy¹⁴. Another study by Linn et al. demonstrated that administration of probiotic capsules containing Lactobacillus acidophilus LA-5 plus Bifidobacterium animalis subsp. lactis BB-12 in cervical cancer patients diminished diarrhea following the use of radiotherapy¹⁵. In colon cancer mouse model, Lactobacillus fermentum ZS40 reduced the level of intestinal inflammation and showed promising therapeutic effects against colon cancer induced by azomethane-dextran sulfate sodium¹⁶. In patients with head and neck cancer, Lactobacillus brevis CD2 minimized oral mucositis resulting from radiation therapy and CP treatment¹⁷. To date, few studies have evaluated the ability of Bifidobacterium and Lactobacillus to improve CP-induced cardiotoxicity¹⁸. According to a survey that we conducted, only one study by Zhao et al. reported that Lactobacillus gasseri supplementation protects cardiac function and delays CP-induced cardiotoxicity by suppressing the inflammatory process⁴, although screening for the oxidative stress parameters and the proteins that regulate apoptosis pathways were not conducted. Another study by Cetik Yildiz et al. demonstrated that administration of kefir (fermented milk product) improves cardiac enzymes and the histopathological integrity in a rat model of cyclophosphamide-induced cardiotoxicity¹⁹, without screening for the oxidative stress and inflammation parameters and the proteins that regulate apoptosis pathways in cardiac tissues. Additionally, administration of Lactobacillus fermentum (Pro5) and Lactobacillus casei (Pro3), either individually or in combination, exhibited protective and regenerative effects against cyclophosphamide-induced liver, kidney, and spleen injury by modulating inflammatory and immune responses²⁰. Recent studies also reported that a mixture consisting of Lactobacillus rhamnosus, Lactobacillus fermentum, and Lactobacillus brevis suppressed nephrotoxicity and hepatotoxicity resulting from treatment with CP through inhibition oxidative stress, inflammation, and apoptosis in the renal²¹ and hepatic²² tissues of rats. Based on this, probiotic Lactobacillus strains are hypothesized to prevent CP-induced cardiotoxicity through their antioxidant, anti-inflammatory, and anti-apoptotic potentials, despite evidence of the therapeutic effects of probiotics Lactobacillus on CP-induced cardiotoxicity is insufficient.

Considering these promising results and in keeping with the global efforts to develop novel effective drugs reducing cardiotoxicity in cancer patients, we evaluated the prophylactic potential of the mixture of probiotics Lactobacillus (Lactobacillus rhamnosus, Lactobacillus fermentum, and Lactobacillus brevis) against CP-induced cardiotoxicity in rats by targeting oxidative stress, inflammation, and apoptosis.

Materials and methods

Probiotic preparation

The kefir sample was diluted with sterile Ringer’s solution and appropriate dilutions were inoculated onto Man, Rogosa and Sharpe (MRS, Merck, Germany) agar to isolate the lactobacilli. The mixture was incubated at 37 °C for 72 h under anaerobic conditions. At the end of incubation, Gramme staining, catalase and oxidase tests were performed on white and opaque colonies. Gramme-positive, rod-shaped and catalase- and oxidase-negative colonies were streaked on MRS agar. After the appropriate MacFarland adjustments were made with the pure bacteria obtained, each well of the API CH50 kit was inoculated and covered with mineral oil to create an anaerobic environment. The selected strains found in the wells of the API CH50 kit are glycerol, erythrol, D- and L-arabinose, D-ribose, D- and L-xylose, D-adonitol, methyl-beta-D-xylopyranoside, D-galactose, D-glucose, D-fructose, D-mannose, D-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl-alpha-D-mannopyranoside, methyl-alpha-D-glucopyranoside, N-acetylglucosamine, amygdalin, Arbutin, esculin/iron citrate, salicin, D-cellobiose, D-maltose, D-lactose, D-melibiose, D-sucrose, D-trehalose, inulin, D-melizitose, D-reaffinose, starch, glycogen, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D- and L-fucose, D- and L-arabitol, potassium gluconate, potassium 2-ketoguconate ribose, potassium 5-ketoguconate were evaluated after 24 and 48 h with regard to the use of substrates. The data obtained was then analysed using the biochemical profiles recorded in the APIweb^® database (bioMerieux). As a result of the analysis, the selected strains were determined to be Lactobacillus rhamnosus, Lactobacillus fermentum and Lactobacillus brevis lactic acid bacteria. A MacFarland adjustment to 10⁵/ml was made for each bacterium and a bacterial solution mix was prepared. The supernatant part of this prepared bacterial mixture was discarded by centrifugation and the bacterial mixture was formed again at a density of 10⁵/ml with PBS^21,22.

Study groups, experimental protocol, and samples

The study was carried out at Ataturk University Medical Experimental Application and Research Center following approval by the Animal Experiments Local Ethics Committee at Ataturk University (Number of consent: 20, Date of consent: February 28, 2019). Sprague–Dawley rats were provided by the medical experimental application and research center at Ataturk University. All methods and experiments were performed in accordance with the relevant guidelines and regulations by the Animal Experiments Local Ethics Committee at Ataturk University. Additionally, this study was conducted accordance with ARRIVE guidelines. The experimental procedure and used doses were determined based on previous articles^21,22. For this study, 28 male Sprague Dawley rats weighing 220–250 g were divided into four experimental groups with seven rats per group as follows: (1) Control: received distilled water; (2) CP: received CP; (3) Lactobacillus + CP: received the mixture of probiotics Lactobacillus and CP; and (4) Lactobacillus: received the mixture of probiotics Lactobacillus. The rats were allowed a standard pellet diet for laboratory rodents and free access to water. The study groups, the experimental protocol, and the timeline are shown in Fig. 1.

Fig. 1.

The experimental protocol of the study. CP: Cisplatin.

Daily treatment continued for seven days. At the end of the experimental application, blood samples were collected from the rats under ketamine-xylazine anesthesia by cardiac puncture using sterile syringes. The heart tissue was taken after cervical dislocation. Heart tissues were collected from all groups and immediately placed in 10% formalin solution for subsequent histopathological, immunohistochemical, and double immunofluorescence examination. For biochemical analyses, blood samples were transferred to gel and clot activator tubes and centrifuged for 10 min at 3500–4000 rpm at 4 °C. Sera were stored at − 80 °C until analysis.

Biochemical analyses in heart tissues and sera

Biochemical parameters in heart tissues and sera were measured using the following standard commercial kits according to manufacturer’s instructions.

Biochemical indicators in heart tissues, malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and nitric oxide (NO) and those in the serum [Creatine Kinase (CK), Lactate Dehydrogenase (LDH), Creatine Kinase Myocardial Band (CK-MB), and troponin I] were measured using the ELK8612, ELK8577, ELK8178 (Biotech USA), E-BC-K135-M, E-EL-0058, E-BC-K046-M (Elabscience USA), ab285275, and ab246529 (Abcam USA) test kits, respectively.

Histopathological analysis

Routine tissue procedures were applied for histopathological examination²³. In brief, the histopathological changes were examined in hematoxylin and eosin (H&E)-stained sections by light microscopy. Five regions from each tissue were evaluated. According to the number and proportion of cells in the region, the condition was scored as no (‒), mild (+), moderate (++), and severe (+++).

Immunohistochemical analysis

All sections prepared on adhesive (poly-L-Lysin) slides for immunoperoxidase examination were deparaffinized and dehydrated by passing through xylol and an alcohol series. Primary antibodies [Tumor necrosis factor-alpha (TNF-α) Catholock: sc-52746, Caspase-3 Catholock: sc-56053, B-cell lymphoma 2 (Bcl-2) Catholock: sc-7382] were applied to the tissues using a routine procedure and 3–3’diaminobenzidine was added to the sections as a chromogen. The sections were examined under a light microscope (ZEISS AXIO, Germany) and evaluated as no (−), mild (+), moderate (++), and severe (+++) according to their immune positivity.

Double immunofluorescence analysis

All sections prepared on adhesive (poly-L-Lysin) slides were deparaffinized and dehydrated by passing through xylol and an alcohol series. Using a standard procedure, primary antibody [8-hydroxydeoxyguanosine (8-OHdG) Catholock: sc-66036] was applied to the tissues and incubated based on the manufacturer’s instructions. FITC Catholock: ab6785, which was used as a secondary immunofluorescence antibody, was added and the tissues were incubated in the dark for 45 min. The second primary antibody [Bcl-2-associated X protein (Bax) Catholock: sc-7480] was applied to the tissues and incubated according to the manufacturer’s instructions. Texas Red Catholock: ab6719, which was used as a secondary immunofluorescence antibody, was added and the tissues were incubated in the dark for 45 min. DAPI with mounting medium (Catholock: D1306) was applied to the sections and incubated in the dark for 5 min. Finally, the stained sections were examined under a fluorescent microscope (ZEISS AXIO, Germany).

Statistical analysis

One-way ANOVA followed by Duncan’s test was conducted to determine the significant differences in the biochemical results (p < 0.05) using the SPSS (Version 20.00) statistical data program. GraphPad Prism (Version 8.0.2) software was used for histopathological, immunohistochemical, and immunofluorescent examinations. Kruskal–Wallis and Mann–Whitney U tests were used to evaluate the histopathological results, whereas a one-way ANOVA followed by Tukey’s test was used to analyze the immunohistochemical and immunofluorescent results. Differences were considered to be significant at p < 0.05. All statistical analyses were expressed as the mean ± SD. Details regarding the statistical tests performed in this study are described in a previous report²⁴.

Results

Biochemical tests

Biochemical markers in heart tissue

The biochemical markers in heart tissue are shown in Fig. 2 as the mean ± SD. Based on these results, CP application resulted in a significant increase (p < 0.01) in MDA (Fig. 2A) and NO (Fig. 2B) levels in the CP group (1.53 ± 0.21 and 77.83 ± 4.26, respectively) compared with those in the control group (0.88 ± 0.19 and 53.67 ± 5.32, respectively). CP administration also resulted in a significant decrease (p < 0.01) in GSH levels (Fig. 2C) and SOD activity (Fig. 2D) in the CP group (1.87 ± 0.20 and 0.93 ± 0.15, respectively) compared with those in the control group (2.64 ± 0.28 and 1.39 ± 0.29, respectively). In contrast, the Lactobacillus probiotic administration significantly reduced (p < 0.01) MDA (Fig. 2A) and NO (Fig. 2B) levels in the Lactobacillus + CP group (1.04 ± 0.21 and 56.33 ± 5.39, respectively) compared with those in the CP group, and significantly increased (p < 0.01) GSH levels (Fig. 2C) in the Lactobacillus + CP group (2.33 ± 0.14) compared with that in the CP group.

Fig. 2.

Effects of probiotic Lactobacillus on MDA, NO, GSH, and SOD in heart tissues in rats with cardiotoxicity. The results are presented as mean ± SD. Different small letters indicate significant differences (p < 0.05). CP: Cisplatin.

Biochemical markers in serum

The activities of CK, CK-MB, and LDH and the level of troponin I were determined in the serum as cardiac markers. The biochemical markers in the serum are shown in Fig. 3 as the mean ± SD. CP application resulted in a significant increase (p < 0.01) of CK (Fig. 3A), CK-MB (Fig. 3B), LDH (Fig. 3C), and troponin I (Fig. 3D) in the CP group (2443.66 ± 233.96, 1198.83 ± 171.48, 1511.50 ± 190.34, and 8.12 ± 0.57, respectively) compared with those in the control group (749.51 ± 61.59, 522.00 ± 80.70, 611.50 ± 66.24, and 5.04 ± 0.39, respectively). In contrast, Lactobacillus probiotic administration significantly reduced CK (Fig. 3A), LDH (Fig. 3C) (p < 0.05), CK-MB (Fig. 3B), and troponin I (Fig. 3D) (p < 0.01) in the Lactobacillus + CP group (1287.17 ± 97.44, 785.50 ± 82.96, 671.33 ± 157.65, and 5.97 ± 0.56, respectively) compared with those in the CP group.

Fig. 3.

Effects of probiotic Lactobacillus on serum CK, CK-MB, LDH, and troponin I in rats with cardiotoxicity. The results are presented as mean ± SD. Different small letters indicate significant differences (p < 0.05). CP: Cisplatin.

Histopathological evaluation of heart tissue

Severe hyaline degeneration and zenker necrosis were observed in the muscle fibers of the CP group. In addition, severe hyperemia and hemorrhage were observed in the interstitial vessels of the CP group (Fig. 4; Table 1). These changes were reduced to non (‒) in the case of zenker necrosis, to a mild (+) in the case of hyaline degeneration, and to moderate (++) in the case of hyperemia and hemorrhage in the interstitial vessels of the Lactobacillus + CP group (Fig. 4; Table 1). The results of the statistical analysis of these histopathological changes are presented in Table 2.

Fig. 4.

Photomicrography of histopathological and immunohistochemical evaluation of heart tissues in rats with cardiotoxicity

Table 1.

Scoring of histopathological findings in heart tissues.

	Control	CP	Lactobacillus + CP	Lactobacillus
Hyaline degeneration in muscle fibers	−	+++	+	−
Zenker necrosis in muscle fibers	−	+++	−	−
Hyperemia in the veins	−	+++	++	−

Sections were evaulated according to lesions as no (‒), mild (+), moderate (++) and severe (+++). CP: Cisplatin.

Table 2.

Findings of histopathological, immunohistochemical, and Immunofluorescence analyses in heart tissues.

		Control	CP	Lactobacillus + CP	Lactobacillus
Histopathological	Degeneration	0.00 ± 0.00	5.00 ± 0.63a	1.50 ± 0.54b	0.16 ± 0.40
	Necrosis	0.00 ± 0.00	5.33 ± 0.81a	0.50 ± 0.54b	0.00 ± 0.00
Immunohistochemical	TNF-α	19.67 ± 1.36	89.17 ± 4.66a	28.00 ± 2.82b	21.33 ± 2.16
	Caspase-3	21.67 ± 0.81	80.67 ± 2.94a	27.50 ± 2.81b	23.33 ± 1.96
	Bcl-2	0.00 ± 0.00	0.66 ± 0.51b	5.16 ± 0.40a	0.00 ± 0.00
Immunofluorescence	8-OHdG	21.67 ± 0.81	80.50 ± 1.64a	29.33 ± 4.63b	23.17 ± 2.71
	Bax	0.00 ± 0.00	4.66 ± 0.81a	0.50 ± 0.54b	0.00 ± 0.00

The results are presented as mean ± SD. Different small letters indicate significant difference for each parameter in the same row (p < 0.05). a, b: p = 0.0022. Bcl-2: B-cell lymphoma 2, 8-OHdG: 8-Hydroxydeoxyguanosine, Bax: Bcl-2-associated X protein, CP: Cisplatin.

Histopathological images: Hyaline degeneration (arrows), zenker necrosis (arrowheads), and hyperemia (stars) H&E, Bar: 50 μm. Immunohistochemical images: TNF-α, Caspase-3, Bcl-2 expression in muscle fibers (arrowheads), IHC-P, Bar: 50 μm. CP: Cisplatin.

Immunohistochemical evaluation of heart tissue

The intense expression of TNF-α between damaged muscle fibers and around vessels and increased cytoplasmic caspase-3 expression in damaged muscle fibers were detected in the CP group compared with the control group (Fig. 4; Table 3). Mild expression of TNF-α and caspase-3 and high expression of cytoplasmic Bcl-2 in the muscle fibers were observed in the Lactobacillus + CP group (Fig. 4; Table 3). The results of a statistical analysis of the immunohistochemical findings are presented in Table 2.

Table 3.

Scoring immunohistochemical and Immunofloresence findings of heart tissue.

	Control	CP	Lactobacillus + CP	Lactobacillus
TNF-α	−	+++	+	−
Caspase-3	−	+++	+	−
Bcl-2 8-OHdG Bax	− − −	+ +++ +++	+++ + +	− − −

Sections were evaluated as no (−), mild (+), moderate (++) and severe (+++) according to their immune positivity.CP: Cisplatin.

Double immunofluorescence evaluation of heart tissue

Negative 8-OHdG and Bax expression were measured in the heart tissues of the control and Lactobacillus groups (Fig. 5; Table 3). High cytoplasmic 8-OHdG and Bax expression was observed in the damaged muscle fibers of the CP group (Fig. 5; Table 3). Mild cytoplasmic 8-OHdG and Bax expression was observed in the muscle fibers in the Lactobacillus + CP group (Fig. 5; Table 3). The results of a statistical analysis of the immunofluorescence findings are presented in Table 2.

Fig. 5.

Effects of probiotic Lactobacillus on 8-OHdG (FITC) and Bax (TEXAS RED) expression in heart tissues of rats with cardiotoxicity. IF, Bar: 50 μm. 8-OHdG: 8-Hydroxydeoxyguanosine, Bax: Bcl-2-associated X protein, CP: Cisplatin.

Discussion

Developing effective treatments to reduce cardiotoxicity resulting from CP treatment of cancer patients is an important challenge facing oncologists. Our study differs from other published studies in this area^4,19 (These studies are explained in the introduction section) in the targeted cellular pathways and the used probiotics strains. The present study expands on this field by using a mixture of probiotics Lactobacillus (Lactobacillus rhamnosus, Lactobacillus fermentum, and Lactobacillus brevis) and examining the oxidative stress and inflammation parameters and the proteins that regulate apoptosis pathways in cardiac tissues, thereby providing new insights into the mechanism and potential clinical relevance. In this study, we evaluated the ability of Lactobacillus probiotics to suppress CP-induced cardiotoxicity by targeting the MDA/8-OHdG/TNF-α/Bax/Bcl-2/caspase-3 pathway, which promotes oxidative stress, inflammation, and apoptosis in heart tissues. We hypothesized that the use of probiotics consisting of Lactobacillus (Lactobacillus rhamnosus, Lactobacillus fermentum, and Lactobacillus brevis) and a targeted approach may improve the management of CP-induced cardiotoxicity in cancer patients.

Oxidative stress is an important factor that contributes to the pathophysiology of cardiotoxicity induced by CP²⁵. It is defined as a breakdown in the antioxidant system during an uncontrolled increase in cellular oxidants²⁴. MDA, GSH, and SOD were measured as parameters to evaluate the status of oxidative stress in cardiac tissue. The observed decrease in antioxidants versus an increase in MDA following CP administration is obvious evidence of oxidative stress causing cardiac tissue damage. We contend that the uncontrolled availability of reactive oxygen species (ROS) diminishes SOD activity and GSH levels in biological systems and renders cell membranes vulnerable to ROS attack, which results in cardiac tissue damage and high MDA levels.

NO is a free radical with many beneficial roles such as cell protection and prevention of heart diseases, thus preserving its bioavailability is essential. However, at exaggerated levels in several disease conditions it is a known cytotoxin reacting with superoxide radicals to form peroxynitrite which is the main mediator of cellular damage and is associated with the establishment and progression of different human diseases^26,27. In the oxidative stress condition, this free radical has a great tendency to react with superoxide radicals to form peroxynitrite, which targets GSH and many enzymes and destroys vital components in the cell. Moreover, we believe that decreased SOD activity, which scavenges superoxide radicals, causing an interaction of large amounts of superoxide radicals with NO, resulting in the formation of large amounts of peroxynitrite, which can wreak havoc on cardiac tissues. Our findings are consistent with those of Khadrawy et al.²⁸ and Abudalo et al.⁵. In the present study, the administration of Lactobacillus probiotics suppressed the development of oxidative stress in cardiac tissue by rebalancing the oxidant/antioxidant system. Despite the paucity of evidence highlighting the role of probiotics in suppressing oxidative stress resulting from CP-cardiotoxicity, there is increasing evidence for their role in suppressing oxidative stress caused by insults other than CP. In this context, Lactobacillus rhamnosus, Lactobacillus helveticus, and Lactobacillus casei inhibited oxidative stress by decreasing MDA levels and improving SOD and CAT enzyme activity in the small intestine and lung after cadmium treatment of adult male Wistar rats²⁹. Wang et al.³⁰ reported that probiotics function as antioxidant agents in different ways. Probiotics contain antioxidant enzyme systems consisting of CAT and SOD, stimulate antioxidant system activity in host cells, produce metabolites with antioxidant capacity, such as GSH, butyrate, and folate, and play an important role in enhancing antioxidant signaling. Sengul et al.²¹ demonstrated that a mixture consisting of Lactobacillus rhamnosus, Lactobacillus fermentum, and Lactobacillus brevis suppressed oxidative stress resulting from treatment with CP in the renal tissue of rats.

The inflammatory process is an inevitable pathological change that occurs after tissue damage. CP stimulates the translocation of nuclear factor kappa B (NF-kB) from the cytoplasm to the nucleus as a result of oxidative damage in the cell. NF-kB induces the production of TNF-α in cardiac tissue, which is a pro-inflammatory cytokine that stimulates an inflammatory response during CP-induced toxicity^31,32. This pathological mechanism explains the high level of TNF-α observed in the present study. Moreover, targeting TNF-α with the Lactobacillus probiotics suppressed the inflammatory process in rat cardiac tissue exhibiting cardiotoxicity.

DNA damage, which can be predicted by measuring 8-OHdG expression in damaged tissues, is one of the most important complications resulting from oxidative stress induced by CP. The 8-OHdG levels increase following the attack of the hydroxyl radical on position 8 in DNA. This disorder occurs from the breakdown of the antioxidant system during an uncontrolled increase in ROS. Ince et al.³³ showed CP-induced DNA damage through increasing 8-OHdG levels in a model of CP-induced cardiac toxicity. Increased 8-OHdG expression in cardiac tissue after CP administration is a clear marker of damage to the genetic material of cardiac muscle fibers. In contrast, the observed decrease in 8-OHdG expression following Lactobacillus probiotic administration is an indication of the ability of these probiotics to protect the genetic material by scavenging ROS and restoring cellular homeostasis. A similar result was observed in a study applied by Sengul et al.²¹ using a model of CP-induced renal toxicity.

DNA damage resulting from oxidative stress, which is activated by CP, leads to mitochondrial membrane damage through the activation of tumor suppressor protein p53-induced Bax. Activated Bax stabilizes in the mitochondrial membrane and forms channels that cause cytochrome c leakage from the mitochondria^34,35. This pathway is strengthened through the suppression of Bcl-2, which is an anti-apoptotic mediator that inhibits Bax and maintains the permeability and integrity of the mitochondrial membrane. On the other hand, the accumulation of CP in mitochondria causes mitochondrial oxidative stress through excessive ROS production in the mitochondrial matrix³⁶, which results in mitochondrial dysfunction and activation of apoptosis factors³⁵. These clearly described pathological mechanisms underlie the observed irregularities in the expression of proteins that regulate apoptosis in the present study. Previous studies reported a disruption in proteins regulating apoptosis in renal²¹ and hepatic²² tissues following CP treatment. By enhancing Bcl-2 expression and suppressing Bax expression, Lactobacillus probiotics modulate proteins regulating apoptosis in a direction that ensures homeostasis in rat cardiac muscle fibers experiencing CP-induced cardiotoxicity. This modification positively affected the integrity of the mitochondrial membrane and prevented cardiac muscle fiber orientation toward apoptosis executioners.

Increased Bax, as a pro-apoptotic mediator, versus decreased Bcl-2, as an anti-apoptotic mediator induces cardiac muscle fibers to undergo apoptosis via caspase activity. Caspase-3 is one of the most important executioners of apoptosis. It is activated through several stages, which are initiated by cytochrome c leakage from damaged mitochondria. Caspase-3 functions inside the nucleus by degrading DNA. We found that high caspase-3 expression is a biomarker for the development of apoptosis in cardiac tissue following CP administration. Previous studies have focused on the role of CP in inducing apoptosis in cardiomyocytes through elevated caspase-3 because of a significant loss in mitochondrial transmembrane potential^31,37. Inhibiting increased caspase-3 expression represents an important ability of Lactobacillus probiotics to reduce CP-induced apoptosis in cardiac tissue. This may be attributed to its role in disrupting the apoptosis pathway by increasing Bcl-2 expression and suppressing Bax. Previously, the oral administration of the Lactobacillus ruteri ZJ617 and LGG probiotics suppressed lipopolysaccharide-induced apoptosis and reduced caspase-3 levels in mice administered lipopolysaccharide-induced barrier dysfunction of the ileum³⁸. In the present study, disrupting the antioxidant system, damage to genetic material, severe inflammatory response, mitochondria injury, and apoptosis induced by caspase-3 were accompanied by histopathological changes and the leakage of cardiac enzymes, such as troponin I, CK-MB, CK, LDH from cardiac muscle fibers into the serum. These enzymes function within cardiac muscle cells and are present in the blood at normal levels. An increase in the activity of these enzymes in the blood serum is a clear indicator of cardiac damage. Increased levels of cardiac enzymes were observed in the serum of rats with CP-induced heart damage³⁹. Suppression of the MDA/8-OHdG/TNF-α/Bax/Bcl-2/caspase-3 pathway by Lactobacillus probiotics improved the structural integrity of the cardiac muscle and prevented the leakage of troponin I, CK-MB, CK, and LDH from cardiac muscle fibers into the blood. The details of this study and the interplay between the studied changes are summarized in Fig. 6.

Fig. 6.

Schematic illustration of the study details.

With respect to the limitations of our study, the inclusion of p53 and cytochrome c within the targeted pathway may have expanded the results; however, examining these biomarkers will be the subject of subsequent studies. Despite these limitations, our conclusions were drawn based on several well-studied biochemical markers of oxidative stress and apoptosis.

Conclusion

Cardiotoxic complications resulting from CP administration, such as angina pectoris, myocarditis, and chronic heart failure, exhaust cancer patients, limit the use of CP and are considered a major challenge for oncologists and cardiologists. This study confirmed that oxidative stress, oxidative DNA damage, mitochondrial damage, severe inflammatory response, and apoptosis are key factors in the pathophysiology of CP-induced cardiotoxicity. To discover new drugs that reduce cardiac symptoms in cancer patients administered CP, our study examined the prophylactic potential of Lactobacillus probiotics against CP-induced cardiotoxicity in rats. This treatment exerted cardioprotective effects against CP-induced cardiotoxicity by inhibiting the MDA/8-OHdG/TNF-α/Bax/Bcl-2/caspase-3 pathway. We believe that the use of Lactobacillus strains against this pathway offers a promising approach for oncologists and cardiologists in this field.

Author contributions

M.A. contributed to writing (original draft, review and editing), software, formal analysis, investigation, and visualization, E.T.D. contributed to writing (review and editing), conceptualization, methodology, software, formal analysis, investigation, resources, data curation, and visualization, E.S. contributed to writing (review and editing), software, formal analysis, investigation, resources, data curation, and visualization, M.U. contributed to writing (review and editing), software, validation, investigation, and visualization, A.Y. contributed to writing (review and editing), conceptualization, methodology, software, investigation, resources, data curation, and visualization, S.A. contributed to writing (original draft, review and editing), software, investigation, and visualization, M.K. contributed to writing (review and editing), software, validation, formal analysis, investigation, resources, and visualization, and S.Y. contributed to writing (original draft, review and editing), conceptualization, methodology, software, formal analysis, investigation, resources, data curation, visualization, and supervised the study. All authors have read and agreed to the published version of the manuscript.

Funding

We thank Ataturk University (Erzurum, Turkey) for providing funding to this study (The project id TYL-2019-7403).

Data availability

The datas of this study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.