Introduction

Aluminum is the most abundant metallic element in the Earth’s crust, which is widely used in our daily life and industry due to its good physical and chemical properties. It has been shown that aluminum is a non-essential element in organisms, which can mediate acute toxicity or chronic toxicity [1]. Among various toxic effects caused by aluminum, neurotoxicity is undoubtedly the most prominent. Studies have confirmed that aluminum toxicity is involved in neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease [2, 3]. In addition, it can also cause liver, blood, immune, kidney, reproduction, and bone tissue damage [4,5,6].

With the development of research, the cardiovascular toxicity of aluminum and its compounds has attracted wide attention [7]. Epidemiological studies have found that aluminum has toxic effects on the cardiovascular system. With the increase of aluminum exposure, the risk of hypertension, coronary heart disease, atherosclerosis, and other cardiovascular diseases increases, and mortality of cardiovascular diseases also increases [8,9,10]. A previous study has found that aluminum can accumulate in the myocardium, leading to myocardial injury and affecting the electrical activity and systolic function of the heart [11]. However, the mechanism of aluminum cardiotoxicity is still unclear and urgent to be clarified.

It has been verified that oxidative stress is the main pathway of metal toxicity [12]. Apoptosis induced by mitochondrial damage is one of the main mechanisms of oxidative stress injury in cardiomyocytes [13]. The Bcl-2 family participates in the mitochondrial pathway of apoptosis and induces apoptosis through a series of processes, ultimately through the activation of caspase-3 [14]. This study aimed to investigate the molecular mechanism of aluminum-induced oxidative damage and apoptosis in rat cardiomyocytes, thereby providing a basis for the risk assessment of cardiovascular toxicity induced by aluminum exposure.

Materials and Methods

Reagent

Al chloride (Howei Chemical Co., Ltd., Guangzhou, China) was diluted with distilled water prior to administration.

Grouping and Modeling of Animals

Thirty-two male Wistar rats of SPF grade, weighing 150–180 g, were provided by Changsha Tianqin Biotechnology Co., Ltd. (License No. SCXK (Xiang) 2019-0014). The rats were reared at 27 °C room temperature and 50% humidity. The rats were fed with standard commercial rodent diet (Changsha Tianqin Biotechnology Co., Ltd., Changsha, China) and drinking water ad libitum. After 1 week of adaptation, thirty-two rats were randomly divided into the blank control group (GC), low-dose group (GL), medium-dose group (GM), and high-dose group (GH), with eight rats in each group. According to the methods in references [15, 16], the rats from the GL, GM, and GH groups were intraperitoneally injected with 5, 10, and 20 mg/(kg·d) AlCl3 solution, which was prepared with distilled water and autoclaved. The rats from the control group were injected with the same volume of normal saline (2 ml/rat/day). The frequency of injection was once a day, 5 days a week, for a total of 28 days. The rats were observed daily for abnormalities in food intake, drinking, behavior, urine, and feces. All animal experiments and procedures used in this study were approved by the Ethics Committee of Animal Experiments of Youjiang Medical University for Nationalities (approval date, 20 July 2020), according to the recommendations of the Guide for the Care and Use of Laboratory Animals in our institutes. All surgery was performed under pentobarbital anesthesia with minimum suffering in the animals.

Sample Collection

After fasting for 12 h at the end of modeling, the rats were anesthetized by i.p. administration of pentobarbital (45 mg·kg−1·BW−1). After anesthesia, 6 to 8 ml of blood was drawn from the abdominal aorta to a common anticoagulant-free biochemical tube. After centrifugation for 5 min at a speed of 4000 rpm, one serum sample was taken to determine the myocardial enzyme and the other sample was utilized to detect the content of aluminum.

The bloodstains of the heart tissue were rinsed with normal saline. Some of them were fixed with formalin for 48 h for routine section and embedding. The sections were then stained with hematoxylin and eosin (H&E) and TUNEL by the Pathology Department technicians. The pathological changes and apoptosis of cardiomyocytes were observed under a light microscope, and the other part was stored at − 80 °C in a cryopreservation tube for subsequent biological tests.

Determination of the Myocardial Enzyme

Lactate dehydrogenase (LDH), creatine kinase (CK), creatine kinase isoenzyme (CKMB), and alpha-hydroxybutyrate dehydrogenase (HBDH) were measured using an automatic biochemical analyzer (BECKMAN COULTER LX20, USA).

The experimental detection was carried out according to the operation instructions of kits (lactate dehydrogenase activity assay kit, creatine kinase activity assay kit, creatine kinase isoenzyme activity assay kit, alpha-hydroxybutyrate dehydrogenase activity assay kit (Colorimetric) (Biovision, Wuhan, China)) and instruments. Only the separated serum needs to be put on the machine. All other operations including sample loading and separation, stirring, and incubation were performed by the automatic biochemical analyzer.

Determination of Aluminum Content

The ICP-OES method was used to identify the level of aluminum in blood and heart tissue. The method for preprocessing the sample was as follows: the weighed sample was put into a digestion tube of polytetrafluoroethylene and added with a mixed solution of 0.5 ml hydrofluoric acid and 7 ml nitric acid. The reaction system was placed in a digestion tank and digested for 8 h at 180 °C. After complete dissolution and cooling, the sample was transferred to a constant volume apparatus for testing.

Determination of Malondialdehyde

The extent of lipid peroxidation by measuring thiobarbituric acid reactive substances (TBARS) in terms of malondialdehyde (MDA) formation was measured according to the method of Draper and Hadley [17], and the content of MDA was determined according to the methods indicated in the kit instructions (Beyotime Bio, Beijing, China). Take 100 mg of myocardial tissue into a cryopreservation tube for use. Normal saline homogenate of myocardial tissue was prepared under the condition of 1 to 4 °C, and 0.5 ml of a heart extract supernatant was mixed with 1 ml of trichloroacetic acid solution and centrifuged at 2500g for 10 min. The resulting supernatant (0.5 ml) and 1 ml of a solution containing 0.67% thiobarbituric acid (TBA) were incubated for 15 min at 90 °C and cooled. The mixture was measured for absorbance at 532 nm using a spectrophotometer (Jenway UV-6305, Shimadzu, Japan). The malondialdehyde values were calculated using 1,1,3,3-tetraethoxypropane as a standard and expressed as nanomoles of malondialdehyde per milligram of protein.

Histopathological Examination

Rat myocardial tissue was fixed in formalin solution, followed by paraffin embedding, conventional 4-μm section, and H&E staining. The morphological changes of myocardial tissue were observed under an optical microscope (BX53, Olympus, Japan).

TUNEL Staining

Myocardial tissue was embedded in paraffin and sectioned routinely. After dewaxing, specimens were treated with protease K to digest membrane protein and nuclear protein and then incubated with the TUNEL reaction solution at 37 °C for 1 h. The anti-fluorescein antibody was incubated and then visualized with DAB. The section was dehydrated, transparent, and mounted after nucleus counterstaining. The cells whose nuclei were stained brown-yellow were positive apoptotic cells. At high magnification, three fields were randomly selected from each section to calculate the apoptosis rate of cardiomyocytes in each field. The apoptosis rate (%) was equal to the number of apoptotic cells divided by the total number of cells.

Western Blot Analysis of Bcl-2, Bax, and Caspase-3

The heart tissue was ground with a liquid nitrogen grinder, and then, 100 μl of RIPA lysis buffer (Beyotime Bio, Beijing, China) per 10 mg of tissues was added. After shaking and mixing, the lysate was placed on ice for 5 min and centrifuged at 4 °C for 10 min (13,000 rpm). The supernatant was collected and the total myocardial protein was extracted. The BCA method was used to determine the protein concentration. After the loading concentration was adjusted, the protein was denatured and stored at −20 °C. Next, 5% stacking gel and 12% separation gel were prepared to load protein samples. GAPDH (Abbkine, Wuhan, China) was used as the internal reference. After loading the samples, a constant voltage of 80 V was first applied to electrophoresis until the bromphenol blue indicator runs to the junction of the two gels in a line. Then, change to a constant voltage of 120 V until the bromphenol blue runs to the bottom of the gel. The gel was transferred to the filter paper wet in transfer buffer solution at a constant current of 300 mA. After blocking, the membrane was placed in a shaker and incubated with rabbit polyclonal antibody (1:3000, Proteintech, Wuhan, China) of Bcl-2, Bax, and caspase-3 at 4 °C overnight. After rinsing the membrane thoroughly, the secondary antibody was hereby added and incubated at room temperature for 1.5 h. The film was exposed in the dark room after rinsing again. The film was then scanned after drying. The gray values were analyzed with Image-Pro Plus 6.0 (Media Cybemetics, USA).

Statistical Analysis

Statistical analyses were carried out using SPSS 20.0. The quantitative data were expressed as mean ± standard deviation (\( \overline{x}\pm s \)). One-way analysis of variance (ANOVA) was used for comparison among groups and the LSD test was used for comparison between any two of these groups. The non-parametric test (Kruskal-Wallis H test) was used when the variance was not uniform. The difference was statistically significant when P < 0.05. Data analysis and graph construction were performed with the aid of GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA).

Results

Myocardial Enzyme Levels

The myocardial enzyme levels of rats in this study were determined to understand the pathogenesis of cardiac toxicity and the effect of aluminum, and the results are listed in Table 1. With the increase of aluminum exposure concentration, the levels of myocardial enzymes tended to increase. Compared with that of the GC group, the serum LDH level of the GH group was significantly increased (P < 0.05); compared with that of the GC group, the serum CK level of the GM group was significantly increased (P < 0.05); the serum CKMB and HBDH levels of the GM and GH groups were significantly higher than those of the GC group (P < 0.05) (Table 1).

Table 1 The level of serum myocardial enzymes in different groups
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Levels of Aluminum in Serum and Tissues

After 28 days of aluminum exposure, the aluminum content in the myocardium and serum of rats was detected. Our results showed that the concentration of aluminum in the myocardium and serum showed an upward trend with the increase of aluminum exposure concentration. Compared with that of the GC group, the content of aluminum in the myocardium of the GL, GM, and GH groups was significantly increased (P < 0.05), showing a significant dose-response relationship in the myocardium (Fig. 1).

Fig. 1
figure 1

Aluminum concentrations in the serum (a) and myocardium (b). GC, GL, GM, and GH refer to 0, 5, 10, and 20 mg/(kg·d) of AlCl3 solution, respectively. Results are expressed as mean ± SEM. Statistical significance was determined by one-way analysis of variance (ANOVA) (a) and Kruskal-Wallis H test (b) (n = 8, aP < 0.05 vs GC; bP < 0.05 vs GL; cP < 0.05 vs GM)

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The Level of MDA in Myocardium

To investigate the oxidative stress level, MDA was used as an indicator of lipid peroxidation in the myocardium. As shown in Fig. 2, with the increase of aluminum exposure concentration, the concentration of MDA showed an upward trend; compared with that of the GC group, the myocardial aluminum content of the GM and GH groups was increased significantly (P < 0.05) (Fig. 2).

Fig. 2
figure 2

Concentration of MDA. Results are expressed as mean ± SEM. GC, GL, GM, and GH refer to 0, 5, 10, and 20 mg/(kg·d) of AlCl3 solution, respectively. Statistical significance was determined by one-way analysis of variance (ANOVA) (n = 8, aP < 0.05 vs GC; bP < 0.05 vs GL; cP < 0.05 vs GM)

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H&E Staining of Myocardial Tissue

The effect of aluminum exposure on myocardial morphological changes was observed by H&E staining. The cardiomyocytes of the GC rats were arranged closely with clear boundary, uniform staining, and abundant cytoplasm; no obvious thickening was seen in the pericardium, and no obvious inflammation was found in the tissues. A few hypertrophic cardiomyocytes were scattered in the GL and GM groups (black arrow). As for the GH group, a small number of cardiomyocytes underwent vacuolar degeneration, which was manifested by small round vacuoles in the cytoplasm (blue arrow); a few hypertrophic cardiomyocytes were scattered, and some cardiac muscle fibers broken (Fig. 3).

Fig. 3
figure 3

Pathological changes of myocardium tissues of rats in each group (H&E ×400). GC, GL, GM, and GH refer to 0, 5, 10, and 20 mg/(kg·d) of AlCl3 solution, respectively. The scale bar represents 50 μm. Hypertrophic cardiomyocyte cells (black arrow) and vacuolar degeneration (blue arrow)

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TUNEL Staining

The apoptosis of cardiomyocytes was detected by TUNEL staining. As shown by the TUNEL staining in Fig. 4, the apoptosis rate of cardiomyocytes showed an upward trend with the increase of aluminum exposure concentration; the apoptosis rate of cells in the GL, GM, and GH groups was significantly higher than that in the GC group, and the difference was statistically significant (P < 0.05) (Fig. 4).

Fig. 4
figure 4

The TUNEL-positive renal cells for four groups of rats (TUNEL staining, ×200) and TUNEL-positive cell count in rat myocardium. GC, GL, GM, and GH refer to 0, 5, 10, and 20 mg/(kg·d) of AlCl3 solution, respectively. Results are expressed as mean ± SEM. Statistical significance was determined by one-way analysis of variance (ANOVA) (n = 3, aP < 0.05 vs GC; bP < 0.05 vs GL; cP < 0.05 vs GM)

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Comparison of the Expression of Bcl-2, Bax, and Caspase-3

To investigate the effects of aluminum exposure on the levels of Bcl-2, Bax, and caspase-3, we detected the levels of these proteins using western blot. Our results showed that the level of Bcl-2 in the myocardium of the GL, GM, and GH groups was significantly decreased compared with that of the GC group (P < 0.05). With the increase of aluminum exposure concentration, the levels of Bax and caspase-3 in cardiomyocytes showed an upward trend; compared with that of the GC group, the level of Bax in the myocardial tissue was significantly increased in each dose of the aluminum exposure groups (P < 0.05); the level of caspase-3 in the myocardial tissue of the GM and GH groups was higher than that of the GC group (P < 0.05) (Fig. 5). Taken together, these results suggested that aluminum exposure could activate apoptosis by increasing the pro-apoptotic proteins Bax and caspase-3, and inhibiting anti-apoptotic protein Bcl-2.

Fig. 5
figure 5

The protein expression of Bax, Bcl-2, and caspase-3 in the myocardium tissues of each group. GC, GL, GM, and GH refer to 0, 5, 10, and 20 mg/(kg·d) of AlCl3 solution, respectively. Statistical significance was determined by one-way analysis of variance (ANOVA) (n = 8, aP < 0.05 vs GC; bP < 0.05 vs GL; cP < 0.05 vs GM)

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Discussion

Aluminum in drinking water, food, aluminum cookers, tableware, and drugs can enter the body through the skin, respiratory tract, and especially the digestive tract [18]. The majority of aluminum ions absorbed into the blood is combined with plasma proteins, and a small part forms free aluminum [19]. The combined aluminum and free aluminum can not only be eliminated by kidney but also be deposited in various tissues and organs with blood flow through the capillary wall. With the increase of aluminum intake, the amount of aluminum deposition is also accumulating, which aggravates the aluminum load in tissues and serum and eventually causes damage to tissues and organs [20, 21]. The blood aluminum level directly reflects the degree of aluminum exposure, while the tissue aluminum level can reflect the degree of aluminum accumulation and the possible damage to the tissues. This study demonstrated that the rats exposed to aluminum at different doses showed increased blood aluminum level compared with the control rats; the aluminum accumulation in the myocardium was enhanced with the increase of aluminum exposure dose, showing an obvious dose dependence, which was consistent with the study of Novaes RD et al. [11]. It was suggested that the heart was also one of the target organs of aluminum toxicity.

The early reaction of the heart caused by the short-term effect of cardiovascular toxicants is biochemical alternation, such as the changes of myocardial enzyme activity, energy metabolism, and ion homeostasis [22]. A series of cellular and molecular events of cardiomyocytes can be triggered by the continuous action of toxicants. Myocardial hypertrophy can be induced by the activation of hypertrophy genes and upregulation of transcription factors [23]. Myocardial hypertrophy in a non-physiological state at the initial stage belongs to the compensatory response of the heart to the changes of cardiac function caused by toxic effects, and such myocardial injury is reversible [24]. If the toxic substances still cannot be removed, a series of changes in the shape and function of the heart will appear, eventually leading to cardiomyocyte death characterized by cardiomyocyte apoptosis and necrosis [25].

Myocardial enzymes are important markers for the diagnosis of myocardial injury. Cardiomyocyte injury leads to the leakage of enzymes in cardiomyocytes to the outside of cells, so the release of these enzymes can be used as an indicator of the degree of cell injury [26]. This study showed that the levels of CKMB and HBDH in the middle- and high-dose groups of rats were significantly higher than those in the control rats. The LDH level in the high-dose group of rats was higher than that in the control rats, which indicated that aluminum load could induce cardiomyocyte injury. Additionally, H&E staining revealed that the cardiomyocytes of aluminum-exposed rats showed pathological damages, mainly manifested as vacuolar degeneration and myocardial hypertrophy. Cardiac hypertrophy attenuates the ability of cells to transport ions, including the relative deficiency of Ca2+ influx, which reduces the function of cardiomyocytes [27]. Moreover, the biological oxidation in hypertrophic myocardium is relatively weakened, resulting in insufficient myocardial energy generation and decreased cardiac function [28]. The pathological results further demonstrated the direct toxic effect of aluminum on rat myocardium.

Oxidative damage plays a vital role in the mechanism of aluminum toxicity. Aluminum is a positive trivalent element with relatively active properties, which can generate a large amount of ROS by interfering with the mitochondrial electron transport chain [29]. When the production of ROS exceeds the clearance rate of the body, the oxidation system and antioxidant system are out of balance; the excessive ROS participates in the intracellular reaction, causes lipid peroxidation, and finally generates MDA [30]. MDA can damage the biomembrane and cause cross-linking of proteins, nucleic acids, and other macromolecules, leading to cell metabolism dysfunction and even death. Due to the short half-life of ROS and the high determination requirement, this study adopted MDA to measure the degree of oxidation reaction. The results showed that the MDA level in the serum of rats exposed to aluminum was significantly higher than that of the control rats, which indicated that aluminum exposure triggered the oxidative stress reaction and activated the process of lipid peroxidation in vivo, thus increasing the production of MDA, the end product of lipid peroxidation. The result of this study was consistent with the finding of Ghorbel I. et al. [17], suggesting that aluminum caused myocardial injury through oxidative stress.

Oxidative stress is considered a critical regulatory signal of apoptosis [31]. The heart is composed primarily of long-lived and post-mitotic cells, which prefer fatty acids as substrate for energy production, so it becomes more susceptible to oxidative damage than other tissues. Oxidative damage leads to Ca2+ overload, MDA content increase, mitochondrial matrix over-swelling, permeability increase, and cytochrome C release. The free cytochrome C in the cytoplasm can activate caspase-related proteins to initiate the intracellular apoptosis signaling pathway, thus causing irreversible cardiomyocyte injury and apoptosis [32, 33]. Due to the nonrenewable nature of cardiomyocytes, apoptosis may lead to the permanent loss of some cardiac functions. The caspase family is one of the core proteins in apoptosis regulation. Emerging evidence has found that the caspase family proteins involved in cardiomyocyte apoptosis are mainly caspase-8 and caspase-3. Caspase-8 is located upstream of the apoptotic pathway, which can stimulate the downstream caspase-3 to cause apoptosis [34]. In this study, western blot results revealed that the myocardial tissues of rats exposed to aluminum showed increased levels of pro-apoptotic proteins (Bax and caspase-3) and decreased level of anti-apoptotic protein (Bcl-2), suggesting that Bcl-2, Bax, and caspase-3 might be implicated in cardiomyocyte apoptosis induced by aluminum. In the apoptosis signal transduction pathway, Bcl-2 is an anti-apoptotic protein and related to the stability of mitochondria, which can protect the stability of the cell membrane and prevent the transduction of apoptosis signal. The increase of Bax can form a heterodimer with Bcl-2 protein, which inactivates Bcl-2 and triggers the cascade reaction of caspase [35]. Caspase-3, the key executive molecule of apoptosis in the caspase family, is activated at the early stage of apoptosis and induces apoptosis [36]. A previous study has demonstrated that the number of early and late apoptotic cells is increased significantly after ALP treatment through flow cytometry assay, and apoptosis is mainly induced via the mitochondrial pathway [37], which is consistent with the conclusion of this study. TUNEL staining also showed that the apoptosis rate of cardiomyocytes in rats exposed to aluminum was significantly increased, which confirmed the toxicity of aluminum load on the myocardial injury.

In conclusion, the experimental results showed that aluminum could accumulate in myocardial tissues and cause damage to cardiomyocytes. It could induce oxidative stress damage by increasing the content of MDA in cardiomyocytes and trigger cardiomyocyte apoptosis by activating the pro-apoptotic proteins caspase-3 and Bax and reducing the anti-apoptotic protein Bcl-2.