Role of autophagy and apoptosis in aluminum exposure-induced liver injury in rats

doi:10.21203/rs.3.rs-2125719/v1

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Role of autophagy and apoptosis in aluminum exposure-induced liver injury in rats

https://doi.org/10.21203/rs.3.rs-2125719/v1

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published 05 Jan, 2023

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Aluminum exposure can lead to different degrees of damage to various organ systems of the body. It has been previously revealed that that aluminum exposure can damage the liver, causing liver dysfunction. However, the specific mechanism remains unclear. This research aims to uncover the damaging effect of aluminum exposure on rat liver and to demonstrate the role of autophagy and apoptosis in this effect. Thirty-two Wistar rats were randomly divided into the control group (C group), low-dose aluminum exposure group (L group), middle-dose aluminum exposure group (M group), and high-dose aluminum exposure group (H group) (n = 8). The rats respectively received intraperitoneal injection of 0, 5, 10 and 20 mg/(kg·d) AlCl₃ solution for 4 w (5 times/w). After the experiment, changes in the ultrastructure and autolysosome in rat liver were observed; the liver function, apoptosis rate, as well as levels of apoptosis-associated proteins and autophagy-associated proteins were detected. The results indicated that aluminum exposure damaged rat liver function and structure and resulted in an increase of autolysosomes. TUNEL staining revealed an elevated number of apoptotic hepatocytes after aluminum exposure. Moreover, we found from Western blotting that the levels of autophagy-associated proteins Beclin1 and LC3-II increased; apoptotic protein Caspase-3 level elevated and Bcl-2/Bax ratio reduced. Our research suggested that aluminum exposure can lead to high autophagy and apoptosis levels of rat hepatocytes, accompanied by hepatocyte injury and impaired liver function. This study shows that autophagy and apoptosis pathways participate in aluminum toxication-induced hepatocyte injury.

Aluminum exposure

autophagy

apoptosis

oxidative damage

hepatocyte injury

Aluminum (Al) is an abundant metal element in nature and is widely used in our life, industrial production, and healthcare industry due to its excellent characteristics. It can enter the human body through food, drinking water, air, medicine, and other ways [1]. The liver is the largest gland in the body and the most important organ for the metabolism and biotransformation of foreign chemicals, making it the most vulnerable organ [2]. The main absorption site of Al is the duodenum. The absorbed Al combines with proteins and enters the blood, by which is transported to various organs. Al accumulation in the liver can lead to pathological changes and liver dysfunction [3–4]. It has been reported that Al exposure can cause cristae rupture, dissolution, and collapse in mitochondria of hepatocytes. Moreover, it can lead to hepatic interstitial fibrosis and inflammatory cell infiltration, and also can result in abnormal synthetic and metabolic functions [5–6]. Patients taking Al nutrient solution for a long time may develop cholestatic liver disease [7]. Al-induced liver injury has been confirmed in vivo and in vitro, while its mechanism has not been elucidated yet [8–9].

Autophagy and apoptosis are crucial catabolic processes that maintain cell and tissue homeostasis. As the first recognized programmed cell death (PCD) procedure, the role and regulatory network of apoptosis have become clear gradually. However, apoptosis is not the only factor determining the fate of cell death. In recent years, autophagy, known as type II PCD, has been confirmed to regulate cell death together with apoptosis [10–11]. There is a close and complex relationship in determining cell fate between autophagy and apoptosis [12]. Currently, there are few studies on the effects of aluminum exposure on autophagy and apoptosis of hepatocytes, and its role in the process of aluminum-induced liver injury in rats remains unknown.

Our previous studies have also implied that Al exposure can damage the rat liver structure and function. This research focuses on the possible role of autophagy and apoptosis in the occurrence and development of Al-induced liver injury to further provide clues for the prevention and treatment of Al-induced liver injury.

Reagent

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

Animals and experimental design

Thirty-two male Wistar rats aging 5–6 w and weighing 150–180 g (Changsha Tianqin Biotechnology Co., Ltd.) were fed under standard conditions (4 rats in each cage, temperature of 24–27℃, relative humidity of 45%-65% and 12 h day/night cycle). The rats were provided with abundant food and water throughout the study process. This research was strictly in accordance with the Guide to the Management and Use of Laboratory Animals and was approved by the Ethics Committee of Youjiang Medical University for Nationalities (approval date, 20 July 2020). All surgery was performed under pentobarbital anesthesia with minimum suffering in the animals.

Acclimated for 7 d, Thirty-two Wistar rats were randomly divided into the control group (C group), low-dose aluminum exposure group (L group), middle-dose aluminum exposure group (M group), and high-dose aluminum exposure group (H group) (n = 8). The rats respectively received intraperitoneal injection of 0, 5, 10 and 20 mg/(kg·d) AlCl₃ solution for 4 w (5 times/w) [13–14]. At the end of 4 w treatment, blood was collected and allowed to clot to collect serum. Rats were killed to excise out the liver which was then soaked between filter papers to remove excess fluid.

Determination of aluminum

The aluminum level in liver tissue was determined using the ICP-OES method. The sample pretreatment methods were as follows: weigh the sample in the polytetrafluoroethylene digestion tube, add the mixture of 0.5 mL hydrofluoric acid and 7 mL nitric acid, and place it in the digestion tank at 180℃ for 8 h. After complete cooling, the sample was transferred to the constant volume instrument for testing.

Measurement of alanine aminotransferase (ALT) and aspartate aminotransferase (AST)

The contents of indices of the liver function ALT and AST were determined using an automatic biochemical analyzer.

Histopathological examination

For HE staining, the rat liver tissue was fixed in paraformaldehyde for 48 h, embedded in paraffin, sectioned, and stained with HE. Then, the morphological changes of the liver were observed under the light microscope.

Transmission electron microscopy (TEM) observation

Liver tissue blocks were rinsed with normal saline and cut into small cubes, which were placed into prepared fixation liquid, sectioned, and stained. The ultrathin sections were put onto a single-hole copper mesh box and a TEM was used for observation and photographing.

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay

The liver tissue samples were embedded using paraffin and regularly sectioned. The dewaxed sections were treated with proteinase K to detach the membrane proteins and nucleoproteins and then were incubated with TUNEL reaction solution at 37℃ for 1 h. Incubated with the anti-fluorescein antibody, the sections were developed using diaminobenzidine (DAB), counterstained, dehydrated, permeabilized, and sealed. Judgment of the results: cells with brownish-yellow-stained nuclei were positively expressed apoptotic cells. Three fields were randomly selected from each section to calculate the apoptosis rate in each of them based on the formula: apoptosis rate (%) = the number of apoptotic cells/the number of total cells.

Western blot assay

The liver tissues (100 mg) were homogenated with 1 mL lysis solution containing proteinase inhibitor under low temperature. The fully lysed cells were centrifuged at 4℃ and 13,000 r/min for 10 min with the supernatant collected. Bicinchoninic acid (BCA) kit (Beyotime Bio, Shanghai, China) was used to detect the protein concentration and adjust it to be consistent. The samples were added with protein loading buffer with the loading amount balanced, performed with 70℃ water bath for 10 min, and subpackaged after protein denaturation. Then, gels with different concentrations based on the proteins to be determined were prepared: 5% spacer gel, 15% separation gel for LC3-II (Abcam, Cambridge, UK), and 12% separation gel for Beclin1 (Abcam, Cambridge, UK), Bcl-2, Bax, caspase3 (ProteinTech, Wuhan, China) respectively showing luminescence on different membranes. The proteins were loaded and the constant voltage was regularly set to 80 V, which was then adjusted to 120 v after the bromophenol blue indicator became linear at the junction of spacer gel and separation gel with the electrophoresis gone on. The electrophoresis was continued until the bromophenol blue reached the bottom of the gel, and the electricity was adjusted to 250 mA constant current. The samples were transferred onto membranes in the prepared and precooled electroporation buffer (45 min for LC3-II and 80 min for Beclin1, Bcl-2, Bax, caspase3), and then the membranes were blocked with 5% blocking buffer and incubated with relative primary antibodies at 4℃ overnight. Subsequently, the membranes were washed and incubated with secondary antibodies at room temperature for 2 h. Finally, a moderate amount of chemiluminescent liquid was prepared at a ratio of A liquid: B liquid = 1: 1. After development, the films were scanned and the gray values were analyzed using image-pro plus 6.0 (Media Cybemetics, U.S.A).

Statistical analysis

All data analyses were conducted using SPSS 22.0 software and the measurement data conforming to the normal distribution were expressed as mean ± standard deviation. The results were analyzed statistically using a one-way analysis of variance followed by Student–Newman–Keuls test. The level of significance was set at p < 0.05 for all statistical analysis.

Aluminum content in liver tissue of rats

After 28 days of aluminum exposure, the aluminum content in liver tissue of rats was detected. With the increase of aluminum exposure concentration, the aluminum content in liver tissue presented an upward trend. Compared with that of the C group, the aluminum content in liver tissue of the L, M, and H groups was significantly increased (P < 0.001), showing a significant dose-effect manner (Fig. 1).

Effect of Al exposure on ALT and AST

The levels of ALT and AST under different concentrations of Al exposure were detected in this study to understand the effect of Al exposure on rat liver function. As shown in Fig. 2, different decrees of liver dysfunction appeared in the Al exposure groups with the increased Al exposure concentration. The higher Al exposure concentration was associated with the higher ALT level. Compared with the C group, the rat serum ALT level was significantly increased in the M group and the H group (P < 0.05); the M group had the highest AST level, and the M group as well as the H group had significantly higher AST level than the C group (P < 0.05).

Pathological changes of rat liver

The effect of aluminum exposure on hepatic histopathological changes was observed by H&E staining. The histopathological results showed that the hepatocytes of the C group were closely arranged, with clear boundary, abundant cytoplasm, uniform staining, round nucleus, normal size, intact and normal venous endothelium, and no obvious abnormality was found in the tissue. The aluminum exposure groups showed loose and light stained cytoplasm of hepatocytes (black arrow), some vacuolated cells, necrotic hepatocytes, nuclear condensation and deep staining, and enhanced acidophilia (red arrow), with the group H the most serious (Fig. 3).

Effect of Al exposure on rat hepatocyte ultrastructure

It was observed under a TEM that there were varying differences in hepatocytes among the groups. Rat hepatocytes in the C group showed a fair overall structure, with complete membranes, abundant organelles in the cytoplasm, round nuclei, complete nuclear membranes, normal perinuclear space, and large nucleolus. There were abundant mitochondria with complete membrane and cristae. The ribosomes attaching to the rough endoplasmic reticulum surface could be observed and there showed many evenly distributed glycogen particles in the cytoplasm. There were no typical autophagosomes and autophagosomes. Rat liver tissues in the L, M, and H groups showed different degrees of fibrosis. Detailedly, there were slight edema and high electron density in hepatocytes, many collagenous fibers around the cells, tortuous dilation of bile canaliculi and many medullary substances and cholestasis in the lumen. The nuclei were irregular in shape with vague nuclear membrane structure; we found that the mitochondria were in large quantity with slight swelling, irregular shape, and ruptured and decreased cristae; the lipid droplets were diffusely distributed and there were many autolysosomes (ASS), the number of which was 8 in the H group (Fig. 4).

Effect of Al exposure on rat hepatocyte apoptosis

TUNEL staining was performed for the histological confirmation to explore the effect of Al exposure on hepatocyte apoptosis. As shown in Fig. 5, cells with brownish-yellow-stained nuclei were apoptotic cells. No apoptotic hepatocyte was found in the C group and the other 3 groups under different doses of Al showed a small number of apoptotic hepatocytes. The result of further comparison suggested that the apoptosis rate in the H group was significantly higher than that in other groups (P < 0.05).

Effect of Al exposure on expression of apoptosis-associated proteins and autophagy-associated proteins in rat liver tissues

We performed Western blotting to determine the expression of apoptosis-associated proteins and autophagy-associated proteins in rat liver tissues and the results were shown in Fig. 6.

(1) There was no significant difference in the expression of Beclin1 proteins between the C group and aluminum exposed group. Beclin1 protein expression in the H groups was obviously increased versus the L group (P < 0.01); LC3-II was elevated with the increased exposure concentration of Al and the L, M and H groups showed significantly higher LC3-II expression in comparison to the C group (P < 0.01). These data indicated that Al exposure can lead to an increased autophagy level in hepatocytes (Fig. 6A).

(2) In contrast to the C group, the M group had markedly higher Bcl-2 protein expression (P < 0.05); With the increase of Al concentration, Bax and caspase-3 expression levels were on the rise, which were increased in the M and H groups versus the C group (P < 0.01); while Bcl-2/Bax ratio was decreased and this decrease was much significant in the H group than the C group (P < 0.05). These results implied that Al exposure can induce hepatocyte apoptosis (Fig. 6B).

Al is not an essential trace element for the human body, excessive intake of which has potential toxicity to the human body [15]. Part of Al can be metabolized in the liver after it entered the body, thus excessive Al intake can cause damage to the liver [16–17]. Our study showed that the Al concentration in rat liver tissues exposed to Al was increased significantly compared with the control group, suggesting that Al may accumulate in the liver and result in damage. ALT and AST are liver function enzymes closely related to liver function, which can be used as the basis for the diagnosis of hepatocyte injury when too many liver function enzymes were transferred into the blood circulation. Therefore, ALT and AST are biomarkers reflecting the severity of many liver diseases [18]. This research revealed that the levels of ALT and AST in rats from the M and H groups were enhanced significantly after Al exposure. As observed under a TEM, there were hepatocyte edema, slight swelling in mitochondria, irregular shape, and ruptured and decreased cristae in rats exposed to Al, which were consistent with the results of a study by Al-Hazmi MA [19]. These data indicated that Al exposure does cause damage to rat liver function and structure.

The mechanism of Al toxicity may be related to the following aspects: oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress, autophagy disorder, apoptosis, and disturbed homeostasis of other metal ions [20–21]. However, the accumulation of Al in different tissues is different [22] and the toxicity of Al to different tissues and organs is not the same as well [23]. The specific mechanism of Al toxicity to the liver is not clear and needs to be further explored. Al is a positive trivalent element, which is more active and easy to lose electrons. It can promote the formation of reactive oxygen species (ROS), improve the oxidation level and reduce the function of the antioxidant defense system and induce the oxidative stress response in cells [24]. Moreover, ROS can also lead to endoplasmic reticulum stress and mitochondrial dysfunction through a series of pathways [25]. It has been reported that oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum stress can eventually induce abnormal autophagy or excessive apoptosis, thus damaging cells and tissues and further affecting their biological activities [26–28]. As a key way of affecting tissue function, it is particularly important to explore the significance of autophagy and apoptosis in liver injury. There are few studies on autophagy disorder and apoptosis in Al-induced liver injury. Therefore, this study mainly discussed the mechanism of Al-induced liver injury based on autophagy and apoptosis.

Apoptosis is a process of autonomous cell death controlled by genes. Among many factors regulating apoptosis, the Bcl-2 family and caspase family play an important role [29]. B-cell lymphoma-2 gene (Bcl-2) is an inhibitor of the apoptosis gene [30], which can inhibit cell apoptosis. Bcl-2 protein is related to the stability of mitochondria, which can protect the stability of cell membrane and prevent the transmission of apoptosis signals; Bax is an apoptosis-promoting protein and the dimer formed by Bax itself can directly promote cell apoptosis [31]. In addition, the increase of Bax protein can lead to the formation of heterodimers with Bcl-2 protein, thus inactivating Bcl-2. The lower the ratio of Bcl-2 to Bax, the higher the probability of cell apoptosis, which leads to the cascade reaction of caspase [32]. Apoptotic cell death is ultimately completed by the caspase family [33]. Caspase-3 is located in the center of the caspase cascade reaction and is the executor of apoptosis. When Caspase-3 is activated by the upstream signal, it will cleave itself into an active state, hydrolyze downstream substrate, and cut off DNA, leading to apoptosis [34–35]. Once Caspase3 is activated, cell death cannot be reversed [36]. The results of our research showed that the expression level of Caspase-3 and Bax were increased and the anti-apoptotic protein Bcl-2 was also slightly increased, whereas the ratio of Bcl-2 to Bax was decreased, suggesting that Al exposure can promote the occurrence of apoptosis. TUNEL staining showed that the apoptosis of the H group was higher than that of the control group. These results were further verified by histology, which confirmed that apoptosis was involved in the liver injury effect. However, the number of apoptotic cells was small and the liver has a strong compensatory ability, thus apoptosis may not have a significant impact on liver function damage. Furthermore, it is not ruled out that there are other pathways participating in the liver injury effect.

Autophagy is a lysosome-mediated process of protein and organelle degradation and is involved in the regulation of cell metabolism and survival. Autophagy dysfunction is related to many diseases, such as tumorigenesis [37], aging [38], and neurodegeneration [39]. Proper autophagy is a self-protection mechanism, while excessive autophagy can lead to cell death [40]. Beclin1, a protein encoded by the BECN1 gene, is an important molecule regulating autophagy, which can mediate related autophagy proteins to act on autophagic vesicles and react with a variety of proteins to regulate the formation and maturation of autophagosomes [41]. Microtubule-associated protein LC3 is a marker protein related to autophagy and the LC3-II positive quantification can be used to evaluate the number of autophagosomes in cells [42]. In this study, it was found through Western blotting that the expression levels of LC3-II and Beclin1 proteins in hepatocytes of rats exposed to Al were significantly increased. It was observed under a TEM that autophagosomes in hepatocytes of rats exposed to Al were significantly increased, mitochondria were obviously damaged, and undegraded lysosomes were increased. These results suggest that the autophagy pathway is implicated in Al-induced liver injury.

Autophagy and apoptosis are two important catabolic processes maintaining cell and tissue homeostasis and widely exist in the eucaryotic organism. Although the characteristics and mechanisms of apoptosis and autophagy are different, these two pathways are not independent of each other. They share the same stimulating factors and regulatory proteins, and between them are complex dialogues [43]. The regulation modes of autophagy and apoptosis can be roughly divided into two types: cooperative relationship and antagonistic relationship [44–45]. In our research, the expression of autophagy- and apoptosis-related factors in hepatocytes showed that the effect of Al exposure on hepatocytes was reflected by the upregulation of autophagy level and apoptosis level, suggesting that autophagy and apoptosis showed a cooperative relationship. However, the specific mechanism remains to be further explored.

In conclusion, the results of our 4 weeks experiments showed that autophagy and apoptosis pathways are involved in Al-induced liver injury, while their role in liver injury and the relationship between them are still unclear. Our next step is to intervene and regulate autophagy and apoptosis and extend the time of our research to further observe the liver-toxic effects of apoptosis and autophagy under different exposure time and intervention levels and to further explore the regulatory mechanism between apoptosis and autophagy.

Author Contribution All authors made contributions to the research design. Xi Wei and Dong Li established the animal model and analyzed the samples. Yueling Luo performed a statistical analysis of the data. Biaoliang Wu directed the design of the research. Xi Wei drafted the manuscript. All authors read and approved the final manuscript.

Funding This work was co-financed by the Guangxi Science Natural Foundation for Young Scientists (No. 2019GXNSFBA245045) and the Young and Middle-aged Backbone Talents Scientific Research Projects of the Affiliated Hospital of Youjiang Medical University for Nationalities in 2021 (No. Y20212606)

Conflict of Interest The authors declare no competing interests.

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No competing interests reported.

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published 05 Jan, 2023

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Role of autophagy and apoptosis in aluminum exposure-induced liver injury in rats

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Journal Publication

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Abstract

Figures

Introduction

Materials And Methods

Reagent

Animals and experimental design

Determination of aluminum

Measurement of alanine aminotransferase (ALT) and aspartate aminotransferase (AST)

Histopathological examination

Transmission electron microscopy (TEM) observation

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay

Western blot assay

Statistical analysis

Results

Aluminum content in liver tissue of rats

Effect of Al exposure on ALT and AST

Pathological changes of rat liver

Effect of Al exposure on rat hepatocyte ultrastructure

Effect of Al exposure on rat hepatocyte apoptosis

Discussion

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1