Abstract
Aluminum (Al) alters iron regulatory factors content and leads to the changes in iron-related proteins causing iron accumulation. But limited evidence ascertains this hypothesis. Therefore, our experiment was conducted and four groups of male Wistar rats were orally administrated of 0, 50, 150, and 450 mg/kg BW/d aluminum chloride (AlCl3) for 90 days by drinking water, respectively. The cognitive function, pathological lesion of hippocampus, oxidative stress, as well as iron-related proteins and iron regulatory factors expression were detected. The results showed that AlCl3 remarkably induced the oxidative stress and pathological lesion in the hippocampus and impaired the learning-memory ability. The contents of Al and iron increased in all AlCl3-exposed groups. Meanwhile, the increased divalent metal transporter 1 (DMT1) expression enhanced iron import and the decreased ferroportin 1 (Fpn1) expression reduced iron export in AlCl3-exposed groups. The iron accumulated and ferritin heavy chains (Fth) expression decreased in all AlCl3-exposed groups led to an increase in free iron. The study also showed that iron regulatory factor iron regulatory protein 2 (IRP2) was decreased and hepcidin was increased in AlCl3-exposed groups. The results indicated that AlCl3 induces iron dyshomeostasis presenting as iron accumulation, the disordered expression of iron import, export, store, and regulatory proteins in rat hippocampus accompanied with oxidative stress, pathological lesion, and impaired learning-memory ability.
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Introduction
The presence of Aluminum (Al) has increased in our life through water, food, medicine and various utensils [1,2,3,4]. Al is less toxic than a heavy metal, and so it is often overlooked. However, potential Al accumulation can cause neurotoxicity in animals and humans [5]. Al can cross the blood-brain-barrier quickly, and the relatively slow rates of elimination contribute to the accumulation of Al in the brain [6]. Al accumulation in the brain has been implicated in the etiology of a variety of neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) [5, 7]. The specific molecular mechanism for neurodegeneration induced by Al is incompletely understood.
The main region of Al accumulation in the brain was the hippocampus, a brain regional related to learning-memory ability [8]. Al exposure impairs learning-memory function in humans and animal models [9]. Impaired learning-memory ability and histopathological alteration reflected the neurotoxicity of aluminum [10, 11]. Thus, the Morris water maze and histopathological alteration were essential indicators of neurotoxicity in Al-exposed animal models.
The neurotoxic mechanisms of Al have been implicated gradually. The oxidative lesion was one of the neurotoxicity mechanisms of Al [12]. The oxidative stress (levels of ROS, MDA, and protein carbonyl) and antioxidative activity (the activities of TAC, SOD, and CAT) were considered to reveal the oxidative lesion [13]. Fe-induced oxidative stress might contribute to the oxidative injury during Al exposure [14]. Evidence showed that Al caused iron accumulation in the brain [15, 16]. However, the mechanism remains unclear.
The homeostasis of iron is vital in the brain, which is tightly regulated by iron-related proteins, including transferrin receptor (TfR) and divalent metal transporter 1 (DMT1) for iron uptake, ferritin (Fn) for iron storage and ferroportin 1 (Fpn1) for iron export. Moreover, the iron-related proteins are regulated by iron regulatory proteins (IRPs) and hepcidin [17]. Under the stimulation of external factors, the change of the above proteins would lead to iron dyshomeostasis and neurotoxicity. Lead decreases the expression of Fpn1, causing iron accumulation in the brains of aged rats [18]. Manganese accumulation increases the expression of DMT1 and reduces the expression of FP1, which probably involved in the processes of dopaminergic neuron loss in the rats with Mn-induced parkinsonism. [19] Similarly, inflammation and intracerebral hemorrhage alter the expression of DMT1, FPN1, and hepcidin, which causes iron accumulation in nervous cells [20, 21]. The roles of TfR, DMT1, Fn, IRPs were focused in the neurotoxicity of Al in vitro [22, 23]. However, they have not been examined systematically to clarify the mechanism of iron accumulation induced by Al. Thus we speculate that Al changes iron-related proteins levels by regulating iron regulatory factors content, eventually causing iron accumulation.
The iron accumulation mechanism was investigated in this study by evaluating the effect of Al on iron-related proteins and iron regulatory factors in the hippocampus of rats. These endpoints were evaluated along with the effect of Al on cognitive function, hippocampal histopathology, and oxidation and antioxidant markers after 90 days of exposure to aluminum chloride (AlCl3) in deionized water.
Materials and Methods
Animals and Treatment
One hundred and twenty male Wistar rats (SPF, three weeks old) were purchased from Harbin Medical University (license number: SCXK-2013-001), were divided equally into four groups: control group (CG, 0 mg/kg BW AlCl3), low-dose group (LG, 50 mg/kg BW AlCl3), mid-dose group (MG, 150 mg/kg BW AlCl3), and high-dose group (HG, 450 mg/kg BW AlCl3) after acclimatized for one week. The rats were orally treated daily with 99.99%AlCl3·6H2O (Aladdin, China) dissolved in deionized water in AlCl3-exposed groups, and deionized water in CG for 90 d by drinking water. The cage was polypropylene PP material and stainless steel wire without Al. A standard laboratory diet was fed to all rats with ad libitum access to feed and water. Feed is the standard clean grade pellet diet (Xietong Organism, China). The nutrient table of pellet diet is presented in supplemental information. All rats received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory animals”. The rats were housed in the Biomedical Research Center, Northeast Agricultural University, China. We have made all efforts to minimize the number of animals used and their suffering. The experimental protocol was carried out according to the Guiding Principles in the Use of Animals in Toxicology, and adopted by the Chinese Society of Toxicology. The experimental procedures were approved by the Animal Ethics Committee of the Northeast Agricultural University (Harbin, China).
Dosage Information and Dosage Regimen
AlCl3 was administered according to our previous dose that exerted neurotoxicity for 90 days [24]. We adopted the amount of AlCl3 administered according to European Food Safety Authority recommended Al doses for children (EFSA, 2013) and the values of oral uptake that promoted neurotoxicity [25]. The dose of AlCl3 in rats was given on a basis of body weight of rats. During Al exposure, the water consumption was increased gradually with the increased of rat body weight. Therefore, in order to maintain a constant dose of AlCl3 (mg/kg BW), the concentration of AlCl3 (g/L) in drinking water was varies accordingly. The water consumption was recorded every day and the body weight was measured every five days. We calculated the concentration of AlCl3 to be configured and then adjusted the dose accordingly.
Morris Water Maze
Briefly, the Morris Water Maze (MWM) test was conducted in a black circular pool with a diameter of 160 and 30 cm depth. The pool was filled with 20 ± 1 °C water and the water depth was 1 cm higher than the escape platform. The water was made opaque white using carbon to hide the escape platform. The water maze was divided into four quadrants and placed in a quiet room decorated with visual cues. Before the tests, rats were first tested for their performance to exclude the possibility that the rats had impairment in visual ability or in swimming capacity, and to facilitate their habituation to the black circular water pool. In the acquisition trial, the platform location was kept constant and the rats were placed randomly into the three starting quadrants except the quadrant on the platform, avoiding track memorization. When the rats found the platform within 90 s, the time of arrival was recorded. They were then made to stay on the platform for 30 s. When the rats failed to reach the platform during the 90 s, we recorded the escape latency as 90 s. Subsequently, rats were guided to the platform and then allowed to remain for 30 s. After the completion of the 5 days learning phase, rats were tested for memory retention of platform location. On the 6th day, the escape platform was removed, and each rat was allowed to swim freely for 90 s to record the frequency crossing original platform location and the swimming time and the path length in target quadrant. The behavior tests were performed after AlCl3 exposure of 90 days. The SuperMaze software (version 2.0; Shanghai Xinruan Information Tech Co., Shanghai, China) was used to analyze the data. All rats were in a normal state before and during the behavior acquisition.
Sample Collection
After 90 days of AlCl3 exposure, rats were deeply anesthetized with sodium pentobarbital (50 mg/kg, i. p.) [26]. One part of the rats (n = 10 per group) was transcardially perfused with 150 mL of normal saline followed by 200 mL of 4% paraformaldehyde in PBS. And then the whole brain was immediately removed from the cranial cavity and fixed in 4% paraformaldehyde for hematoxylin and eosin (HE) staining and immunohistochemical staining. The hippocampus was isolated immediately and washed in ice-cold saline. Hippocampus of part of rats was homogenized for kit determination. Hippocampus of the rest of rats was stored at −80 °C for determination of Al and iron content and protein extraction.
Histopathology Examination
After 4% paraformaldehyde fixed for 24 h, five brains per group were embedded in paraffin using standard histological procedures. After that, brains were sectioned coronally in 5 mm thickness, and then, brain slices containing the hippocampus were underwent dewaxing and hydration procedures. The sections were then routinely stained with HE Staining kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions. The final images were captured by using an Olympus light microscope (Olympus BX51, Tokyo, Japan) with an attached photographic machine (BX-FM; Olympus Corp, Tokyo, Japan). For quantification, three different views of hippocampal CA1 region were selected from each brain slice at 200× magnification. The necrosis rates of the necrotic neuron in the total neuron were neurons were manually counted along the ipsilateral of hippocampal CA1 region.
Determination of Al and Iron Content in the Hippocampus
Iron and Al content in hippocampus were determined using inductively coupled plasma mass spectroscopy (ICP-MS) (Thermo Fisher, X Series, FL, USA). Approximately 200 mg of fresh hippocampus tissue was added to 8 mL of 70% ultra-pure nitric acid at 200 °C for 4 h. The completely digested samples were cooled to room temperature, diluted to 50 mL with metal-free double distilled water, and analyzed by ICP-MS for metal content. Metal concentrations were recorded as μg/mg wet weight.
Determination of the Oxidation and Antioxidant Markers in the Hippocampus
Hippocampus samples were cryogenically homogenized in phosphate buffer saline (w/v, 1: 9). The homogenized solution was centrifuged at 3500 g for 15 min at 4 °C and the content of the protein sample was quantified using assay kits (Beyotime Institute of Biotechnology, Jiangsu, China). Protein concentration of spleen homogenates and single cell suspensions were determined by the BCA analysis kit (Beyotime Institute of Biotechnology, Jiangsu, China). ROS content was detected using chemiluminescence method. MDA content was detected using visible light method. Protein carbonyl content and CAT activity were detected using visible light method. SOD activity was detected using thiobarbituric acid method. T-AOC activity was detected using rapid ABTS method. All commercial kits were purchased from Nanjing Jiancheng Bioengineering Institute. The fluorescence of ROS was determined using a fluorescence microplate reader (FP-6500, JASCO, Japan) at 488 nm excitation and 525 nm emission wavelength. The absorbance of MDA, SOD CAT, and TAC was recorded at 532, 414, 405, and 520 nm by a 318 MC microplate reader (Shanghai Sanco instrument Co., Ltd., China), respectively. The absorbance of protein carbonyl was recorded at 370 nm by a ultraviolet spectrophotometer (T6, Beijing PGENERAL instrument Co., Ltd., China).
Determination of Protein Expression in the Hippocampus by Western Blot
Total protein from the hippocampus was extracted by superactive RIPA lysis buffer (Beyotime, China) with PMSF and Protease inhibitor cocktail (Beyotime, China), and then protein concentration was determined by BCA analysis kit (Beyotime, China). Western blot analysis was carried out according to previous study with modification [27]. Proteins (30–50 μg) of each lane were separated on sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, USA). After blocking in 5% fat-free milk in Tris Buffered Saline (TBS) containing 0.1% Tween-20 for 1 h, the membranes were then incubated overnight at 4 °C with the following primary antibodies: anti-IRP1 (1:600, Santa Cruz, CA, USA), anti-IRP2 (1:600, Santa Cruz, CA, USA), anti-TfR (1:600, Santa Cruz, CA, USA), anti-Fth (1:600, Santa Cruz, CA, USA), anti-Ftl (1:600, Santa Cruz, CA, USA). anti-DMT1(1:600, Santa Cruz, CA, USA), anti-Fpn1 (1:600, Bioss, Beijing, China), anti-β- actin (1: 6000 ZSGB-BIO, Beijing, China). Following washes using TBST (3 × 10 min), membranes were stained with the appropriate horseradish peroxidase-conjugated second antibody (1:5000, ZSGE-BIO, Beijing, China) for 2 h at room temperature, then washed 5 times with TBST. The object protein was detected using the enhanced ECL reagent (P0018 Beyotime institute of biotechnology, Jiangsu, China). β-actin was used as the internal control. Quantitative analysis was carried out using Amersham Imager 600 (Fairfield, USA).
Determination of Hepcidin Content by Immunohistochemical Staining
For immunohistochemical staining, brain specimens were fixed in 4% paraformaldehyde for 4–6 h at 4 °C and then immersed in 30% sucrose in 0.1 mol/L PBS solution for 24 h. Five brains per group were embedded in paraffin, cut into 4-μm thick sections and mounted on slides. Briefly, the paraffin-embedded tissue sections were deparaffinized with xylene and rehydrates with alcohols series. Prior to staining, sections were treated with 3% hydrogen peroxide followed by washing with PBS and incubation with 3% BSA for 30 min at room temperature. Primary antibody of anti-Hepcidin (1:100, Abcam, MA, USA) was added and incubated overnight at 4 °C. After washed 3 times with PBS for 10 min, the brain sections were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:200, Servicebio, Wuhan, China) for 60 min at RT. The slides were then washed three times with PBS for 5 min. The staining reaction was carried out using DAB (Servicebio, Wuhan, China) as the chromogen and counterstained with hematoxylin. The sections were dehydrated in ethanol, cleared in xylene, and cover-slipped with neutral balsam. The final images were captured by using an Olympus light microscope (Olympus BX51, Tokyo, Japan) with an attached photographic machine (BX-FM; Olympus Corp, Tokyo, Japan). The integral optical density (IOD) in the positive areas of the rat hippocampus was measured using Image-Pro Plus 6.0 software.
Data Analysis
All analyses were performed as described [28]. Data were expressed as mean ± standard error (mean ± SEM). Statistical significance was analyzed using one-way ANOVA followed by LSD test as the post-hoc test (SPSS 22.0 software; SPSS Inc., Chicago, IL, USA). In all statistical comparisons, values of p < 0.05 were considered significantly, and values of p < 0.01 were considered markedly significantly.
Results
The Contents of Al and Iron in Hippocampal
As shown in Fig.1, the content of Al and iron of all AlCl3-exposed groups were significantly increased than those of the CG in the hippocampus (p < 0.05, p < 0.01), and iron content is proportional to Al content.
Effects of AlCl3 on Learning-Memory Abilities
As shown in Fig.2, the time to reach platform was no differences among all groups on first day (p > 0.05). Over 5 days of training, all groups improve their ability to find the submerged platform. Regarding AlCl3 effect, the escape latency of the MG and the HG was significantly longer than that of the CG (p < 0.05, p < 0.01), but the time to reach platform of the LG was similar to the CG (p > 0.05). In probe trials, the swimming time and path length in target quadrant in the CG were significantly higher than that in the MG and the HG (p < 0.05, p < 0.01), and there was no significantly difference between the LG and the CG (p > 0.05). These results indicate AlCl3 impair learning-memory abilities.
Effects of AlCl3 on Hippocampal Structure
As shown in Fig.3, HE staining revealed that the hippocampal neurons were distinct and regular in structure, and arranged densely in the CG. In contrast, the AlCl3-exposed groups revealed significant injuries including cell loss and karyopycnosis as well as loose and disorderly arrangement in hippocampal neurons in AlCl3-exposed groups. Quantitative analysis of necrotic neurons showed that AlCl3 significantly increased neuronal loss compared with the CG (p < 0.05, p < 0.01). Cumulatively, these observations suggest that AlCl3 remarkably caused pathological changes in the hippocampus, accompanied with the neurodegeneration and neuronal cell damage/death.
Effects of AlCl3 on Oxidation and Antioxidant Markers in the Hippocampus
As shown in Fig.4, compared with the CG, the levels of ROS, MDA were significantly increased in AlCl3-exposed groups (p < 0.05, p < 0.01), and level of protein carbonyl was significantly increased in the MG and HG (p < 0.01). The activities of TAC and SOD were significantly decreased in AlCl3-exposed groups (p < 0.05, p < 0.01), and the activity of CAT was significantly decreased in the MG and HG compared with the CG (p < 0.01). The results indicate AlCl3 induces oxidative stress in the hippocampus.
Effects of AlCl3 on Iron-Related Proteins in the Hippocampus
As shown in Fig.5, compared with the CG, the protein expression of Fpn-1, TfR and Fth was significantly decreased in the MG and HG (p < 0.01); and the protein expression of DMT1 was significantly increased in AlCl3-exposed groups (p < 0.05, p < 0.01). There was no significant change in the Ftl protein content of all groups (p > 0.05).
Effect of AlCl3 on Iron Regulatory Factors in the Hippocampus
As shown in Fig. 6, the expression of IRP1 was not significantly changed when compared with the CG (p > 0.05), and the expression of IRP2 was significantly decreased in AlCl3-exposed groups (p < 0.01) as determined by western blot. The expression of hepcidin in the hippocampus was performed by immunohistochemical staining. The content of hepcidin was significantly increased in AlCl3-exposed groups than that in the CG (p < 0.05, p < 0.01).
Discussion
Previous studies reported that Al accumulated in all regions of brain and the hippocampus was the main target organ [29, 30]. Our studies treated male rats using drinking water containing AlCl3 for 90 days, as this is the most common route for human exposure. Our results demonstrated that AlCl3 caused structural damage and oxidative stress in the hippocampus, a brain region that plays a vital role in learning-memory processes. Meanwhile, AlCl3 induces iron dyshomeostasis presenting as iron accumulation, higher DMT1 expression, and lower TfR, Fpn1 and Fth expression. Moreover, AlCl3 decreases IRP2 expression and increases hepcidin expression, which may result in perturbation of iron metabolism.
Here, we demonstrated the neurotoxicity of Al. Our present results displayed that Al accumulated in the hippocampus, and caused the learning-memory impairment, which can be explained by the increased escape latency to find the hidden platform from the Morris water maze test. Comparable outcomes from cognition testing supported our results [31]. Meanwhile, the hippocampal neurons decreases, damages, and dies in CA1 regions of AlCl3-exposed groups, which can impair cognitive function, including learning-memory. Neurodegenerative diseases have been characterized by higher levels of oxidative stress biomarkers and by lower levels of antioxidant defense biomarkers in the brain [32]. Although Al is not a transition metal, excessive ROS generation and reduced antioxidant enzymes activities have been reported in AlCl3-exposed rat [33, 34]. In our study, augmented ROS, as well as reduced antioxidant enzymes SOD and CAT activities are observed after AlCl3 exposure. Meanwhile, AlCl3 increased MDA and protein carbonyl representing lipid peroxidation and protein oxidation. It indicates that AlCl3 causes oxidative stress and damages neurocytes, processes that could lead to a decline in learning-memory.
The abnormal accumulation of brain iron can induce various neurodegenerative diseases [35]. Our study found that AlCl3 caused iron dyshomeostasis in the hippocampus, including elevation of iron content and iron-related protein expression disorders. Iron-related proteins include TfR, DMT1, Fpn1, and Fn. There are the mainly two pathways for iron uptake in brain. TfR ligand binding transferrin-bound iron (TBI) can be incorporated into cells by an endocytic process. DMT1 transports non-transferrin-bound iron (NTBI) from the extracellular matrix and/or recycling endosomes [36]. It is reported that Al stimulates uptake of TBI and NTBI in human glial cells [22]. But in our study, AlCl3 caused a significant reduction in TfR and augment DMT1 in the hippocampus. Changes of DMT1 and TfR were also found in the previous study without Al, which showed an age-dependent decrease in TfR1 expression in the hippocampus and progressive increase in DMT1 expression [37]. Moreover, DMT1, not TfR, participate in the increase of iron in the parkinsonian substantia nigra [38]. Therefore, the expression of iron importer DMT1, but not TfR1, may be associated with the increased iron content in the hippocampus with AlCl3. The inconsistent changes of DMT1 and TfR may be due to its expression in different types of nerve cell. DMT1 is mainly expressed in astrocytes and oligodendrocytes [39, 40] and probably mediates iron influx into these glial cells. However, neurons and microglia can uptake iron via TfR [41]. Astrocytes are largely responsible for distributing iron in the brain [42], which may lead to the fact that the increase of DMT1 reversed the reduction of TfR, and then enhanced the iron uptake in hippocampus. Fpn1 is the currently only known iron transporter on the cell surface that exports ferrous iron from the cytosol across the plasma membrane [43]. We found that AlCl3 caused a decrease of Fpn1 expression, which inhibited iron export and then aggravated iron accumulation. Therefore, it is reasonable to propose that the increase of DMT1 and decrease of Fpn1 might be responsible for the increase of iron content in the hippocampus with AlCl3. Ferritin, a 24-subunit heteropolymer composed of ferritin heavy chains (Fth) and ferritin light chains (Ftl), is the major iron storage protein in the brain, which plays a role in the protection against oxidative damage [17, 44]. The hippocampal Fth expression decreased, and the content of iron increased with AlCl3. Thus, we speculate that the iron concentration exceeds the iron sequestration capacity of Fn and caused the increase of uncombined iron, which might be available to induce oxidative damage in the hippocampus.
To further confirm the detailed mechanism underlying the effects of AlCl3 on the iron homeostasis in hippocampus, we determined the alteration of iron regulatory factors. It has been suggested that the regulation of iron-related proteins mainly involves two processes, posttranscriptional repression by iron IRPs and posttranslational degradation by hepcidin. The expression of IRP1 is dominant in the kidney, liver and brown fat, whereas the expression of IRP2 is dominant in the central nervous system [45]. IRPs, including IRP1 and IRP2, affect post-transcriptional regulation of cellular iron metabolism by binding to iron-responsive elements (IREs) [46]. Targeted deletion of the gene encoding IRP2 causes dysregulation of iron metabolism and neurodegenerative disease in mice [47, 48]. In the present study, there was no significant difference in the IRP1 content, while the IRP2 content was significantly decreased in AlCl3-exposed groups. This may due to the IRE-binding form of IRP1 converts to an active cytosolic aconitase in iron-replete cells, whereas IRP2 is degraded by the ubiquitin proteasome system [49]. IRPs, which bind to IREs within 3’UTRs of transcripts such as TfR and DMT1, result in stabilization of the transcript [50]. As DMT1 mRNAs only contains a single IRE in the 3’ UTR [51], TfR1 mRNA contains five IREs in its 3 ‘UTR [52, 53]. Compared with DMT1, TfR is more sensitive to IRP. In our study, the decrease of TfR induced by AlCl3 may be due to a decrease in IRP2.
Hepcidin, a 25 amino acid iron regulatory hormone produced mainly by hepatocytes, has been shown to be the central player in body iron homeostasis [54, 55]. Recent studies have revealed that hepcidin is also widely distributed in the brain and has the ability to suppress brain iron accumulation by down-regulating iron transport proteins [56, 57]. However, our study showed hepcidin increases accompanied iron accumulation in the hippocampus of animals exposed to AlCl3. This was similar to that seen in aged rats and in an intracerebral hemorrhage model [37, 58]. Additionally, increase of hepcidin and DMT1 and decrease of Fpn1 induced by inflammation were reported [20]. Our previous results showed that AlCl3 increased interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) content [59]. Although not test here, the increase of hippocampal hepcidin and DMT1 and decrease of Fpn1 in AlCl3-exposed rats might result from inflammation. In our opinion, the contradictory effects of hepcidin on brain iron accumulation may be due to different experimental models or conditions. It is also possible that the amount of hepcidin, although increased, is not enough to restore brain iron to a normal state.
In summary, this paper suggests that AlCl3 induces iron accumulation in hippocampus partly due to the increase of DMT1 and reduction of Fpn1, which may be coordinately regulated by IRP2 and hepcidin. Meanwhile, iron accumulation causes oxidative stress, injures hippocampus structure, and contributes to learning-memory dysfunction. To verify whether the synergy of IRP2 and hepcidin lead iron accumulation in AlCl3-exposed rat hippocampus, future studies could explore this issue further by using gene knockout rats.
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The study was supported by a grant from The National Natural Science Foundation of China (31872530).
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Zhang, J., Huang, W., Xu, F. et al. Iron Dyshomeostasis Participated in Rat Hippocampus Toxicity Caused by Aluminum Chloride. Biol Trace Elem Res 197, 580–590 (2020). https://doi.org/10.1007/s12011-019-02008-7
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DOI: https://doi.org/10.1007/s12011-019-02008-7