Volume 7, Issue 4 (Journal of Clinical and Basic Research (JCBR) 2023)
jcbr 2023, 7(4): 15-19 |
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Azhir M, Gazmeh S, Elyasi L, Jahanshahi M, Bazrafshan B. The effect of apelin-13 on memory of scopolamine-treated rats and accumulation of amyloid-beta plaques in the hippocampus. jcbr 2023; 7 (4) :15-19
URL: http://jcbr.goums.ac.ir/article-1-412-en.html
URL: http://jcbr.goums.ac.ir/article-1-412-en.html
1- Neuroscience Research Center, Golestan University of Medical Sciences, Gorgan, Iran
2- Neuroscience Research Center, Golestan University of Medical Sciences, Gorgan, Iran , lyasy_leila@yahoo.com
3- School of Advanced Technologies in Medicine, Golestan University of Medical Sciences, Gorgan, Iran
2- Neuroscience Research Center, Golestan University of Medical Sciences, Gorgan, Iran , lyasy_leila@yahoo.com
3- School of Advanced Technologies in Medicine, Golestan University of Medical Sciences, Gorgan, Iran
Keywords: Alzheimer disease, Apelin-13 peptide, Scopolamine, Plaque, Amyloid, Hippocampus, Avoidance learning
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Introduction
Neurodegenerative disorders (NDDs) are a group of diseases characterized by progressive neuronal loss in the brain; the clinical manifestations differ based on the brain region(s) involved and proteins aggregated in neurons and glia (1, 2). Alzheimer's disease (AD) is the most common type of NDD, involving over 50 million patients today. The rate is expected to double by 2050, considering population aging (3). This progressive disease starts with an irreversible decline in episodic memory and in the overall cognitive ability of the patients, which deteriorates and finally results in death (4-8).
Research is continued on the most effective treatment for AD, mainly those tackling the disease pathology to halt its progression (9). An important pathology of AD is related to the accumulation of intracellular neurofibrillary nodules (NFTs) and extracellular amyloid-beta (Aβ) plaques produced by hyper-phosphorylation of the tau protein in normal neurons (10-12). Accumulation of Aβ reduces the number of synapses and synaptic flexibility, resulting in ongoing neuronal damage in the brain and ultimately leading to cell death (13). Another mechanism involved in apoptotic death, expressed and activated in the AD brain, is the caspases, the activation of which is mediated through the mitochondrial release of cytochrome C into the cytosol. Caspase-3 is more likely to be related to neuronal apoptosis regulation in AD (14). Other inflammatory processes have also been suggested to play a role in AD pathogenesis and are used as therapeutic targets (15).
Neuropeptides are signaling molecules used by neurons for information transmission and communicating with each other (16), with the potential to treat human brain disorders (17). Apelin is an endogenous neuropeptide, first isolated from bovine gastric tissue in 1998 (17, 18), found in several human tissues, including the cell body of neurons and nerve cell fibers, such as the hypothalamus, piriform cortex, substantia nigra, striatum, hippocampus, olfactory system, gray matter, amygdala, cerebellum, corpus callosum, pituitary gland, medulla, and the spinal cord. The neuroprotective effect of apelin and its widespread distribution in the central nervous system suggests its potential therapeutic role in neurological disorders (19, 20). Animal studies have confirmed its efficacy on ischemic stroke (21) and Parkinson's disease (22), possibly acting through the inhibition of oxidative stress and neuroinflammation.
Concerning AD, research has suggested the efficacy of apelin on apoptosis, autophagy, synaptic plasticity, neuro-inflammation (23), and direct or indirect prevention of Aβ production by increasing its degradation (16). Apelin-13 is the most abundant member of the apelin family, with high neuroprotective function (24, 25). The role of apelin-13 in the inhibition of Aβ-induced injury and apoptosis in SH–SY5Y cells and increasing cell survival by reducing oxidative stress confirm the critical role of apelin-13 as an anti-AD agent (26). Different mechanisms have been suggested for this role, including its effect on glucocorticoid receptor and FKBP5 (17), brain-derived neurotrophic factor, and tyrosine receptor kinase B in the hippocampus (27). Considering the important role of Aβ and oxidative stress in the pathology of AD and the existing evidence on the possible effect of apelin-13 on AD, there is a need for further studies to confirm its effect on molecular and cognitive functions.
For induction of AD in this animal study, we used scopolamine, which accumulates Aβ in the brain, impairs mitochondrial function, cholinergic function, and antioxidant defense, and increases oxidative stress, apoptosis, and neuro-inflammation (5, 28). The similarity of these pathological changes, induced by scopolamine, to that in AD resulted in its use for inducing AD in experimental models for studying cellular and molecular changes associated with the pathogenesis of AD (5, 18). Considering the evidence stated above on the potential of apelin-13 in the treatment of AD and the need for more investigations to confirm its efficacy, we assessed the effect of apelin-13 on memory impairment, plaque density, and apoptosis in scopolamine-treated rats.
Methods
Chemicals
The used drugs and chemical agents were apelin-13 (Phoenix Pharmaceuticals, Mannheim, Germany), scopolamine hydrobromide (Tocris, Great Britain), primary polyclonal anti caspase-3, Congo red, and primary monoclonal anti-b-actin antibodies, provided by Cell Signaling Technology, USA.
Study populations
Forty male Wistar rats (10 weeks old; 120 to 180 g) were obtained from the Pasteur Institute of Amol and located in clear plastic cages; 8 rats were kept in each cage at room temperature (22 ± 3° C) during the 12-hour light-dark period (7 am to 7 pm) without any noise pollution in the animal lab of Golestan University of Medical Sciences. Animals were fed ad libitum with no restrictions on food and water, except during the experiments. After adaptation to the laboratory environment and following the available references, the following groups (n=8 per group) were considered randomly:
Group 1, control group: no surgery or drug.
Group 2 (sham group), scopolamine group: Following 7 days of recovery from stereotaxic surgery, this group received an intraperitoneal (IP) injection of scopolamine (3 mg/kg), followed by an intraperitoneal injection of 5 μL normal saline on the next day daily for 7 days.
Groups 3, 4, and 5 interventional groups: In these three groups, scopolamine was injected in the same manner as the previous group. On the next day (24 hours later), apelin-13 was injected. The dose of apelin-13 differed among the groups: group 3 received 1.25 μg, group 4 received 2.5 μg, and group 5 received 5 μg apelin-13. Apelin-13, soluble in normal saline, was injected into the right lateral brain ventricles (10 μL Hamilton syringe; needle number 21) at 9 am, once daily for 7 days. The study by Haghparast et al. was used for apelin dose (29). The Ethics Committee of the Neuroscience Research Center of the Golestan University of Medical Sciences approved the protocol of this research.
Passive avoidance learning test
Before the intervention, animals were trained using a passive avoidance device for 1 day, and 16 days later, the memory test was conducted again. Accordingly, after locating the rats in the light compartment, the guillotine door was opened for 5 seconds into the dark box, and the latency was recorded; rats with a latency of >120 seconds were excluded from the study. After half an hour, the door was opened, and animals received a foot shock (50 Hz, 1 mA, 3 seconds) upon entering the dark box. The animals were then transferred to their cages (for 120 seconds). The rats were located in the lightbox again, and the latency was recorded for up to 2 minutes. Successful passive avoidance was considered as no entrance to the dark chamber for at least 120 seconds. On the test day, animals were first located in the lightbox, followed by opening the guillotine door (for 5 seconds) with no foot shock, and the duration of delay was recorded (maximum time: 300 seconds).
Surgery
Animals were anesthetized by IP injections of 2% xylazine and 10% ketamine 48 hours after the experiment to remove their brains. After placing the rats in a stereotaxic device, their hair was shaved and disinfected with 70% alcohol, followed by making a longitudinal incision between the ears and eyes using a scalpel. Connective tissue was isolated, and the device marker was fixed on Bergma. The stereotactic coordinates of the right lateral ventricles were determined on the skull (AP = -0/8 and DV = -4/2; based on the Atlas of Paxinos and Watson) and drilled with a tooth drill. After inserting the guide cannula into the skull, 2 small screws were implanted into the skull (to secure the cannula to the skull bone) and secured using dental acrylic. After drying and hardening the acrylic teeth, the rats were removed from the apparatus, transferred to a warm place, and maintained in separate cages until they regained consciousness (29).
Histology
After brain removal, the brain tissues were fixed in phosphate buffer saline, including a 4% paraformaldehyde solution, for 1 week. An automated tissue processor (Green Vision, Urmia, Iran) was used for the histological processing, and the brains were embedded in paraffin blocks. A rotating microtome (Pooyan MK 1110, Iran) cut the brain sagittally (6-μm-thick) and formed the hippocampus (lateral, 0.90 to 4.32 mm).
After deparaffinization and hydration with xylene and ethanol, 6-μm-thick brain sections were stained for 5 minutes with 1% Congo red (Sigma, MO, USA), washed with distilled water twice, and dehydrated in 96% and 100% ethanol. After cleaning in xylene, they were covered with Entellan. Images were taken from samples using a Tokyo Olympus BX51 microscope with a DP 72 digital camera. We only examined right-hemispheric hippocampal neurons. First, the hippocampus was identified using a 4× microscope lens, and 40 areas were imaged using a magnifying glass. After images, the hippocampal CA1, CA2, CA3, CA4, and dentate gyrus (DG) areas were first graded by Image J software; the same software was used for counting the 30 000-μm area for each hippocampal area (29).
Canola approval
Animals were randomly selected to confirm the appropriateness of cannula placement in the lateral ventricle. In addition, the cannula was injected with 0.5 μL methylene blue. Animals were exterminated 60 minutes later, and after brain removal, they were placed in a formaldehyde solution (10%). The next day, a microtome (Leitz, Germany) cut serial 3-μm incisions, and the cannula tip was placed in the lateral ventricle under microscopic control.
Western blot analysis for cytochrome C and cleaved caspase–3
Carbon dioxide-induced anesthesia was administered. The rats' brains were isolated immediately, washed using cold normal saline, and kept on ice-cold glass plates. The right striatum was promptly removed, and its tissue homogenization was performed in ice-cold buffer including EDTA (1mM), 10mM Tris–HCl (pH 7.4), 0.1% SDS, 0.1% sodium–deoxycholate, 1% nonyl phenoxypolyethoxylethanol–40, and protease inhibitors, including 1mM phenyl methyl sulfonyl fluoride, 2.5mg/mL leupeptin, and 10mg/mL aprotinin, and 1mM sodium orthovanadate. After centrifuging the homogenate at 14 000g/15 min/4 °C, the supernatant was collected as the whole tissue sample, and the total protein level was calculated by the Bradford approach (Bio-Rad Laboratories, Germany). Electrophoresis was performed using similar levels of protein (40 mg) on 12% sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) transmitting into polyvinylidene difluoride (PVDF) membrane (Roche, Germany). Following 2 h of blocking at room temperature by 5% non-fat dried milk in Tris-buffered saline 0.1% Tween 20 (TBST), the membranes were probed via initial polyclonal cytochrome C antibody (sc–376861, Santa Cruz Biotechnology) and monoclonal anti-caspase-3 antibodies (Cell Signaling Technology, USA) (1:1000) for 1 night at 4 °C. After being washed 3 times by TBST (in 5 min), the blots were incubated for 60 min at room temperature using diluted horse-radish peroxidase-conjugated secondary antibodies (1:10000, Santa Cruz Biotechnology). We applied a pre-stained protein ladder (SM7012, Cinagen Co, Iran) to monitor protein purification and estimate the molecular weight and united intensity of the blotting bands. An ECL system was used to identify the antibody-antigen complex, subjected to Lumi–Film chemiluminescent detection film (Roche, Germany). To evaluate the blotting bands' intensity, we used LabWorks Software (UVP, UK). Beta-actin (1:10 000) was applied as a loading control, and the expression scores were expressed as studied proteins/b-actin proportion per animal (29).
Data analysis
SPSS v. 21.0 (IBM Corp., Armonk, NY, USA) was used to analyze the data. Mean and standard deviation (SD) were applied to describe the numerical variables. One-way analysis of variance was employed to compare the means between groups. Further significant differences were assessed using the Student-Newman-Keuls (S-N-K) test. A P-value <0.05 was regarded as significant.
Results
Discussion
We investigated the effect of apelin-13 neuropeptide on several parameters; the first and most important parameter was the density of Aβ plaques in the hippocampus (CA1, CA2, CA3, CA4, and DG areas). The results showed the highest effect in the highest dose (5 μg). These results confirmed our hypothesis, implying that apelin had neuroprotective effects against AD by reducing the density of the Aβ plaques accumulated in the hippocampus. Considering the previous evidence, Aβ is primarily responsible for cognitive impairment and neurological death in AD, and Aβ antagonists can treat AD by reducing Aβ production by inhibiting β and γ–secretase enzymes and eliminating cerebral Aβ plaques (30). This mechanism may be the same mechanism of action for the effect of apelin-13 on Aβ concentration, which suggests it is a promising candidate for AD treatment. Other mechanisms have also been introduced for the direct or indirect effect of apelin on inhibiting the production of Aβ (31), including reducing the amount of APP and decreasing β–secretase activity. It has been suggested that by increasing the level of ATP-binding cassette subfamily A1 (ABCA1) and the activity of neutral endopeptidase, it degrades and reduces Aβ (16, 32). Luo et al. (2019) investigated the impact of apelin on various physiological processes of AD and found that apelin plays an essential role in suppressing inflammatory responses, inhibiting oxidative stress, reducing calcium (Ca2+) signaling, suppressing apoptosis, and inducing autophagy. Thus, apelin was presented as a new strategy for managing neurological diseases (33).
In a previous study, our team showed the protective effect of apelin-13 on human neuroblastoma cells (SH–SY5Y) (34). Chen et al. reported the neuroprotective effect of apelin-13 against rotenone–related neurotoxicity through activation of AMPK/mTOR/ULK–1 medicated autophagy (35). Others have also shown the effect of apelin-13 on oxidative stress-induced apoptosis (36). All these studies are consistent with the findings of the present study and imply that apelin-13 can be considered a practical therapeutic agent in AD.
The second important parameter evaluated in the present study was memory impairment, evaluated by passive avoidance latency. The results showed improved memory in therapeutic groups after the substantial decrease in scopolamine. Other studies have shown the positive effect of apelin-13 on memory as well. In 2019, Yavari et al. examined the effect of apelin-13 on Aβ-induced memory impairment and autophagy and apoptotic processes in the hippocampus. They found that apelin-13 significantly protected against the destructive effects of Aβ. Apelin-13 also significantly protects against the destructive effects of Aβ on memory and improves Aβ–induced functional disorders and spatial memory by inhibiting autophagy and suppressing apoptosis (31). These results, in line with our study, showed the favorable effect of apelin-13 on memory impairment, implying that this agent is not only effective in the molecular phase but also influences the clinical symptoms. Removal or reduction of amyloid plaques from the hippocampus of rats with AD is possibly the underlying mechanism of increased memory (37).
In order to evaluate how apelin-13 can influence or treat AD, we evaluated another mechanism, which has been suggested to play a role in AD in addition to Aβ plaque formation. Considering the significance of oxidative stress and apoptosis in the pathogenesis of NDDs and AD, we evaluated caspase-3 and cytochrome C values. The results showed that the increased mean values in the scopolamine group were attenuated in groups receiving 1.25 and 2.5 µg/kg/day apelin-13. In the previous study from our team, we demonstrated the effect of apelin-13 on the inhibition of caspase-3 and cytochrome C increase (34). A similar effect was observed in hair cells derived from bone marrow; as suggested, protein expression of caspase-3 and Bax/Bcl-2 were significantly lower in the group pretreated with apelin-13 (36). Activation of the caspase cascade is important for apoptosis, initiated by caspases 2, 8, and 9, and followed by executive caspases, such as 3, 7, and 14 (38); therefore, inhibition of caspase-3 can be an important step for reducing or inhibiting apoptosis, the main mechanism of cell death in AD. Zang et al. also showed that apelin-13 decreased cytochrome C release and inactivated caspase-3 in cultured mouse cortical neurons (39), which confirms the results of the present study. Decreased levels of cleaved caspase-3 and caspase-9, induced by apelin-13, were also detected in the SHSY5Y cell line in the rat vascular dementia model (40). Different pathways have been suggested for the mechanism of action for this effect, including inhibiting the activation of ATF6/CAAT enhancer binding proteins homologous protein-CHOP pathway (41) and glucagon-like peptide-1 receptor/PI3K/AKT signaling pathway (42). The effect of apelin-13 on cytochrome C efflux and apoptosis has also been suggested in regulating mitochondrial dysfunction for the prevention of lung injury (43). Further studies are required to determine how apelin-13 decreases caspase-3 and cytochrome c levels.
In this study, in addition to the effect of apelin-13, we also showed the effect of scopolamine in rats by evaluating the parameters in the sham group; as shown, intraperitoneal injection of 3 mg/kg scopolamine can cause increased amyloid plaques in CA1, CA2, CA3, CA4, and DG regions in rat hippocampus, and staining with Congo red confirmed the accumulation of Aβ plaques in the hippocampal tissue. These findings suggest that scopolamine injection causes similar changes as AD. Previous evidence has also suggested that scopolamine is an appropriate model of AD (44), resulting in atrophy and degeneration of the brain's nerves, mitochondrial dysfunction, increased apoptosis of hippocampal neurons, neuroinflammation, and oxidative disorders in rats through Aβ accumulation in the brain (5). Therefore, scopolamine injection has been used in several studies for inducing AD in animals (45, 46). Our results also showed the successful induction of the AD model in rats by scopolamine injection; this finding confirms its use in further studies for the rat AD model.
An important strength of the present study was the simultaneous evaluation of cellular and behavioral (memory function) tests in one study, which can provide strong evidence for the effect of apelin-13 on AD. However, we only evaluated 3 cellular mechanisms in this study, namely Aβ accumulation, caspase-3, and cytochrome C, and performed only 1 behavioral test, while other mechanisms may also be involved, which were not evaluated in this study.
Conclusion
In this study, with the help of scopolamine, we successfully induced an AD model in rats and showed that apelin-13 alone could significantly reduce the scopolamine-induced cell damage by preventing the accumulation of Aβ plaques on the hippocampus neurons and reducing the destructive effects of Aβ on neurons. We also showed that apelin-13 could reduce the levels of caspase-3 and cytochrome c, important agents in the apoptosis cascade. The favorable effect of apelin-13 on AD was also confirmed by improving the memory impairment induced by scopolamine in the rats. Therefore, apelin-13 should be considered in future research on novel treatments for AD to define whether this treatment can improve memory loss and other AD symptoms in humans.
Acknowledgement
None.
Funding sources
This research was financially supported by the Golestan University of Medical Sciences Neuroscience Research Center.
Ethical statement
The protocol of this research was confirmed by the Ethics Committee of Golestan University of Medical Sciences Neuroscience Research Center IR.GOUMS.REC.1399.156 .NA (This study was not conducted on humans).
Conflicts of interest
The authors have no conflict of interest
Author contributions
L. Elyasi was involved in idea formation and writing the manuscript. M. Jahanshahi was involved in statistical analysis and writing the manuscript. Behnaz Bazrafshan, Maryam Azhir, and Sara Gazme performed the study on rats, collected the data, and were involved in preparing the materials. All the authors confirmed the final version of the manuscript.
Neurodegenerative disorders (NDDs) are a group of diseases characterized by progressive neuronal loss in the brain; the clinical manifestations differ based on the brain region(s) involved and proteins aggregated in neurons and glia (1, 2). Alzheimer's disease (AD) is the most common type of NDD, involving over 50 million patients today. The rate is expected to double by 2050, considering population aging (3). This progressive disease starts with an irreversible decline in episodic memory and in the overall cognitive ability of the patients, which deteriorates and finally results in death (4-8).
Research is continued on the most effective treatment for AD, mainly those tackling the disease pathology to halt its progression (9). An important pathology of AD is related to the accumulation of intracellular neurofibrillary nodules (NFTs) and extracellular amyloid-beta (Aβ) plaques produced by hyper-phosphorylation of the tau protein in normal neurons (10-12). Accumulation of Aβ reduces the number of synapses and synaptic flexibility, resulting in ongoing neuronal damage in the brain and ultimately leading to cell death (13). Another mechanism involved in apoptotic death, expressed and activated in the AD brain, is the caspases, the activation of which is mediated through the mitochondrial release of cytochrome C into the cytosol. Caspase-3 is more likely to be related to neuronal apoptosis regulation in AD (14). Other inflammatory processes have also been suggested to play a role in AD pathogenesis and are used as therapeutic targets (15).
Neuropeptides are signaling molecules used by neurons for information transmission and communicating with each other (16), with the potential to treat human brain disorders (17). Apelin is an endogenous neuropeptide, first isolated from bovine gastric tissue in 1998 (17, 18), found in several human tissues, including the cell body of neurons and nerve cell fibers, such as the hypothalamus, piriform cortex, substantia nigra, striatum, hippocampus, olfactory system, gray matter, amygdala, cerebellum, corpus callosum, pituitary gland, medulla, and the spinal cord. The neuroprotective effect of apelin and its widespread distribution in the central nervous system suggests its potential therapeutic role in neurological disorders (19, 20). Animal studies have confirmed its efficacy on ischemic stroke (21) and Parkinson's disease (22), possibly acting through the inhibition of oxidative stress and neuroinflammation.
Concerning AD, research has suggested the efficacy of apelin on apoptosis, autophagy, synaptic plasticity, neuro-inflammation (23), and direct or indirect prevention of Aβ production by increasing its degradation (16). Apelin-13 is the most abundant member of the apelin family, with high neuroprotective function (24, 25). The role of apelin-13 in the inhibition of Aβ-induced injury and apoptosis in SH–SY5Y cells and increasing cell survival by reducing oxidative stress confirm the critical role of apelin-13 as an anti-AD agent (26). Different mechanisms have been suggested for this role, including its effect on glucocorticoid receptor and FKBP5 (17), brain-derived neurotrophic factor, and tyrosine receptor kinase B in the hippocampus (27). Considering the important role of Aβ and oxidative stress in the pathology of AD and the existing evidence on the possible effect of apelin-13 on AD, there is a need for further studies to confirm its effect on molecular and cognitive functions.
For induction of AD in this animal study, we used scopolamine, which accumulates Aβ in the brain, impairs mitochondrial function, cholinergic function, and antioxidant defense, and increases oxidative stress, apoptosis, and neuro-inflammation (5, 28). The similarity of these pathological changes, induced by scopolamine, to that in AD resulted in its use for inducing AD in experimental models for studying cellular and molecular changes associated with the pathogenesis of AD (5, 18). Considering the evidence stated above on the potential of apelin-13 in the treatment of AD and the need for more investigations to confirm its efficacy, we assessed the effect of apelin-13 on memory impairment, plaque density, and apoptosis in scopolamine-treated rats.
Methods
Chemicals
The used drugs and chemical agents were apelin-13 (Phoenix Pharmaceuticals, Mannheim, Germany), scopolamine hydrobromide (Tocris, Great Britain), primary polyclonal anti caspase-3, Congo red, and primary monoclonal anti-b-actin antibodies, provided by Cell Signaling Technology, USA.
Study populations
Forty male Wistar rats (10 weeks old; 120 to 180 g) were obtained from the Pasteur Institute of Amol and located in clear plastic cages; 8 rats were kept in each cage at room temperature (22 ± 3° C) during the 12-hour light-dark period (7 am to 7 pm) without any noise pollution in the animal lab of Golestan University of Medical Sciences. Animals were fed ad libitum with no restrictions on food and water, except during the experiments. After adaptation to the laboratory environment and following the available references, the following groups (n=8 per group) were considered randomly:
Group 1, control group: no surgery or drug.
Group 2 (sham group), scopolamine group: Following 7 days of recovery from stereotaxic surgery, this group received an intraperitoneal (IP) injection of scopolamine (3 mg/kg), followed by an intraperitoneal injection of 5 μL normal saline on the next day daily for 7 days.
Groups 3, 4, and 5 interventional groups: In these three groups, scopolamine was injected in the same manner as the previous group. On the next day (24 hours later), apelin-13 was injected. The dose of apelin-13 differed among the groups: group 3 received 1.25 μg, group 4 received 2.5 μg, and group 5 received 5 μg apelin-13. Apelin-13, soluble in normal saline, was injected into the right lateral brain ventricles (10 μL Hamilton syringe; needle number 21) at 9 am, once daily for 7 days. The study by Haghparast et al. was used for apelin dose (29). The Ethics Committee of the Neuroscience Research Center of the Golestan University of Medical Sciences approved the protocol of this research.
Passive avoidance learning test
Before the intervention, animals were trained using a passive avoidance device for 1 day, and 16 days later, the memory test was conducted again. Accordingly, after locating the rats in the light compartment, the guillotine door was opened for 5 seconds into the dark box, and the latency was recorded; rats with a latency of >120 seconds were excluded from the study. After half an hour, the door was opened, and animals received a foot shock (50 Hz, 1 mA, 3 seconds) upon entering the dark box. The animals were then transferred to their cages (for 120 seconds). The rats were located in the lightbox again, and the latency was recorded for up to 2 minutes. Successful passive avoidance was considered as no entrance to the dark chamber for at least 120 seconds. On the test day, animals were first located in the lightbox, followed by opening the guillotine door (for 5 seconds) with no foot shock, and the duration of delay was recorded (maximum time: 300 seconds).
Surgery
Animals were anesthetized by IP injections of 2% xylazine and 10% ketamine 48 hours after the experiment to remove their brains. After placing the rats in a stereotaxic device, their hair was shaved and disinfected with 70% alcohol, followed by making a longitudinal incision between the ears and eyes using a scalpel. Connective tissue was isolated, and the device marker was fixed on Bergma. The stereotactic coordinates of the right lateral ventricles were determined on the skull (AP = -0/8 and DV = -4/2; based on the Atlas of Paxinos and Watson) and drilled with a tooth drill. After inserting the guide cannula into the skull, 2 small screws were implanted into the skull (to secure the cannula to the skull bone) and secured using dental acrylic. After drying and hardening the acrylic teeth, the rats were removed from the apparatus, transferred to a warm place, and maintained in separate cages until they regained consciousness (29).
Histology
After brain removal, the brain tissues were fixed in phosphate buffer saline, including a 4% paraformaldehyde solution, for 1 week. An automated tissue processor (Green Vision, Urmia, Iran) was used for the histological processing, and the brains were embedded in paraffin blocks. A rotating microtome (Pooyan MK 1110, Iran) cut the brain sagittally (6-μm-thick) and formed the hippocampus (lateral, 0.90 to 4.32 mm).
After deparaffinization and hydration with xylene and ethanol, 6-μm-thick brain sections were stained for 5 minutes with 1% Congo red (Sigma, MO, USA), washed with distilled water twice, and dehydrated in 96% and 100% ethanol. After cleaning in xylene, they were covered with Entellan. Images were taken from samples using a Tokyo Olympus BX51 microscope with a DP 72 digital camera. We only examined right-hemispheric hippocampal neurons. First, the hippocampus was identified using a 4× microscope lens, and 40 areas were imaged using a magnifying glass. After images, the hippocampal CA1, CA2, CA3, CA4, and dentate gyrus (DG) areas were first graded by Image J software; the same software was used for counting the 30 000-μm area for each hippocampal area (29).
Canola approval
Animals were randomly selected to confirm the appropriateness of cannula placement in the lateral ventricle. In addition, the cannula was injected with 0.5 μL methylene blue. Animals were exterminated 60 minutes later, and after brain removal, they were placed in a formaldehyde solution (10%). The next day, a microtome (Leitz, Germany) cut serial 3-μm incisions, and the cannula tip was placed in the lateral ventricle under microscopic control.
Western blot analysis for cytochrome C and cleaved caspase–3
Carbon dioxide-induced anesthesia was administered. The rats' brains were isolated immediately, washed using cold normal saline, and kept on ice-cold glass plates. The right striatum was promptly removed, and its tissue homogenization was performed in ice-cold buffer including EDTA (1mM), 10mM Tris–HCl (pH 7.4), 0.1% SDS, 0.1% sodium–deoxycholate, 1% nonyl phenoxypolyethoxylethanol–40, and protease inhibitors, including 1mM phenyl methyl sulfonyl fluoride, 2.5mg/mL leupeptin, and 10mg/mL aprotinin, and 1mM sodium orthovanadate. After centrifuging the homogenate at 14 000g/15 min/4 °C, the supernatant was collected as the whole tissue sample, and the total protein level was calculated by the Bradford approach (Bio-Rad Laboratories, Germany). Electrophoresis was performed using similar levels of protein (40 mg) on 12% sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) transmitting into polyvinylidene difluoride (PVDF) membrane (Roche, Germany). Following 2 h of blocking at room temperature by 5% non-fat dried milk in Tris-buffered saline 0.1% Tween 20 (TBST), the membranes were probed via initial polyclonal cytochrome C antibody (sc–376861, Santa Cruz Biotechnology) and monoclonal anti-caspase-3 antibodies (Cell Signaling Technology, USA) (1:1000) for 1 night at 4 °C. After being washed 3 times by TBST (in 5 min), the blots were incubated for 60 min at room temperature using diluted horse-radish peroxidase-conjugated secondary antibodies (1:10000, Santa Cruz Biotechnology). We applied a pre-stained protein ladder (SM7012, Cinagen Co, Iran) to monitor protein purification and estimate the molecular weight and united intensity of the blotting bands. An ECL system was used to identify the antibody-antigen complex, subjected to Lumi–Film chemiluminescent detection film (Roche, Germany). To evaluate the blotting bands' intensity, we used LabWorks Software (UVP, UK). Beta-actin (1:10 000) was applied as a loading control, and the expression scores were expressed as studied proteins/b-actin proportion per animal (29).
Data analysis
SPSS v. 21.0 (IBM Corp., Armonk, NY, USA) was used to analyze the data. Mean and standard deviation (SD) were applied to describe the numerical variables. One-way analysis of variance was employed to compare the means between groups. Further significant differences were assessed using the Student-Newman-Keuls (S-N-K) test. A P-value <0.05 was regarded as significant.
Results
- Behavioral Results
- Density of congophilic amyloid-beta plaque in the hippocampus region
- Cytochrome C, assessed by Western blotting
- Caspase-3, assessed by Western blotting
Discussion
We investigated the effect of apelin-13 neuropeptide on several parameters; the first and most important parameter was the density of Aβ plaques in the hippocampus (CA1, CA2, CA3, CA4, and DG areas). The results showed the highest effect in the highest dose (5 μg). These results confirmed our hypothesis, implying that apelin had neuroprotective effects against AD by reducing the density of the Aβ plaques accumulated in the hippocampus. Considering the previous evidence, Aβ is primarily responsible for cognitive impairment and neurological death in AD, and Aβ antagonists can treat AD by reducing Aβ production by inhibiting β and γ–secretase enzymes and eliminating cerebral Aβ plaques (30). This mechanism may be the same mechanism of action for the effect of apelin-13 on Aβ concentration, which suggests it is a promising candidate for AD treatment. Other mechanisms have also been introduced for the direct or indirect effect of apelin on inhibiting the production of Aβ (31), including reducing the amount of APP and decreasing β–secretase activity. It has been suggested that by increasing the level of ATP-binding cassette subfamily A1 (ABCA1) and the activity of neutral endopeptidase, it degrades and reduces Aβ (16, 32). Luo et al. (2019) investigated the impact of apelin on various physiological processes of AD and found that apelin plays an essential role in suppressing inflammatory responses, inhibiting oxidative stress, reducing calcium (Ca2+) signaling, suppressing apoptosis, and inducing autophagy. Thus, apelin was presented as a new strategy for managing neurological diseases (33).
In a previous study, our team showed the protective effect of apelin-13 on human neuroblastoma cells (SH–SY5Y) (34). Chen et al. reported the neuroprotective effect of apelin-13 against rotenone–related neurotoxicity through activation of AMPK/mTOR/ULK–1 medicated autophagy (35). Others have also shown the effect of apelin-13 on oxidative stress-induced apoptosis (36). All these studies are consistent with the findings of the present study and imply that apelin-13 can be considered a practical therapeutic agent in AD.
The second important parameter evaluated in the present study was memory impairment, evaluated by passive avoidance latency. The results showed improved memory in therapeutic groups after the substantial decrease in scopolamine. Other studies have shown the positive effect of apelin-13 on memory as well. In 2019, Yavari et al. examined the effect of apelin-13 on Aβ-induced memory impairment and autophagy and apoptotic processes in the hippocampus. They found that apelin-13 significantly protected against the destructive effects of Aβ. Apelin-13 also significantly protects against the destructive effects of Aβ on memory and improves Aβ–induced functional disorders and spatial memory by inhibiting autophagy and suppressing apoptosis (31). These results, in line with our study, showed the favorable effect of apelin-13 on memory impairment, implying that this agent is not only effective in the molecular phase but also influences the clinical symptoms. Removal or reduction of amyloid plaques from the hippocampus of rats with AD is possibly the underlying mechanism of increased memory (37).
In order to evaluate how apelin-13 can influence or treat AD, we evaluated another mechanism, which has been suggested to play a role in AD in addition to Aβ plaque formation. Considering the significance of oxidative stress and apoptosis in the pathogenesis of NDDs and AD, we evaluated caspase-3 and cytochrome C values. The results showed that the increased mean values in the scopolamine group were attenuated in groups receiving 1.25 and 2.5 µg/kg/day apelin-13. In the previous study from our team, we demonstrated the effect of apelin-13 on the inhibition of caspase-3 and cytochrome C increase (34). A similar effect was observed in hair cells derived from bone marrow; as suggested, protein expression of caspase-3 and Bax/Bcl-2 were significantly lower in the group pretreated with apelin-13 (36). Activation of the caspase cascade is important for apoptosis, initiated by caspases 2, 8, and 9, and followed by executive caspases, such as 3, 7, and 14 (38); therefore, inhibition of caspase-3 can be an important step for reducing or inhibiting apoptosis, the main mechanism of cell death in AD. Zang et al. also showed that apelin-13 decreased cytochrome C release and inactivated caspase-3 in cultured mouse cortical neurons (39), which confirms the results of the present study. Decreased levels of cleaved caspase-3 and caspase-9, induced by apelin-13, were also detected in the SHSY5Y cell line in the rat vascular dementia model (40). Different pathways have been suggested for the mechanism of action for this effect, including inhibiting the activation of ATF6/CAAT enhancer binding proteins homologous protein-CHOP pathway (41) and glucagon-like peptide-1 receptor/PI3K/AKT signaling pathway (42). The effect of apelin-13 on cytochrome C efflux and apoptosis has also been suggested in regulating mitochondrial dysfunction for the prevention of lung injury (43). Further studies are required to determine how apelin-13 decreases caspase-3 and cytochrome c levels.
In this study, in addition to the effect of apelin-13, we also showed the effect of scopolamine in rats by evaluating the parameters in the sham group; as shown, intraperitoneal injection of 3 mg/kg scopolamine can cause increased amyloid plaques in CA1, CA2, CA3, CA4, and DG regions in rat hippocampus, and staining with Congo red confirmed the accumulation of Aβ plaques in the hippocampal tissue. These findings suggest that scopolamine injection causes similar changes as AD. Previous evidence has also suggested that scopolamine is an appropriate model of AD (44), resulting in atrophy and degeneration of the brain's nerves, mitochondrial dysfunction, increased apoptosis of hippocampal neurons, neuroinflammation, and oxidative disorders in rats through Aβ accumulation in the brain (5). Therefore, scopolamine injection has been used in several studies for inducing AD in animals (45, 46). Our results also showed the successful induction of the AD model in rats by scopolamine injection; this finding confirms its use in further studies for the rat AD model.
An important strength of the present study was the simultaneous evaluation of cellular and behavioral (memory function) tests in one study, which can provide strong evidence for the effect of apelin-13 on AD. However, we only evaluated 3 cellular mechanisms in this study, namely Aβ accumulation, caspase-3, and cytochrome C, and performed only 1 behavioral test, while other mechanisms may also be involved, which were not evaluated in this study.
Conclusion
In this study, with the help of scopolamine, we successfully induced an AD model in rats and showed that apelin-13 alone could significantly reduce the scopolamine-induced cell damage by preventing the accumulation of Aβ plaques on the hippocampus neurons and reducing the destructive effects of Aβ on neurons. We also showed that apelin-13 could reduce the levels of caspase-3 and cytochrome c, important agents in the apoptosis cascade. The favorable effect of apelin-13 on AD was also confirmed by improving the memory impairment induced by scopolamine in the rats. Therefore, apelin-13 should be considered in future research on novel treatments for AD to define whether this treatment can improve memory loss and other AD symptoms in humans.
Acknowledgement
None.
Funding sources
This research was financially supported by the Golestan University of Medical Sciences Neuroscience Research Center.
Ethical statement
The protocol of this research was confirmed by the Ethics Committee of Golestan University of Medical Sciences Neuroscience Research Center IR.GOUMS.REC.1399.156 .NA (This study was not conducted on humans).
Conflicts of interest
The authors have no conflict of interest
Author contributions
L. Elyasi was involved in idea formation and writing the manuscript. M. Jahanshahi was involved in statistical analysis and writing the manuscript. Behnaz Bazrafshan, Maryam Azhir, and Sara Gazme performed the study on rats, collected the data, and were involved in preparing the materials. All the authors confirmed the final version of the manuscript.
Article Type: Research |
Subject:
Neuroscience
References
1. Wray S. Modelling neurodegenerative disease using brain organoids. Semin Cell Dev Biol. 2021;111:60-6. [View at Publisher] [DOI] [PMID] [Google Scholar]
2. Zhu J, Dou S, Jiang Y, Chen J, Wang C, Cheng B. Apelin-13 protects dopaminergic neurons in MPTP-induced Parkinson's disease model mice through inhibiting endoplasmic reticulum stress and promoting autophagy. Brain Res. 2019;1715:203-12. [View at Publisher] [DOI] [PMID] [Google Scholar]
3. Budelier MM, Bateman RJ. Biomarkers of Alzheimer Disease. J Appl Lab Med. 2020;5(1):194-208. [View at Publisher] [DOI] [PMID] [Google Scholar]
4. Chen Z-R, Huang J-B, Yang S-L, Hong F-F. Role of Cholinergic Signaling in Alzheimer's Disease. Molecules. 2022;27(6):1816. [View at Publisher] [DOI] [PMID] [Google Scholar]
5. Tang KS. The cellular and molecular processes associated with scopolamine-induced memory deficit: A model of Alzheimer's biomarkers. Life Sci. 2019;233:116695. [View at Publisher] [DOI] [PMID] [Google Scholar]
6. 2020 Alzheimer's disease facts and figures. Alzheimers Dement. 2020;16(3):391-460. [View at Publisher] [DOI] [PMID]
7. Terranova JI, Ogawa SK, Kitamura T. Adult hippocampal neurogenesis for systems consolidation of memory. Behav Brain Res. 2019;372:112035. [View at Publisher] [DOI] [PMID] [Google Scholar]
8. Puzzo D, Fiorito J, Purgatorio R, Gulisano W, Palmeri A, Arancio O, et al. Chapter 1 - Molecular Mechanisms of Learning and Memory**The authors declare no competing financial interests. In: Lazarov O, Tesco G, editors. Genes, Environment and Alzheimer's Disease. San Diego: Academic Press; 2016. p. 1-27. [View at Publisher] [DOI] [Google Scholar]
9. Kumar A, Singh A, Ekavali. A review on Alzheimer's disease pathophysiology and its management: an update. Pharmacol Rep. 2015;67(2):195-203. [View at Publisher] [DOI] [PMID] [Google Scholar]
10. Ulep MG, Saraon SK, McLea S. Alzheimer Disease. The Journal for Nurse Practitioners. 2018;14(3):129-35. [View at Publisher] [DOI] [Google Scholar]
11. Derby CA. Trends in the public health significance, definitions of disease, and implications for prevention of Alzheimer's disease. Curr Epidemiol Rep. 2020;7(2):68-76. [View at Publisher] [DOI] [PMID] [Google Scholar]
12. Esposito Z, Belli L, Toniolo S, Sancesario G, Bianconi C, Martorana A. Amyloid β, glutamate, excitotoxicity in Alzheimer's disease: are we on the right track? CNS Neurosci Ther. 2013;19(8):549-55. [View at Publisher] [DOI] [PMID] [Google Scholar]
13. DeTure MA, Dickson DW. The neuropathological diagnosis of Alzheimer's disease. Mol Neurodegener. 2019;14(1):32. [View at Publisher] [DOI] [PMID] [Google Scholar]
14. Roth KA. Caspases, apoptosis, and Alzheimer disease: causation, correlation, and confusion. J Neuropathol Exp Neurol. 2001;60(9):829-38. [View at Publisher] [DOI] [PMID] [Google Scholar]
15. Onyango IG, Jauregui GV, Čarná M, Bennett Jr JP, Stokin GB. Neuroinflammation in Alzheimer's disease. Biomedicines. 2021;9(5):524. [View at Publisher] [DOI] [PMID] [Google Scholar]
16. Masoumi J, Abbasloui M, Parvan R, Mohammadnejad D, Pavon-Djavid G, Barzegari A, et al. Apelin, a promising target for Alzheimer disease prevention and treatment. Neuropeptides. 2018;70:76-86. [View at Publisher] [DOI] [PMID] [Google Scholar]
17. Aminyavari S, Zahmatkesh M, Khodagholi F, Sanati M. Anxiolytic impact of Apelin-13 in a rat model of Alzheimer's disease: Involvement of glucocorticoid receptor and FKBP5. Peptides. 2019;118:170102. [View at Publisher] [DOI] [PMID] [Google Scholar]
18. Lv SY, Chen WD, Wang YD. The Apelin/APJ System in Psychosis and Neuropathy. Front Pharmacol. 2020;11:320. [View at Publisher] [DOI] [PMID] [Google Scholar]
19. Zhou JX, Shuai NN, Wang B, Jin X, Kuang X, Tian SW. Neuroprotective gain of Apelin/APJ system. Neuropeptides. 2021;87:102131. [View at Publisher] [DOI] [PMID] [Google Scholar]
20. Zhang Y, Jiang W, Sun W, Guo W, Xia B, Shen X, et al. Neuroprotective Roles of Apelin-13 in Neurological Diseases. Neurochem Res. 2023:48(6):1648-62. [View at Publisher] [DOI] [PMID] [Google Scholar]
21. Xin Q, Cheng B, Pan Y, Liu H, Chen J, Bai B. Neuroprotective effects of apelin-13 on experimental ischemic stroke through suppression of inflammation. Peptides. 2015;63:55-62. [View at Publisher] [DOI] [PMID] [Google Scholar]
22. Zhu J, Gao W, Shan X, Wang C, Wang H, Shao Z, et al. Apelin-36 mediates neuroprotective effects by regulating oxidative stress, autophagy and apoptosis in MPTP-induced Parkinson's disease model mice. Brain Res. 2020;1726:146493. [View at Publisher] [DOI] [PMID] [Google Scholar]
23. Wan T, Fu M, Jiang Y, Jiang W, Li P, Zhou S. Research Progress on Mechanism of Neuroprotective Roles of Apelin-13 in Prevention and Treatment of Alzheimer's Disease. Neurochem Res. 2022;47(2):205-17. [View at Publisher] [DOI] [PMID] [Google Scholar]
24. Antushevich H, Wójcik M. Review: Apelin in disease. Clin Chim Acta. 2018;483:241-8. [View at Publisher] [DOI] [PMID] [Google Scholar]
25. Yildiz Z, Eren N, Orcun A, Münevver Gokyigit F, Turgay F, Gündogdu Celebi L. Serum apelin-13 levels and total oxidant/antioxidant status of patients with Alzheimer's disease. Aging Med (Milton). 2021;4(3):201-5. [View at Publisher] [DOI] [PMID] [Google Scholar]
26. Wan T, Fu M, Jiang Y, Jiang W, Li P, Zhou S. Research progress on mechanism of neuroprotective roles of Apelin-13 in prevention and treatment of Alzheimer's disease. Neurochem Res. 2022:47(2):205-17. [View at Publisher] [DOI] [PMID] [Google Scholar]
27. Luo H, Xiang Y, Qu X, Liu H, Liu C, Li G, et al. Apelin-13 suppresses neuroinflammation against cognitive deficit in a streptozotocin-induced rat model of Alzheimer's disease through activation of BDNF-TrkB signaling pathway. Front Pharmacol. 2019;10:395. [View at Publisher] [DOI] [PMID] [Google Scholar]
28. Paul J. Chapter 5 - Experimental Medicine Approaches in CNS Drug Development. In: Nomikos GG, Feltner DE, editors. Handbook of Behavioral Neuroscience.US: Elsevier; 2019. Vol 29. p 63-80. [View at Publisher] [DOI] [Google Scholar]
29. Haghparast E, Esmaeili-Mahani S, Abbasnejad M, Sheibani V. Apelin-13 ameliorates cognitive impairments in 6-hydroxydopamine-induced substantia nigra lesion in rats. Neuropeptides. 2018;68:28-35. [View at Publisher] [DOI] [PMID] [Google Scholar]
30. Folch J, Petrov D, Ettcheto M, Abad S, Sánchez-López E, García ML, et al. Current Research Therapeutic Strategies for Alzheimer's Disease Treatment. Neural Plast. 2016;2016:8501693. [View at Publisher] [DOI] [PMID] [Google Scholar]
31. Aminyavari S, Zahmatkesh M, Farahmandfar M, Khodagholi F, Dargahi L, Zarrindast MR. Protective role of Apelin-13 on amyloid β25-35-induced memory deficit; Involvement of autophagy and apoptosis process. Prog Neuropsychopharmacol Biol Psychiatry. 2019;89:322-34. [View at Publisher] [DOI] [PMID] [Google Scholar]
32. Eren N, Den Z, Yildiz Z, Go N, Gu L, Karabiyik T. P200-levels of Apelin-13 and total oxidant/antioxidant status in sera of Alzheimer patients. Turkish Journal of Biochemistry/Turk Biyokimya Dergisi. 2012;37(S1):341. [View at Publisher] [Google Scholar]
33. Luo H, Han L, Xu J. Apelin/APJ system: a novel promising target for neurodegenerative diseases. J Cell Physiol. 2020;235(2):638-57. [View at Publisher] [DOI] [PMID] [Google Scholar]
34. Samandari-Bahraseman MR, Elyasi L. Apelin-13 protects human neuroblastoma SH-SY5Y cells against amyloid-beta induced neurotoxicity: Involvement of anti oxidant and anti apoptotic properties. J Basic Clin Physiol Pharmacol. 2021;33(5):599-605. [View at Publisher] [DOI] [PMID] [Google Scholar]
35. Chen P, Wang Y, Chen L, Song N, Xie J. Apelin-13 protects dopaminergic neurons against rotenone-induced neurotoxicity through the AMPK/mTOR/ULK-1 mediated autophagy activation. Int J Mol Sci. 2020;21(21):8376. [View at Publisher] [DOI] [PMID] [Google Scholar]
36. Niknazar S, Abbaszadeh H-A, Peyvandi H, Rezaei O, Forooghirad H, Khoshsirat S, et al. Protective effect of [Pyr1]-apelin-13 on oxidative stress-induced apoptosis in hair cell-like cells derived from bone marrow mesenchymal stem cells. Eur J Pharmacol. 2019;853:25-32. [View at Publisher] [DOI] [PMID] [Google Scholar]
37. Khaledi S, Ahmadi S. Amyloid Beta and Tau: from Physiology to Pathology in Alzheimer's Disease. The Neuroscience Journal of Shefaye Khatam. 2016;4(4):67-88. [View at Publisher] [DOI] [Google Scholar]
38. Respekta N, Pich K, Dawid M, Mlyczyńska E, Kurowska P, Rak A. The Apelinergic System: Apelin, ELABELA, and APJ Action on Cell Apoptosis: Anti-Apoptotic or Pro-Apoptotic Effect? Cells. 2022;12(1):150. [View at Publisher] [DOI] [PMID] [Google Scholar]
39. Zeng X, Yu SP, Taylor T, Ogle M, Wei L. Protective effect of apelin on cultured rat bone marrow mesenchymal stem cells against apoptosis. Stem Cell Res. 2012;8(3):357-67. [View at Publisher] [DOI] [PMID] [Google Scholar]
40. Wang D, Wang Y, Shan M, Chen J, Wang H, Sun B, et al. Apelin receptor homodimer inhibits apoptosis in vascular dementia. Exp Cell Res. 2021;407(1):112739. [View at Publisher] [DOI] [PMID] [Google Scholar]
41. Xu W, Li T, Gao L, Zheng J, Yan J, Zhang J, et al. Apelin-13/APJ system attenuates early brain injury via suppression of endoplasmic reticulum stress-associated TXNIP/NLRP3 inflammasome activation and oxidative stress in a AMPK-dependent manner after subarachnoid hemorrhage in rats. J Neuroinflammation. 2019;16(1):247. [View at Publisher] [DOI] [PMID] [Google Scholar]
42. Liu Y, Zhang T, Wang Y, Wu P, Li Y, Wang C, et al. Apelin-13 attenuates early brain injury following subarachnoid hemorrhage via suppressing neuronal apoptosis through the GLP-1R/PI3K/Akt signaling. Biochem Biophys Res commun. 2019;513(1):105-11. [View at Publisher] [DOI] [PMID] [Google Scholar]
43. Zhang L, Li F, Su X, Li Y, Wang Y, Fang R, et al. Melatonin prevents lung injury by regulating apelin 13 to improve mitochondrial dysfunction. Exp Mol Med. 2019;51(7):1-12. [View at Publisher] [DOI] [PMID] [Google Scholar]
44. Chen WN, Yeong KY. Scopolamine, a Toxin-Induced Experimental Model, Used for Research in Alzheimer's Disease. CNS Neurol Disord Drug Targets. 2020;19(2):85-93. [View at Publisher] [DOI] [PMID] [Google Scholar]
45. Safar MM, Arab HH, Rizk SM, El-Maraghy SA. Bone Marrow-Derived Endothelial Progenitor Cells Protect Against Scopolamine-Induced Alzheimer-Like Pathological Aberrations. Mol Neurobiol. 2016;53(3):1403-18. [View at Publisher] [DOI] [PMID] [Google Scholar]
46. Bajo R, Pusil S, López ME, Canuet L, Pereda E, Osipova D, et al. Scopolamine effects on functional brain connectivity: a pharmacological model of Alzheimer's disease. Sci Rep. 2015;5(1):9748. [View at Publisher] [DOI] [PMID] [Google Scholar]
47. Gazmeh S, Azhir M, Elyasi L, Jahanshahi M, Nikmahzar E, Jameie SB. Apelin-13 protects against memory impairment and neuronal loss, Induced by Scopolamine in male rats. Metab Brain Dis. 2022;37(3):701-9. [View at Publisher] [DOI] [PMID] [Google Scholar]
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