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Introduction
Alzheimer’s disease (AD) is the one of the most common age-related neurodegenerative disorders, a multifactorial dementia type with personality change, cognitive domain disorder, and inappropriate behavior. The clinical symptoms are the loss of selected cognitive domains, especially those related to memory, which begins with episodic memory loss, impaired concentration, a sudden mood change, and difficulty performing daily tasks. As the disease progresses, symptoms such as forgetfulness, inability to communicate, confusion, difficulty swallowing or speaking, and even movement appear [1].
The use of statins in treating AD is a topic of controversy due to potential side effects, optimal dosage, and long-term effects on brain health [2, 3]. Statins have been shown to affect the development of AD and cognitive function [4, 5], while their beneficial effects are still up for debate [2, 3]. On one hand, statins can decrease the production of beta-amyloid by inhibiting the beta-secretase enzyme involved in its production [6]. Also, statins, rich in antioxidant properties, have been found to reduce neuroinflammation associated with AD by inhibiting the activation of microglia, immune cells in the brain [7]. On the other hand, some studies suggest that higher doses of statins may be more effective, while others suggest that lower doses may be sufficient and have fewer side effects [8, 9].
Neurogenesis in the adult’s hippocampus dentate gyrus (DG) occurs constitutively throughout postnatal life. Various physiological and pathophysiological conditions can alter the rate of neurogenesis within the DG. Different gene expression levels can help diagnose the AD treatment process, including doublecortin (DCX) [10, 11], calbindin [10], and NeuroD [12]. These genes are markers of differentiating neurons [13]. Whereas DCX labels immature neurons in the granule cell layer of the DG, calbindin is a marker of mature cells [11]. NeuroD has been identified as a differentiation factor for neurogenesis which is important for the proper development of the DG since studies of NeuroD-deficient mice have revealed that NeuroD is crucial for the proliferation of neurons in the DG and for postnatal differentiation [14].
Recent studies show that elevated cholesterol levels increase the risk of developing AD and investigations illustrate the potential use of lipid-lowering agents, particularly statins, as preventive or therapeutic agents for AD [15]. Impaired regulation of cholesterol homeostasis in the brain significantly increases the risk of AD. Thus, dysregulation of lipid homeostasis may increase amyloid beta (Aβ) levels by affecting amyloid precursor protein (APP) which is the most critical risk factor for the pathogenesis of AD [16]. Previous research has shown that Aβ can induce neuronal insulin resistance, essential in responding to Aβ-induced neurotoxicity in AD [17]. Besides inhibiting cholesterol metabolism by Statins, recent studies also show that they have neuroprotective effects including the effect on nitric oxide by upregulating endothelial nitric oxide synthase; anti-inflammatory effects by attenuating essential mediators of inflammation and immunological responses; antioxidant effect by reducing lipoprotein oxidation [18]. Rosuvastatin (RZV) is a type of statin that inhibits the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and is effective on plasma lipids [19]. It decreases low-density lipoprotein cholesterol, apolipoprotein B, and triglycerides and increases high-density lipoprotein cholesterol [20]. Absorption of RZV is sufficiently from the gastrointestinal tract, and the peak plasma concentration of the drug is reached 5 hours after oral administration [19]. It is mainly metabolized in the liver by the cytochrome P450 isoenzyme (CYP2C9). Up to 90% of it binds to plasma proteins and the plasma half-life of the drug is 19 hours. In general, lipophilic statins have more side effects than hydrophilic ones [21]. Significant side effects of statins include elevated serum transaminases and muscle inflammation (myositis). Indigestion and mild, transient, and reversible abdominal pain are other side effects of statins [22]. Since a large part of the world’s pharmaceutical research is devoted to the drug delivery system and operating the extent of drug penetration into the central nervous system (CNS) and, consequently, evaluating drug efficacy in the CNS, we used chitosan nanoparticles (NPs) to increase or decrease drug access to the brain. This research aimed to survey the effect of RZV coated with chitosan NPs on a model of Alzheimer’s (Alz) induction in rats. Thus, we evaluate neuronal survival, alteration of behavioral indicators, DCX, calbindin, and NeuroD1 gene expressions as neuronal markers following drug administration.
Materials and Methods
Animals and ethics
Adult male Wistar rats were purchased from the Pasteur Institute (Tehran, Iran), weighing approximately 200–230 g. Animals were housed under standard controlled conditions (temperature 22°C–24°C, relative humidity of 30% to 50%, 12 hours light-dark cycle) and allowed free access to standard rat chow and drinking water during the experiment. Duo to the Institutional Animal Care and Use Committee recommendations (code number; IR.IAU.K.REC.1399.026), forty rats were randomly divided into five groups, or a power analysis using inclusion, exclusion, and mortality indicated that each of the five experimental groups required 8 animals. (1) Control: no treatment was performed in this group and animals received 1 μl normal saline into hippocampus bilaterally. (2) Alz: rats in this group were injected bilaterally with 1 μg/μl of Aβ1-42 into hippocampal CA1 area to induce the model. (3) Alz+NP: Alz rats received 200 mg/kg body weight NP by oral gavage for 30 consecutive days. (4) Alz+RZV: Alz rats received 20 mg/kg body weight with RZV by oral gavage for 30 consecutive days. (5) Alz+RZV+NP: Alz rat Alz rats received RZV (20 mg/kg) coated with chitosan NPs (200 mg/kg) by oral gavage for 30 consecutive days.
Aβ1-42 and induction of Alz
Powder of Aβ (Sigma-Aldrich Co.) was dissolved in normal saline (pH=7.2) at the concentration of 1 μg/μl and incubated at 37°C for one week before application.
Shortly, animals were anesthetized with injection of the mixture of ketamine (90 mg/kg) and xylazine (10 mg/kg; Bremer pharma GmBH) intraperitoneally. After cutting and drilling the skull, Aβ1-42 (1 μg/μl in each site) was infused into the CA1 area of the hippocampus bilaterally (1 μl/5 minutes) using a 10-μl Hamilton syringe (Hamilton; catalog number: 80300); after infusion, the cannula was remained in place for an additional 3 minutes to allow the complete diffusion of the drug. The infusion was done with coordinates of anteroposterior (AP)=−4.8 mm from the bregma, mediolateral=±3.5 mm, dorsoventral=−4 mm from dura mater, according to the Paxinos and Watson rat brain atlas for stereotaxic surgery All animals were allowed the recovery period before testing (7–10 days) [23].
Rosuvastatin and chitosan-nanoparticles
RZV-encapsulated chitosan was prepared by the Chandirika et al. [24] method. First of all, 10 mg of RZV was diluted in 1 ml ethanol to obtain a stock. To access a homogeneous solution, RZV particles were dissolved by sonication for 2 minutes and stirred for 5 minutes with a magnetic stirrer. Chitosan (0.1% w/v) was diluted in 1% acetic acid and cross-linked by sodium tripolyphosphate 2% (w/v) very gently at room temperature. The RZV solution was loaded 10 mg/ml into the chitosan-NP in distilled water. The solution was added gently to 10 ml of chitosan solution in magnetic stirring. It was further stirred for two hours and afterward centrifuged at 1,000 rpm for 10 minutes. The supernatant was removed, and then the pellet was used as a final product.
Analysis of rosuvastatin nanosuspensions by dynamic light scattering
The size distribution of droplets for the RZV-encapsulated chitosan structure, was assessed using the dynamic light scattering (DLS) approach [25] using a Malvern ZetaSeizer Nano-series instrument (HORIBA SZ-100 for Windows [Z type] ver 2.20; HORIBA). Approximately 30 µl of the sample was filtered using a standard syringe filter with a pore size of 0.2 µm or smaller or through spin-filtering by SpinX® Cat. #8160 (Corning Inc.). Subsequently, the filtered sample was placed into the cuvette and analyzed using the ZetaSizer Nano-series instrument.
Behavioral testing: Morris water maze
The Morris water maze (MWM) was performed to survey spatial learning and reference memory according to Morris [26] The MWM consisted of a black pool (a diameter of 180 cm and a depth of 60 cm) filled with water (26°C±2°C). A circular black platform was submerged 2 cm below the water surface, in the middle of the target quadrant. The behavior of the rats in the pool could be tracked with a camera connected to the Ethovision system (Ethovision XT 7; Noldus Inc.) allowing us to measure swim speed, travelled distance, and escape latency and spending time to find the platform. The rats in each group were tested (one at a time). The rats were trained for four days prior to the formal experiments. Each rat was subjected to 4 consecutive trials on each day with an interval of 1 minute. Each trial was initiated by placing the rat randomly at 1 of the 4 starting points. The rats were allowed to swim in the pool for a period of 90 seconds to locate the hidden platform. If a rat did not locate the hidden platform within this period, it was manually guided to the platform by the investigator. The rats were allowed to remain on the platform for 30 seconds. All the trials were performed at 9 A.M. [27].
Tissue collection and processing
One day after the behavioral test, the rats were euthanized and brain rapidly removed on ice and kept at –80°C until use. These dissected pieces were fixed and separated with paraffin. The sections were made by rotary microtome machine (Leica RM2265 Rotary Microtome; Leica) in different thicknesses (μm) and then placed on slides covered with polylysine and used for for the cresyl violet, thioflavin staining and immunohistochemical staining.
Thioflavin S staining of amyloid beta
The analysis of beta-amyloid plaque in brain tissue was done using thioflavin staining. The anterior brain region, which includes the hippocampal region, was preserved in 4% formalin following rat sacrificing. As a result, tissue sections were cleaned in 70% ethanol after being stained with 1% thioflavin S (Sigma-Aldrich). After washing with H2O, the sections were mounted in 75% glycerol in H2O. Stained areas are expressed as a percentage of the hippocampus, respectively. For CA1, thioflavin S-positive blood vessels in three to four right hemisphere sections for each rat are enumerated and expressed as a number of thioflavin S-positive blood vessels per square millimeter. Parts were examined visually with a fluorescence microscope (Labomed LX-400 Binocular Fluorescent microscope; Labomed) [28].
Cresyl violet staining
We analyzed cell density and intact cell count of the cornu ammonis (CA) 1 sections of the brain with 10-μm thickness by cresyl violet staining according to Nissl stained method [29]. After rinsing the pieces in distilled water, they were dried in a series of ethanol grades. Then, they were mounted in entellan and submerged in xylene. After being cover-slipped, neuronal loss identified by Nissl-positive cells was investigated and evaluated. The overall count of the representative sections is defined as the number of pyramidal cells in the CA1 region of the hippocampus. A light microscope was used to visually evaluate each section (DP12) with the magnification of ×400 (in the area of 133,530 μm²) using OLYSIA Bio Report Soft Imaging System GmbH, Version: 3.2 (Build 670).
Hematoxylin and eosin staining
Hematoxylin and Eosin (H&E) staining detects morphological changes in the rat hippocampus. After the entire brain was stored overnight in 10% formalin, the brain was embedded in paraffin for 4 hours. Paraffin blocks were prepared, and 5 μm CA of hippocampus were cut with a rotarty microtome (Leitz 1512; Leica). Units were mounted on silane-coated slides, washed in xylene to dewax, rehydrated in graded ethanol, and finally stained with H&E (Servicebio). The hippocampal neurons morphology observed at ×400 magnification and the number of intact neurons in 1 mm length of the middle portion of the hippocampal CA1 region was counted using a light microscopy (Nikon Eclipse E100; Nikon) connected to a camera (Olympus, DP12; Olympus), and quantitatively analyzed by Image J software [30].
Gene expression
To assess the expression of the DCX, calbindin, and NeuroD1 genes, real-time polymerase chain reaction (PCR) is employed in the investigation of brain tissue. Total RNA was extracted from about 100 mg of brain tissue (hippocampus) in a sterilized RNase-free tube using the Qiazol (Qiazol Lysis Reagent; Qiagen) method.
The absorbance ratio was used to determine the content and purity of RNA at 260 nm and 280 nm (A260/A280) by employing a NanoDrop ND-100 spectrophotometer (Thermo Scientific). The RevertAid cDNA synthesis kit converted RNA into cDNA (Fermentas) in a volume of 25 μl according to the manufacturer’s structure. The process of PCR amplification was done by 2 μl of the synthesized cDNA, 0.2 μl of each forward and reverse primers (at the concentration of 100 μmol/L), 12.5 μl AccuPrime SuperMix I (Fermentas), and 10.1 μl of distilled water. Primer 3 software was utilized in the design and confirmation of primers by the NCBI BLAST Tool. The primers used during this study are displayed in Table 1. To operate real-time PCR to assess the relative gene expression, 500 ng of the newly-synthesized cDNA was used. PCR reactions were carried out in a total volume of 25 μl containing 12.5 μl of SYBR Green Premix 2X (Takara Bio) and 10 pico-molar of mixed primers. To standardize the expression levels of the genes under investigation, the reference gene GAPDH was utilized. Initial denaturation at 95°C for 15 minutes, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds were the parameters of the PCR program. In the end, a melting curve analysis was carried out. All tests were run three times, and determining of relative expression of each gene was performed by the 2–∆∆Ct technique.
Immunohistochemistry staining
Hippocampal coronal slices from both control and treatment rats were stained with IHC to examine the expression of neuronal nuclei (NeuN) and neurofilament proteins near the injection site. First, the sections of the hippocampus/choroid plexus were blocked with 0.3% Triton X-100 and 10% goat serum in phosphate buffer saline (PBS) (pH 7.3) for 30 minutes. The primary antibodies for NeuN (rabbit anti-NeuN, 1:100; Abcam) and neurofilament (anti-neurofilament, 1:1,000; Abcam) were added to tissue sample with PBS and the slices were then refrigerated at 4°C overnight. After washing with 0.01 M PBS, the slices were incubated with a secondary antibody at 37°C for 90 minutes in a dark place. Peroxidase-conjugated secondary antibodies were used for chromogenic detection by oxidizing 3, 30-diaminobenzidin (1:200, ab205718) and it was utilized in accordance with the manufacturer’s guidelines. The sections were then rinsed with 0.01 M PBS and stuck to glass slides. The stained tissue sections of the rat brains were exposed to quantification and immunohistochemistry analysis after obtaining digital images at ×400 magnification using a Zeiss Axioplan 2 fluorescent microscope fitted with digital video camera system (Diagnostic Instruments). Three portions that are similarly spaced along the injection site’s level (AP: –3.3 mm) were examined. Using the Image J software, the digital images were analyzed (version 1.52h) by a blind observer.
Results
Dynamic light scattering
The size measurements of all the conjugates were performed using a Malvern ZetaSizer Nano-series equipment (HORIBA SZ-100). DLS measurements were obtained at a temperature of 25°C using a 3-mm light path cuvette and employing SBL as the algorithm. A refractive index of 1.331 was utilized. The outcomes obtained from the DLS analysis results indicate that the RZV-encapsulated chitosan-NPs have an average size of 50±5.1 nm (Fig. 1).
Morris water maze
To survey the cognitive function of rats, the MWM test was performed (Figs. 2, 3). The results of the escape latency test (second) on days 1 to 4 (F [4, 12]=4.908, P<0.0141) between the healthy group and the Alz showed a significant difference (P<0.05), which shows the correct induction of disease. Also, there is no significant difference between the group treated with Alz+NP and the group treated with Alz+RZV compared to the Alz group (P>0.05) (Fig. 2A, B). In contrast, a significant difference was observed between the Alz group, and the group treated with Alz+RZV+NP (P<0.05). The results of travelled distance on days 1 to 4 (Fig. 2C), F (4, 8)=11.24, P<0.0023 showed that in the Alz group, compared to the healthy group, the travelled distance increased significantly, indicating that the cognitive impairment was correctly induced in the rats (P<0.05). Also, results demonstrated no significant difference in Alz+NP and Alz+RZV groups compared to Alz group. On the other hand, there is significant difference between Alz+RZV+NP with Alz group (P<0.05).
In the test day, we also measured escape latency, travelled distance, swimming speed, and spending time (Fig. 3). The rats in Alz, Alz+NP, and Alz+RZV group showed a significant increase in escape latency compared to the control group. Compared to the Alz group, escape latency significantly decreased in Alz+RZV+NP (P<0.05) (Fig. 3A). The travelled distance (F [4, 10]=10.51, P<0.0013) significantly increased in Alz group compared to healthy controls (P<0.05). Treatment with RZV, NP, and NP+RZV significantly decreased the total distance travelled (F [4, 10]=28.47, P<0.0001) to reach the platform in compared to Alz group (P<0.05), which suggested improvement in memory associated with treatments (Fig. 3B). As shown in Fig. 3C, there is a significant decrease in swimming speed (F [4, 8]=12.71, P<0.0015) in the probe trial of Alz, Alz+NP, and Alz+RZV groups compared to the control group and these results indicate that there were motor activity disturbances in Alz rats with different treatments. On the other hand, there is a significant increase between Alz+RZV+NP and Alz group (P<0.05). As shown in Fig. 3D, animals in Alz group showed a significant decrease in time spent (F [4, 8]=12.24, P<0.0017) in the target quadrant compared to the control group (P<0.05). Also, there is a significant difference between Alz+RZV and Alz+RZV+NP with Alz group (P<0.05).
Thioflavin S staining of amyloid beta
According to our data, Aβ plaques percentage (F [4, 10]=120.3, P<0.0001) in Alz and Alz+NP groups significantly increased compared to control group in the brain sections (P<0.001). On the other hand, there is a significant decrease in Aβ plaques in the brain sections of Alz+RZV+NP (P<0.001) and Alz+RZV (P<0.01) groups in compared to Alz and Alz+NP groups (Fig. 4).
Cresyl violet staining for dead cell counting
Cresyl violet staining was used to evaluate changes in dead cells in the CA1 hippocampus of rats. The results showed (Fig. 5) that the percentage of dead cells increased significantly (F [4, 10]=166.7, P<0.0001) in Alz and Alz+NP groups in comparison with the control group (P<0.05). Comparing with the Alz group, Alz+RZV, and Alz+RZV+NP revealed a significant decrease in the number of dead cells. The lowest number of dead cells was associated with the Alz+RZV+NP group (P<0.001). In contrast, no significant difference can be seen between ALZ and ALZ+NP groups (P>0.05).
Hematoxylin and Eosin staining of hippocampal CA1 neurons
The H&E staining results demonstrated hippocampal CA1 neurons in three layers (Fig. 6). In these layers, nerve cells are heterochromatin, with specific cytoplasm, transparent nuclear membrane, and cell membrane. One or more nucleoli characterize the active and healthy neurons from round to oval. The neurons with dark cytoplasm, unclear nucleus, absence of membrane, and cytoplasm are considered dead or dying neurons. In Alz and Alz+NP groups the number of dead cells gradually increased. In addition, the cells with small sizes and dark nuclei around the neurons were considered oligodendrocyte or astrocyte cells. Also, in Alz and Alz+NP groups, many single cells have fewer cytoplasmic nuclei, characterized as microglial cells, which inthe Alz+RZV and Alz+RZV+NP, this amount decreased significantly (Fig. 6A). In addition, we investigated the number of intact neuron in the hippocampal CA1 region. According to our data, the number of intact neurons (F [4, 10]=37.94, P<0.0001) in Alz and Alz+NP groups was significantly lower than control group (P<0.001). In contrast, the number of intact neurons the hippocampal CA1 sections of Alz+RZV+NP (P<0.001) and Alz+RZV (P<0.001) groups was significantly more than Alz and Alz+NP groups (Fig. 6B).
The calbindin, DCX, and NeuroD1 genes expression
In regard to Fig. 7, through establishing the Alz model in rats, expression of calbindin (F [4, 10]=7.154, P<0.0055), DCX (F [4, 10]=9.261, P<0.0021), and NeuroD1 (F [4, 10]=3.849, P<0.038) genes considerably lower than in the control group (P<0.01). While all three of these genes’ expression rose following therapy, this rise is not statistically significant when compared to the Alz group. Only for the DCX gene, there is a noticeable difference in Alz+RZV+NP compared to the Alz group.
Protein expression of NeuN and neurofilament
Protein expression of NeuN (F [4, 10]=107.1, P<0.0001) and neurofilament (F [4, 10]=76.48, P<0.0001) in brain tissue was determined using the immunohistochemical technique (Fig. 8). According to Fig. 8A, B, when compared to the control group, the induction of AD resulted in a significant drop in NeuN protein expression in all groups’ brain tissue. Compared to the Alz group, Alz+RZV (P<0.001), and Alz+RZV+NP (P<0.001) groups shown a notable rise in NeuN. Also, the rise in the expression of the NeuN protein of the Alz+RZV+NP group was significant compared to the Alz+RZV group (P<0.01).
Neurofilament protein expression changes in brain tissue were similar to NeuN (Fig. 8C, D). When compared to the control group, the expression of the Neurofilament protein was significantly lower in all groups. Alz+RZV (P<0.01) and Alz+RZV+NP (P<0.001) groups demonstrated a noteworthy rise in neurofilament in contrast to the Alz group. Furthermore, compared to the Alz+RZV group, the Alz+RZV+NP group had a significant rise in neurofilament protein expression (P<0.01).
Discussion
Alz is one of the progressive diseases of the nervous system, which has become more challenging today due to the aging of the human population. Further studies on the mechanisms of Alz pathogenesis demonstrated a direct relationship between the increase of blood cholesterol and the accumulation of Aβ due to the prevalence of AD. Epidemiological studies also indicate that using statins, which are blood cholesterol-lowering drugs, reduces the incidence of AD [31]. On the other hand, statins improve learning and memory ability by inhibiting inflammatory response in AD progression by decreasing cytokines such as IL-1β, IL-6, and TNF-α, which can attenuate nerve cell damage. Also, statins can reduce the expression and function of molecules on the leukocytes’ surface. In addition, they can inhibit transendothelial migration and chemotaxis of neutrophils, which can explain the anti-inflammatory effect of these compounds [32]. So, the theory of the effect of blood cholesterol controlling drugs on Alz treatment was proposed. Therefore, we evaluated the effect of RZV coated with chitosan NPs in an Alz induced model of rats.
The therapeutic potential of statins on AD was reported by Jick et al. [3] and Wolozin et al. [33] for the first time. They surveyed 284 cases with dementia and 1,080 control subjects. Among the control group, 13% had untreated hyperlipidemia, 11% were prescribed statins, 7% were prescribed other cholesterol-lowering drugs, and 69% did not have hyperlipidemia or did not take cholesterol-lowering drugs. Their results demonstrated that people aged fifty years and older who received statins had a lower risk of developing dementia, regardless of the presence or absence of untreated hyperlipidemia and receiving blood cholesterol-lowering drugs other than statins [3]. In the same year, another study revealed that Lovastatin and Pravastatin reduce the progression of AD, but simvastatin does not.
Our findings of the spending time and travel distance behavioral tests showed a significant difference between the control group and the Alz group, which means correctly induced Alz model. Comparing the results of the spending time test in the RZV groups with and without chitosan NPs have significant differences compared to the Alz group. As a result, the memory and learning ability of animals treated with RZV as well as chitosan-RZV NP has increased compared to the untreated groups.
Also, an in vivo behavioral study indicated that in the scopolamine-induced Alz rat model, intranasal cerium oxide NPs dose-dependently reversed the cognitive ability [34]. On the other hand, the results of another research group demonstrated that treating rosuvastatin in a high-salt and cholesterol diet in rats would ameliorate cognitive impairment and reduce cholesterol deposition, acetylcholinesterase activity, and A1-42 peptide aggregation [35].
Our histopathological studies using H&E, cresyl violet, and thioflavin revealed a decrease in dead neurons, apoptotic cells, and percentage of Aβ1-42 plaque accumulation, respectively, in the AD rat model. The widely used cholesterol-lowering drugs simvastatin and lovastatin decrease intracellular and extracellular levels of Abeta42 and Abeta40 peptides in primary cultures of hippocampal neurons and mixed cortical neurons. Likewise, guinea pigs treated with high doses of simvastatin revealed a potent and reversible drop of cerebral Abeta42 and Abeta40 levels in the cerebrospinal fluid and brain homogenate [36].
Besides, in a research which used the cresyl violet staining, treatment with pomegranate extract-loaded NPs was more effective in recovering Nissl’s granules of cerebral neurons than the control group in aluminum chloride-induced Alz rat model [37]. Wang et al. [38] indicated that the estradiol-loaded chitosan NPs significantly improved estradiol transported into the central nervous system. These NPs can be attached to negatively charged materials such as mucus and cell surfaces. Due to having various chemical compounds, such as sialic acid, which has a negative charge, mucins can have a solid electrostatic interaction with chitosan. Chitosan, as a bioadhesive material, significantly increases the half-time of clearance.
In this study, we also considered the expression levels of two proteins, NeuN and neurofilament, and both markers significantly decreased after the model’s induction compared to the control group. After treatment with RZV coated with chitosan NPs, both protein markers’ expression percentages increased significantly compared to the Alz group. In the research conducted in 2018, the therapeutic effect of Simvastatin was investigated in 3xTg-AD Alz model mice. The results revealed that Simvastatin prevents cognitive disorders and improves the ability to learn and recall by reducing the apoptosis of hippocampal cells. Simvastatin treatment also increases anti-apoptotic gene expression and decreases apoptotic gene expression, which is probably associated with improved motor senses and cognitive ability in Alz induced mice [39].
Also, NeuN and glial fibrillary acidic protein immunofluorescence revealed that neurotropin treatment increased neuronal survival and reduced gliosis in tissue samples obtained from the lesion’s epicenter in rats subjected to contusion-induced spinal cord injury [40]. On the other hand, a group of researchers indicated that the neurofilament and SOX2 cell markers were modulated during the differentiation of the PC12 cell line into neurons by valproic acid encapsulated in the stabilized core-shell liposome-chitosan nanocarriers. So, their findings suggest a promising potential for the lip-valproic acid-chitosan nanocarrier in inducing the differentiation of PC12 into neurons for treating neurodegenerative disorders [41].
Assessment of calbinidin, DCX, and NeuroD genes in the Alz group revealed a significant decrease compared to the control group. In contrast, the expression of all three genes increased after treatment by RZV coated with chitosan NPs. Tong et al. [11] indicated that simvastatin improves memory and granule cell maturation via the Wnt/β-catenin signaling pathway in an Alz mouse model. Whereas calbindin-immunolabelled mature granule cells were decreased in APP mice and not restored, their dendritic arborizations were normalized to control levels by simvastatin treatment. Another study described that dihydroartemisinin upregulated the NeuN, NeuroD, MAP2, and synaptophysin levels and promoted neurite outgrowth in APP/PS1 mice [42].
On the other hand, some studies indicate the adverse effects of statins on memory. The treated astrocytes with Simvastatin leads to the induction of apoptosis in a dose- and time-dependent manner [43]. Furthermore, orally treated healthy rats with Simvastatin for forty-five days causes deficits in spatial memory using the Barnes maze. Also, it was reported that simvastatin interferes with myelin repair in mice with myelin damage. Since cholesterol synthesis is necessary for the normal functioning of the brain, there is a possibility that inhibiting cholesterol synthesis following the use of simvastatin leads to cognitive impairment [44].
In conclusion, the data reported in this study illustrate that RZV coated with chitosan NPs in an Alz induced model of rats decreased apoptotic, dead cells, and beta-amyloid aggregations. On the other hand, the treatment can alter behavioral indicators and increase NeuN and neurofilament protein synthesis and DCX, calbindin, and NeuroD gene expression. The results also indicate that if preclinical tests and clinical trials would success, this combination can be a suitable candidate along with other effective drugs in the prevention or treatment of Alz.
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