Therapeutic potential of SSZ in modulating Alzheimer’s disease pathology: A multi-targeted experimental approach

Shams Tabrez; Zuber Khan; Nasimudeen R Jabir; Syed Kashif Zaidi; Sidharth Mehan; Mohammad Abid; Torki A. Zughaibi; Mohammad Hassan Alhashmi; Rahaf F Khoja; Md Nasiruddin Khan; Ravi Rana; Mohd Suhail; Shazi Shakil

doi:10.25259/JKSUS_1564_2025

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Research Article

ARTICLE IN PRESS

doi:

10.25259/JKSUS_1564_2025

Therapeutic potential of SSZ in modulating Alzheimer’s disease pathology: A multi-targeted experimental approach

Shams Tabrez^a,b,*,#, Zuber Khan^c,#, Nasimudeen R Jabir^d, Syed Kashif Zaidi^e, Sidharth Mehan^c,*, Mohammad Abid^f, Torki A. Zughaibi^a,b, Mohammad Hassan Alhashmi^a,b, Rahaf F Khoja^a, Md Nasiruddin Khan^c, Ravi Rana^c, Mohd Suhail^a,b, Shazi Shakil^e

aKing Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia

bDepartment of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia

cDivision of Neuroscience, Department of Pharmacology, ISF College of Pharmacy, Moga, Punjab, India

dDepartment of Biochemistry, Research and Development Cell, PRIST University, Vallam, Thanjavur, Tamil Nadu, India

eInstitute of Genomic Medicine Sciences, King Abdulaziz University, Jeddah, Saudi Arabia

fMedicinal Chemistry Laboratory, Department of Biosciences, Jamia Millia Islamia, New Delhi, India

^#Equal contributing author

* Corresponding author: E-mail address: shamstabrez1@gmail.com (S. Tabrez), sidh.mehan@gmail.com (S. Mehan)

Received: 2025-10-06, Accepted: 2025-12-22, Epub ahead of print: 2026-01-30,

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Abstract

Alzheimer’s disease (AD) is a common neurological disorder marked by progressive cognitive decline and memory loss, and it remains a major global health concern due to the limited effectiveness of current symptomatic treatments. This study used SSZ, a novel, chemically synthesized compound featuring pyrrolopyridine and N-cyclohexyl groups, designed as a multi-targeted inhibitor with anti-AD pharmacophore properties. We investigated the therapeutic potential of SSZs using an experimental rat model of AD produced by amyloid-beta (Aβ). SSZ demonstrated substantial pharmacological activity by targeting key enzymes implicated in AD pathogenesis, including BACE-1, gamma-secretase, MAO-B, and acetylcholinesterase. In addition, SSZ therapy demonstrated neuroprotective benefits by significantly reducing apoptotic markers (Bax, caspase-3) and upregulating the anti-apoptotic protein Bcl-2. This compound also restored myelin basic protein (MBP) levels and reduced pathological markers such as neurofilament light chain (NEFL) and microtubule-associated protein (MAP). Moreover, SSZ increased antioxidant defences, such as glutathione (GSH) and superoxide dismutase (SOD), decreased oxidative stress markers, such as lactate dehydrogenase (LDH), nitric oxide (NO), and malondialdehyde (MDA), and regulated inflammatory cytokines, such as TNF-α and IL-1β. Notably, SSZ restored neurotransmitter levels, dopamine, acetylcholine, and glutamate, essential for cognitive function. Histopathological analyses revealed that SSZ mitigated neuronal and myelin damage across critical brain regions, including the midbrain, hippocampus, and cortex. When combined with standard treatments such as donepezil and memantine, SSZ demonstrated synergistic effects, further enhancing its therapeutic efficacy. These results highlight SSZ’s potential as a multi-targeted therapeutic option to combat AD pathogenesis and improve therapy approaches.

Keywords

Acetylcholinesterase

Alzheimer’s disease

BACE-1

Butyrylcholinesterase

Neuroprotection

SSZ

γ-secretase

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1. Introduction

Alzheimer’s disease (AD) is the leading cause of chronic neurological conditions and remains a significant unmet medical need worldwide (Huang et al., 2023). Over 55 million individuals worldwide currently have AD, and by 2030, that number is expected to reach 82 million, posing a serious global health emergency, according to the World Alzheimer Report (Nayak et al., 2024; Huang et al., 2023). Although AD is mainly classified as an age-related illness, those over 65 are typically affected. There has been a rise in AD cases among younger individuals, with the condition beginning at around the age of 40 (Long et al., 2024; Zeliger, 2023). Due to variations in sex hormones, genetic vulnerability, and neuroinflammatory pathways, the course of AD differs in men and women. Therefore, recognizing sex-specific mechanisms may increase the translational significance of treatment research (Lopez-Lee et al., 2024).

The increasing prevalence of AD is closely linked to various risk factors, including aging, depression, smoking, diabetes, obesity, and hypertension, all of which together contribute to its rising incidence (Li et al., 2022). Addressing these risk factors and implementing effective strategies to manage AD is essential for reducing its long-term public health impact. Urgent actions are needed to prevent the onset of AD, slow its progression, and ease its severe symptoms, which not only affect patients but also place a significant burden on global healthcare systems and caregivers.

Neuronal death in the cerebral cortex and hippocampus, a region essential for learning and memory, is a clinical hallmark of AD, which worsens over time (Rao et al., 2022). Beyond memory deterioration, AD manifests with various mental and cognitive symptoms, including deviant behavior, aphasia, and agnosia. These symptoms collectively impose a substantial psychological and financial burden on patients, caregivers, and healthcare systems worldwide (Li et al., 2022; Hoque et al., 2023). The pathogenesis of AD is intricate and diverse. Still, they primarily include dysregulation in neurotransmission machinery, aberrant buildup of β-amyloid, aggregation of tau proteins, oxidative stress, mitochondrial failure, loss of synapses, and neuronal cell death (Makhaeva et al., 2021). Additionally, aging-related biological mechanisms significantly contribute to the disease’s pathophysiology (Guo et al., 2022).

The incredibly high failure rate of AD therapy development during clinical trials, roughly 99.6%, demonstrates the tremendous challenge of restoring the function of degraded neurons in AD (Cao et al., 2022). This failure is due to the disease’s complex nature and its multifactorial, nonspecific causes, highlighting the urgent need for a comprehensive, multidimensional treatment approach.

Over the past decade, considerable efforts have been made to discover disease-modifying therapies targeting the underlying neurological abnormalities related to AD (Golde, 2022). Among these, cholinergic enhancement through cholinesterase enzyme inhibition has proven effective. The USFDA currently approves cholinesterase inhibitors as first-line treatments for mild-to-moderate AD symptoms, including donepezil, galantamine, and rivastigmine (Tabrez, 2019; Zhang et al., 2024; Obrenovich et al., 2020). Concurrently, several studies on amyloid and tau-pathology-based theories are underway or have been completed (Cummings et al., 2023; Zhang et al., 2023; Lane-Donovan & Boxer, 2024). However, the limitations of these approaches have highlighted other promising therapeutic targets, such as inflammation, metabolic dysfunction, and vascular contributions, which are increasingly recognized as important factors in AD pathology (Cummings et al., 2023; UI Islam et al., 2017; Islam & Shams, 2017). Given the involvement of numerous molecular pathways in AD, concurrent modulation of multiple targets has been suggested as a promising therapeutic approach to attain more effective results (Drakontaeidi & Pontiki, 2024; Jabir et al., 2023).

Using in silico screening approaches, our team has previously identified and reported several multi-targeted ligand molecules capable of modulating multiple critical enzymes, including beta-site amyloid precursor protein cleaving enzyme (BACE-1), acetylcholinesterase, γ-secretase, butyrylcholinesterase etc. These molecules hold potential as effective anti-AD therapeutic agents and form the foundation for further exploration and development (Jabir et al., 2021).

Building upon the pharmacophoric features of our previously reported lead compounds, the current study investigates the molecular mechanism of SSZ in an experimental rat model. SSZ has N-cyclohexyl groups and pyrrolopyridine, which are known to have potential against AD and show promising pharmacological characteristics (Konecny et al., 2020; Haghighijoo et al., 2020). Pyrrolopyridine scaffolds have long been the focus of drug development efforts due to their presence in various naturally occurring polyheterocyclic compounds containing pyrrole and pyridine pharmacophores. Among the six different structural isomers of pyrrolopyridine, pyrrolo[2,3-b]pyridine, which is present in SSZ, is a heterocyclic compound that has a six-membered pyridine ring fused to a five-membered pyrrole ring (Wójcicka & Redzicka, 2021). This structural motif has been widely studied in anti-AD drug discovery and is linked to important drug targets, including monoamine oxidase B and phosphodiesterase (Vadukoot et al., 2020).

Furthermore, N-cyclohexyl groups are commonly found in both synthetic and natural drugs as major components or side chains. These fragments are reported as functional groups in anti-AD drugs that target enzymes, such as BACE1 and butyrylcholinesterase (Haghighijoo et al., 2020). Including these pharmacologically relevant scaffolds in SSZ has likely contributed to its enhanced pharmacological profile, providing improved efficacy against AD compared to previously reported compounds. These characteristics highlight SSZ’s promise as a beneficial candidate with several targets in the management of AD.

2. Materials and Methods

2.1 Chemicals and drugs

The synthesis and detailed characterization of SSZ have been described in our earlier publications, providing a strong foundation for its development and application (Tong et al., 2013; Khan et al., 2025a). The neurotoxic amyloid beta (Aβ) was purchased from Sigma-Aldrich (USA), while donepezil (DON) and memantine (MEM) were obtained from Sun Pharma, New Delhi, India. All compounds were prepared and supplied according to established protocols to ensure consistency and reproducibility. Specifically, MEM and DON were administered intraperitoneally after dissolving in 1% DMSO, following previously documented protocols (Guo et al., 2020; Anoush et al., 2023). Similarly, SSZ was dissolved in DMSO for intraperitoneal administration, following well-established methods (Guzmán-Ruiz et al., 2021; Sharma et al., 2016). All the compounds used in this investigation were classified as analytical grade to ensure high-quality experimental standards. Thorough preparation protocols were followed before administering all drugs and substances, guaranteeing the accuracy and reliability of the experimental results. These strict methods reinforce the credibility of the study’s findings and enhance confidence in their reproducibility.

2.2 Animals

This study involved fifty-six adult Wistar rats, consisting of 28 males and 28 females, as experimental subjects. The animals were obtained from the Central Animal House Facility of ISF College of Pharmacy, Moga, Punjab, India. To maintain consistent living conditions, each animal was housed separately in 38 × 32 × 16 cm polyacrylic cages that could accommodate two rats, with comfortable bedding. Following accepted ethical standards, husbandry procedures provided animals with unlimited access to water and food as needed. To ensure optimal conditions, environmental factors were carefully controlled. These included an automatic light-dark cycle replicating a 24-h rhythm, an average temperature of 22.2 ± 2°C, and a humidity level of 55 ± 10%. To ensure the animals’ well-being and minimize stress throughout the study, a 5-day acclimatization period was implemented before beginning the experimental procedures.

2.3 Animal ethical approval

The Institutional Animal Ethics Committee (IAEC) reviewed and approved the experimental procedure in accordance with the rules established by the Indian government. The study was conducted under registration number 816/PO/ReBiBt/S/04/CPCSEA. This protocol (ISFCP/IAEC/CPCSEA/Meeting number 03/2022/Protocol number 21) was carefully reviewed by the IAEC to ensure it complied with the ethical guidelines established by the CPCSEA. This comprehensive review process confirmed adherence to all ethical standards for the humane treatment and use of animals in scientific research.

2.4 Experimental animal classification

Animals were divided into eight groups based on the study’s design, which used a randomized allocation method: Group 1 received a sham control intervention, Group 2 received a vehicle control, and Group 3 was administered SSZ Perse intraperitoneally at 15 mg/kg. Group 4 was exposed to Aβ (200 ng/5 µL) via intracerebroventricular (ICV) administration; Group 5 received a combination of SSZ (15 mg/kg, i.p.) and Aβ (200 ng/5 µL, ICV); Group 6 was given Aβ (200 ng/5 µL, ICV) along with DON (5 mg/kg, i.p.); and Group 7 was treated with MEM (10 mg/kg, i.p.) in combination with Aβ (200 ng/5 µL, ICV). This detailed grouping aimed to thoroughly compare the effects of these interventions on the experimental results.

2.5 Experimental design schedule

The 42-day study was carefully designed to minimize the effects of the circadian cycle by limiting all activities to the hours of 9:00 a.m. to 2:00 p.m. Amyloid-beta (Aβ) was administered intracerebroventricularly (ICV) to the test animals from day 1 to day 5 to induce AD. To evaluate its therapeutic potential, 15 mg/kg of SSZ was given intraperitoneally (i.p.) from day 6 to day 42. During the same period, from day 6 to day 42, the NMDA receptor blocker, memantine (MEM; 10 mg/kg, i.p.) and the acetylcholinesterase inhibitor, donepezil (DON; 5 mg/kg, i.p.) were administered to assess how effectively these medications worked alongside SSZ. A series of behavioral and physiological assessments were performed to evaluate the phenotypic features of AD and have been reported in our earlier study (Khan et al., 2025a).

On day 43, after completing the study regimen, animals were anesthetized and sacrificed to obtain brain specimens. The entire brain was carefully removed and stored in formalin solutions for further analysis. These brain samples underwent gross pathological examinations, biochemical tests, and histological evaluations to investigate changes in white matter fibers and other pathological markers associated with AD. The experimental procedure is summarized in Fig. 1 and is based on previously established methodologies and findings. (Guzmán-Ruiz et al., 2021; Sharma et al., 2016; Zhang et al., 2023).

2.6 Methods

2.6.1 ICV-amyloid-β administration induced AD in an animal model

We administered the rat an intraperitoneal (i.p.) dose of 75 mg/kg ketamine to induce anesthesia. Once anesthetized, their heads were immobilized for surgery by placing them on a heated surgical pad with stereotaxic equipment (Stoelting Co., Wood Dale, IL, USA). After shaving and cleaning the scalp with 70% ethanol, the skull was exposed via a mid-sagittal incision. The bregma and lambda landmarks on the exposed skull were identified to determine the precise locations for ICV injections. Cotton swabs soaked in saline were used to control bleeding during the surgery.

Stereotaxic coordinates were used to drill a hole in the parietal bone to reach the right lateral cerebral ventricle: anteroposterior (AP) -0.92 mm from the bregma area, mediolateral (ML) ±1.5 mm from the mid-sagittal suture, and ventral position (VP) -3.0 mm from the skull surface (Kumar et al., 2018; Chhabra et al., 2023). A plastic ear pin was used to secure the 2.5-centimeter cannula after it was carefully inserted through the incision. To minimize complications after surgery, dental cement was applied to seal the opening, and a readily absorbed surgical suture was used to close the wound.

On the first day of the experiment, 5 μL of amyloid-beta (Aβ; 200 ng/kg body weight) mixed in a 0.1M citrate buffer (pH 4.5) was slowly injected into the right side of the brain’s ventricle using a 10-μl Hamilton microlitre syringe with a thin needle (0.4 mm wide) at a rate of 5 μL/min. The injection was done slowly at a rate of 5 μL/min. An injection was given steadily at 5 μL/min, (Alkandari et al., 2023; Prajapati et al., 2023). Following the injection, the microneedle was kept in place for 5 min to ensure adequate diffusion of Aβ into the cerebrospinal fluid (CSF). Using the same technique, a second injection of Aβ was administered into the left lateral cerebral ventricle 48 h later (Sharma et al., 2016; Prajapati et al., 2023; Upadhayay et al., 2022; Kapoor et al., 2022).

2.6.2 Cannula implantation procedure

As part of the experimental methodology, ketamine (80 mg/kg, i.p.) was used to anesthetize the entire experimental animal group to ensure immobilization and reduce pain. The scalp was carefully cut with a 1 cm midline incision, starting halfway between the eyes and extending beyond the lambda region at the back. A cotton swab was used to gently remove the delicate tissue covering the skull, revealing all cranial landmarks. A Hamilton syringe linked to an injector pump delivered 200 ng/5 μL of Aβ 1-42 directly into the right lateral ventricle of the brain. The stereotaxic coordinates used to place the needle properly concerning the bregma were anteroposterior (AP) = 0.92 mm, mediolateral (ML) = 1.5 mm, and ventral position (VP) = 3.9 mm. To ensure accurate injection needle placement, a small cranial burr hole was drilled at the designated entry point. A volume of 5 μL of Aβ (1-42) was slowly injected at a controlled rate to guarantee proper delivery. After the injection, dental cement was used to seal the burr hole, and surgical sutures were applied to close the incision, promoting normal healing. To ensure consistency across all experimental conditions, sham animals were injected with the same volume of saline solution in the control group. (Guzmán-Ruiz et al., 2021; Khera et al., 2022; Singh et al., 2021).

2.6.3 Post-surgical treatment

After the surgery, each rat was placed in a polyacrylic housing lined with a warm towel to ensure comfort and maintain body temperature throughout recovery. Special attention was given to the animals for 2-3 h post-anaesthesia until they regained spontaneous movement (Duggal et al., 2020; Sharma et al., 2022). For the first 2-3 days after surgery, glucose water and milk were provided in the animal cage to prevent harm and support healing. Gentamicin (35 mg/kg, i.p.) was administered three times daily for three days in sequential doses to lower the risk of sepsis. Additionally, lignocaine gel was applied to ease local discomfort, and neosporin powder was applied to the sutured area to prevent bacterial skin infections. Clinical parameters, including overall body health and hydration levels, were regularly monitored to ensure the animals’ well-being during recovery. By the seventh day post-surgery, the rats resumed spontaneous movement, displayed normal feeding behavior, and consumed a nutritious diet and water, indicating successful recovery (Upadhayay et al., 2022; Khera et al., 2022; Khan et al., 2025b).

2.7 Neurochemical analysis

2.7.1 Procedure for acquiring and processing biological specimens

A 30-gauge insulin syringe was modified for cerebrospinal fluid (CSF) collection by bending the needle to an angle of 90–120° using 4–5 mm metal forceps. After removing the infusion catheter, the curved needle was attached to narrow-cut tubing from an intravenous infusion set. The other end of the tubing was attached to a 1 mL needleless syringe to aid fluid collection. Rats received sodium phenobarbital (270 mg/mL, i.p.) to induce anesthesia, and the full extent of the anesthesia was confirmed using the toe-pressing reflex test.

To collect CSF minimally invasively, the rats were placed on a specialized platform that simplified the procedure. To protect the animal’s eyes, erythromycin eye ointment was applied before inserting its head into a conical hole. The head was placed vertically to locate the foramen magnum, then cleaned with 75% ethanol. A precisely measured, curved needle was carefully inserted along the margin of the foramen magnum to access the cerebello-medullary cistern without damaging the brain’s parenchyma. A syringe with flexible tubing was used to collect CSF. The syringe plunger was carefully withdrawn to aspirate CSF into the tube after the needle was vertically inserted through the epidermis of the foramen magnum. At the same time, the skull was steadied with one hand. Once the required volume of CSF (70–120 μL) was collected, the syringe and tubing were carefully detached, and the curved needle was removed. After collecting CSF, the plunger was depressed to transfer the contents to a microcentrifuge tube. The minimally invasive procedure allowed the animals to recover and resume normal activities within 5–10 min.

Blood and CSF were collected from the anaesthetized animals on day 43 after the treatment concluded. The researchers used the retro-orbital method to obtain blood samples by carefully inserting a capillary tube behind the nictitating membrane at the medial canthus of the eye. The plasma was separated by centrifuging the collected blood for 15 min at 10,000 × g in a capillary tube. In preparation for further neurochemical analysis, plasma samples were stored at -80°C. CSF was collected as previously described. Rats were euthanized, and the entire brains were carefully removed after collecting the CSF. After rinsing with freezing isotonic saline, the brains were cleaned using 0.1 M (w/v) stored phosphate-buffered saline solution (PBS, pH 7.4). Centrifugation at 10,000 × g for 15 min separated the supernatant following homogenization of the brain samples. The supernatant was stored at -80°C for further biochemical analysis (Chhabra et al., 2023; Sharma et al., 2022; Kumar et al., 2024).

2.7.2 Enzyme-linked immunosorbent assay (ELISA)

The brain homogenates, blood plasma, and CSF were spun in a centrifuge at 10,000 rpm and 4°C for 5 min after homogenizing the brain tissue samples with PBS. The enzyme-linked immunosorbent assay (ELISA) kits were then used to test the supernatant liquid according to the manufacturer’s instructions (Guzmán-Ruiz et al., 2021; Sharma et al., 2016; Zhang et al., 2023).

Specific enzymes and biomarkers were quantified in the brain homogenates. These included BACE-1 (orb777177, Biorbyt, Cambridge, United Kingdom), γ-secretase (orb782816, Biorbyt, Cambridge, United Kingdom), and MAO-B (orb782186, Biorbyt, Cambridge, United Kingdom). Additionally, neurofilament light chain (NEFL) (E-EL-R2536; Elabscience, Wuhan, China) and myelin basic protein (MBP) (E-EL-R0642; Elabscience, Wuhan, China) were measured, as performed in previous studies [52,58]. Apoptotic markers, including bcl-2 (KLR1880; Krishgen Biosystem), Bax (KLR0034; Krishgen Biosystem) and caspase-3 (KLR1648; Krishgen Biosystem, Mumbai, India), were also analyzed to assess cellular apoptosis (Khan et al., 2025b). Neurotransmitter levels of acetylcholine (KLR0722), glutamate (KLR1474), serotonin (KLR0866), and dopamine (KLR0219) were quantified using ELISA kits (Chhabra et al., 2023). Furthermore, neuroinflammatory cytokines, including TNF-α (KB1145) and IL-1 beta (KLR0119), were evaluated using kits from Krishgen Biosystem, Mumbai, India (Prajapati et al., 2023; Albekairi et al., 2022).

2.7.3 Histopathological analysis

To assess the impact of treatments on neuronal tissue, experimental animals were euthanized on day 43 following anesthesia induced by sodium phenobarbital (270 mg/mL, i.p.). The brain samples were carefully extracted and prepared for processing for histopathological analysis. Coronal sections were selected for detailed analysis, covering areas, such as the cerebral cortex, midbrain, hippocampus, and striatum. Hematoxylin and eosin (H&E) staining was used to detect potential abnormalities, including inflammatory infiltration or other pathological changes, in these specific brain regions.

After removing the cerebral organs from the cranial cavity, they were carefully cleaned and sliced into 5-mm-thick sections. To preserve the tissue structure, they were fixed in a solution of 4% paraformaldehyde in PBS with a pH of 7.4 and kept at room temperature for 8–12 h. Following fixation, tissue samples were rinsed with PBS and then soaked in 70% ethanol. During the paraffin embedding process, tissues were kept at 37°C to ensure proper embedding.

A rotary microtome cuts thin slices (5 µm) from paraffin-embedded tissue blocks. These sections were stained with H&E to facilitate histopathological evaluation. Morphological assessments were conducted using a MOTICAM-Ba310 image plus 2.0 digital microscope at 40× magnification, enabling detailed observation of neuronal structures. Additionally, a fluorescence microscope with an ocular reticule was used at 40× magnification to count the average number of neuronal cells in different areas of the coronal slices. This detailed protocol enabled a thorough assessment of neuronal morphology and structural changes in the experimental rat model, offering insights into how the treatments affect brain tissue integrity (Prajapati et al., 2023; Upadhayay et al., 2022; Kumar et al., 2024).

2.8 Statistical analysis

GraphPad Prism 8.0.1 (San Diego, CA, USA) was used to analyze the experimental data to ensure a thorough statistical assessment. To look at the neurochemical details and see how the groups compared, we used Tukey’s post-hoc test after doing a one-way analysis of variance (ANOVA) with repeated measurements. Bonferroni’s post hoc test was used after a two-way ANOVA to assess interactions between treatment groups and experimental conditions and to determine specific differences among the treatment groups. For consistency and clarity, all experimental results are presented as mean ± standard deviation (SD). This statistical approach allowed for robust comparison and interpretation of the neurochemical changes observed in the different treatment groups.

3. Results

3.1 Effect of SSZ compound on BACE-1, γ-secretase, and MAO-B enzyme levels

We measured BACE-1, γ-secretase, and MAO-B levels in hippocampal and cortical brain samples to evaluate the neuroprotective effect of SSZ in animals exposed to Aβ. Rats treated with Aβ alone showed significantly elevated BACE-1, γ-secretase, and MAO-B levels compared to the sham control, vehicle control, or SSZ15-treatment animal groups. On the other hand, MAO-B, γ-secretase, and BACE-1 enzyme levels in the homogenates of the brain were significantly decreased by the simultaneous delivery of donepezil (DON) (5 mg/kg) and SSZ (15 mg/kg). Interestingly, the combined treatment of SSZ15 and memantine (MEM) (10 mg/kg) further reduced the levels of these proteins in rats exposed to Aβ compared to SSZ15 treatment alone.

These results were validated by statistical analysis. In the cerebral cortex, the Aβ-treated group showed much higher BACE-1, γ-secretase, and MAO-B levels than the SSZ15-treated group. The amounts of BACE-1 [one-way ANOVA: F(6, 49) = 558.1, p < 0.05], γ-secretase [one-way ANOVA: F(6, 49) = 635.9, p < 0.05], and MAO-B [one-way ANOVA: F(6, 49) = 151, p < 0.05] showed a similar trend in the hippocampus. Also, when comparing SSZ15 and DON5 with Aβ, the SSZ15/MEM10 mix with Aβ showed slightly lower levels of BACE-1, γ-secretase, and MAO-B. These findings indicate that the combination of SSZ15 and MEM10 provides greater protection against Aβ-induced increases in BACE-1, γ-secretase, and MAO-B than SSZ15 alone or with DON5 Figs. 2(a-c).

(a-c) Effect of SSZ compound on BACE-1, γ-secretase, and MAO-B enzyme levels in Aβ-induced Alzheimer’s rats A one-way ANOVA and post hoc Tukey’s tests were utilized for statistical evaluation of data from eight Wistar rats per experimental group. Results are presented as mean values with standard deviation (p < 0.05). Comparisons were made between various groups: a versus sham control, vehicle control, and SSZ15 perse; b versus Aβ; c versus SSZ15 and Aβ; and d versus Aβ, DON5, and SSZ15.

3.2 Effect of SSZ compound on apoptotic markers

Compared to the sham control, vehicle control, and SSZ-perse-treated groups, the Aβ-injected group’s brain homogenates had significantly higher levels of Bax and Caspase-3. Bax levels in the hippocampus [one-way ANOVA: F(6, 42) = 1.448, p < 0.05] and cerebral cortex [one-way ANOVA: F(6, 42) = 0.9813, p < 0.05] were considerably decreased by regular administration of SSZ (15 mg/kg). Likewise, there was a substantial decrease in Caspase-3 levels in the hippocampus [one-way ANOVA: F(6, 42) = 0.6944, p < 0.05] and cerebral cortex [one-way ANOVA: F(6, 42) = 1.074, p < 0.05]. Interestingly, compared to rats treated with Aβ, animals treated with 15 mg/kg SSZ had considerably lower levels of caspase-3 and BAX. Furthermore, the combination of Aβ, SSZ15, and MEM10 demonstrated greater effectiveness in restoring apoptotic markers than Aβ, SSZ15, and DON5 Figs. 3(a and b).

(a-c) Effect of SSZ compound on Caspase-3, Bax, and Bcl-2 levels in Aβ-induced Alzheimer’s rats. A one-way ANOVA and post hoc Tukey’s tests were utilized to assess results from eight Wistar rats per experimental group. Findings are presented as mean values with standard deviation (p < 0.05). The groups compared included: a vs vehicle control, SSZ15 perse, and sham control; b versus Aβ; c versus Aβ and SSZ15; and d versus SSZ15, DON5, and Aβ.

When comparing the brain samples from rats that received a long Aβ injection to those that received a vehicle control, sham control, and SSZ15-perse treatment, it was found that the levels of the anti-apoptotic protein Bcl-2 were significantly lower in the hippocampus [one-way ANOVA: F(6, 42) = 4.217, p < 0.05] and cerebral cortex [one-way ANOVA: F(6, 42) = 1.447, p < 0.05]. Bcl-2 levels were much higher after treatment with DON 5 mg/kg than after treatment with Aβ alone. However, when Aβ, MEM10, and SSZ15 were given together, Bcl-2 levels rose more significantly than when DON5, SSZ15, or Aβ have been given alone Fig. 3(c). These findings demonstrate the superior effectiveness of the combination therapy with MEM10 and SSZ15 in reducing Aβ-induced apoptosis and restoring anti-apoptotic markers, compared to other treatment options.

3.3 Effect of SSZ compound on MBP, NEFL, and MAP levels

The levels of NEFL and MBP in CSF were evaluated to assess the neuroprotective impact of SSZ in animals exposed to Aβ. Aβ-treated animals had much higher levels of protein markers, such as NEFL and MBP, than the vehicle control, sham control, or SSZ15-treated animals. However, NEFL and MBP protein levels in CSF samples were markedly reduced by the simultaneous administration of SSZ (15 mg/kg) and DON (5 mg/kg). Notably, the combination of SSZ15 and MEM10 further reduced NEFL and MBP levels in Aβ-treated rats compared with SSZ15 alone. Statistical analysis revealed that NEFL levels [one-way ANOVA: F(6, 42) = 0.5662, p < 0.05] and MBP levels [one-way ANOVA: F(6, 42) = 2.684, p < 0.05] were significantly lower in the SSZ-treatment group in comparison to the Aβ-exposed experimental group. Additionally, compared to rats that received Aβ, SSZ15, and DON5, those given Aβ, SSSZ15, and MEM10 exhibited slightly lower MBP and NEFL levels Fig. 4(a).

(a-d) Effect of SSZ compound on MBP and NEFL (4a); MBP (4b); NEFL (4c) and MAP (4d) levels in CSF and brain homogenate in Aβ-induced Alzheimer’s rats A one-way analysis of variance (ANOVA) and post hoc Tukey’s tests were used to examine the data in the statistical evaluation. Each experimental group had eight Wistar rats, and the results are shown as mean values with standard deviation (p < 0.05). The following groups were statistically compared: a vs vehicle control, SSZ15 perse, and sham control; b versus Aβ; c versus Aβ and SSZ15; and d versus SSZ15, DON5, and Aβ.

MBP concentration in brain tissue was notably decreased after prolonged exposure to Aβ. However, when SSZ15 was administered, MBP levels markedly increased in the hippocampus [one-way ANOVA: F(6, 42) = 0.3954, p < 0.05] and cerebral cortex [one-way ANOVA: F(6, 42) = 1.609, p < 0.05]. MBP concentrations were also noticeably greater in rats treated with DON5 than in the group treated with Aβ. Furthermore, MBP protein levels in the cortex and hippocampus of rats receiving SSZ15, MEM10, and Aβ were significantly higher than in those receiving SSZ15, DON5, and Aβ Fig. 4(b).

The Alzheimer’s model exposed to Aβ showed significantly higher NEFL levels in brain samples after treatment compared to the vehicle control, sham control, and SSZ-perse-treatment groups. When SSZ15 was administered, NEFL levels were notably lower than in the Aβ-treated group. Similarly, when SSZ15 was given, NEFL levels remained substantially lower than in the group treated with Aβ. NEFL levels were significantly lower in the group receiving Aβ treatment compared to the group receiving SSZ15. Compared to Aβ treatment alone, DON5 therapy significantly reduced NEFL levels in the hippocampus [one-way ANOVA: F (6, 42) = 0.8749, p < 0.05] and cerebral cortex areas of the brain [one-way ANOVA: F (6, 42) = 2.453, p < 0.05]. Additionally, the SSZ15, MEM10, and Aβ treatments resulted in significantly lower NEFL levels compared to rats treated with Aβ alone or the Aβ, SSZ15, and DON5 groups Fig. 4(c). Microtubule-associated protein ( MAP) levels were higher in animals given Aβ than in sham control, vehicle control, and SSZ-perse-treated groups. When SSZ15 was given, MAP levels were much lower than in the Aβ-treated group in both the hippocampus [one-way ANOVA: F (6, 42) = 0.4259, p < 0.05] and cerebral cortex [one-way ANOVA: F (6, 42) = 1.515, p < 0.05]. When SSZ15 was administered, MAP levels were considerably lower than in the Aβ-treated group. In the brain sample, the DON5 group had much lower MAP levels than the Aβ group in both the hippocampus and the cerebral cortex. Additionally, those treated with Aβ, SSZ15, and MEM10 had lower MAP levels than those treated with SSZ15, DON5 Fig. 4(d).

3.4 Effect of SSZ compound on cytokine levels

Rats that received Aβ showed much higher levels of the pro-inflammatory cytokines, TNF-α and IL-1β in their blood compared to the control groups that received a placebo, vehicle, or SSZ-perse. However, TNF-α and IL-1β levels were significantly reduced by SSZ15 therapy at a dose of 15 mg/kg. According to statistical analysis, the SSZ-treated group had significantly lower levels of TNF-α [one-way ANOVA: F(6, 42) = 0.7116, p < 0.05] and IL-1β [one-way ANOVA: F(6, 42) = 1.151, p < 0.05] than the Aβ-treated group. Additionally, animals given Aβ, SSZ15 (15 mg/kg), and DON5 showed considerably lower plasma levels of TNF-α and IL-1β than those given Aβ, SSZ15, and MEM10 Fig. 5(a). TNF-α and IL-1β levels in brain homogenates were assessed equally across all treatment groups. TNF-α and IL-1β levels in the brain homogenates were significantly higher after chronic Aβ treatment than in the control groups. However, SSZ15 (15 mg/kg) therapy markedly decreased these pro-inflammatory cytokines in the hippocampus and cerebral cortex. In the cerebral cortex, the SSZ15-treated group had substantially lower levels of TNF-α [one-way ANOVA: F (6, 42) = 1.174, p < 0.05] and IL-1β [one-way ANOVA: F (6, 42) = 1.065, p < 0.05] than the Aβ-treated group. Similarly, when SSZ15 was administered, there was a substantial decrease in IL-1β levels [one-way ANOVA: F (6, 42) = 1.091, p < 0.05] and TNF-α levels [one-way ANOVA: F (6, 42) = 0.3552, p < 0.05] and in the hippocampus. IL-1β and TNF-α levels in brain homogenates were also somewhat lower in rats that received a combination of SSZ15, MEM10, and Aβ compared to those that received SSZ15, DON5, and Aβ Figs. 5(b and c).

(a-c) Effect of SSZ compound on TNF-α & IL-1β [5A]; TNF- α [5B] and IL-1β [5C] levels in blood plasma and brain homogenate in Aβ-induced Alzheimer’s rats A one-way ANOVA analysis and post-hoc Tukey’s testing were employed in the statistical evaluation to examine the data. Each experimental group had eight Wistar rats, and the results are shown as mean values with standard deviation (p < 0.05). The following groups were statistically compared: a vs vehicle control, sham control, and SSZ15 perse; b versus Aβ; c versus SSZ15 and Aβ; and d versus SSZ15, DON5, and Aβ.

3.5 Effect of SSZ compound on neurotransmitter alterations

The levels of acetylcholine, glutamate, and dopamine in experimental rat brain homogenates were measured to assess the neuroprotective effectiveness of SSZ. Compared to the sham control, vehicle control, and SSZ-perse-treated groups, rats exposed to Aβ showed significantly lower levels of these neurotransmitters. However, the intervention with SSZ at a dose of 15 mg/kg significantly restored neurotransmitter levels. Specifically, dopamine concentrations increased notably in the hippocampus [one-way ANOVA: F(6, 42) = 0.8265, p < 0.05] and cerebral cortex [one-way ANOVA: F(6, 42) = 0.0794, p < 0.05]. Similarly, acetylcholine levels were significantly restored in the hippocampus [one-way ANOVA: F(6, 42) = 1.712, p < 0.05] and cerebral cortex [one-way ANOVA: F(6, 42) = 1.145, p < 0.05], while glutamate levels were also substantially increased in the hippocampus [one-way ANOVA: F(6, 42) = 0.7657, p < 0.05] and cerebral cortex [one-way ANOVA: F(6, 42) = 1.062, p < 0.05] of SSZ15-treated rats compared to those subjected to Aβ treatment alone.

Additionally, rats treated with Aβ, MEM (10 mg/kg), and SSZ (15 mg/kg) showed a significantly greater restoration of neurotransmitters, especially glutamate and dopamine levels, compared to rats treated with Aβ, DON (5 mg/kg), and SSZ (15 mg/kg). However, unlike the groups that received Aβ, SSZ15, and MEM10, the levels of acetylcholine were much higher in the groups treated with Aβ, SSZ15, and DON5, see Figs. 6(a-c). These results suggest that SSZ therapy at 15 mg/kg effectively restored neurotransmitter levels disrupted by Aβ exposure, with combined therapies improving specific neurotransmitter recovery depending on the treatment combination.

(a-c) Effect of SSZ compound on neurotransmitter alterations in Aβ-induced Alzheimer’s rats A one-way analysis of variance (ANOVA) and post hoc Tukey’s tests were employed to examine the data in the statistical evaluation. Each experimental group had eight Wistar rats, and the findings are shown as mean values with standard deviation (p<0.05). The following groups were statistically compared: a vs vehicle control, SSZ15 perse, and sham control; b versus Aβ; c versus SSZ15 and Aβ; and d versus SSZ15, DON5, and Aβ.

3.6 Effect of SSZ compound on oxidative stress markers

Superoxide dismutase (SOD) and glutathione (GSH) levels in rat brain samples were measured to evaluate the neuroprotective effects of SSZ. Compared to the sham control, vehicle control, and SSZ-alone-treated groups, rats given Aβ exhibited significantly lower levels of GSH and SOD (p < 0.05). Conversely, administering SSZ at 15 mg/kg significantly elevated both antioxidant levels. In particular, the hippocampus [one-way ANOVA: F(6, 42) = 0.6507, p < 0.05] and cerebral cortex [one-way ANOVA: F(6, 42) = 0.9874, p < 0.05] had significantly higher SOD levels. When comparing rats that received SSZ to those that only got Aβ, the levels of GSH were also significantly increased in the hippocampus and cerebral cortex. Also, when comparing the treatment with Aβ alone to the combination of Aβ, SSZ15, and MEM10, the combined treatment led to a significantly greater increase in GSH and SOD levels. These results suggest that the antioxidant defenses compromised by Aβ exposure can be effectively restored by SSZ therapy at 15 mg/kg. Furthermore, the combination of SSZ and MEM10 provides superior neuroprotective benefits compared to other treatment regimens (Fig 7).

(a and b) Effect of SSZ compound on GSH and SOD levels in Aβ-induced Alzheimer’s rats A one-way ANOVA analysis and post-hoc Tukey’s tests were employed in the statistical evaluation to examine the data. Each experimental group had eight Wistar rats, and the results are shown as mean values with standard deviation (p < 0.05). The following groups were statistically compared: a vs vehicle control, SSZ15 perse, and sham control; b versus Aβ; c versus Aβ and SSZ15; and d versus SSZ15, Aβ, and DON5.

The neuroprotective effect of SSZ was further assessed by measuring the levels of lactate dehydrogenase (LDH), acetylcholinesterase (AChE), nitric oxide (NO), and malondialdehyde (MDA) in rat brain homogenates. The rats treated with Aβ had much higher amounts of MDA, LDH, AChE, and NO than the vehicle control, SSZ-only treated, and sham control groups (p < 0.05). Compared with Aβ treatment alone, SSZ at 15 mg/kg significantly reduced these markers of oxidative stress in brain samples. Unlike the Aβ treatment, administering SSZ at a dose of 15 mg/kg significantly reduced these signs of oxidative stress in brain samples.

Specifically, SSZ15 treatment significantly reduced AChE levels in the hippocampus [one-way ANOVA: F(6, 42) = 0.3653, p < 0.05] and cerebral cortex [one-way ANOVA: F(6, 42) = 0.7171, p < 0.05]. Similarly, reductions in LDH levels were observed in the hippocampus [one-way ANOVA: F(6, 42) = 1.634, p < 0.05] and cerebral cortex [one-way ANOVA: F(6, 42) = 2.427, p < 0.05], as well as MDA levels in the hippocampus [one-way ANOVA: F(6, 42) = 0.3932, p < 0.05] and cerebral cortex [one-way ANOVA: F(6, 42) = 1.255, p < 0.05]. NO levels were also significantly reduced in the hippocampus [one-way ANOVA: F(6, 42) = 1.731, p < 0.05] and cerebral cortex [one-way ANOVA: F(6, 42) = 1.427, p < 0.05] following SSZ15 treatment compared to Aβ-treated rats.

Furthermore, compared to the combination of Aβ, SSZ15, and DON5, the combination of Aβ, MEM10, and SSZ15 more effectively restored MDA, LDH, and NO levels. Rats treated with SSZ15, DON5, and Aβ recovered AChE enzyme level more successfully than rats treated with Aβ, SSZ15, and MEM10 Fig. 8(a-d). These findings demonstrate SSZ’s ability to mitigate oxidative stress and restore key biomarkers disrupted by Aβ exposure, with specific combinations of SSZ and DON or MEM yielding differential effects on oxidative stress and acetylcholinesterase activity.

(a-d) Effect of SSZ compound on AChE, MDA, Nitrite, and LDH levels in Aβ-induced Alzheimer’s rats A one-way ANOVA analysis and post-hoc Tukey’s tests were employed in the statistical evaluation to examine the data. Each experimental group had eight Wistar rats, and the results are shown as mean values with standard deviation (p<0.05). The following groups were statistically compared: a vs vehicle control, SSZ15 per se, and sham control; b versus Aβ; c versus Aβ and SSZ15; and d versus SSZ15, DON5, and Aβ.

3.7 Effect of SSZ compound on histopathological abnormalities in Aβ-induced Alzheimer’s rats (Coronal sections - Striatum)

Histological analysis was performed on adult Wistar rats given intraperitoneal SSZ injections to assess its neuroprotective effects. Typical histological characteristics, such as intact, regularly formed blood capillaries, healthy oligodendrocytes, and undamaged oval neurones with rounded nuclei, were seen in the vehicle control (A), sham control (B), and SSZ-alone-treated (C) groups. These groups appeared to have the same distribution of oligodendrocytes, capillaries, and striatal neurons. The upper panel of the Aβ-treated group (D) showed notable histological abnormalities, such as pronounced demyelination, severe neuronal degeneration, and occluded capillaries. However, there was a slight reduction in neuronal degeneration and some improvement in the shapes of neurones and oligodendrocytes in the group treated with Aβ and SSZ (E) (15 mg/kg). Aβ, SSZ15, and DON5 (F) slightly improved the shape of neurons, reduced the damaged area, and enhanced the appearance of oligodendrocytes. However, despite these improvements, this group still suffered significant neuronal loss and damage. This group experienced severe neuronal degeneration despite these advances.

The combined administration of SSZ15, Aβ, and MEM10 (G) notably demonstrated significant neuroprotective effects. This group showed intact oligodendrocytes and capillaries, along with notable improvements in neuronal count and morphology. The combination therapy was more effective in healing than the other treatments and efficiently reduced Aβ-related damage to the striatal tissue in adult Wistar rats. Overall, SSZ15 considerably minimized Aβ-induced striatal tissue damage, with the most protective effects observed with the combination of SSZ15 and MEM10 (Fig. 9).

Effect of SSZ compound on histopathological abnormalities in Aβ-induced Alzheimer’s rats (Coronal sections - Striatum). Hematoxylin and Eosin (H&E) staining was used to examine striatal tissues in adult Wistar rats, utilizing a digital microscope. The results shown in panels a-g depict the structural features of nerve cells, blood vessels, and oligodendrocytes after SSZ administration. Key tissue features were identified using specific markers: brown circles indicate normal oligodendrocytes, black circles indicate blood vessels, and red circles show healthy oval neurons with distinct, round nuclei. No structural abnormalities were seen in oligodendrocytes, blood vessels, or neurons of the sham control (a), vehicle control (b), and SSZ-perse (c) groups. However, the Aβ-treated group (d) exhibited significant histopathological changes, such as demyelination, altered neuronal structure, congested blood vessels, and irregular oligodendrocyte morphology, indicating severe degenerative effects caused by Aβ. In contrast, the Aβ + SSZ15-treated group (e) showed some improvement in oligodendrocyte morphology (brown circle), blood vessel integrity (black circle), and neuronal structure (red circle). These findings suggest a neuroprotective effect of SSZ at a dose of 15 mg/kg. The Aβ + SSZ15 + DON5 group (f) displayed less neuronal degeneration (red circle), a slight improvement in the shape of neurons and oligodendrocytes (brown circle), and a moderate reduction in vascular congestion (black circle). The Aβ + SSZ15 + MEM10 group (g) demonstrated a significant improvement in degenerative areas (black circle), restoration of neuronal count and structure (red circle), and the health of oligodendrocytes and blood vessels. Overall, the results indicate that Aβ-induced degenerative changes in the striatal tissues were reduced by SSZ at the 15 mg/kg dose. The combination of SSZ15 and MEM10 showed the most notable restorative effects, highlighting its potential as an effective therapy for neurodegenerative diseases. (Magnification = 40X; Scale bar = 50 µm).

3.8 Effect of SSZ compound on histopathological abnormalities in Aβ-induced Alzheimer’s rats (Coronal sections - Cortex)

We used hematoxylin and eosin (H&E) staining to examine the brain’s nerve cells and observed the images with a fluorescence microscope at 40× magnification. In the exterior pyramidal layer of the brain, cortical neurons in the sham control, vehicle control, and SSZ-perse-treated groups displayed typical histological features, such as spherical vesicular nuclei and well-organized pyramidal cell patterns. The Aβ-treated group, however, showed significant histological abnormalities. These included altered pyramidal cell morphology, decreased neuronal counts, and hypertrophied astrocytes with limited cytoplasm, indicating extensive neuronal damage. Compared to the Aβ-treated group, the group that received Aβ + SSZ exhibited improved pyramidal cell and cortical neuron morphology. Nonetheless, some structural issues persisted, and the improvements were not complete. The group treated with Aβ + SSZ + DON exhibited many congested capillaries, structural damage, and abnormalities in pyramidal and inflammatory cells, suggesting that this treatment combination provided limited neuroprotection. Some structural abnormalities still remained, and the repairs were not entirely successful. The Aβ + SSZ + MEM-treated group showed significant improvements in cortical tissue structure. Pyramidal cell reshaping and astrocyte morphology restoration were observed. These findings indicate that the combination of SSZ and MEM offered superior neuroprotective effects compared to other treatment protocols. Overall, these results demonstrate the ability of SSZ to partially restore cortical neuronal morphology damaged by Aβ, with the combination of SSZ and MEM showing the marked restorative effects (Fig. 10).

Effect of SSZ compound on histopathological abnormalities in Aβ-induced Alzheimer’s rats (Coronal sections - Cortex). Panels (a-g) show digitally enhanced images of cortical tissue slices from adult Wistar rats stained with H&E, highlighting the histopathological changes following SSZ therapy. The upper panel displays the cortical tissues of the sham control (a), vehicle control (b), and SSZ-perse (c) groups, all exhibiting normal histological features. Neurons have spherical, vesicular nuclei, while astrocytes display well-defined nuclei (green circle). Pyramidal cells are orderly arranged in the outer pyramidal layer (red circle), with prominent nucleoli and intact cytoplasm, and blood capillaries (brown circle) appear normal and undamaged. In contrast, cortical sections from the Aβ-treated group (d) reveal significant histopathological disruptions, including altered pyramidal neuron arrangement and morphology (red circle), congestion in blood capillaries (brown circle), and irregular neuron structures and inflammatory cells (black circle), emphasizing the damaging effects of Aβ on cortical tissues. The lower panel illustrates improved cortical tissue morphology with different treatment protocols. The Aβ + SSZ15-treated group (e) shows recovery of pyramidal neurons, cortical neurons, astrocytes, and capillaries compared to the Aβ group, especially in severely affected cortical areas. The Aβ + SSZ15 + MEM10-treated group (g) exhibits substantial structural recovery, with clear restoration of pyramidal cell morphology (red circle), astrocytes (green circle), and capillaries (brown circle), making their cortical tissue resemble that of the control groups. This indicates a significant improvement in Aβ-induced cortical damage with combined SSZ and MEM10 treatment. Overall, these findings highlight the neuroprotective effects of SSZ (15 mg/kg) in reducing Aβ-related cortical damage, with the combined SSZ and MEM10 regimen producing the most notable restorative outcomes, suggesting its therapeutic potential for cortical tissue repair in adult Wistar rats. (Magnification = 40×; Scale bar = 50 µm).

3.9 Effect of SSZ compound on histopathological abnormalities in Aβ-induced Alzheimer’s rats (Coronal sections – hippocampus)

The multifaceted, molecular layers and pyramidal neuronal cells were among the distinctive layers identified during hippocampus tissue examination. Pyramidal neurons in the pyramidal layer of the vehicle control (A), sham control (B), and SSZ-perse-treated (C) groups displayed typical features, including prominent nucleoli, limited cytoplasm, euchromatic nuclei, and well-organized capillary patterns without structural abnormalities. In contrast, the Aβ-treated group (D) showed significant histopathological changes, such as abnormal pyramidal neuron morphology, enlarged capillaries, and inflammatory cell presence. These findings suggest damage to hippocampal tissue architecture caused by Aβ treatment. The Aβ+SSZ (15 mg/kg) group (E) showed slight improvements in the organization of pyramidal neurons within the pyramidal cell layer compared to the Aβ group. Although some abnormalities remained, neuronal structure and alignment appeared to improve. The Aβ + SSZ15 + DON5-treated group (F) demonstrated further enhancement, partially correcting structural abnormalities and achieving better organization of pyramidal neurons. These results underscore the role of SSZ with donepezil in supporting neuronal recovery. Notably, the Aβ + SSZ15 + MEM10-treated group (G) showed substantial restoration of pyramidal neurons and capillary structures, with restored morphology, normal alignment, and improved structural integrity. SSZ treatment, especially combined with memantine (MEM10), significantly boosted neuronal and capillary recovery compared to other regimens. These findings highlight the importance of SSZ at 15 mg/kg in alleviating Aβ-induced hippocampal damage, with the combination of SSZ and memantine (MEM10) exhibiting the most potent neuroprotective effects, restoring neuronal and vascular structures within the hippocampus (Fig. 11).

Effect of SSZ compound on histopathological abnormalities in Aβ-induced Alzheimer’s rats (Coronal sections – hippocampus). Panels (a-g) show digital microscope images of hippocampus tissue slices from adult Wistar rats, analyzed with H&E staining. The hippocampus has three distinct layers: pyramidal, molecular, and polymorphic cells. This panel focuses on the pyramidal cell layer, which contains densely packed pyramidal neurons with limited cytoplasm, prominent nucleoli indicating high activity, and nuclei rich in genetic material. Healthy tissue samples from the sham control (a), vehicle control (b), and SSZ-perse (c) groups display a well-organized, thick layer of pyramidal neurons (red circles) and normal blood vessels (brown circles), indicating preserved cell structure and vasculature. In contrast, the Aβ-treated group (d) shows significant tissue damage, with fewer pyramidal neurons and dark, condensed nuclei, suggesting cell injury (red circle). Congested blood vessels (brown circle) and inflammatory cells (black circle) reveal the adverse effects of Aβ on the tissue. The Aβ + SSZ15-treated group (e) shows slight improvements, with a thicker pyramidal cell layer and partially repaired capillaries (brown circles). The Aβ + SSZ15 + DON5 group (f) shows further recovery, with better organization of pyramidal neurons (red circle) and restored capillaries (brown circle). These results suggest a stronger neuroprotective effect when SSZ and DON5 are combined. The Aβ + SSZ15 + MEM10 group (g) exhibits the highest recovery, with clear repair of granule and pyramidal cells, indicated by dark nuclei. Increased molecular cells and reformed capillaries (brown circles) reflect tissue healing. Overall, these findings demonstrate the neuroprotective effects of SSZ15, especially when combined with MEM10, indicating potential for treatment of Aβ-induced hippocampal damage. (Magnification = 40X; Scale bar = 50 µm).

3.10 Effect of SSZ compound on histopathological abnormalities in Aβ-induced Alzheimer’s rats (Coronal sections – Midbrain)

Panels A–G display photomicrographs of histological slices that highlight the morphology of neurons and their surrounding components. The groups treated with vehicle control (A), sham control (B), and SSZ-perse (C), exhibited healthy-looking neurons with clear nuclei. There were no signs of inflammation or neurodegeneration in these groups, and the grey matter surrounding the white matter maintained its well-preserved structure. In contrast, the Aβ-treated group (D) showed clear pathological changes, such as the presence of microglial cells and noticeable clusters of immune cells around blood vessels. These features suggest neurodegeneration and demyelination, indicating severe neuronal damage caused by Aβ exposure. The Aβ + SSZ15-treated group (E) showed a slight reduction in damaged areas and neuroglial cell infiltration compared to the Aβ group.

Additionally, moderate improvements in neuronal structure were observed, indicating a partial restoration of neuronal morphology with SSZ therapy. The SSZ15 + DON5+ Aβ-treated group (F) showed a more pronounced reduction in deteriorated areas and neuroglial cell infiltration. Neuronal nuclei appeared clearer, and the overall structural integrity of neurons improved steadily, suggesting enhanced neuroprotection with the combined treatment of SSZ and DON5. The Aβ + SSZ15 + MEM10-treated group (G) exhibited the most significant recovery, with a notable reduction in neuronal degeneration, restoration of neuronal morphology, and less neuroglial cell infiltration. Damaged areas were substantially repaired, and neuronal structure closely resembled that of the control groups. Our results indicate that SSZ has neuroprotective effects, especially at a dose of 15 mg/kg, which reduced the severity of Aβ-induced neurodegeneration. The combination of SSZ with MEM10 produced the most pronounced restorative effects, effectively reducing neuronal degeneration and restoring neuronal morphology to nearly normal levels (Fig. 12).

Effect of SSZ compound on histopathological abnormalities in Aβ-induced Alzheimer’s rats (Coronal sections – Midbrain). Panels a-g show midbrain tissue sections from adult Wistar rats, stained with H&E and viewed under a digital microscope, focusing on histological evaluation after SSZ therapy. The sham control (a), vehicle control (b), and SSZ-perse (c) groups display normal tissue structure with well-organized neuronal populations, as indicated by red circles highlighting nuclei. The grey matter appears well-preserved without structural abnormalities. In contrast, the Aβ-treated group (d) exhibits significant pathological changes, including microglial cell infiltration and perivascular mononuclear cell cuffing, indicating neurodegeneration and inflammation. Aberrant neuronal morphology is also observed (red circle). The Aβ + SSZ15-treated group (e) shows a slight reduction in neuronal degeneration and some improvement in neuronal shape, suggesting a neuroprotective effect of SSZ15. The SSZ15 + DON5 + Aβ-treated group (f) demonstrates further benefits, with decreased degeneration, fewer neuroglial cells, and partial restoration of neuronal nuclei (red circle), indicating SSZ-dependent repair mechanisms. The Aβ + SSZ15 + MEM10-treated group (g) shows the most significant recovery, with notable decreases in neuronal degeneration, higher neuronal counts, and restored morphology (red circle). These findings indicate a positive impact on neuronal structure and reduced inflammation, highlighting the synergistic effects of combining SSZ15 with MEM10. The results confirm that SSZ treatment at 15 mg/kg effectively reduces Aβ-induced neurodegeneration in midbrain tissues. Combining SSZ15 and MEM10 provides more potent neuroprotective effects compared to other treatments. (Magnification = 40X; Scale bar = 50 µm).

3.11 Effect of SSZ compound on histopathological abnormalities in Aβ-induced Alzheimer’s rats (Coronal sections – Cerebellum)

Panels (A–H) show the histological evaluation of myelinated neurons in different groups undergoing experimentation, emphasizing the impact of exposure to Aβ and subsequent SSZ and combination therapy. In the vehicle control (B), SSZ-perse-treated (C), and sham control (A) groups, the neurons had normal myelination, with healthy and organized myelin sheaths surrounding the axons. These groups showed no structural abnormalities or degradation, indicating standard neuronal architecture. In contrast, the Aβ-treated group (D) displayed significant disruption of the myelin sheath, indicative of severe demyelination. These findings demonstrate the degenerative effects of Aβ on neuronal tissues. The myelin sheath was partially preserved in the Aβ + SSZ15-treated group (E), which had less demyelination than the Aβ group. This evidence implies that 15 mg/kg of SSZ somewhat reduces Aβ-induced myelin deterioration. Myelin sheath repair was evident in the SSZ15+DON5+Aβ-treated group (F), and this improvement was further evident. The results indicate that combining DON5 and SSZ15 enhances the protection and restoration of myelinated neurons. Image H notably highlights myelin sheath recovery in the Aβ + SSZ15 + MEM10-treated group (G). This group displayed the most pronounced restoration of myelin integrity with well-formed and continuous myelin sheaths. When combined with MEM (10 mg/kg), SSZ (15 mg/kg) showed significant healing and protective effects against Aβ-induced demyelination. These findings suggest SSZ treatment effectively inhibits Aβ-induced demyelination, particularly when combined with MEM10 or DON5. After 21 days of treatment, SSZ demonstrated a robust ability to preserve and restore myelin sheaths in myelinated neurons, with the Aβ + SSZ15 + MEM10 group showing the most substantial recovery (Fig. 13).

Effect of SSZ compound on histopathological abnormalities in Aβ-induced Alzheimer’s rats (Coronal sections – Cerebellum). Panels (a-g) display H&E-stained cerebellum tissue slices from adult Wistar rats, analyzed via digital microscopy. The images assess neural organization and myelination after SSZ therapy. In the sham control (a), vehicle control (b), and SSZ-perse-treated (c) groups, the cerebellum showed normal neuronal arrangement and healthy myelinated neurons, indicated by red circles highlighting preserved myelin sheaths and structured neuronal morphology, with no signs of abnormalities or myelin damage. In contrast, the Aβ-treated group (d) presented demyelination, indicating significant myelin sheath deterioration due to Aβ exposure, resulting in neuronal damage. The Aβ + SSZ15-treated group (e) showed slight recovery in demyelinated areas, with brown circles indicating healthier myelin and decreased macrophage presence, suggesting healing with SSZ (15 mg/kg). The Aβ + SSZ15 + DON5-treated group (f) demonstrated further improvement and partial restoration of myelin structure, while the Aβ + SSZ15 + MEM10-treated group (g) showed the most notable recovery, with fully restored myelin sheaths and healthy myelinated neurons, indicating a marked improvement in cerebellar tissue structure. These findings suggest that SSZ can prevent Aβ-induced demyelination and support myelin recovery, especially when SSZ15 and MEM10 are combined (Magnification = 40X; Scale bar = 50 µm).

4. Discussion

AD is a progressive neurodegenerative disorder predominantly characterized by cognitive decline and memory impairment. Its pathological hallmark involves the formation of senile plaques, particularly in hippocampal regions, composed of phosphorylated tau protein, amyloid-beta (Aβ), and neurofibrillary tangles (Abyadeh et al., 2024). We examined the potential therapeutic effects of a novel multi-targeted inhibitor, SSZ/SSZ (6-chloro-N-cyclohexyl-4-(1H-pyrrolo [2, 3-b] pyridin-3-yl) pyridin-2-amine), in mitigating AD-related pathology. An AD-like condition was induced in rats through intracerebral injections of Aβ (1-42), followed by neurochemical and histological assessments to evaluate the anti-AD potential of SSZ and its possible mechanisms of action. Previous studies have shown that the brains of AD patients contain higher levels of MAO-B, BACE1, and γ-secretase, especially in astrocytes and pyramidal neurons (Schedin-Weiss et al., 2017). These enzymes worsen neurodegeneration by promoting the formation and accumulation of Aβ plaques. Aβ plaques in genetically modified mice with AD support Kim et al. (2016)‘s finding that there is a strong connection between monoamine oxidase activity and AD development. Similarly, it has been shown that giving rats Aβ (1-42), increases the levels of BACE-1 and amyloid precursor protein (APP) in the CA1 and DG regions of the hippocampus (Kim et al., 2016). Our investigation revealed elevated levels of the enzymes MAO-B, γ-secretase, and BACE-1 in the brain tissue homogenates of rats with AD, which is consistent with earlier findings. However, SSZ treatment significantly lowered the levels of these enzymes, whether used alone or in combination with common medications (DON5 and MEM10), demonstrating that it effectively influences key disease processes in AD.

Higher amounts of pro-apoptotic markers, such as Bax and caspase-3, along with a lower concentration of the anti-apoptotic protein Bcl-2, indicate that AD is associated with increased apoptosis (Saddam et al., 2024). Research indicates that injecting Aβ (1-42) causes the hippocampus and other brain regions to overexpress apoptotic markers (Ahmad et al., 2021). Similarly, we found that rats with AD had lower levels of Bcl-2 and higher levels of Bax and caspase-3. However, SSZ therapy effectively modulated these signs of cell death by restoring Bcl-2 levels and reducing caspase-3 and Bax levels. This study shows that SSZ can decrease Aβ-induced cell death and preserve neuronal integrity.

Problems in white matter for AD patients are connected to higher amounts of NEFL found in blood and CSF (Lewczuk et al., 2018). Furthermore, improper modification of tau protein causes issues with myelination, evidenced by fewer myelinating oligodendrocytes that test positive for MBP and increased presence of MAP (Bacioglu et al., 2016). These results align with our investigation, which shows that AD rats’ brain tissues have lower MBP levels and higher NEFL levels in CSF and brain homogenates (Brureau et al., 2017). Additionally, the brain homogenates of AD rats have higher levels of MAP protein. SSZ therapy effectively restored MBP, NEFL, and MAP protein levels, indicating that it promotes white matter health and reduces damage to the protective nerve coverings. Due to the activation of glial cells, such as astrocytes and microglia, neuroinflammation plays a significant role in AD development. Since these cells produce inflammatory cytokines like IL-6, IL-12, IL-1β, and TNF-α, their activation causes further damage to neurons (Guzman-Martinez et al., 2019). People with moderate AD have been shown to have higher levels of these inflammatory markers in their brain tissues and plasma (Rani et al., 2023). Consistent with other research, our investigation found elevated IL-1β and TNF-α levels in AD-induced rats. However, SSZ treatment significantly reduced cytokine levels, and the combination of SSZ, DON5, and MEM10 further enhanced this effect, indicating a strong anti-inflammatory response.

Significant neurotransmitter imbalances, such as lower acetylcholine and dopamine levels and higher glutamate levels, are another characteristic of AD (Chen et al., 2022; Pan et al., 2019). In the cerebral cortex and hippocampus of AD rats, we observed higher glutamate and lower dopamine and serotonin levels. However, SSZ treatment, especially when combined with DON5 and MEM10, restored these neurotransmitter levels, indicating improved neurotransmitter balance. Furthermore, oxidative stress, a hallmark of AD, causes neuronal damage by increasing levels of oxidative stress markers, such as acetylcholinesterase (AChE), MDA, LDH, and nitric oxide (NO₂), while decreasing antioxidants like SOD and GSH (Long et al., 2020; Azargoonjahromi, 2023). Our results support these changes, showing that AD rats had higher levels of MDA, LDH, AChE, and NO2 and lower levels of GSH and SOD. SSZ treatment effectively modulated these parameters, reducing oxidative stress and restoring antioxidant levels.

Histological analysis revealed significant morphological changes in the brains of AD-induced rats, including irregular neuronal populations and structural damage in coronal and midbrain regions (Baerends et al., 2022). However, SSZ treatment over 35 days restored neuronal morphology and structural integrity, especially in the hippocampal and midbrain regions. Continued therapy enhanced neuronal organization and reduced Aβ-induced lesions, emphasizing SSZ’s neuroprotective effects.

These findings from the present study support the idea that SSZ may possess neuroprotective and anti-AD properties. SSZ effectively mitigates AD-related damage by targeting multiple pathogenic pathways, including Aβ accumulation, enzyme activity, apoptosis, neuroinflammation, neurotransmitter imbalance, and oxidative stress. Overall. the results from the present study demonstrate that SSZ is a promising multi-targeted therapeutic for treating AD, and further research is needed to validate its effectiveness.

5. Limitations

The study’s findings are promising, but there are some limitations too. The dose-response relationship and therapeutic range of SSZ are limited due to single dose of 15 mg/kg, requiring a broader range for improved efficacy and safety. Additionally, the rat model used in the study may not accurately represent human AD disease. The study’s limitations also include inadequate pharmacokinetic profiling, a lack of advanced tools, and insufficient evaluation of potential toxicity. Future research should investigate SSZ’s therapeutic effects across various dosages and time points, incorporating pharmacokinetic and pharmacodynamic studies as well as advanced molecular techniques. Assessing efficacy and toxicity in human clinical trials is essential for determining its potential as a therapeutic agent for AD.

6. Conclusions

According to our study findings, SSZ seems to be a promising multi-targeted inhibitor with substantial therapeutic promise for AD. We found that SSZ affects key processes in AD by effectively modulating essential enzymes involved in the disease, like acetylcholinesterase, BACE-1, MAO-B, and γ-secretase. SSZ effectively prevents cell death by elevating protective protein, Bcl-2, and reducing harmful proteins, like Bax and caspase-3, enhancing neuronal survival. Moreover, SSZ further demonstrated its neuroprotective advantages by restoring MBP levels, normalizing NEFL levels, and controlling inflammatory cytokines (TNF-α and IL-1β), which are known to contribute to neuroinflammation in AD. Furthermore, by raising dopamine and serotonin levels and normalizing high glutamate levels, SSZ restored neurotransmitter equilibrium. Increases in GSH and SOD, as well as decreases in oxidative stress markers such as MDA, LDH, and NO₂ were indicative of its antioxidant qualities.

Histological analyses further confirmed that SSZ mitigated Aβ-induced neuronal damage, evidenced by improved structural integrity in brain areas including midbrain, hippocampus, and cerebellum. These findings underscore the neuroprotective and multi-targeted therapeutic potential of SSZ for AD treatment. Although these findings are encouraging, further research is needed to fully assess the translational potential of SSZ. Clinical studies are necessary to assess its effectiveness, safety, and long-term effects in human populations. Additionally, investigating its dose-response relationships, pharmacokinetics, and potential toxicity will offer further insights into its potential as a disease-modifying therapy for AD. Collectively, our findings support the idea that SSZ might be part of an effective multi-targeted therapy strategy for AD.

Acknowledgement

The authors gratefully acknowledge technical and financial support from the Ministry of Education and King Abdulaziz University, Jeddah, Saudi Arabia.

CRediT authorship contribution statement

Shams Tabrez: Conceptualization, formal analysis, validation, supervision, editing. Zuber Khan: Investigation, methodology, data curation, visualization, resources, writing—original draft. Nasimudeen R. Jabir: Investigation, methodology, data curation, visualization, resources, writing—original draft. Syed Kashif Zaidi: Investigation, methodology, data curation, visualization, resources, writing—original draft. Sidharth Mehan: Conceptualization, formal analysis, validation, supervision, editing. Mohammad Abid: Conceptualization, formal analysis, validation, supervision, editing. Torki A. Zughaibi: Conceptualization, formal analysis, validation, supervision, editing. Mohammad Hassan Alhashmi: Conceptualization, formal analysis, validation, supervision, editing. Rahaf Fawaz Khoja: Conceptualization, formal analysis, validation, supervision, editing. Md Nasiruddin Khan: Investigation, methodology, data curation, visualization, resources, writing—original draft. Ravi Rana: Investigation, methodology, data curation, visualization, resources, writing—original draft. Mohd Suhailf: Investigation, methodology, data curation, visualization, resources, writing—original draft. Shazi Shakil: Conceptualization, formal analysis, validation, supervision, editing. All authors have read and agreed to the published version of the manuscript.

Declaration of competing interest

The authors declare that they have no competing financial interests or personal relationships that could have influenced the work presented in this paper.

Data availability

The data sets generated during this study are available from the corresponding author upon reasonable request.

Declaration of generative AI and AI-assisted technologies in the writing process

The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.

Funding

This research work was funded by Institutional Fund Projects under grant number (IFPRC-058-142-2020)

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1. Introduction

2. Materials and Methods

2.1 Chemicals and drugs
2.2 Animals
2.3 Animal ethical approval
2.4 Experimental animal classification
2.5 Experimental design schedule
2.6 Methods
2.7 Neurochemical analysis
2.8 Statistical analysis

3. Results

3.1 Effect of SSZ compound on BACE-1, γ-secretase, and MAO-B enzyme levels
3.2 Effect of SSZ compound on apoptotic markers
3.3 Effect of SSZ compound on MBP, NEFL, and MAP levels
3.4 Effect of SSZ compound on cytokine levels
3.5 Effect of SSZ compound on neurotransmitter alterations
3.6 Effect of SSZ compound on oxidative stress markers
3.7 Effect of SSZ compound on histopathological abnormalities in Aβ-induced Alzheimer’s rats (Coronal sections - Striatum)
3.8 Effect of SSZ compound on histopathological abnormalities in Aβ-induced Alzheimer’s rats (Coronal sections - Cortex)
3.9 Effect of SSZ compound on histopathological abnormalities in Aβ-induced Alzheimer’s rats (Coronal sections – hippocampus)
3.10 Effect of SSZ compound on histopathological abnormalities in Aβ-induced Alzheimer’s rats (Coronal sections – Midbrain)
3.11 Effect of SSZ compound on histopathological abnormalities in Aβ-induced Alzheimer’s rats (Coronal sections – Cerebellum)

4. Discussion

5. Limitations

6. Conclusions

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Therapeutic potential of SSZ in modulating Alzheimer’s disease pathology: A multi-targeted experimental approach

Abstract

Keywords

Acetylcholinesterase

Alzheimer’s disease

BACE-1

Butyrylcholinesterase

Neuroprotection

SSZ

γ-secretase

1. Introduction

2. Materials and Methods

2.1 Chemicals and drugs

2.2 Animals

2.3 Animal ethical approval

2.4 Experimental animal classification

2.5 Experimental design schedule

2.6 Methods

2.6.1 ICV-amyloid-β administration induced AD in an animal model

2.6.2 Cannula implantation procedure

2.6.3 Post-surgical treatment

2.7 Neurochemical analysis

2.7.1 Procedure for acquiring and processing biological specimens

2.7.2 Enzyme-linked immunosorbent assay (ELISA)

2.7.3 Histopathological analysis

2.8 Statistical analysis

3. Results

3.1 Effect of SSZ compound on BACE-1, γ-secretase, and MAO-B enzyme levels

3.2 Effect of SSZ compound on apoptotic markers

3.3 Effect of SSZ compound on MBP, NEFL, and MAP levels

3.4 Effect of SSZ compound on cytokine levels

3.5 Effect of SSZ compound on neurotransmitter alterations

3.6 Effect of SSZ compound on oxidative stress markers

3.7 Effect of SSZ compound on histopathological abnormalities in Aβ-induced Alzheimer’s rats (Coronal sections - Striatum)

3.8 Effect of SSZ compound on histopathological abnormalities in Aβ-induced Alzheimer’s rats (Coronal sections - Cortex)

3.9 Effect of SSZ compound on histopathological abnormalities in Aβ-induced Alzheimer’s rats (Coronal sections – hippocampus)

3.10 Effect of SSZ compound on histopathological abnormalities in Aβ-induced Alzheimer’s rats (Coronal sections – Midbrain)

3.11 Effect of SSZ compound on histopathological abnormalities in Aβ-induced Alzheimer’s rats (Coronal sections – Cerebellum)

4. Discussion

5. Limitations

6. Conclusions

Acknowledgement

CRediT authorship contribution statement

Declaration of competing interest

Data availability

Declaration of generative AI and AI-assisted technologies in the writing process

Funding

References

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