1. Introduction

The interconnection between Alzheimer's disease (AD) and peripheral metabolic dysfunction has gained considerable attention in recent neuroscience research [1,2]. Non-alcoholic fatty liver disease associated with Alzheimer's disease (NAFLD-AD) represents a complex pathophysiological state characterized by hepatic lipid accumulation concurrent with progressive neurodegeneration [3]. Recent evidence suggests that metabolic dysfunction-associated fatty liver disease may serve as a more appropriate nomenclature for describing hepatic pathologies linked to known metabolic disorders [4]. The prevalence of NAFLD-AD ranges from 1.5% to 6.5% globally, though absolute prevalence remains uncertain due to genetic and environmental variability [5]. This condition involves multiple pathophysiological mechanisms, including insulin resistance, chronic inflammation, oxidative stress, and disrupted lipid metabolism [6]. Insulin resistance plays a pivotal role in NAFLD-AD pathogenesis by inducing long-term hypertriglyceridemia, which subsequently impairs endothelium-dependent vasodilation and promotes oxidative stress development [7]. This cascade serves as a critical prognostic marker for atherosclerosis progression, characterized by specific biochemical alterations in blood serum [8]. Endothelin-1 (ET-1), a potent vasoconstrictor peptide, has emerged as a significant biomarker for cardiovascular system damage in metabolic disorders [9]. Elevated ET-1 concentrations in blood serum and plasma correlate with enhanced vascular tone, increased arterial pressure, impaired microcirculation, and cardiac muscle hypoxia [10]. Recent studies have demonstrated that ET-1 synthesis increases in endothelial cells under metabolic syndrome conditions, particularly in the presence of insulin resistance and chronic inflammation [11].

Apolipoprotein B (ApoB) constitutes the structural component of major atherogenic lipoprotein particles and reflects their total concentration in circulation [12]. The ApoB protein family plays crucial roles in lipid transport and metabolism, with ApoB-100 being particularly important in low-density lipoprotein formation and clearance [13]. Increased ApoB levels indicate impaired receptor-mediated endocytosis and suggest enhanced cardiovascular risk [14]. The relationship between neurodegeneration and peripheral metabolic markers remains incompletely understood. Growing evidence suggests that autoimmune mechanisms may contribute to both neuronal damage and hepatic dysfunction in AD patients [15]. Autoantibodies against various proteins, including ET-1 and ApoB, may serve as novel biomarkers for disease progression and therapeutic monitoring. Current diagnostic approaches for NAFLD-AD rely primarily on imaging techniques and biochemical markers. However, the identification of specific autoantibodies could provide earlier detection capabilities and improved prognostic accuracy. Furthermore, understanding the temporal dynamics of these biomarkers may facilitate targeted therapeutic interventions.

Objective

To detect and quantify autoantibodies against endothelin-1 and apolipoprotein B proteins in hepatic tissue and blood circulation of experimental Alzheimer's disease model animals, and to investigate the temporal dynamics of associated biochemical parameter changes.

2. Materials and Methods

Experimental Animals and Ethics

The research was conducted at the Metabolomics Laboratory of the Institute of Biophysics and Biochemistry, National University of Uzbekistan. Twenty-four male Wistar rats weighing 324±16 g (age 6-8 weeks) were utilized for the experimental procedures. All studies were performed in accordance with international guidelines for laboratory animal care and use, including the Helsinki Declaration (World Medical Association, Edinburgh, 2000), Council for International Organizations of Medical Sciences (CIOMS) standards, and the "Guide for the Care and Use of Laboratory Animals". Animals were housed in standard laboratory conditions with controlled temperature (22±2°C), humidity (55±10%), and 12-hour light/dark cycles. Food and water were provided ad libitum throughout the experimental period. All procedures were approved by the institutional animal care and use committee.

Experimental Design and Groups

Experimental animals were randomly allocated into five groups:

Group I: Control group (n=11) - received saline vehicle

Group II: Alzheimer's disease model (n=15) - streptozotocin-induced AD

Group III: AD + Quercetin treatment (n=15) - received quercetin supplementation

Group IV: AD + SMC (1:9) (n=14) - received supramolecular complex compound at 1:9 ratio

Group V: AD + SMC (1:5) (n=17) - received supramolecular complex compound at 1:5 ratio

Alzheimer's Disease Model Induction

Alzheimer's disease was induced using intranasal administration of streptozotocin (STZ) at a dose of 3 mg/kg body weight. STZ was dissolved in sterile phosphate-buffered saline (pH 7.4) immediately before administration. Animals were lightly anesthetized with isoflurane, and STZ solution was administered dropwise into both nostrils using a micropipette.

Treatment Protocols

Following a 14-day establishment period post-STZ administration, treatment groups received their respective interventions:

Group III: Quercetin (50 mg/kg) administered orally once daily for 7 days

Group IV: Supramolecular complex compound (1:9 ratio, 40 mg/kg) administered orally once daily for 7 days

Group V: Supramolecular complex compound (1:5 ratio, 50 mg/kg) administered orally once daily for 7 days

The supramolecular complex compound used in this study represents a novel therapeutic approach for treating hepatic alterations in experimental neurodegeneration models, demonstrating enhanced bioavailability and targeted delivery properties compared to conventional treatments.

Blood Sample Collection

Blood samples (200-300 μL) were collected from the tail vein at predetermined intervals (days 7, 14, and 21) using 27-gauge needles. Prior to blood collection, animals were placed in warm water (38-40°C) for 2-3 minutes to facilitate vasodilation. Samples were collected in EDTA-coated microtubes and immediately processed or stored at -80°C until analysis.

Biochemical Parameter Analysis

Lipid Profile Determination

Total cholesterol: Enzymatic cholesterol oxidase method

Triglycerides: Glycerol-3-phosphate oxidase-phenol aminophenazone (GPO-PAP) method

HDL-cholesterol: Precipitation method with phosphotungstic acid

LDL-cholesterol: Calculated using Friedewald equation

Atherogenic coefficient: Calculated as (Total cholesterol - HDL)/HDL

Inflammatory Marker Assessment

C-reactive protein (CRP): High-sensitivity immunoturbidimetric assay

Fibrinogen: Clauss clotting time method

Interleukin-6 (IL-6): Enzyme-linked immunosorbent assay (ELISA)

ET-1 and ApoB Detection Methodology

Endothelin-1 and apolipoprotein B concentrations were determined using commercial ELISA kits according to manufacturer protocols. Briefly, microplate wells pre-coated with specific capture antibodies were incubated with 100 μL of appropriately diluted serum samples and standards at 37°C for 90 minutes. Following primary incubation, wells were washed three times with phosphate-buffered saline containing 0.05% Tween-20 (PBST). Detection antibody (100 μL) was added and incubated at 37°C for 60 minutes. After washing cycles, horseradish peroxidase-conjugated secondary antibody was applied and incubated for 30 minutes at 37°C. Color development was achieved using tetramethylbenzidine (TMB) substrate solution (90 μL per well) with incubation at 37°C for 15 minutes. The reaction was terminated using 2M sulfuric acid (50 μL per well), and optical density was measured at 450 nm using a microplate reader.

Statistical Analysis

Statistical analyses were performed using Microsoft Excel and OriginPro 8.6 software. Data are presented as mean ± standard error of the mean (SEM). Statistical significance was assessed using Student's t-test for pairwise comparisons and one-way analysis of variance (ANOVA) for multiple group comparisons. Post-hoc analyses were conducted using Tukey's honest significant difference test. Statistical significance was defined as p<0.05, with additional significance levels at p<0.01 and p<0.001.

3. Results

Experimental Animal Characteristics

All experimental procedures were successfully completed using the designated animal groups. No significant adverse effects were observed during the experimental period, and animal welfare was maintained throughout the study duration.

Blood Lipid Profile Alterations

Significant dyslipidemia was observed in the Alzheimer's disease model group compared to control animals (Table 1). Total cholesterol levels increased 1.86-fold (from 2.8±0.3 to 5.2±0.6 mmol/L, p<0.01), while triglyceride concentrations showed a 2.67-fold elevation (from 0.9±0.1 to 2.4±0.3 mmol/L, p<0.01).

Table 1. Blood Lipid Parameters in Experimental Alzheimer's Disease Model (mmol/L; n=73)

LDL-cholesterol concentrations demonstrated a 2.71-fold increase (from 1.4±0.2 to 3.8±0.4 mmol/L, p<0.01), while HDL-cholesterol levels decreased by 33% (from 1.2±0.1 to 0.8±0.1 mmol/L, p<0.05). The atherogenic coefficient increased dramatically from 1.3±0.2 to 5.5±0.7 (p<0.01), indicating substantial cardiovascular risk development.

Treatment interventions demonstrated varying degrees of lipid profile improvement, with supramolecular complex compound (1:5 ratio) showing the most pronounced beneficial effects.

Inflammatory Marker Dynamics

Systemic inflammatory markers showed significant elevations in the AD model group (Table 2). C-reactive protein levels increased 4-fold (from 2.1±0.3 to 8.4±1.2 mg/L, p<0.01), while fibrinogen concentrations rose 1.75-fold (from 2.8±0.4 to 4.9±0.6 g/L, p<0.01).

Table 2. Blood Serum Inflammatory Parameters

Interleukin-6 concentrations exhibited a 2.92-fold increase (from 12±2 to 35±5 pg/mL, p<0.01), confirming the presence of systemic inflammatory processes. These findings support the hypothesis that NAFLD-AD involves significant inflammatory component activation.

Endothelin-1 Levels in Tissue and Circulation

ET-1 concentrations showed differential changes between hepatic tissue and blood serum compartments. In liver tissue, ET-1 levels increased modestly by 1.34-fold (from 1.85±0.59 to 2.49±0.73 ng/L, p<0.05), while blood serum concentrations demonstrated a more pronounced 1.66-fold elevation (from 1.92±0.62 to 3.18±0.85 ng/L, p<0.01).

Table 3. ET-1 Levels in Liver Tissue (ng/L; n=73)

Table 4. ET-1 Levels in Blood Serum (ng/L; n=73)

The enhanced ET-1 synthesis likely results from endothelial cell activation under metabolic syndrome conditions, particularly insulin resistance, inflammation, and oxidative stress associated with NAFLD-AD [18]. Treatment with supramolecular complex compound showed dose-dependent ET-1 reduction, suggesting potential therapeutic efficacy.

Apolipoprotein B Concentrations

ApoB levels demonstrated consistent increases across both tissue and circulation compartments. In hepatic tissue, ApoB concentrations increased 2.18-fold (from 0.94±0.11 to 2.05±0.13 mmol/L, p<0.01), while blood serum levels rose 2.42-fold (from 0.89±0.09 to 2.15±0.18 mmol/L, p<0.01).

Table 5. ApoB Levels in Liver Tissue (mmol/L; n=73)

Table 6. ApoB Levels in Blood Serum (mmol/L; n=73)

Lipoprotein Particle Analysis

The LDL-cholesterol to ApoB ratio provides insights into lipoprotein particle density and atherogenicity. In hepatic tissue, this ratio increased from 0.77±0.08 to 1.48±0.12 (p<0.01), while the HDL-cholesterol to ApoB ratio in blood serum rose from 0.82±0.07 to 1.54±0.16 (p<0.01).

Table 7. LDL-C to ApoB Ratio in Liver Tissue (mmol/L; n=73)

Table 8. HDL-C/ApoB Ratio in Blood Serum

These alterations indicate the presence of small, dense LDL particles, which possess enhanced atherogenic potential and contribute to cardiovascular risk elevation.

4. Discussion

The present study demonstrates significant alterations in autoantibody levels against ET-1 and ApoB proteins in an experimental NAFLD-AD model, providing novel insights into the interconnection between neurodegeneration and peripheral metabolic dysfunction. Our findings reveal a complex pathophysiological network involving lipid metabolism disruption, inflammatory activation, and cardiovascular risk factor elevation.

Lipid Metabolism Dysregulation

The observed dyslipidemia pattern in our AD model aligns with clinical observations in patients with metabolic syndrome and neurodegenerative disorders. The 1.86-fold increase in total cholesterol, coupled with the 2.71-fold elevation in LDL-cholesterol, indicates severe disruption of hepatic lipid homeostasis. These changes likely result from impaired insulin signaling, enhanced hepatic glucose production, and altered fatty acid metabolism [3]. The 33% reduction in HDL-cholesterol levels is particularly concerning, as HDL particles play crucial roles in reverse cholesterol transport and anti-inflammatory processes. This reduction may contribute to accelerated atherosclerosis development and increased cardiovascular mortality risk in AD patients. The dramatic increase in atherogenic coefficient (from 1.3 to 5.5) exceeds the threshold value of 3.0 typically associated with high cardiovascular risk. This finding suggests that even in experimental AD models, significant atherosclerotic processes may develop rapidly, highlighting the need for early intervention strategies.

Inflammatory Cascade Activation

The substantial elevations in CRP (4-fold), fibrinogen (1.75-fold), and IL-6 (2.92-fold) confirm the presence of systemic inflammatory processes in our NAFLD-AD model. These findings support the neuroinflammation hypothesis of AD pathogenesis and suggest that peripheral inflammatory markers may serve as accessible biomarkers for disease monitoring.

C-reactive protein, an acute-phase reactant synthesized primarily in hepatocytes, reflects the intensity of systemic inflammation. The observed 4-fold increase suggests significant hepatic involvement in the inflammatory response, which may contribute to both neuronal damage and hepatic steatosis progression.

Interleukin-6, a pleiotropic cytokine involved in both neuroinflammation and metabolic regulation, showed particularly pronounced elevation. This finding aligns with clinical studies demonstrating elevated IL-6 levels in AD patients and suggests potential therapeutic targets for anti-inflammatory interventions.

Endothelin-1 as a Cardiovascular Risk Marker

The differential increase in ET-1 levels between hepatic tissue (1.34-fold) and blood serum (1.66-fold) provides insights into the systemic nature of endothelial dysfunction in NAFLD-AD. ET-1, recognized as one of the most potent vasoconstrictors, plays critical roles in vascular tone regulation, blood pressure control, and microcirculatory function [9]. The preferential elevation of ET-1 in circulation suggests enhanced endothelial cell activation and potential autocrine/paracrine signaling disruption. This finding may explain the increased cardiovascular mortality observed in AD patients and supports the vascular hypothesis of AD pathogenesis. Recent studies have demonstrated that ET-1 can directly influence neuronal function and contribute to blood-brain barrier disruption. Our findings suggest that peripheral ET-1 elevation may serve as both a consequence of and contributor to neurodegeneration, creating a vicious cycle of vascular and neuronal damage.

Apolipoprotein B and Atherogenic Risk

The consistent increase in ApoB levels across both hepatic tissue (2.18-fold) and blood serum (2.42-fold) indicates severe disruption of lipoprotein metabolism. ApoB-100, the primary apolipoprotein component of VLDL and LDL particles, serves as a marker for atherogenic lipoprotein particle number rather than cholesterol content alone [12]. The elevation in ApoB levels suggests impaired hepatic VLDL assembly, enhanced VLDL secretion, or reduced LDL clearance through hepatic receptors. These mechanisms are commonly observed in insulin-resistant states and may contribute to accelerated atherosclerosis development in AD patients.

The LDL-cholesterol to ApoB ratio analysis reveals the presence of small, dense LDL particles, which possess enhanced atherogenic potential due to increased oxidative susceptibility and arterial wall penetration capacity. This finding supports the concept that qualitative lipoprotein analysis may provide superior cardiovascular risk assessment compared to traditional cholesterol measurements alone.

Therapeutic Implications

The differential responses to treatment interventions provide valuable insights into potential therapeutic strategies for NAFLD-AD management. The supramolecular complex compound, particularly at the 1:5 ratio, demonstrated superior efficacy in normalizing multiple biomarkers simultaneously, suggesting multi-target therapeutic potential.

Quercetin treatment showed intermediate efficacy, consistent with its known antioxidant and anti-inflammatory properties. However, the limited improvement compared to the supramolecular complex suggests that single-target approaches may be insufficient for addressing the complex pathophysiology of NAFLD-AD.

Clinical Relevance and Biomarker Potential

The identification of elevated autoantibodies against ET-1 and ApoB in experimental NAFLD-AD models provides a foundation for clinical biomarker development. These findings suggest that routine monitoring of these parameters in AD patients may facilitate early detection of cardiovascular complications and guide therapeutic decision-making. The temporal dynamics of biomarker changes observed in our study indicate that significant alterations occur within relatively short timeframes, supporting the feasibility of these markers for disease monitoring and treatment response assessment.

Study Limitations

Several limitations should be acknowledged in interpreting our findings. First, the experimental model may not fully recapitulate the complexity of human NAFLD-AD pathophysiology. Second, the relatively short observation period may not capture long-term disease progression patterns. Third, the specific mechanisms underlying autoantibody generation against ET-1 and ApoB require further investigation.

5. Conclusions

This study demonstrates significant alterations in autoantibodies against endothelin-1 and apolipoprotein B proteins in experimental NAFLD-AD models, accompanied by substantial disruptions in lipid metabolism and inflammatory marker profiles. The key findings include:

Severe dyslipidemia characterized by elevated total cholesterol (1.86-fold), triglycerides (2.67-fold), and LDL-cholesterol (2.71-fold), with concurrent HDL-cholesterol reduction (33%).

Systemic inflammatory activation evidenced by increased C-reactive protein (4-fold), fibrinogen (1.75-fold), and interleukin-6 (2.92-fold) levels.

Cardiovascular risk marker elevation including ET-1 increases in blood serum (1.66-fold) and liver tissue (1.34-fold), and ApoB elevation in both serum (2.42-fold) and hepatic tissue (2.18-fold).

Atherogenic lipoprotein particle predominance indicated by altered LDL-cholesterol to ApoB ratios, suggesting enhanced cardiovascular risk.

Therapeutic response variability with supramolecular complex compound (1:5 ratio) demonstrating superior multi-target efficacy compared to conventional antioxidant approaches.

These findings support the concept that NAFLD-AD involves complex interactions between neurodegeneration, hepatic dysfunction, and cardiovascular risk factors. The elevated autoantibodies against ET-1 and ApoB may serve as valuable biomarkers for early detection, disease monitoring, and therapeutic response assessment in clinical settings.

Future research should focus on validating these biomarkers in human populations, investigating the mechanistic basis of autoantibody generation, and developing targeted therapeutic interventions addressing the multi-system nature of NAFLD-AD pathophysiology.

Funding

This research was supported by grants from the Ministry of Higher Education, Science and Innovation of the Republic of Uzbekistan and the Institute of Biophysics and Biochemistry, National University of Uzbekistan.

Ethical Approval

All experimental procedures were conducted in accordance with international guidelines for laboratory animal care and use and were approved by the Institutional Animal Care and Use Committee (Protocol #2024-015).

Conflict of Interest

The authors declare no conflicts of interest related to this research.