1. INTRODUCTION:

Dementia is a chronic and progressive disorder associated with impaired memory, learning, thinking, and comprehension, which interferes with daily activities¹.

On a global scale, the estimated prevalence of dementia stands at around 36 million individuals, and it is anticipated to rise to 66 million by 2030, followed by a further increase to 115 million by the year 2050². AD is an intricate and progressive neurodegenerative disorder within the realm of dementia. It has the highest incidence among various forms of dementia; however, its etiology remains largely elusive³. AD is distinguished by the presence of intracellular neurofibrillary tangles (NFTs) composed of hyperphosphorylated Tau protein, as well as extracellular senile plaques (SPs) and synaptic decline^4-6. Clinical manifestations arise due to enduring neuronal degeneration and the emergence of aberrant extracellular aggregates of amyloid beta (Aβ) protein, which represents a notable neuropathological signature^7,8. Regrettably, viable therapeutic interventions to arrest the progression of Alzheimer's disease (AD) remain elusive. Consequently, researchers are compelled to exert substantial efforts toward comprehending plausible targets implicated in pathological alterations and innovating novel pharmacotherapeutic agents tailored for individuals afflicted by AD.

In accordance with epidemiological investigations and studies conducted in animal models, the deposition of aluminum within the hippocampus causes anomalous aggregation of amyloid and triggers neuronal apoptosis through the promotion of amyloidogenesis and initiation of neuroinflammatory processes. Consequently, this sequence of events culminates in disrupting hippocampal-dependent learning processes and impairing memory capabilities^9,10. Previous investigations have revealed the critical role of metal ion equilibrium in facilitating cognitive processes. Disruption of homeostatic mechanisms has emerged as the primary cause of neurodegenerative disorders¹¹. Aluminum infiltration via designated transferrin receptors across the blood-brain barrier causes disruption of neuronal function, consequently leading to memory deterioration and the onset of Alzheimer's disease¹². Notably extended periods of aluminum exposure have the capacity to exert an impact on the intricate signaling pathways associated with brain neurotransmitters. This contributes to the degradation of cognitive function by interrupting the transmission of signals to neurons and eliciting neuronal injury¹³. Aluminum has demonstrated its capacity to induce neurodegeneration through the initiation of oxidative stress (OS), leading to the buildup of iron and the generation of reactive oxygen species (ROS)^14,15. Moreover, Aluminum (Al) is recognized for its capability to accelerate oxidative stress, facilitate the cross-linking and deposition of Aβ oligomers, and contribute to the formation of plaques within the cerebral cortex and hippocampal regions. Hence, the induction of Alzheimer's disease (AD) in rats using aluminum chloride (AlCl3) serves as a valuable model for investigating the neuroprotective potential of various phytochemicals and chemical compounds in countering AD-related effects¹⁶.

Ayurveda, a longstanding medical tradition originating in India, has been practiced for centuries. In contrast, archaic Indian medical manuscripts such as the Charaka-Samhita expound a spectrum of botanical compositions and the integration of metallic elements employed for the alleviation of varied physiological afflictions. Furthermore, established methodologies such as Swarna Prashana, which incorporates gold, have been shown to have considerable efficacy in ameliorating cognitive impairments^17,18. As documented in the scientific literature, compounds comprising gold salts, namely auranofin, gold sodium thiomalate, and aurothioglucose (ATG), exhibit noteworthy anti-inflammatory properties and have shown efficacy in the management of individuals afflicted by rheumatoid arthritis¹⁹. Aurothioglucose (ATG) has demonstrated a modulatory effect on diverse inflammatory mediators implicated in arthritis, a cascade often linked to cognitive and motor impairments. Nevertheless, its limited capacity to traverse the blood-brain barrier (BBB) results in restricted cerebral delivery²⁰.

To overcome this constraint, the use of poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) has emerged as a prominent strategy for mitigating unfavorable side effects, augmenting lipophilic characteristics, and improving the bioavailability profile of ATG. Poly lactic-co-glycolic acid (PLGA) functions as a carrier system, augmenting the properties of pharmaceutical compounds owing to its inherent biocompatibility and biodegradability^21-24. FDA endorsement includes PLGA for medical applications, such as drug delivery devices and biomaterials. Notably, PLGA-based nanoparticles offer an extended half-life, enhanced bioavailability, and efficacy, and can be effectively administered to the brain through oral delivery.

Consequently, the utilization of ATG-loaded PLGA nanoparticles presents a prospective avenue for therapeutic intervention in Alzheimer's disease (AD). Hence, this investigation focused on the assessment of the neuroprotective efficacy of ATG-loaded PLGA nanoparticles against AlCl3-induced AD in a rat model.

2. MATERIAL AND METHODS:

2.1 Animals:

In this experiment, adult healthy Wistar rats (230-270 g and 12 weeks old) were purchased from ISF College of Pharmacy, Moga, (Pb). Rats were grouped and entrained under standard conditions (Temp. 24 to 25°C), normal light-dark cycle, RH 55 to 65%, free access animal pellet, and water. Animal handling protocols were carried out as per The Institutional Animal Ethical Committee (IAEC) with approval number CPCSEA/LIPH/2019/10.

2.2 Chemicals:

Aurothioglucose provided Pro Lab, New Delhi, India, [Cayman chemical, >95% High-Performance Liquid Chromatography] CAS No. 15316). Nomisma Healthcare, Gujarat, India, supplied PLGA (50:50). Aluminium Chloride (AlCl3) (CAS No.15630-89-4), and other chemicals (analytical grade) and kits are procured from authorized distributors.

2.3 In-vivo study:

2.3. 1 Experimental design:

After acclimation (7 days), the 36 rats were weighed and evenly sorted into six groups (n=6). All experimental rats underwent a seven-day training regimen encompassing sessions on the actophotometer, Morris water maze (MWM), and object recognition test (ORT). Subsequently, oral AlCl₃ was administered over a span of 21 days. Following this phase, ATG and ATG NPs was administered for a duration of 14 days, commencing from the 8^th day and continuing until the 20^th day (Table 1). Throughout the course of the experimental protocol, behavioral traits were evaluated employing an actophotometer, MWM, and ORT. On the 21^stday after the behavioral assessments, the rats were euthanized and their hippocampal regions were extracted for the preparation of homogenates, which were subsequently subjected to biochemical, inflammatory and histological analyses.

Table 1. Experimental groups

Group 1: Control (5% DMSO)

Group 2: AlCl₃ (100 mg/kg/p.o.)

Group 3: AlCl₃ (100 mg/kg/p.o.) + ATG (5 mg/kg/s.c.)

Group 4: AlCl₃ (100 mg/kg/p.o.) + ATG (10 mg/kg/s.c.)

Group 5: AlCl₃ (100 mg/kg/p.o.) + ATG NPs (2.5 mg/kg/s.c.)

Group 6: AlCl₃ (100 mg/kg/p.o.) + ATG NPs (5 mg/kg/s.c.)

2.3.2 Behavioral parameters:

Each animal was habituated in an activity cage for more than 3min to measure locomotor activity. Subsequently, the animals were placed individually in a square-covered arena for 5min, equipped with an electronic actophotometer containing infrared light-sensitive photocells (IMCORP, India). The actophotometer measured the animals’ spontaneous locomotor activity, reported in counts per 5min²⁵. TheMWM test was conducted to evaluate spatial memory and learning abilities of the rats, as previously described by Sharma et al. During the trial, escape latency time (ELT) and time spent in the targeted quadrant (TSTQ) were recorded²⁶. Furthermore, a novel object recognition test was conducted, with modifications to a previously described approach. During the trial, the time spent exploring the familiar object and the time spent exploring the novel object was recorded²⁷.

2.3.3 Tissue homogenate preparation:

On the 21^st day, six rats from each group were euthanized and their brains were rapidly removed for hippocampal dissection using a rat brain atlas. The brain tissue was collected and washed with isotonic saline. Following this, cerebral tissues were homogenized in ice-cold 10mM phosphate buffer (pH 7.4). Upon centrifugation of the homogenate at 10,000 × g for 15 minutes, the resultant solution was employed for subsequent biochemical analysis.

2.3.4 Biochemical Parameters:

In the hippocampal tissue homogenate, malondialdehyde (MDA) level was measured using the methods described by Wills 1966 at 532nm²⁸. The given outline measured GSH in the hippocampal homogenate using the Ellman method 1959 at 412nm²⁹. Superoxide dismutase (SOD) activity was determined using the Kono method. A spectrophotometer set at 560nm was used to measure the change in absorbance every 30s for 2min³⁰.

Table 2: Effect of ATG and ATG NPs on oxidative parameters in AlCl3-induced Rats

Groups/ Parameters

Control

AlCl₃

(100 mg/kg/p.o.)

AlCl₃ + ATG

(5mg/kg/s.c.)

AlCl₃+ ATG

(10mg/kg/s.c.)

AlCl₃+ ATG NPs

(2.5mg/kg/s.c.)

AlCl₃ + ATG NPs (5mg/kg/s.c.)

GSH (μmol/mg protein)

1.498±0.09

0.362±0.18

^aP<0.001

0.469± 0.15

1.086± 0.13

^bP<0.05

0.601± 0.14

1.203± 0.11

^cdP<0.01

MDA

(nmol/mg protein)

9.96±0.32

16.17±1.15

^aP<0.001

15.14± 0.99

12.35±0.62

^bP<0.05

14.42± 0.91

11.32± 0.50

^cdP<0.05

SOD

(U/min/mg protein)

20.15±0.55

13.02± 1.22 ^aP<0.001

14.12± 1.11

17.03± 1.04

15.87± 1.01

18.78± 0.66

^cdP<0.05

Catalase

(U/mg protein)

1.045±0.05

0.243± 0.14 ^aP<0.001

0.377± 0.12

0.698± 0.10

0.487± 0.11

0.876 ± 0.80

^cdP<0.05

ATG-Aurothioglucose, NPs-nanoparticles, GSH- Reduced glutathione, MDA- Malondialdehyde, SOD- Superoxide Dismutase.

Data are expressed as Mean ± Control ^aP<0.001 AlCl₃ vs Sham, ^bP<0.05 ATG vs AlCl₃,^cP<0.05 ATG NPs vs AlCl₃^dP<0.05 ATG NPs vs ATG.

Table 3. Effect of ATG and ATG NPs on TNF-α and IL-6 level in AlCl3-induced Rats

Groups/ Proinflammatory Markers

Control

AlCl₃

(100 mg/kg/p.o.)

AlCl₃ + ATG

(5 mg/kg/s.c.)

AlCl₃+ ATG

(10 mg/kg/s.c.)

AlCl₃+ ATG NPs

(2.5 mg/kg/s.c.)

AlCl₃ + ATG NPs

(5 mg/kg/s.c.)

TNF-α

14.87 ± 1.08

36.88 ± 2.69

^aP<0.001

34.29 ± 1.35

29.05 ± 1.12

^bP<0.05

31.64 ± 1.42

26.31 ± 1.12 ^cdP<0.001

IL-6

12.69 ± 0.83

40.00 ±1.91

^aP<0.001

37.78 ± 1.27

32.53 ± 1.22

^bP<0.05

34.68 ± 1.26

30.49 ± 1.14

^cdP<0.01

ATG-aurothioglucose, NPs-nanoparticles, TNF-α- Tumor necrosis factor-alpha, IL-6 -Interleukin 6.

Data are expressed as Mean ± SEM ^aP<0.001 AlCl₃ vs Control, ^bP<0.05 ATG vs AlCl₃,^cP<0.01 ATG NPs vs AlCl₃^dP<0.05 ATG NPs vs ATG.

All parameters were measured using an ELISA microplate reader. Catalase activity was tested using the Luck 1978 technique at 240nm and measured using a spectrophotometer at 30-second intervals for 2min (Luck 1978). Additionally, levels of TNF-α and IL-6 in hippocampal homogenates were determined using an ELISA kit (Krishgen Biosystems, India).

2.3.5 Statistical analysis:

Statistical analysis was performed using GraphPad Prism software, and values are expressed as the mean± Standard Error of the Mean (SEM). Two-way analysis of variance (ANOVA) followed by Tukey's post hoc multiple comparisons tests were used to analyze MWM, locomotor activity, and ORT outcomes. One-way ANOVA was used to evaluate the results of TSQT, biochemical, and proinflammatory markers, and the significance level was set at P<0.05.

3 RESULTS:

3.1 Effect of ATG NPs on behavioural parameters in AlCl₃induced Rats:

The rats administered AlCl₃ showed a significant (P<0.001) reduction in spontaneous locomotor activity compared with the normal control group (Fig. 2). In contrast, the administration of ATG and ATG NPs (10 and 5mg/kg, s.c.) significantly (P<0.05 and P<0.01, respectively) increased locomotor activity. Notably, ATG NPs (5mg/kg, s.c.) exhibited a more significant effect (P<0.05) than ATG alone.

In the MWM, AlCl₃ administered rats displayed a significant cognitive deficit, as evidenced by their significantly greater ELT and reduced TSTQ compared with the sham group (P<0.001 and P<0.001, respectively), as shown in Fig. 3 and 4. However, treatment with ATG alone significantly decreased ELT (P<0.05) compared to that in the AlCl₃ treated group. Treatment with ATG NPs also significantly decreased ELT and increased TSQT (P<0.05) compared with the AlCl₃treated group. Moreover, the effects of ATG NPs (5mg/kg, s.c.) were more significant than those of ATG alone.

Figure 5a displays the results of the object and location recognition tests on the ORT. On day 20^th, the first test (T1) was conducted, which involved examining comparable objects for 15 seconds. The time required for object exploration did not differ significantly between the groups. On day 21^st, AlCl₃administered rats could not distinguish between familiar and novel objects compared with the sham group (P<0.01). However, treatment with ATG (10mg/kg, s.c.) and ATG NPs (5 mg/kg, s.c.) shows non-significantly spending more time on the novel object than AlCl₃ treatment (Fig. 5b).

Fig.1 Protocol schedule

AF- Aurothioglucose, NPs- Nanoparticles, AlCl₃- Aluminum Chloride, DNP- Donepezil, OFT- Open Field Test, ORT, Object Recognizing Test, MWM- Morris Water Maze, SAC- Sacrifice, BE- Biochemical Estimation, NIA- Neuroinflammatory analysis.

Fig. 2: Effect of ATG and ATG NPs on beam cross time using actophotometer in AlCl₃- induced rats (n=6). Data was analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. Data are expressed as Mean±SEM ^aP<0.001 AlCl₃ vs control, ^bP<0.05 ATG vs AlCl₃, ^cP<0.01 ATG NPs vs AlCl₃, ^dP<0.05 ATG NPs vs ATG. ATG- Aurothioglucose, NPs- Nanoparticle, AlCl₃- Aluminum Chloride.

Fig. 3: Effect of ATG and ATG NPs on ELT in AlCl₃-induced rats (n=6). Data were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons test. Data are expressed as Mean ± SEM ^aP<0.001 AlCl₃ vs control, ^cP<0.01 ATG NPs vs AlCl₃, ^dP<0.05 ATG NPs vs AF. ATG- Aurothioglucose, NPs- Nanoparticle, AlCl₃- Aluminum Chloride.

Fig. 4: Effect of ATG and ATG NPs on time spent in the target quadrant in AlCl₃-induced rats (n=6). Data was analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. Data are expressed as Mean ± SEM ^aP<0.001 AlCl₃ vs control, ^cP<0.01 ATG NPs vs AlCl₃, ^dP<0.05 ATG NPs vs ATG. ATG- Aurothioglucose, NPs- Nanoparticle, AlCl₃- Aluminum Chloride.

Fig. 5a) Effect of ATG and ATG NPs on memory performance (acquisition phase, trial 1) in object recognition test in AlCl₃-induced rats (n=6). Data were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons test. Data are expressed as Mean ± SEM. Ns- non-significant.

b) Effect of ATG and ATG NPs (retention phase, trail 2) in object recognition test in AlCl₃-induced ratsData are expressed as Mean ± SEM ^aP<0.01 AlCl₃ vs control. ATG- Aurothioglucose, NPs- Nanoparticle, AlCl₃- Aluminum Chloride.

3.2 Effect of ATG NPs biochemical parameters in AlCl₃induced Rats:

In AlCl₃-administered rats, MDA levels were significant (P<0.001). GSH, SOD, and catalase levels were reduced compared with those in the control group. However, treatment with ATG and ATG NPs (10 and 5 mg/kg, s.c.) effectively reduced MDA levels, Catalase, SOD, and glutathione levels restored compared to the AlCl₃-treated group. The reduction in MDA and restoration of glutathione levels were significant (P<0.05, P<0.01) for ATG and ATG NPs, respectively but restoration of catalase and SOD were found significant (P<0.05) with administered ATG NPs only. However, ATG NPs (5 mg/kg, s.c.) were more effective (P<0.05) than ATG alone (Fig. 6,7,8,9).

3.3 Effect of ATG NPs on proinflammatory cytokines (TNF-α and IL-6) levels in AlCl₃induced Rats:

The levels of proinflammatory cytokines (TNF- α and IL-6) were found to be significantly (P<0.001) greater in AlCl₃ rat's brains in contrast to the sham group (Fig. 10). Subsequently, ATG and ATG NPs treated group had significantly (P<0.05 and P<0.01, respectively) reduced TNF-α, and IL-6 levelsin the hippocampus against AlCl₃alone treated group. Nevertheless, ATG NPs (5 mg/kg, s.c.) were more significant (P<0.05) than ATG alone.

4. DISCUSSION:

Alzheimer's disease denotes a gradual decline in cognitive capacity and neuronal degradation, instigated by the accretion of 𝛽-amyloid peptides, subsequently accompanied by the conglomeration of tau proteins^31-33. Presently, the affliction impacts a populace exceeding 50 million individuals, with projections anticipating a surge to approximately 152 million by the year 2050, as indicated by Christina Patterson in 2018. Aluminum (Al) is a neurotoxic agent that precipitates the formation of neurofibrillary tangles and amyloid aggregates³⁴. Furthermore, aluminum has been documented to exert deleterious effects on neurological functionality by inducing degradation of Aβ within brain tissue, instigating neuronal apoptosis, and eliciting symptoms reminiscent of Alzheimer's disease^35-37.

A behavioral assessment, encompassing methodologies such as the actophotometer, Morris maze test, and objective recognition test, stands as the favored approach for investigating cognitive processes. Furthermore, an array of biochemical and inflammatory markers was assessed to provide a comprehensive evaluation of the pathological alterations linked to the presented condition. Antecedent investigations have established a causal association between extended exposure to aluminum chloride (AlCl₃) and the impairment of memory and cognitive functions. This phenomenon stems from neuronal modifications incited by the aforementioned exposure^38,39. The current study examined the impacts of ATG and ATG NPs on cognitive behavior, biochemical parameters, and levels of inflammatory cytokines in rats subjected to aluminum chloride (AlCl₃) exposure.The findings elucidate the potential of ATG and ATG NPs to mitigate the deleterious effects instigated by exposure to aluminum chloride (AlCl₃). As evidenced by preceding studies, the administration of aluminum chloride (AlCl₃) led to pronounced deficits in memory and behavioral functions, a phenomenon conspicuously corroborated by the outcomes of the present investigation^40,41. Similarly, the administration of ATG and AF NPs exhibited noteworthy enhancements in locomotor activity, memory,and learning performance impaired by AlCl₃.

Aluminum (Al) is widely acknowledged for its ability to traverse the blood-brain barrier (BBB) and accumulate within diverse cerebral regions. Moreover, it has the potential to induce the generation of free radicals, thereby precipitating brain injury, particularly affecting regions associated with memory and learning functions⁴². Oxidative stress-induced neurotoxicity represents a fundamental pathological occurrence in the primary neurodegenerative progression of Alzheimer's disease⁴³. In the present study, it was observed that exposure to aluminum chloride (AlCl₃) led to an elevated accumulation of malondialdehyde (MDA) and a concomitant decrease in levels of glutathione, superoxide dismutase, and catalase within the rat brain. This decrement in antioxidant enzyme concentrations correspondingly contributed to heightened oxidative damage, in line with outcomes documented in prior investigations^44-46. Similarly, ATG and ATG NPs administered to AD rats showed a significant augmentation in reduced glutathione. superoxide dismutase, and catalase status in brain tissue. This was accompanied by a marked decrease in malondialdehyde status. These observations denote that both ATG and ATG NPs interventions effectively bolstered antioxidant defenses while mitigating oxidative damage.

Inflammation represents the initial event in the pathogenesis of Alzheimer's disease (AD), contributing significantly to its pathological progression. Moreover, neuroinflammation assumes a pivotal role in instigating and advancing neuronal disorders, thereby contributing to the manifestation of memory and cognitive impairments⁴⁷.The transcription factor NF-κB intricately participates in the activation of a multitude of genes associated with inflammatory processes. Amid neuroinflammatory processes, NF-κB becomes localized within the nucleus through the phosphorylation of IκBα, thereby facilitating the activation of proinflammatory cytokine transcription (including TNF-α, IL-6, IL-1β, iNOS, COX-2). This activation sequence initiates a robust inflammatory response^48-49. A previous study has well documented that exposure to metals, including aluminum, led to heightened levels of TNF-α and IL-6, alongside the pro-inflammatory cytokine IL-1β⁵⁰. Nonetheless, administration of ATG and ATG NPs yielded a significant reduction in the concentrations of TNF-α and IL-6 within the hippocampus of the rat brain. These observations allude to a plausible anti-inflammatory characteristic exhibited by both AF and AF NPs, potentially mediated by the modulation of cerebral cytokine levels.

5. CONCLUSION:

In the current investigation, both ATG and ATG NPs have exhibited a mitigating effect on thecognitive decline induced by aluminum chloride (AlCl₃), along with a reduction in various biochemical markers and proinflammatory indicators. The therapeutic results observed in this study suggest that ATG and ATG NPs provide a mitigation of AlCl₃-induced neurotoxicity in rats, likely mediated by mechanisms that encompass antioxidative and anti-inflammatory effects.Our findings provide novel insights, indicating that ATG NPs exhibit enhanced efficacy when compared to unmodified ATG, thereby warranting further investigations aimed at elucidating theunderlying molecular mechanisms involved. Hence, ATG NPs could be a potential drug for correcting Alzheimer’s disease.

ABBREVIATIONS:

AD - Alzheimer’s disease; AlCl3 – Aluminum Chloride; PLGA - Poly lactic-co-glycolic acid; p.o. – Per oral; ATG – Aurothioglucose; ATG NPs – Aurothioglucose Nanoparticles; mg – milligram; kg – kilogram; Aβ - Amyloid-β; Ca – Calcium; NMDA - N-methyl-D-aspartate; FDA - Food and Drug Administration; BBB - Blood-brain barrier; DMSO - Dimethyl sulfoxide; DTNB - 5,5´-dithiobis (2-nitro benzoic acid); TNF- α - Tumor necrosis factor; GSH - Reduced glutathione; TBA - Thiobarbituric acid; MWM - Morris water maze; ORT - Object recognition test; MDA – Malondialdehyde; SOD - Superoxide dismutase; IL-6 – Interleukin; ELISA - Enzyme-linked immunosorbent assay; S.E.M - Standard error mean; SD - Standard deviation; ELT - Escape latency time; TSTQ -Time spent in time quadrant.

ACKNOWLEDGMENT:

The authors are thankful to Dr. Ran Singh, Managing Director, Laureate Institute of Pharmacy for providing resources and facilities for this research work, Dr. Rahul K. Verma, and Dr. Manish Sinha for technical and moral support.

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Received on 18.10.2023 Modified on 16.11.2023

Research J. Pharm. and Tech 2024; 17(2):756-762.

DOI: 10.52711/0974-360X.2024.00118