Glycyrrhizic Acid Attenuates Aβ₄₂-Induced Neurodegeneration Through Coordinated Regulation of Oxidative Stress, Synaptic Markers, and Key Alzheimer’s Signaling Pathways

Abstract

Alzheimer’s disease (AD) is a catastrophic neurodegenerative disorder marked by progressive decline of cognitive function, memory loss, and neuronal death. Its pathology is characterized by the formation of extracellular amyloid-beta (Aβ) plaques and intracellular neurofibrillary tangles from tau hyperphosphorylation. Despite extensive research, current treatments are limited to symptomatic relief and are associated with significant side effects. This accentuates the critical need for alternative therapeutic strategies with potent neuroprotective effects and minimal toxicity. This study investigates the neuroprotective potential of glycyrrhizic acid, as the precise molecular mechanisms by which it might improve AD pathology remain poorly understood. Using an Aβ₄₂-induced IMR-32 cell model of AD, our research revealed that Aβ₄₂ treatment caused significant protein alterations associated with AD pathology, mitochondrial dysfunction, cell cycle re-entry, and synaptic activity. Co-treatment with glycyrrhizic acid not only restored these protein levels, but also mitigated the hyperactivation of several key signaling pathways and rescued neurons from apoptosis. These findings suggest that glycyrrhizic acid exerts neuroprotective effects by preventing mitochondrial dysfunction and apoptosis via modulation of critical signaling pathways. This study provides strong evidence for glycyrrhizic acid’s neuroprotective properties in AD, paving the way for further research into its potential as a promising therapeutic agent for Alzheimer’s disease.

1. Introduction

Alzheimer’s disease (AD) is the most common cause of dementia and is a major medical, social, and economic burden worldwide. More than 55 million people worldwide are affected by AD or related conditions, as per the World Alzheimer Report 2022, and this number is expected to rise to 82 million by 2030 and 138 million by 2050 [1]. The etiology of AD is governed by several interconnected hypotheses, spanning from traditional amyloid and tau protein models to more advanced perspectives involving neuroinflammation, oxidative stress, abnormal autophagy, and the microbiota–gut–brain axis. Central to its pathology is the deposition of Aβ plaques and the formation of tau-based neurofibrillary tangles (NFTs), which collectively trigger other pathological events. These processes include cognitive impairment, acetylcholine deficiency, neuroinflammation, synaptic dysfunction, gut microbiome abnormalities, autophagy abnormalities, disruption of cholesterol homeostasis, oxidative stress, glutamate imbalance, insulin resistance, disruptions in mitochondrial functions and bioenergetics, and vascular abnormalities. Together, these changes can ultimately lead to neuronal death [2,3,4,5,6].

The onset of AD frequently involves behavioral shifts, including social isolation, depressive moods, and heightened anxiety alongside disrupted sleep–wake cycles. As the AD condition advances, these symptoms intensify, leading to progressive memory loss and neuropsychiatric symptoms like hallucinations and delusions. In its advanced stages, patients often face intensified behavioral and emotional issues. Some people with non-amnestic cognitive impairment may exhibit deficits in visual–spatial ability, language, executive processes, and motor skills [7,8,9,10,11]. Additional complications include thrombosis [12] dysphagia [13], malnutrition [14], and pneumonia [15], all of which substantially reduce quality of life and increase mortality risk [16].

Currently available drugs for AD, including donepezil [17,18], rivastigmine [19,20], galantamine [21,22], memantine [23,24], and namzaric [25], are employed in clinical settings to provide symptomatic relief; they are often accompanied by side effects [26,27]. The recently FDA-approved aducanumab and lecanemab show promise for disease-modifying effects; however, they are also linked with substantial side effects, and their long-term efficacy and safety require further validation [28,29,30,31].

Glycyrrhizic acid (GA), the primary active compound in Glycyrrhiza glabra, is also known as glycyrrhizin, which is a major triterpene glycoside widely used in traditional Chinese medicine [32]. A wide range of products, like sweets, medications, drinks, chewing gum, chewing tobacco, and toothpaste, use glycyrrhizic acid salts as sweeteners and flavoring agents [33,34]. It has a wide variety of pharmacological activities, such as anti-inflammatory [35,36,37], anti-ulcer [38], anti-allergic [39], antioxidant [40], anti-viral [41], anti-aging [42], anti-tumor [43,44], neuroprotective [45], hepatoprotective [46,47], and cytokine-inducing effects [48]. The dual stereoisomeric nature of glycyrrhizic acid, comprising 18α and 18β forms, has been considered as a key contributor to its diverse biological activities, which have been investigated for their efficacy against various neurological pathologies, including Parkinson’s disease (PD), Alzheimer’s disease, dementia, and ischemic stroke [49,50,51].

Research suggests that glycyrrhizic acid has potent neuroprotective properties. The neuroprotective potential of glycyrrhizic acid has been demonstrated in SH-SY5Y cells, where it counteracts the toxic effects of corticosterone and 6-hydroxydopamine through regulating autophagy, which is linked to Parkinson’s disease depression [52]. It has also been shown to improve scopolamine-induced acetylcholinesterase activity, as well as superoxide dismutase and catalase activity, thereby enhancing cognitive function in mice [49,53]. Furthermore, glycyrrhizic acid treatment has been shown to suppress the overexpression of the pro-inflammatory cytokines IL-1β and TNF-α in the brains of C57 mice by inhibiting the toll-like receptor 4 (TLR4) signaling pathway, thereby improving neuroinflammation and cognitive impairment [54]. The neuroprotective effects of glycyrrhizic acid are primarily attributed to its ability to inhibit reactive oxygen species (ROS) and reduce neuroinflammation [33,55]. The diverse pharmacological profile of glycyrrhizic acid makes it a compelling alternative for addressing the limitations of current therapies for neurological disorders [42].

Although glycyrrhizic acid has shown promising neuroprotective properties, its potential therapeutic benefits for AD remain largely unexplored, as no studies have specifically investigated its effects on the pathological characteristics of the disease. Given the growing incidence of AD among the elderly and the lack of effective treatments, there is an urgent need for research into novel, cost-effective interventions with minimal side effects. The current study aims to address this critical gap by investigating the therapeutic efficacy of glycyrrhizic acid and its ability to target and modify the molecular mechanisms underlying AD. This research provides an innovative and crucial step toward developing a new treatment strategy for this devastating neurodegenerative disorder.

2. Materials and Methods

2.1. Reagent Procurement

The following reagents were utilized in this study: Glycyrrhizic acid (Cat# PHR1516), retinoic acid (Cat# R2625), collagen (Cat# C9791), thiazolyl blue tetrazolium bromide (MTT, Cat# M5655), bisBenzimide H 33342 (HOECHST, Cat# B2261), 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA, Cat# D6883), TRIzol™ Reagent (Cat# T9424) and propidium iodide (Cat# 537059), all sourced from Sigma-Aldrich, St. Louis, MO, USA. rPeptide (Watkinsville, GA, USA) supplied the oligomeric Aβ₄₂ peptide (Cat# A-1163-2). Dulbecco’s modified Eagle medium (DMEM, Cat# 12100046), high glucose, fetal bovine serum (FBS, Cat# A5256701), Trypsin EDTA (Cat# 25200-056), 100× antibiotic/antimycotic solution (Cat# 15240062), BCA protein estimation assay kit (Pierce, Cat# 23225), and FxCycle™ PI/RNase Staining Solution (Cat# F10797) were purchased from Thermo Fisher Scientific, Waltham, MA, USA. Dimethyl sulfoxide (DMSO, Cat# PCT1303) was obtained from Himedia Laboratories, Mumbai, India. PrimeScript™ 1st strand cDNA synthesis kit (Cat# 6110A) and TB Green^® Premix Ex Taq™ II (Tli RNaseH Plus) (Cat# RR820A) were procured from TAKARA BIOINC, Kusatsu, Japan. Primers were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nitrocellulose membrane (Cat. No. 1620115) and Clarity ECL Substrate (Cat. No. 170-5061) were purchased from Bio-Rad Laboratories, Hercules, CA, USA. Primary and secondary antibodies were sourced from Sigma-Aldrich (St. Louis, MO, USA) and Cell Signaling Technology (Danvers, MA, USA).

2.2. Cell Culture and Treatment

In this study, IMR-32 neuroblastoma cells (ATCC^® CCL-127™) obtained from the National Centre for Cell Sciences, Pune, India, were cultured in DMEM supplemented with high glucose, L-glutamine, 10% FBS, and 1× antibiotic/antimycotic solution and incubated at 37 °C with 5% CO₂. Cells were seeded at a density of 3 × 10⁴ cells per well onto collagen-coated 6-well plates and differentiated with 10 μM retinoic acid in DMEM containing 2% FBS for 7 days. Following differentiation, cells were subjected to 48 h treatments with glycyrrhizic acid (100 μM), Aβ₄₂ (0.5 μM in DMEM), co-treatment of Aβ₄₂ with glycyrrhizic acid, and the control group consisted of cells that received no treatment.

2.3. Assessment of Cell Cytotoxicity in IMR-32 Cells

The cytotoxicity of glycyrrhizic acid on IMR-32 cells was assessed using an MTT assay. Five thousand cells were seeded per well in a 96-well plate and treated with varying concentrations of glycyrrhizic acid (10–1000 μM) for 48 h. Following treatment, MTT dye was added and incubated for 4 h; the resulting formazan crystals were then dissolved in a 50:50 ethanol/DMSO mixture. Absorbance was measured at 570 nm with background subtraction at 650 nm, allowing for the calculation of cell cytotoxicity as a percentage relative to untreated control cells [56].

2.4. Determination of Glycyrrhizic Acid’s Effect on Reactive Oxygen Species Levels in a Cellular Model of Alzheimer’s Disease

Previous studies suggested that elevated reactive oxygen species (ROS) production is a vital hallmark of Alzheimer’s disease, contributing to neuronal apoptosis [57]. Therefore, we tested the effect of glycyrrhizic acid on ROS production in differentiated IMR-32 cells using the DCFDA method. IMR-32 cells were seeded at a density of 3 × 10⁴ cells per well onto 1× collagen-coated 6-well plates. Differentiation of cells was carried out with 10 μM retinoic acid for seven days and subsequently treated for 48 h with Aβ₄₂ (0.5 μM), Aβ₄₂ (0.5 μM) co-treated with glycyrrhizic acid (100 μM), glycyrrhizic acid alone (100 μM), and untreated cells served as the control. Following treatment, the cells were washed twice with 1× PBS, stained with DCFDA (25 μM) and HOECHST (5 μg/mL) in serum-free media for 20 min in the dark, imaged using a ZOE™ Fluorescent Cell Imager (BioRad Laboratories, Hercules, CA, USA) in green (480 nm excitation, 517 nm emission) and blue (355 nm excitation, 433 nm emission) channels, and analyzed with ImageJ software Version 1.54p 17 February 2025 (https://imagej.net/ij/) (NIH, Bethesda, MD, USA) to quantify ROS intensity [58], which was then expressed as fold change relative to the control, with the assay performed in biological triplicate [59].

2.5. Evaluation of the Effect of Glycyrrhizic Acid on Neuronal Apoptosis in a Cellular Model of Alzheimer’s Disease Using HOECHST-PI Staining

Neuronal apoptosis plays a crucial role in the pathology of Alzheimer’s disease [60]. HOECHST-PI staining was carried out to evaluate the apoptotic effects of Aβ₄₂ treatment in differentiated IMR-32 cells and to assess the potential of glycyrrhizic acid to mitigate this apoptosis. Specifically, 3 × 10⁴ cells per well were seeded in collagen-coated 6-well plates and differentiated with 10 μM retinoic acid for seven days. Following differentiation, cells were subjected to 48 h treatments, including Aβ₄₂ (0.5 μM), Aβ₄₂ (0.5 μM) co-treated with glycyrrhizic acid (100 μM), glycyrrhizic acid alone (100 μM), and an untreated control. After treatment, cells were washed twice with 1× PBS and stained with 20 μg/mL propidium iodide (PI) for dead-cell detection and 5 μg/mL Hoechst for nuclear counterstaining in serum-free medium for 20 min in the dark [61]. The assay was performed in biological triplicate. Cellular imaging was carried out using a ZOE™ Fluorescent Cell Imager (BioRad Laboratories, Hercules, CA, USA) with red (excitation 556 nm, emission 615 nm) and blue (excitation 355 nm, emission 433 nm) channels, and subsequent image analysis was performed using ImageJ software Version 1.54p 17 February 2025 (https://imagej.net/ij/) (NIH, Bethesda, MD, USA) [58]. Cell viability was then calculated relative to the untreated control and expressed as a fold change.

2.6. Analysis of the Effect of Glycyrrhizic Acid on Cell Cycle Dysregulation in a Cellular Model of Alzheimer’s Disease

This study investigated the potential of glycyrrhizic acid to target neuronal cell cycle reentry in Alzheimer’s disease, based on research suggesting its involvement in AD pathology [62]. To achieve this, IMR-32 cells were seeded at 3 × 10⁴ cells/well in collagen-coated 6-well plates, differentiated with 10 μM retinoic acid for seven days, and then treated for 48 h with Aβ₄₂ (0.5 μM), glycyrrhizic acid (100 μM), and Aβ₄₂ + glycyrrhizic acid (100 μM) co-treatment. Untreated cells served as the control. Following treatment, cells were washed twice with 1× PBS, trypsinized, and resuspended in FxCycle™ PI/RNase Staining Solution for 20 min in the dark. Red fluorescence was then measured using a Guava^® easyCyte Flow Cytometer (Merck Millipore, Burlington, MA, USA), and cell cycle data were analyzed using FCS Express (version 7).

2.7. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Analysis

To investigate the molecular mechanisms underlying the potential therapeutic effects of glycyrrhizic acid in an AD model, qRT-PCR analysis was performed. Specifically, IMR-32 cells were seeded at a density of 3 × 10⁴ cells per well in collagen-coated 6-well plates and differentiated for 7 days with 10 μM retinoic acid. Following differentiation, cells were subjected to various treatments for 48 h: Aβ₄₂ (0.5 μM), co-treatment of glycyrrhizic acid with Aβ₄₂ (0.5 μM), and glycyrrhizic acid alone (100 μM). Untreated cells served as the control. Subsequently, cells were washed with 1× PBS, harvested with TRI reagent, and total RNA was isolated following the protocol provided by the manufacturer. RNA concentration was then determined using a Colibri microvolume spectrometer (Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany). To perform gene expression analysis, reverse transcription was carried out with the PrimeScript 1st Strand cDNA Synthesis kit (Takara Bio Inc., Kusatsu, Japan) using 1 µg of RNA to generate cDNA. Finally, qRT-PCR was carried out in CFX96 Touch Real-Time PCR Detection System to quantify mRNA levels of key neuronal proteins implicated in AD, including AChE, MAO-B, BACE1, MAP2, BDNF, and ChAT. Furthermore, the expression of synaptic proteins such as SNAP23, synaptophysin, NLGN1, NLGN3, and ENO2 (Supplementary Table S1) was assessed, with β-actin serving as the internal reference gene for normalization [63]. RNA expression was quantified by threshold cycle (CT), and the ΔΔCT method was used to calculate relative expression levels. Results were expressed as fold change with respect to control cells, and all experiments were carried out in biological triplicate.

2.8. Western Blotting

To determine changes in protein expression related to mitochondrial dysfunction, cell apoptosis, cell cycle re-entry, Alzheimer’s disease pathophysiology, and cellular signaling, the following proteins were analyzed by Western blotting: superoxide dismutase 1 (SOD1), pyruvate dehydrogenase E1 alpha 1 (PDHA1), voltage-dependent anion channel (VDAC), heat-shock protein 60 (HSP60), cytochrome c, B-cell lymphoma 2 (BCL-2), BCL2-antagonist/killer 1 (BAK1), Bcl-2-associated X protein (BAX), cleaved caspase 3 (cl-Cas-3), cleaved caspase 9 (cl-Cas-9), proliferating cell nuclear antigen (PCNA), beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), phosphorylated tau (p-tau, Thr181 and Ser262), synaptosomal-associated protein 25 (SNAP-25), phosphorylated cyclin-dependent kinase 5 (p-CDK5, Ser159), phosphorylated glycogen synthase kinase-3 (p-GSK-3β, Ser21), phosphorylated protein kinase B (p-Akt, Ser473), phosphorylated extracellular-signal regulated kinase-1/2 (p-ERK-1/2, T202/Y204), and β-Catenin.

For Western blot analysis, IMR-32 cells (3 × 10⁴ cells/well) were seeded onto collagen-coated 6-well plates and differentiated with 10 μM retinoic acid for 7 days. Differentiated cells were then treated for 48 h with 0.5 µM Aβ₄₂, 0.5 µM Aβ₄₂ + 100 µM glycyrrhizic acid, 100 µM glycyrrhizic acid alone, and untreated cells served as the control. Upon completion of the treatment, cells were thoroughly washed twice with 1× PBS before the addition of lysis buffer (2% SDS, 50 mM TEABC, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate) for harvesting. The lysate was sonicated on ice (Q-Sonic, Cole-Parmer, India; 3 cycles of 3 min at 20% amplitude) and centrifuged at 12,000× g for 20 min at 4 °C. The supernatant was used for Western blotting. The BCA assay method was used to estimate protein concentration, with BSA as the standard. For optimal separation, proteins were resolved using SDS-PAGE gels (Bio-Rad Laboratories, Hercules, CA, USA) of varying percentages (Supplementary Table S2). Following electrophoresis, proteins were transferred onto nitrocellulose membranes. The membrane was incubated in 5% skim milk in 1× PBST for one hour to block non-specific binding. Primary antibodies diluted in 3% BSA in 1× PBST were added to the membranes at a 1:1000 dilution and incubated overnight (4 °C). After washing with 1× PBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies diluted in 3% skimmed milk in 1× PBST for 2 h at room temperature. β-actin was used as the loading control. Immunoreactive bands were developed using an enhanced chemiluminescence reagent using Fusion© FX chemiluminescence imaging system (Vilber, Collégien, France). The ImageJ (NIH) tool was used for densitometric analysis to quantify band intensities, which were normalized with β-actin. Fold change in protein expression was calculated in relation to the untreated control [56].

2.9. Statistics and Reproducibility

The statistical analysis was performed using GraphPad Prism 8 software. Data generated from three independent experiments were presented as mean ± SEM. A one-way ANOVA followed by Bonferroni post hoc test was used for group comparison. A p-value ≤ 0.05 was taken as significant.

3. Results

3.1. In Vitro Cytotoxicity Assessment of Glycyrrhizic Acid

Glycyrrhizic acid (GA) was dissolved in DMSO. To assess its cytotoxicity, IMR-32 cells were treated with varying concentrations of glycyrrhizic acid, ranging from 10 μM to 1000 μM, for 48 h, followed by an MTT assay. The results demonstrated no significant toxicity at any tested glycyrrhizic acid concentration, indicating that glycyrrhizic acid is non-toxic to IMR-32 cells (Supplementary Figure S1). However, we observed significant cell growth (proliferation) in 50 μM (1.18-fold ± 0.04, p ≤ 0.05), 100 μM (1.19-fold ± 0.03, p ≤ 0.01), 250 μM (1.23-fold ± 0.05, p ≤ 0.01), 500 μM (1.27-fold ± 0.04, p ≤ 0.01), and 1000 μM (1.32-fold ± 0.005, p ≤ 0.001) when compared to control (IMR-32 cells treated with DMSO). Consequently, based on these results and also from a previously reported study [64], a concentration of 100 μM glycyrrhizic acid was selected for subsequent cell culture experiments. Aβ₄₂ (soluble oligomers) was used at 0.5 μM, dissolved in DMSO, based on previously established doses [65].

3.2. Glycyrrhizic Acid Restores Mitochondrial Dysfunction and ROS Generation in a Cellular Model of Alzheimer’s Disease

Our results quantifying ROS across all four conditions show that Aβ₄₂ treatment significantly increased ROS levels by 6.62-fold ± 0.28 relative to untreated cells (p ≤ 0.001), indicating a substantial induction of oxidative stress. However, when cells were co-treated with glycyrrhizic acid, ROS levels were effectively reduced to 1.09-fold ± 0.15 (p ≤ 0.001), as illustrated in Figure 1A,B. This suggests a protective role for glycyrrhizic acid against Aβ₄₂-induced damage. Further analysis indicated that Aβ₄₂ treatment increased ROS and disrupted several cellular processes. Specifically, it led to a significant decline in the expression of superoxide dismutase 1 (SOD1, 0.51-fold ± 0.008, p ≤ 0.001), an antioxidant enzyme, and the proteins associated with mitochondrial dysfunction showed a concurrent increase in expression, including pyruvate dehydrogenase-E1 alpha-1 (PDHA1, 1.60-fold ± 0.07, p ≤ 0.01), voltage-dependent anion channel (VDAC, 1.56-fold ± 0.06, p ≤ 0.01), heat shock protein 60 (Hsp60, 1.68-fold ± 0.13, p ≤ 0.01), and cytochrome c (1.80-fold ± 0.04, p ≤ 0.01). Notably, glycyrrhizic acid co-treatment reversed these detrimental effects, restoring SOD1 activity (1.08-fold ± 0.02, p ≤ 0.001) and normalizing the expression of PDHA1 (0.79-fold ± 0.03, p ≤ 0.001), VDAC (0.91-fold ± 0.01, p ≤ 0.001), Hsp60 (0.81-fold ± 0.03, p ≤ 0.01), and cytochrome c (1.02-fold ± 0.11, p ≤ 0.01). These results are detailed in Figure 1C–H, provide a comprehensive picture of glycyrrhizic acid’s ability to mitigate Aβ₄₂-induced cellular damage, primarily by reducing ROS production and restoring mitochondrial homeostasis.

Figure 1.

Glycyrrhizic acid protects differentiated IMR-32 cells from Aβ₄₂-induced oxidative stress and mitochondrial dysfunction. (A) DCFDA (green) were used to stain IMR-32 cells to detect ROS and HOECHST (blue) to identify nuclei, (B) graph depicting ROS production in IMR-32 cells treated with glycyrrhizic acid, (C) immunoblot analysis expressing the levels of SOD1, PDHA1, VDAC, HSP60, and Cytochrome C, with β-actin serving as a loading control, (D) densitometry graph of SOD1, (E) densitometry graph of PDHA 1, (F) densitometry graph of VDAC, (G) densitometry graph of HSP 60, (H) densitometry graph of Cytochrome C. (** p ≤ 0.01, *** p ≤ 0.001, n = 3). Labels: C: untreated cells, Aβ₄₂: Aβ₄₂ treatment, Aβ₄₂ + GA: Aβ₄₂ + Glycyrrhizic acid co-treatment, GA: Glycyrrhizic acid treatment, PDHA1: pyruvate dehydrogenase E1, HSP 60: heat shock proteins, VDAC: voltage-dependent anion channel, SOD1: superoxide dismutase type 1.

3.3. Glycyrrhizic Acid Prevents Aβ₄₂-Mediated Apoptosis in a Cellular Model of Alzheimer’s Disease

Given that accumulating data indicate neuronal cell loss in AD is significantly driven by the activation of the apoptotic pathway [66,67], with oxidative damage being a predominant factor [68,69], we investigated cell apoptosis using the HOECHST-PI staining assay (Figure 2A,B). Our study revealed that Aβ₄₂ treatment significantly elevated cell apoptosis to 8.90-fold ± 1.03 (p ≤ 0.001). This detrimental effect was effectively rescued by co-treatment with glycyrrhizic acid, which reduced cell death to 2.01-fold ± 0.31 (p ≤ 0.001). Further studies revealed that Aβ₄₂ treatment led to a decrease in the anti-apoptotic protein Bcl-2 (0.47-fold ± 0.04, p ≤ 0.001) and a concurrent increase in pro-apoptotic proteins Bak1 (1.86-fold ± 0.004, p ≤ 0.01), BAX (2.17-fold ± 0.01, p ≤ 0.001), cleaved caspase-3 (2.27-fold ± 0.19, p ≤ 0.01), and cleaved caspase-9 (1.68-fold ± 0.13, p ≤ 0.01). This imbalance, favoring apoptosis, likely contributes to neuronal loss in AD. Notably, co-treatment with Aβ₄₂ and glycyrrhizic acid effectively reversed this abnormal condition, restoring the expression levels of Bcl-2 (1.04-fold ± 0.02, p ≤ 0.001), Bak1 (1.12-fold ± 0.09, p ≤ 0.01), BAX (0.95-fold ± 0.01, p ≤ 0.001), cleaved caspase-3 (1.07-fold ± 0.03, p ≤ 0.01), and cleaved caspase-9 (0.98-fold ± 0.01, p ≤ 0.01) to normal levels, as shown in Figure 2C–H.

Figure 2.

Glycyrrhizic acid protects cells from Aβ₄₂-induced apoptosis in differentiated IMR-32 cells. (A) The viability of the cells was determined using a live-dead staining assay with propidium iodide (PI) to identify dead cells (red) and HOECHST as a counterstain (blue), (B) graph representing the percentage of cell death relative to the untreated control, demonstrating that Aβ₄₂ treatment increases cell death, while co-treatment with glycyrrhizic acid counteracts this effect, (C) immunoblot analysis expressing the levels of Bcl2, BAK1, BAX, cleaved caspase 3, and cleaved caspase 9 with β-actin as the loading control across the control, (D) densitometry graph of Bcl2, (E) densitometry graph of BAK1, (F) densitometry graph of BAX, (G) densitometry graph of cleaved caspase 3, and (H) densitometry graph of cleaved caspase 9. ** p ≤ 0.01, *** p ≤ 0.001, n = 3). Labels: C: untreated cells, Aβ₄₂: Aβ₄₂ treatment, Aβ₄₂ + GA: Aβ₄₂ + Glycyrrhizic acid co-treatment, GA: Glycyrrhizic acid treatment, Bcl 2: B-cell lymphoma-2, BAK1: BCL2 antagonist/killer 1, Bcl 2, BAX: Bcl-2-associated X protein, cl_cas3: cleaved caspase 3, cl_cas9: cleaved caspase 9.

3.4. Glycyrrhizic Acid Mitigates Aβ₄₂-Induced Cell Cycle Aberrations in a Cellular Model of Alzheimer’s Disease

Aberrant neuronal cell cycle reentry is implicated as one of the prime incidents in Alzheimer’s Disease (AD) [62]. To address this, we analyzed cell-cycle distribution in differentiated IMR-32 cells using flow cytometry. Undifferentiated cells showed a distinct distribution: 47.61% in G0/G1, 35.61% in S, and 16.77% in G2/M. In differentiated cells, the control group showed a predominant population in the G0/G1 phase (91.93%), followed by S (5.70%) and G2/M (2.69%). Treatment with glycyrrhizic acid also showed the same pattern as observed in differentiated control cells, resulting in a significant number of cells in the G0/G1 phase (91.47%), with notably lower percentages in the S phase and G2/M phase (5.72% and 2.79%, respectively). Compared to the control, treatment with amyloid-beta 42 (Aβ₄₂) led to a significant increase in cells in the S phase (26.49%) and G2/M phase (12.56%). However, co-treatment with Aβ₄₂ and glycyrrhizic acid significantly reversed this effect, restoring cells to the G0/G1 phase (90.70%), with a corresponding decrease in the S phase (6.5%) and G2/M phase (2.8%) (Figure 3A–F). Western blot investigation of the S-phase indicator PCNA established these findings, demonstrating a 1.60-fold ± 0.09 (p ≤ 0.05) increase in the expression with Aβ₄₂, which was reduced to 0.97-fold ± 0.09 (p ≤ 0.05) by glycyrrhizic acid co-treatment (Figure 3G,H).

Figure 3.

Treatment with glycyrrhizic acid restores the aberrant cell cycle reentry caused by the Aβ₄₂. (A) Undifferentiated IMR32 cells, (B) Differentiated and untreated cells control, (C) Aβ₄₂ (0.5 µM), (D) Aβ₄₂ (0.5 µM) with glycyrrhizic acid (100 μM) co-treatment, (E) Glycyrrhizic acid alone (100 μM), and (F) A graph illustrating the distribution of cells across different cell cycle phases, (G) Immunoblot analysis of PCNA, and (H) densitometry graph of PCNA. Differentiated and untreated cells served as controls. ** p ≤ 0.01, *** p ≤ 0.001, n = 3. Labels: C&Control: Differentiated untreated cells, Aβ₄₂&AB: Aβ₄₂ treatment, Aβ₄₂ + GA&AB + GA100: Aβ₄₂ + Glycyrrhizic acid co-treatment, GA&GA100: Glycyrrhizic acid treatment, PCNA: Proliferating Cell Nuclear Antigen.

3.5. Glycyrrhizic Acid Effectively Reverses the mRNA Expression Changes Caused by Aβ₄₂ in Differentiated IMR-32 Cells

The results of qRT-PCR analysis demonstrated that Aβ₄₂ exposure significantly modulated mRNA expression levels. Specifically, we observed a marked increase in the mRNA levels of genes such as AChE (2.33-fold ± 0.03, p ≤ 0.001), BACE1 (2.41-fold ± 0.13, p ≤ 0.001), and MAO-B (2.00-fold ± 0.21, p ≤ 0.001), while decreasing the expression of ChAT (0.35-fold ± 0.05, p ≤ 0.001), MAP2 (0.52-fold ± 0.05, p ≤ 0.01), and BDNF (0.34-fold ± 0.01, p ≤ 0.001). Furthermore, Aβ₄₂ treatment led to a substantial reduction in the mRNA levels of synaptic proteins: NLGN1 (0.43-fold ± 0.09, p ≤ 0.001), NLGN3 (0.39-fold ± 0.05, p ≤ 0.001), SNAP23 (0.42-fold ± 0.01, p ≤ 0.01), and synaptophysin (0.39-fold ± 0.02, p ≤ 0.001). In contrast, ENO2 mRNA levels were significantly elevated (2.00-fold ± 0.14, p ≤ 0.001). Co-treatment with glycyrrhizic acid effectively mitigated the Aβ₄₂-induced changes. Specifically, glycyrrhizic acid normalized the expression of AChE (0.76-fold ± 0.10, p ≤ 0.001), BACE1 (1.01-fold ± 0.05, p ≤ 0.001), and MAO-B (0.84-fold ± 0.11, p ≤ 0.001) to levels comparable to the control. Notably, glycyrrhizic acid also significantly increased the expression of ChAT (1.11-fold ± 0.09, p ≤ 0.001), MAP2 (1.02-fold ± 0.09, p ≤ 0.001), and BDNF (0.93-fold ± 0.10, p ≤ 0.001) (Figure 4). Similarly, glycyrrhizic acid reversed the Aβ₄₂-mediated reduction in synaptic protein expression, restoring NLGN1 (1.18-fold ± 0.10, p ≤ 0.01), NLGN3 (0.95-fold ± 0.04, p ≤ 0.001), SNAP23 (1.18-fold ± 0.12, p ≤ 0.001), and synaptophysin (0.93-fold ± 0.08, p ≤ 0.01) mRNA levels. Additionally, glycyrrhizic acid significantly decreased the elevated ENO2 mRNA expression (1.05-fold ± 0.04, p ≤ 0.001) (Figure 5).

Figure 4.

Glycyrrhizic acid restored the alterations in gene expression caused by Aβ₄₂. The expression levels of genes (A) AChE, (B) BACE1, (C) MAO-B, (D) ChAT, (E) MAP2, and (F) BDNF were assessed in Aβ₄₂-treated IMR-32 cells to evaluate the effect of Glycyrrhizic acid using qRT-PCR. ** p ≤ 0.01, *** p ≤ 0.001, n = 3. Labels: C: untreated cells, Aβ₄₂: Aβ₄₂ treatment, Aβ₄₂ + GA: Aβ₄₂ + Glycyrrhizic acid co-treatment, GA: Glycyrrhizic acid treatment, AChE: Acetylcholinesterase, BACE1: beta-site amyloid precursor protein cleaving enzyme 1/Beta-secretase 1, MAO-B: monoamine oxidase B, ChAT: choline acetyltransferase, MAP2: microtubule-associated protein 2, and BDNF: Brain-derived neurotrophic factor.

Figure 5.

Glycyrrhizic acid counteracts the Aβ₄₂-induced gene dysregulation in differentiated IMR-32 cells. The expression levels of genes (A) NLGN1, (B) NLGN3, (C) SNAP23, (D) SYP, and (E) ENO2 were assessed in Aβ₄₂-treated IMR-32 cells to evaluate the effect of Glycyrrhizic acid using qRT-PCR ** p ≤ 0.01, *** p ≤ 0.001, n = 3. Labels: C: untreated cells, Aβ₄₂: Aβ₄₂ treatment, Aβ₄₂ + GA: Aβ₄₂ + Glycyrrhizic acid co-treatment, GA: Glycyrrhizic acid treatment, NLGN1: Neuroligin 1, NLGN3: Neuroligin 3, SNAP23: Synaptosomal-associated protein of 23 kDa, SYP: synaptophysin, and ENO2: enolase 2/neuron-specific enolase (NSE).

3.6. Glycyrrhizic Acid Regulates Key Proteins Associated with Alzheimer’s Disease and Synaptic Integrity

To investigate the impact of Aβ₄₂ on key pathological proteins associated with AD, we performed immunoblotting analysis. We examined β-site amyloid precursor protein cleaving enzyme 1 (BACE1), accountable for cleaving amyloid precursor protein (APP) and generating Aβ, which contributes to amyloid plaque formation, and hyperphosphorylated Tau (p-Tau at Thr181 and S262), a component of neurofibrillary tangles. Our results demonstrated that Aβ₄₂ exposure significantly increased protein levels of both BACE1 (1.54-fold ± 0.01, p ≤ 0.001), p-Tau at Thr181 (1.66-fold ± 0.01, p ≤ 0.001), and p-Tau at S262 (2.17-fold ± 0.08, p ≤ 0.001). Notably, co-treatment with glycyrrhizic acid effectively attenuated these increases, substantially reducing BACE1 (0.99-fold ± 0.02, p ≤ 0.001), p-Tau Thr181 (1.18-fold ± 0.004, p ≤ 0.001), and p-Tau at S262 (1.05-fold ± 0.03, p ≤ 0.001) levels, suggesting a preventive effect. Furthermore, we assessed synaptic function by analyzing SNAP 25 levels. Aβ₄₂ treatment significantly decreased SNAP 25 protein expression (0.46-fold ± 0.005, p ≤ 0.001), suggesting synaptic dysfunction. However, co-treatment with glycyrrhizic acid effectively restored SNAP-25 levels (1.17-fold ± 0.01; p ≤ 0.001), demonstrating a protective effect on synaptic integrity. These findings, shown in Figure 6, provide convincing proof for the potential beneficial role of glycyrrhizic acid in mitigating Aβ₄₂-induced neurotoxicity.

Figure 6.

Glycyrrhizic acid restores the aberrant protein activity and synaptic dysfunction caused by Aβ₄₂ in differentiated IMR-32 cells. (A) immunoblot analysis of BACE1, p-Tau (Thr181), p-Tau (S262), T-Tau, and SNAP25, (B) densitometry graph of BACE1 normalized with β-actin, (C) densitometry graph of p-Tau (Thr181) normalized with total Tau, (D) densitometry graph of p-Tau (S262) normalized with total Tau, and (E) densitometry graph of SNAP25 normalized with β-actin. *** p ≤ 0.001, n = 3. Labels: C: untreated cells, Aβ₄₂: Aβ₄₂ treatment, Aβ₄₂ + GA: Aβ₄₂ + Glycyrrhizic acid co-treatment, GA: Glycyrrhizic acid treatment, BACE1: beta-site amyloid precursor protein cleaving enzyme 1, p-Tau: phosphorylated Tau, T-Tau: Total Tau, and SNAP25: Synaptosomal-associated protein of 25kDa.

3.7. Glycyrrhizic Acid Attenuates Aβ₄₂-Driven Dysregulation of Alzheimer’s Disease-Associated Signaling Molecules

Exposure to Aβ₄₂ resulted in a marked elevation in phosphorylation of CDK5 (1.93-fold ± 0.06, p ≤ 0.001), GSK3 β (1.49-fold ± 0.05, p ≤ 0.01), AKT (1.86-fold ± 0.12, p ≤ 0.01), and ERK-1/2 (1.53-fold ± 0.01, p ≤ 0.001). Furthermore, Aβ₄₂ treatment resulted in a significant increase in β-catenin levels (2.25-fold ± 0.13; p ≤ 0.01). Glycyrrhizic acid co-administration successfully mitigated the Aβ₄₂-induced hyperphosphorylation of CDK5 (1.01-fold ± 0.003, p ≤ 0.001), GSK3β (1.10-fold ± 0.007, p ≤ 0.01), AKT (0.99-fold ± 0.04, p ≤ 0.01), and ERK1/2 (1.03-fold ± 0.04, p ≤ 0.01) and effectively restored β-catenin levels (1.10-fold ± 0.05, p ≤ 0.01) (Figure 7). Collectively, these results infer that Aβ₄₂ induces a signaling cascade involving protein phosphorylation and β-catenin, thereby contributing to Alzheimer’s disease pathology. The ability of glycyrrhizic acid to reverse these effects suggests a promising neuroprotective mechanism.

Figure 7.

Glycyrrhizic acid restores the aberrant hyperactivation of signaling molecules caused by Aβ₄₂ in differentiated IMR-32 cells. (A) immunoblot analysis of p-CDK5, p-GSK3β, T-GSK3β, p-Akt, T-Akt, p-ERK-1/2, T-ERK-1/2, and β-Catenin, (B) densitometry graph of p-CDK5 normalized with β-actin, (C) densitometry graph of p-GSK3β normalized with total-GSK3β, (D) densitometry graph of p-Akt normalized with total-Akt, (E) densitometry graph of p-ERK-1/2 normalized with total-ERK-1/2, and (F) densitometry graph of β-Catenin normalized with β-actin. *** p ≤ 0.001, n = 3. Labels: C: untreated cells, Aβ₄₂: Aβ₄₂ treatment, Aβ₄₂ + GA: Aβ₄₂ + Glycyrrhizic acid co-treatment, GA: Glycyrrhizic acid treatment, p-Cdk5: phosphorylated Cyclin-dependent kinase 5, p-GSK3β: phosphorylated Glycogen synthase kinase-3 beta, T-GSK3β: Total Glycogen synthase kinase-3 beta, p-Akt: phosphorylated Protein kinase B, T-Akt: Total Protein kinase B, p-ERK: phosphorylated extracellular signal-regulated kinase, T-ERK: Total extracellular signal-regulated kinase.

4. Discussion

Alzheimer’s Disease (AD) has a complex pathology and involves the interplay among amyloid-beta (Aβ) accumulation, Tau hyperphosphorylation, cell cycle reentry, oxidative stress, and synaptic dysfunction, which together operate as a closely interrelated system. While previous research has identified the neuroprotective potential of glycyrrhizic acid, our results provide a comprehensive mechanistic basis for its action, suggesting that glycyrrhizic acid regulates the Aβ production, hyperphosphorylation of Tau, ROS, cell cycle, synaptic, and apoptotic axes. Rather than viewing the observed changes in mRNA and protein levels as independent phenotypes, our data suggest a coordinated regulatory network in which glycyrrhizic acid-mediated inhibition of BACE1, p-Tau (Thr181 and S262), and ROS functions as an upstream regulator that prevents subsequent synaptic loss and aberrant cell cycle reentry. This integrated perspective is essential as multiple hypotheses are linked with Alzheimer’s disease pathology.

The primary neuroprotective mechanism of glycyrrhizic acid involves the regulation of Aβ production and the stabilization of the cholinergic environment. The Aβ overproduction, resulting from the proteolytic cleavage of APP by BACE1, is a well-known initiator of the Alzheimer’s disease cascade [70]. We observed that glycyrrhizic acid significantly downregulated BACE1 and MAO-B, the latter of which promotes Aβ production via γ-secretase regulation [71]. By reducing the Aβ burden at its source, glycyrrhizic acid prevents the formation of the toxic Aβ-AChE complex, which otherwise promotes amyloid fibril deposition and reduces acetylcholine (ACh) levels [72,73]. The restoration of ChAT levels by glycyrrhizic acid suggests a metabolic “re-tuning” of the cholinergic neuron, ensuring that ACh synthesis and vesicular transport are maintained [74,75]. This upstream stabilization is critical, as the loss of cholinergic integrity is directly linked to the cognitive abnormalities that characterize AD [76].

A key question raised by our findings is how glycyrrhizic acid translates reduced Aβ levels into cellular survival. Our data suggest that it is mediated by regulated mitochondrial function. The Aβ-induced increase in ROS disrupts the mitochondrial electron transport chain, leading to a decline in ATP and an increase in oxidative damage [77,78]. We propose that glycyrrhizic acid protects mitochondria through a dual mechanism involving antioxidant defense and maintenance of structural integrity. By restoring SOD1 levels, glycyrrhizic acid enhances the cell’s capacity to neutralize superoxide radicals, which are known to accelerate Aβ oligomerization and tau hyperphosphorylation [79]. Simultaneously, the observed restoration of VDAC1 and HSP60 levels suggests that glycyrrhizic acid prevents the “clogging” of mitochondrial pores, which is reported to be altered in Alzheimer’s disease [80,81]. In Alzheimer’s disease, elevated VDAC1 interacts with Aβ and p-Tau, blocking mitochondrial protein and metabolite transport, thereby promoting cytochrome c release and initiating the apoptotic cascade [82,83]. By stabilizing PDHA1-mediated glucose metabolism and preventing VDAC1-mediated disruption of the mitochondrial permeability transition pore, glycyrrhizic acid preserves neuronal viability and effectively mitigates oxidative stress before it triggers downstream nuclear and synaptic damage.

This decrease in oxidative stress is a prerequisite for restoring survival signaling pathways, specifically the PI3K/Akt/GSK3β nexus. Aβ-induced ROS triggers prolonged ERK1/2 activation, a known driver of neuronal death and tau phosphorylation [84,85]. Furthermore, studies reported that the ERK signaling pathway regulates the CDK5 activity [86]. Our data show that glycyrrhizic acid-mediated reduction of ROS correlates with the restoration of signaling homeostasis. Specifically, glycyrrhizic acid-mediated activation of Akt serves as a critical inhibitory signal for GSK3β [87,88]. In the AD brain, hyperactive GSK3β is the primary kinase responsible for tau hyperphosphorylation and NFT formation [89]. By promoting the inhibitory phosphorylation of GSK3β at Ser9, glycyrrhizic acid stabilizes β-catenin and prevents the dissociation of MAP2 and Tau from microtubules [90,91,92]. This represents a direct mechanistic link between glycyrrhizic acid treatment and the structural preservation of the neuronal cytoskeleton, as MAP2 and Tau are essential for maintaining the axonal and dendritic integrity required for signal transmission [93,94].

The association between oxidative stress and aberrant cell cycle re-entry provides further evidence of glycyrrhizic acid’s role in preventing terminal neuronal fate. Intern, it also contributes to the hyperphosphorylation of tau and Aβ deposition [95,96,97]. In mature neurons, Aβ-induced DNA damage often forces a lethal attempt to exit the quiescent G0 state [98]. The elevation of PCNA, a S-phase marker, in our Aβ-treated group indicates pathological cell cycle re-entry prior to neuronal death [97,99]. Our comprehensive interpretation suggests that ROS inhibition by glycyrrhizic acid prevents activation of the DNA damage checkpoint, thereby preventing initiation of the abnormal cell cycle re-entry. By maintaining neurons in a quiescent state, glycyrrhizic acid bypasses activation of Bak/Bax and the subsequent Caspase-9/Caspase-3 apoptotic cascade [100,101]. The reduction in cleaved Caspase-3 is not merely an isolated marker; it is the functional outcome of glycyrrhizic acid’s ability to stabilize the mitochondrial membrane and inhibit the upstream kinases, like CDK5, that would otherwise promote Tau-mediated apoptosis [102,103].

The functional outcome of these combined mechanistic actions, such as reduced Aβ levels, stabilized mitochondrial function, and normalized signaling pathways, is the preservation of synaptic integrity. Synaptic loss is a pathological correlate of cognitive decline in Alzheimer’s disease [104]. We observed that glycyrrhizic acid prevents the loss of presynaptic proteins such as SNAP-25 and Synaptophysin, and postsynaptic markers such as Nlgn1 and Nlgn3 [105,106,107,108]. This restoration is the functional consequence of BDNF upregulation, which we found to be restored by glycyrrhizic acid. BDNF promotes synaptic maturation and regulates NMDAR function, acting as a neurotrophic shield against Aβ toxicity [109,110,111]. Furthermore, by reducing ENO2/NSE levels, a distinct marker of neuronal damage, glycyrrhizic acid reduces neuronal stress that otherwise leads to synaptic degeneration [112,113].

In summary, our in vitro findings indicate mechanistic links among the effects of glycyrrhizic acid, suggesting an integrated model in which it works as a multifunctional neuroprotective agent. By simultaneously inhibiting the Aβ/BACE1 pathway, reducing mitochondrial ROS generation, and restoring PI3K/Akt/GSK3β signaling, it prevents the detrimental cascade of cell cycle re-entry and apoptosis while maintaining synaptic connectivity vital for cognitive function (Figure 8).

Figure 8.

A schematic representation summarizing the study’s key findings. Upward arrows indicate an increase, downward arrows indicate a decrease, and the ‘T’ symbol signifies prevention or inhibition.

However, it is significant to acknowledge that these results are based on the IMR-32 cell line, which, as a pediatric neuroblastoma model, has inherent limitations regarding its neoplastic nature, lack of full electrophysiological maturity, and inability to replicate the complex, age-dependent microenvironment of the adult human brain [114,115]. However, we utilize retinoic acid (RA)-differentiated IMR-32 cells. They show a shift from a proliferative neuroblastoma phenotype to a more neuron-like, post-mitotic phenotype, with distinct changes in morphology, gene expression, and protein expression compared with undifferentiated IMR-32 cells. Moreover, our results are mainly derived from the coordinated observation of various pathological markers of Alzheimer’s disease. A significant limitation always remains due to the lack of direct experimental validation concerning particular causal relationships. We identified a distinct correlation among glycyrrhizic acid treatment, reduced oxidative stress, and improved synaptic protein expression; however, this study did not use inhibitor-based assays or gene silencing to conclusively delineate the sequential molecular pathway underlying the neuroprotective effects of glycyrrhizic acid.

While our results offer a compelling basis for upcoming research. This study identifies glycyrrhizic acid as a promising candidate for multi-target intervention in Alzheimer’s disease by demonstrating a hierarchical restoration of cellular homeostasis. The clinical applicability of glycyrrhizic acid remains to be established. Consequently, its therapeutic efficacy and safety must be validated through comprehensive in vivo models and human clinical trials to fully elucidate its mechanisms of action and pave the way for its development as a novel intervention for Alzheimer’s disease.

Abbreviations

PI3K	phosphatidylinositol 3-kinase
PD	Parkinson’s disease
AD	Alzheimer’s disease
IL-1β	Interleukin-1 beta
TNF-α	Tumor Necrosis Factor-alpha
Aβ42	Amyloid Beta 42
qRT-PCR	quantitative reverse transcription polymerase chain reaction
NMDA	N-methyl-D-aspartate
DNA	deoxyribonucleic acid
APP	amyloid precursor protein
CSF	cerebrospinal fluid
FDA	food and drug administration
MTT	thiazolyl blue tetrazolium bromide
HOECHST	bisBenzimide H 33342
EDTA	ethylenediaminetetraacetic acid
RNase	ribonuclease
DMSO	dimethyl sulfoxide
BCA	bicinchoninic acid
ECL	enhanced chemiluminescence
PBS	phosphate-buffered saline
NIH	national institutes of health
RNA	ribonucleic acid
cDNA	complementary deoxyribonucleic acid
BSA	bovine serum albumin
SDS-PAGE	sodium dodecyl sulfate-polyacrylamide gel electrophoresis
HRP	horseradish peroxidase
mRNA	messenger ribonucleic acid
CDK5	cyclin-dependent kinase 5
Apaf-1	Apoptotic Protease Activating Factor 1
CO2	Carbon Dioxide
PI	Propidium Iodide
AChE	Acetylcholinesterase
SNAP23	Synaptosomal-Associated Protein 23
SNAP25	Synaptosomal-Associated Protein 25
ENO2	Enolase 2
TEABC	Triethylammonium bicarbonate
BCL-2	B-cell lymphoma 2
BAX	Bcl-2-associated X protein
BAK1	BCL2-antagonist/killer 1
p-GSK-3β	phosphorylated glycogen synthase kinase-3
p-Akt	phosphorylated protein kinase B
p-ERK-1/2	phosphorylated extracellular-signal regulated kinase-1/2

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cells15050436/s1, Figure S1: MTT assay of Glycyrrhizic acid (GA) at different concentrations (10–1000 µM); Figure S2: Original Uncropped Immunoblots; Table S1: List of the Primers and their sequences used in the study; Table S2: List of the antibodies used in the study.

Author Contributions

P.K.M. conceived the idea, planned the experiments, interpreted the data, and critically reviewed and edited the manuscript. T.S.K.P. conceived the idea, and reviewed and corrected the manuscript. S.A. conducted experiments, executed data analysis, drafted the manuscript, and prepared illustrations. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no relevant financial or non-financial interests.

Funding Statement

The work was funded by Yenepoya (Deemed to be University) as a Seed grant (YU/Seed grant/134–2022).