Revision as of 15:58, 11 February 2026

Abstract

This study investigated the combined efficacy of docosahexaenoic acid (DHA) and Panax ginseng extract for treating Alzheimer's disease (AD). Single-target therapies aimed at clearing amyloid-beta (Aβ) have been largely unsuccessful, shifting attention toward multi-target approaches; among the most promising are DHA and Panax ginseng. Although DHA shows strong efficacy in AD prevention, once neurodegeneration has taken hold, blood-brain barrier (BBB) damage impairs its transport into the brain. Ginseng may counteract this by strengthening the BBB through the Wnt/β-catenin (BAR-1 ortholog in Caenorhabditis elegans) pathway and preventing DHA oxidation via its antioxidant properties, thereby enhancing delivery where it exerts neuroprotective effects. Based on these complementary mechanisms, the present study evaluated whether combined treatment confers benefits beyond either treatment alone using CL2355 (transgenic AD model) and N2 (wild-type) Caenorhabditis elegans. Five experimental groups were compared: two untreated controls (wild-type; AD model) and three AD model groups receiving DHA (5 mM), ginsenoside (10 μg/mL), or both. Treatment effects were assessed through paralysis (mobility), chemotaxis (neurological function), and population growth (reproduction) assays. The combined treatment reduced paralysis by 40% relative to untreated AD worms and 10% beyond either treatment alone, while significantly improving chemotactic function (p<0.01). These results suggest combined treatment holds potential as a multi-target therapeutic strategy, possibly by enhancing DHA absorption by the neurological system and/or targeting AD pathology through compounding mechanisms, though this study cannot distinguish between the two. These promising findings warrant further research using higher-order AD models or assays measuring nervous system DHA absorption.

1.0 Introduction

Alzheimer’s disease (AD) is a neurodegenerative disease and the leading cause of dementia, accounting for 75% of cases (Qiu et al., 2009). As of 2014, AD affects more than 35.6 million people worldwide, a number estimated to quadruple by 2050 (Anstey et al., 2014; Zhou et al., 2019). AD impairs language recognition and cognitive function by damaging the neocortex and hippocampus, reducing life expectancy, and increasing physical impairment in the elderly (Sharma, 2019; Qiu et al., 2009). Although the etiology of AD remains incompletely understood, current research is primarily based on a theory attributing its cause to abnormal accumulation of amyloid-beta (Aβ) aggregates resulting in neuroinflammation and then neurodegeneration (Sadigh-Etaghad et al., 2015; Deng et al., 2020; Bredesen, 2016; Soni & Galluci, 2023).

Currently, FDA-approved AD treatments target cholinergic and glutamatergic systems (Kim et al., 2018). Cholinesterase inhibitors (CHEIs) reduce the breakdown of acetylcholine but are unable to halt disease progression or reverse damage (Sharma, 2019). NMDA receptor antagonists, which reduce glutamate excitotoxicity, the excessive activation of glutamate receptors leading to neuronal atrophy, represent another therapeutic class (Mark et al., 2001). However, dysregulation of NMDA receptors, which play a critical role in synaptic transmission and plasticity underlying learning and memory, is itself implicated in AD pathology (Liu et al., 2019; Paoletti, 2011; Kodis et al., 2018).

More broadly, drugs that solely target Aβ have repeatedly failed to pass Stage 3 trials. Over the years, hundreds of therapies have been proposed and developed to clear Aβ; Yet none, with the exception of Donanemab, has improved clinical outcomes to a significant extent. Researchers attribute Donanemab’s relative effectiveness to its unique ability to specifically target neurotoxic forms of Aβ, unlike treatments that target harmless or even protective Aβ species indiscriminately (Rashad et al., 2022). However, Donanemab is effective only in the early stages of AD, carries a risk of brain swelling, and costs approximately $32,000 per year, placing it out of reach for many patients (Sims et al., 2023). These persistent limitations have shifted attention toward multi-target therapeutics, drugs that target pathology through numerous mechanisms, to better address the multifactorial etiology of AD (Maramai et al., 2020). The present study evaluated the efficacy of the combined treatment of two established multitarget AD therapies, DHA and Panax Ginseng extract, to assess their potential for synergistic effects, such as increased bioavailability, and contribute to the search for more accessible therapies.

Omega-3 fatty acids (ω-3FAs)

DHA is the predominant omega-3 fatty acid (ω-3FA) in the brain (Dighriri et al., 2022). Dietary ω-3FA supplementation, particularly at moderate or high doses, shows promise for slowing AD progression and restoring executive and hippocampal function, with studies reporting up to ~64% reduction in AD risk (Arellanes et al., 2020; Wei et al., 2023; Power et al., 2022). These outcomes can be largely attributed to ω-3FAs multi-target free radical scavenging, anti-inflammatory, BBB-strengthening, antiapoptotic, and neurotrophic properties (Ajith, 2018; Patrick, 2019). Upon metabolism, ω-3FAs generate specialized pro-resolving mediators that facilitate BBB repair and enhance clearance of Aβ and fibrinogens (Valente et al., 2022; Francos-Quijorna et al., 2017). Furthermore, Mfsd2a, the primary DHA transporter at the BBB, plays an integral role in barrier and pericyte health but declines with age and cognitive decline. However, maintaining a high ω-3FA index, especially before significant neurodegeneration has occurred, offsets this progression (Yang et al., 2020; Patrick, 2019). Unfortunately, Epidemiological data indicate that U.S. ω-3FA intake is largely inadequate (~0.1–0.2 g DHA/EPA per day), leading many researchers to identify ω-3FA deficiency as a leading driver of AD prevalence (Kris-Etherton et al., 2000; Wei et al., 2023).

Beyond targeting hallmark AD pathology (Aβ plaques, neurofibrillary tangles), ω-3FAs may attenuate cognitive and motor decline through additional mechanisms. A systematic review of eleven studies (n=698) found that ω-3FA supplementation substantially increased serum brain-derived neurotrophic factor (BDNF) levels at moderate or high doses (Ziaei et al., 2024). BDNF is a neurotrophin critical for synaptic formation and maintenance, with established roles in learning, memory, emotion, sensory integration, motor function, executive function, and stress response—all of which are impaired with AD (Brown et al., 2017; Miranda et al., 2019). Additionally, AD patients exhibit reduced BDNF levels, an association that may both result from and exacerbate disease progression, though whether the relationship is causal remains an area of active investigation (Zhang et al., 2014; Weinstein et al., 2014).

ω-3FAs also possess potent anti-neuroinflammatory properties. In the aging brain, misfolded proteins, primarily Aβ aggregates and neurofibrillary tangles (NFTs), trigger innate immune responses in microglia, initiating a neurodegenerative cascade (Fig. 1). ApoE-4, a major genetic risk factor for AD, further amplifies this process by upregulating glial activation (relative to ApoE-2 or ApoE-3) through lipid modulation (Parhizkar & Holtzman, 2022). Although ω-3FAs have been extensively studied for their anti-inflammatory effects in cardiology and metabolic health (Wierenga & Pestka, 2021), recent evidence demonstrates that dietary ω-3FAs also traverse the BBB and reduce neuroinflammation directly. In a randomized controlled trial of 33 mild AD patients, six months of DHA-based ω-3FA supplementation (2.3 g) reduced phosphorylated tau and lowered neuroinflammatory biomarkers including interleukin-1 and interleukin-6 (Freund et al., 2014).

Figure 1: Pathomechanism sequelae of immune activation (Heneka et al., 2015)

Panax ginseng

Panax ginseng, a perennial plant of the Araliaceae family, has been used in East Asian medicine since approximately 1500 CE and is among the earliest herbal medicines applied to the treatment of dementia and neurological disorders (Kim, 2018; Lin et al., 2020;Wang et al., 2023). Ginsenosides, the primary bioactive compounds isolated from Panax ginseng, exhibit multi-target neuroprotective properties relevant to AD: anticholinesterase activity, neuroplasticity promotion, anti-Aβ and anti-tau effects, anti-inflammatory and antioxidant activity, anti-apoptotic effects, alleviation of insulin resistance, and BBB strengthening (Wang et al., 2023; Zhang et al., 2023). Multiple human trials support ginseng’s efficacy in AD prevention and treatment (Lee et al., 2023), and researchers have emphasized the need for further investigation into ginseng-based drug combinations (Wang et al., 2023).

Evidence from animal models corroborates these findings. In transgenic AD mice, fermented ginseng extract ameliorated memory dysfunction concurrent with reduced Aβ42 brain accumulation (Kim et al., 2013). Red, black, and white ginseng extracts inhibit acetylcholinesterase and reduce hippocampal Aβ oligomer-induced memory impairment (Lee et al., 2011). In the present study, ginseng was administered as Korean red ginseng, which contains the highest concentration of ginsenosides among ginseng preparations. Korean red ginseng treatment has demonstrated improved frontal lobe function via quantitative electroencephalography (Heo et al., 2016), and its cognitive benefits have been sustained for over two years in AD patients (Heo et al., 2011).

Blood-brain barrier (BBB) and nutrient transport

On a fundamental level, ApoE in the brain serves as a regulator of astrocytes and glial cells, helping to modulate inflammation and thus the brain’s ability to fight pathogens and repair. At the beginning of human history, ApoE-4 existed as the sole isoform. Alternate isoforms that are less inflammatory by comparison, ApoE-2 and ApoE-3, appeared in the presence of a more glucose-based diet facilitated by agriculture. Although ApoE-4 was highly evolutionarily advantageous before the advent of modern medicine; it now poses a leading risk factor for AD pathogenesis, raising AD risk by up to 15-fold in double allele cohorts (Belloy et al., 2019; Yamazaki et al., 2019).

Multiple mechanisms underpin ApoE-4’s contribution to AD risk. ApoE-4 binds to receptor LRP1, which is responsible for Aβ clearance, thereby elevating Aβ oligomer levels by up to 2.7-fold (Ba et al., 2016). This binding also triggers the release of MMP-9 and cyclophilin A which further degrades the BBB and renders the brain vulnerable to neurotoxic bloodstream proteins, including plasminogen, auto-antibodies, albumin, and fibrinogen, that trigger a cascade of neuroinflammation (Fig. 2).

The BBB’s role in ω-3FA transport is directly relevant to this study (Platt et al., 2017; Levine, 2016; Valente et al., 2022). Although ω-3FA supplementation shows strong preventive efficacy, contradictory results in post-onset cohorts impede broader clinical recommendations. In a randomized controlled trial of 174 patients with mild to moderate AD, moderate-dose ω-3FAs failed to improve cognitive outcomes relative to placebo over six months (Freund et al., 2006). While the short study duration likely played a role, existing BBB damage in the AD patients may also be a contributing factor. When the BBB becomes compromised, the brain’s ability to uptake of DHA drastically deteriorates because the amount of DHA transporters, Mfsd2a, sharply decreases. Although the brains of young adults readily uptake ω-3FAs regardless of ApoE status, older adults, especially those with ApoE-4 and/or AD, show significantly reduced uptake (Arellanes et al., 2020). A separate trial confirmed this pattern: DHA supplementation was significantly less effective at mitigating AD symptoms in ApoE-4 carriers compared to ApoE-3 carriers (Quinn et al., 2010).

Several approaches have been proposed to increase ω-3FA bioavailability in the brain, including co-administration with antioxidants and polyphenols. For instance, combining ω-3FAs with carotenoid and vitamin E improved cognitive outcomes in otherwise healthy older adults—an effect attributed to antioxidant-enhanced bioavailability (Power et al., 2022). However, ginseng may offer even greater synergistic potential. Ginsenoside Rg1 directly activates the Wnt/β-catenin pathway, the transcriptional regulator of Mfsd2a, thereby upregulating DHA transport across the BBB (Wang et al., 2020). Additionally, ginseng may halt broader BBB degradation through inhibition of MMP-9, an enzyme that downregulates Mfsd2a in ApoE-4 carriers, and by crossing the BBB to reduce the neuroinflammation at the center of the neurodegenerative cascade (Gong et al., 2022; Fig. 1). Preliminary studies in C. elegans further suggest that ginseng modulates lipid metabolism and extends healthspan by upregulating ω-3FA  (in the form of lipoic acid) metabolic signaling pathways; though whether this effect translates to DHA unclear (Shi et al., 2023; Yu et al., 2021). Together these findings suggest that ginseng may increase DHA absorption by the brain, thus bypassing the primary limitation of ω-3FA treatment for patients who have already developed AD. 

Figure 2. Schematic representation of the blood–brain barrier (Abbott et al., 2010)

Caenorhabditis elegans

Caenorhabditis elegans is a widely used model organism for AD research due to its cellular simplicity, genetic amenability, low cost, and substantial homology with humans—83% of its proteome has human homologous genes (Sofela et al., 2021; Lai et al., 2000). Its nervous system of 302 neurons enables mechanisms to be traced at the level of individual neurons, and its 2–3 week lifespan allows observation of neurodegeneration across an entire adult life (Alvarez et al., 2022; Alexander et al., 2014). Previous studies have utilized the organism’s transparent body for in vivo visualization of neuronal death and protein aggregation (Alvarez et al., 2022). Although previous research has tested ginseng and ω-3FAs separately in C. elegans AD models, the combination of both treatments has not been investigated.

Previous research

To date, no AD studies have combined ω-3FAs (DHA, ALA, or EPA) with ginseng. However, two studies in other contexts provide relevant precedent. A double-blind randomized controlled trial found that ω-3FAs combined with ginseng and green tea catechins significantly improved cognitive test performance and increased activation in relevant brain regions via fMRI (Carmichael et al., 2018). A later study in children with ADHD found that the combination significantly alleviated symptoms beyond the sole use of either treatment, possibly indicating a synergistic effect (Lee et al., 2020). However, these studies did not investigate the potential connection between ginseng and increased ω-3FA absorption in the brain.

Research question/hypothesis

The present study researched the efficacy of a combination of Docosahexaenoic acid (DHA) and Panax Ginseng extract for the treatment of Alzheimer's Disease (AD). Based on both the comprehensive mechanisms the combined treatment addresses and the potential for ginseng to enhance DHA absorption in the nervous system, this study hypothesizes that a combination of ginseng extract and DHA would confer benefits in alleviating Aβ toxicity in CL2355 (transgenic AD model) beyond those of either treatment alone. Operationally, this translates to a higher percentage of unparalyzed worms following temperature upshift, greater population growth, and a higher chemotaxis index (attraction to a chemical stimulus)

2.0 Materials and Methods

Creating M9 buffer

Using a top loading balance, 1.5 g of KH₂PO₄, 3 g of Na₂HPO₄, and 2.5 g of NaCl was dissolved in a 500 mL of distilled water in a graduated cylinder. The solution was then sealed with aluminum foil and autoclaved for 20 minutes. Next, 1 mL of 1 M MgSO₄ was added to the solution. The final solution was stored at room temperature and sterilized using an autoclave prior to each use.

Creating ginsenoside stock solution (10 mg/ml)

Five grams of ginseng extract was measured using a top loading balance. Next, 50 mL of distilled water was measured into a graduated cylinder, mixing it with the ginseng extract in the graduated cylinder until fully dissolved. The solution was then autoclaved at 120°C for 2 hours before being transferred to the test tube and stored in a dark and cool environment. If necessary, the solution was repeatedly mixed before each use to avoid contamination.

Creating Omega-3 stock solution (500 mM)

Utilizing a top-loading balance 8.2g of DHA omega-3 was measured in an Erlenmeyer flask using a top-loading balance. In a graduated cylinder, 50 mL of sterilized M9 solution was measured. Using 1.5 mL Tween 80, the measured omega-3 was dissolved. The water and fatty acid emulsion was transferred into a vial and vortexed until it was fully homogeneous. The stock solution was stored in a light blocking tinted vial and placed in a dark and cool environment until its usage. Before each use, a vortex was used to ensure a homogeneous mixture of the treatment.

Creating Nematode Growth Media (NGM) agar plates

The C. elegans were cultured in the Nematode Growth Media (NGM) agar plates with various concentrations of the tested treatments. The Petri dishes were labeled by group names per distinct treatment concentrations and strains according to Table 1. The NGM (provided by Carolina Biological) in solid form was melted into liquid form for usage. First, the bottle was uncapped about halfway to release steam and pressure. The NGM agar was then added into the microwave for 30 seconds in intervals of 15 seconds checking each time for according to if adequately melted (alternatively a water bath at approximately 100℃ was used when time permitted). Due to caution of the heat gloves were used when taking the agar out of the microwave, properly shaking it to ensure no solids remain (agar should be a dark-yellow liquid). Then, the agar was allowed to cool for a few minutes with caution of re-solidification if excessive cooling.

Half of the NGM agar was dedicated to ginseng groups by transferring them to a separate graduated cylinder. A 1000x dilution of ginsenoside solution (0.18 mL) was then performed using 180 mL of NGM agar as the solvent. Then the agar was poured into the Petri dish very quickly, covering the lid of the Petri dish and tapping the bottle to avoid possible contamination. The aforementioned procedure was repeated for all Petri dishes with a waiting time of 15-20 minutes until agar solidified in the dish prior to storage in the refrigerator for 24 hours before usage (keeping the Petri dish upside down to prevent condensation from falling on the agar).

Each plate was then spotted with E. coli OP50 grown for 24 hours in LB Media. Upon transferring, plates were dried for an additional 24 hours. The previously made omega-3 stock solution was diluted from 1mL to 5 mM with sterilized water to a final volume of 100 mL (100x dilution). Then, a dispersion of 0.1 mL of DHA omega-3 aliquot to the solidified and spotted NGM in Groups C, and E was done using a spreader. Upon administration of treatment in each Petri dish, the mediums were allowed to dry for another 24 hours.

Table 1: Experimental Setup

Toothpick technique C. elegans Transfer

A blunt dissecting probe, acting as a pick for C. elegans transfer, was sterilized on a flame. A bead of OP50 was collected from an empty place to make the point sticky. Gently touch the worm selected using the OP50 coated probe. When the worm disappeared, it was on the probe. The worms were placed on the destination place by gently rolling the probe's tip until it became free. The worm was zoomed in on using the microscope to ensure that it was not damaged after the transfer and only the selected worm or egg was transferred.

Creating bacteria agar

The table was cleaned before beginning and preparing the materials for the study. All safety precautions were followed as well. A weighing paper was folded to make it easier to handle. On a weighing boat on a scale, 17.5 g of premix LB Agar powder was weighed. This powder was transferred to an Erlenmeyer flask and distilled water was added until reaching a final volume of 500 mL. The bottle cap and the top were sealed with aluminum foil tightly. The solution was then autoclaved in the liquid setting for 20 minutes. Following the autoclaving, the agar was cooled to about 55℃. A thin layer of LB agar was poured into each Petri dish, ensuring the liquid was properly and evenly spread. After putting on the lid of the Petri dishes, each plate was left to cool down until solidification. After 24 hours of the solidification process, the dish was flipped to avoid the condensation falling onto the agar. After this, the plates were placed in plastic bags in the refrigerator at 4℃.

Bacterial aseptic technique

E. coli OP50 (Source: https://cgc.umn.edu/strain/OP50) was used to feed C. elegans

(Sourced: CL2355 strain: https://cgc.umn.edu/strain/CL2355 and N2 strain: https://cgc.umn.edu/strain/N2) and placed in all Petri dishes. A rubber tube was connected to the valve of the gas and the valve of a Bunsen burner. Once the gas ignited, a match was lit and placed over the gas. The inoculation loop was sterilized by passing the tip and the neck of the inoculation loop through the flame. After the inoculation loop cooled, it was used to scoop the agar from a bacterial colony. The bacteria were streaked across the surface of a new Petri dish, ensuring that there was no excessive pressure put on the agar to refrain from breaking it. After, the lid was placed back on to the Petri dish and flipped upside down in the incubator to ensure that the accumulated condensation does not fall on the agar. Following two days of the Petri dishes being incubated, parafilm was wrapped around the sides of the Petri dish and placed into the fridge to store for later use. Before and after each process, diluted bleach was used to sterilize all surfaces.

Spotting Petri dishes with E. coli OP50

A 0.5 cm x 0.5 cm square chunk of agar with E. coli OP50 was cut using forceps or other sterilized metal. Sterilized forceps were used to grab a piece of agar. The agar was placed into the middle of the premade NGM plates. Each chunk was gently placed into its respective Petri dish. Before and after cutting and transferring each chunk, disinfectant tools were used to prevent contamination.

Conducting the paralysis assay

After 48 hours of C. elegans culturing, the temperature was either adjusted to 27℃ (for Trial 1) or 30℃ (for Trial 2). To ensure that the plates reached the temperature at the same time, the plates were not stacked on each other. After 24 hours of temperature upshift, the scoring of paralysis was performed for every two hours until all nematodes were paralyzed. Paralyzed nematodes were transferred to a part of the plate that was not spotted to refrain from scoring them again. In C. elegans, the head of nematodes is last to become paralyzed while the tail is first, restricting its movement. The C. elegans were considered paralyzed when they were unable to move, excluding its head. Nematodes that rarely voluntarily move were poked using a worm picker. If C. elegans did not show full movement, they were considered paralyzed.

Testing for Population Growth

A 24 well plate’s first column was labeled “WT”, the second column “CL -G/-O”, the third column “CL -G/+O”, the fourth column “CL +G/-O”, and the fifth column “CL +G/+O”. Four wells were used for each group. First, 1.0 mL of ginsenoside NGM was poured into the columns of CL +G/-O and CL +G/+O. Then, 0.1 mL of DHA omega-3 aliquot was pipetted to the solidified and spotted NGM in the columns CL -G/+O and CL +G/+O. Then, C. elegans were chunked into each column containing NGM. Every day for one week, the amount of C. elegans was recorded under a compound microscope. For Trial 1 of the assay, 10 μL ginseng was spread on top of the NGM for the treatment groups that required it. For Trial 2, ginseng was diluted into the NGM following the previous procedure.

Conducting the Chemotaxis Index Assay

Seven days before conducting the chemotaxis, C. elegans from each group were transferred to similar treatment plates to allow the C. elegans to culture. These plates were regarded as the treatment plates. Two days before the chemotaxis, a marker was used to divide each of 5 NGM Petri dishes into 4 equal quadrants, and a circle was created from the origin with a 1 cm radius. Then, another circle was created from the origin with a 2 cm radius. 10 µL of E. coli, acting as an attractant, was pipetted onto quadrant 1 and quadrant 3 of the chemotaxis plate. These 5 Petri dishes with the absence of treatment were regarded as the chemotaxis plates.

A micropipette was used to pipette 2 mL M9 on the treatment plates. The treatment plate was gently angled to cover all surface area to ensure that all nematodes were cleansed. A pipette was used to pipette 1 mL of the previously made worm solution in a microcentrifuge tube. The solution was centrifuged for 6,600 rpm for 10 seconds. After the centrifugation, the remaining M9 was removed. One mL M9 was pipetted in the centrifuge tube and the tube was turned to ensure that all nematodes were cleansed. The process was repeated three times. After, the volume of the supernatant was reduced to 100 μl and centrifuged for another 10 seconds. A pipette was used to transfer 2 μl of nematodes onto the chemotaxis plates.

After the 60 minute cleansing period, the nematodes were used. A micropipette was used to transport 2 μL of the nematode solution from the centrifuged solution to the center of the chemotaxis plate, or the intersection of the perpendicular lines drawn. Immediately after, a recording device was set directed towards the chemotaxis plates in a compound microscope, and a 60 minute video was recorded. After 60 minutes, the chemotaxis plates with the nematodes were placed in an incubator with a controlled temperature of 4 °C. The number of nematodes that passed through the circle with a 1 cm radius and moved towards the attractant zone or control zone and the time it took was recorded in Microsoft Excel corresponding with their respective quadrants. The process was repeated, replacing the treatment solution with sole control treatment. This Petri dish was regarded as the control plate. The chemotaxis assay was performed in quadruplicate.

Safety

Proper lab safety, including wearing gloves, goggles, and tied hair during the experimentation process, was fulfilled especially when conducting bacterial works. The tabletop working area was cleansed with a diluted bleach solution before and after experimentation along with the sterilization of all instruments at the end of the trial period. All Petri plates were sealed with parafilm during experimentation and autoclaved at 120 ℃ and 100 kPa for 20 minutes as part of the disposal procedure. Tools in contact with cultures were also soaked in 10% bleach and sterilized with heat as a segment of the disposing process. Hands were washed after any bacterial work. Three days of safety training specifically for bacterial works and the presence of experienced supervisors were ensured whilst dealing with microorganisms and the flame or gas of a Bunsen burner required for sterilization.

Three days of safety training specifically for bacterial works and the presence of experienced supervisors were ensured while dealing with microorganisms and the flame or gas of a Bunsen burner required for sterilization of tools. Most substances utilized in the study possessed a biosafety level BSL-1 and deemed “not hazardous” under the criteria of the federal OSHA Hazard Communication Standard 29CFR 1910.1200, and Regulation (EC) No 1272/2008 (GHS). Nonetheless, substances specifically can be dangerous when ingested, inhaled, or especially in cases of in contact with eyes (skin contact and inhalation do not pose serious harm unless it is done excessively or if the researcher feels unwell after exposure) so precautionary safety such as protective gloves, clothing, and above all else eye protection was adhered to.

Data Analysis

Data was collected, stored, and analyzed using Microsoft Excel. Data was shown using scatter plots and bar graphs in which the x values were time in hours, time in days, and group names and the y values were not paralyzed C. elegans as a percentage, number of C. elegans, and chemotaxis index. The median paralysis time was conducted using a Kaplan–Meier survival analysis. In the paralysis assay, the number of non-paralyzed C. elegans on each plate was converted into a percentage. The standard mean of error was calculated and represented as error bars added using Microsoft Excel. To calculate significance, one-way ANOVA followed by Tukey HSD was done using https://astatsa.com/OneWay_Anova_with_TukeyHSD/. Significance was determined using p-values in which a p-value less than 0.05 was considered significant. Chemotaxis index was determined using the following formula:

CI = (number of worms in attractant zone - number of worms in control zone)/total number of scored worms

Results

Figure 3. Trial 1 Paralysis Assay of WT and CL2355 with respective treatments. Following 48 h of incubation at 25℃, the temperature was increased to 30℃ for 31 h. The number of unparalyzed nematodes was converted to percentages (n=10).

In the C. elegans strain CL2355, as the temperature rises, Aꞵ peptide production increases in nematode muscles, leading to paralysis. CL-G/-O (the negative control group) had the lowest percentage of unparalyzed worms 48 hours after the temperature upshift. CL -G/+O had the highest percentage of unparalyzed worms compared with the negative control group, at 70% 48 hours after the temperature upshift. Additionally, CL +G/-O and CL +G/+O showed similar effects, with the percentage of unparalyzed worms remaining identical over time. Throughout the assay, all treatment groups exhibited a higher percentage of unparalyzed nematodes, indicating alleviation of the effects of Aβ aggregation. The wild-type C. elegans (WT) does not produce Aꞵ peptides, so the nematodes did not exhibit paralysis following a temperature upshift.

Figure 4. Trial 1 Population Growth with respective treatments. Data collection lasted over 8 days. Error bar values represent mean ±SEM. Statistically significant comparisons (p<0.05) are represented with different letters.

Figure 4 shows population growth throughout the whole assay, with CL +G/-O and CL +G/+O showing higher populations compared to WT from Day 5. At Day 6, CL +G/+O (combined treatments) had a statistically significantly higher population than the CL -G/+O group. Similarly, at Day 7, CL +G/-O had statistically higher growth than WT along with Day 8, demonstrating a significantly higher population compared to CL -G/+O.

Figure 5. Chemotactic response of WT and CL2355 with respective treatments. Chemotactic plot displaying chemotaxis index of WT and CL2355 (+/-) omega-3 and (+/-) ginseng. Error bars = ±SEM. p<0.05 is represented with different letters.

The results shown in Figure 5 indicate that WT (N2 wildtype) had the greatest chemotaxis index compared to all other groups. CL -G/-O, the negative control group, had the lowest chemotaxis index, which was statistically significant when compared to the CL -G/+O group, CL +G/-O, and CL +G/+O. Out of the groups expressing Aꞵ peptides, CL +G/+O had the greatest chemotaxis index, with statistical significance when compared to CL -G/-O and CL -G/+O. The CL +G/-O group had the next greatest chemotaxis index (0.222), followed by CL -G/+O (0.188).

Figure 6.  Trial 2 Paralysis Assay of WT and CL2355 with respective treatments. Following 48 h of incubation at 25℃, the temperature was increased to 30℃ for 31 h. The number of unparalyzed nematodes was converted to percentages (n=10).

The results of Figure 6 are consistent with that of Trial 1 in which all treatment groups had a higher percentage of unparalyzed C. elegans compared to CL -G/-O. WT had the lowest percentage of unparalyzed worms throughout the trial, except at Hour 0. Among all treatment groups with C. elegans exhibiting Aβ aggregation, CL +G/+O (combined treatment) had the highest percentage of unparalyzed C. elegans (80%) at 31 hours following the temperature upshift. The CL-G/+O and CL+G/-O groups both had 70% unparalyzed C. elegans 31 hours after a temperature upshift. Therefore, treatment with sole ginseng, sole omega-3, and a combination of ginseng and omega-3 all showed alleviation of Aβ aggregation effects, with the combination treatment exhibiting the greatest effect in Trial 2 of the paralysis assay.

Figure 7. Trial 2 Population Growth Assay. Data collection lasted over 9 days. Error bar values represent mean ±SEM. Statistically significant comparisons (p<0.05) are represented with different letters.

Figure 7 shows that WT had the highest population number throughout the assay. All groups exhibiting Aβ aggregation had significantly lower populations than WT on Days 8 and 9. CL-G/-O (the negative control group) decreased in population number as the assay progressed. Within the groups exhibiting Aβ aggregation, CL +G/+O (combined treatments) displayed the highest population throughout the whole trial, other than Day 5.

Discussion

The results of this study suggest that a multitarget treatment consisting of Panax ginseng extract and ω-3FAs confers a synergistic, or at minimum additive, effect in alleviating Aβ toxicity. This conclusion is supported by the outcomes in Fig. 6, where CL +G/+O (combined treatment) displayed 40% and 10% lower rates of paralysis relative to untreated CL2355 worms (CL -G/-O) and sole treatment groups (CL -G/+O and CL +G/-O), respectively. Lower rates of paralysis in AD worms indicate mitigation of Aβ oligomer production and/or attenuation of its deleterious effects (Tangrodchanapong et al., 2023). Although researchers proposed the amyloid cascade theory in 1992 as the guiding framework of AD research, many now deviate from or modify this original hypothesis, forming numerous schools of thought, each backed by tumultuous evidence regarding the causes of AD, such as blood-brain barrier breakdown, mitochondrial dysfunction, and the Aβ Oligomer Hypothesis (Bhatia et al., 2022; Sweeney et al., 2018; Cline et al., 2018). The latter directly relates to interpreting paralysis in the C. elegans model of AD. Put simply, the Aβ Oligomer Hypothesis identifies Aβ oligomers, instead of Aβ plaques, as the key neurotoxic species driving neuroinflammation, neuron damage, and eventually AD pathogenesis. In C. elegans models of AD, researchers typically observe paralysis without visible deposition of Aβ plaques, concluding Aβ oligomers as the primary cause of paralysis (Tangrodchanapong et al., 2023). Over the years, hundreds of trials have identified drugs that work to clear Aβ, but none, with the exception of Donanemab, has improved outcomes to a clinically significant extent. Researchers attribute the relative effectiveness of Donanemab to its unique ability to specifically target neurotoxic forms of Aβ, unlike treatments that target harmless or even protective Aβ species indiscriminately (Rashad et al., 2022). However, with the drug currently costing $32,000 per year and proving effective only in the early stages of AD, it has a long way to go before widespread clinical adoption (Sims et al., 2023). As a combination of Panax ginseng extract and ω-3FAs similarly targets neurotoxic forms of Aβ (i.e., Aβ oligomers), these treatments may serve as a viable alternative with benefits beyond Aβ clearance due to their multitarget nature.

However, even with promising preliminary results, the paralysis assays in the present study are not without their limitations. Firstly, Trial 1 of the paralysis assay (Fig. 3) displays results that contradict those of Trial 2 (Fig. 6). Specifically, Fig 3. shows that the combined treatment of ginseng and ω-3FAs (30% less paralysis relative to Cl2355 control) exerts weaker anti-paralytic effects than the sole treatment of omega-3 (40% less paralysis relative to Cl2355 control). These contradictory results are likely due to the method of ginseng delivery. In the first trial, ginseng was infused within NGM (nematode growth medium) agar instead of being spread across the surface (the delivery method used for omega-3). Initially, this approach aimed to control the treatment volume (i.e., amount of water) across groups and address concerns that volatile ginsenosides might denature upon contact with tween 80 (a surfactant and emulsifier used in the omega-3 treatment solution). Since this delivery system likely impeded proper ginseng absorption, it was replaced with a spreading method in Trial 2, as per prior research. With this change, Trial 2 displayed markedly higher efficacy in both the sole ginseng group and combined treatment (10% and 10% reduction in paralysis in Trial 1, compared to 30% and 40%, respectively). Moreover, this theorized improvement in absorption correlates with the increased population in the Trial 2 ginseng group (CL +G/-O; relative to groups within each trial) compared to Trial 1.

Even with this limitation addressed, additional concerns arise regarding the C. elegans strain. Although researchers used Cl2355 for both trials, which demonstrates temperature-induced pan-neuronal expression of Aβ, it ultimately proves nonoptimal for paralysis testing. Initially, researchers of this study chose Cl2355 due to its incomplete sterility (suitable for population assays) and its prior use in a chemotaxis assay (Tangrodchanapong et al., 2020). However, Aβ formation in Cl2355 only partially depends on temperature and thus cannot achieve complete paralysis solely through temperature upshift. In contrast, the CL4176 strain is strictly temperature-dependent and has undergone extensive use in previous studies for testing paralysis. Therefore, future research should use CL4176 to address the limitations of Cl2355.

For both paralysis trials, data significance (p<0.05) could not be determined due to each group having only one sample (n=10/group). Although this experimental design aligns with some previous research, demonstrating significance can allow the experiment to undergo more rigorous scrutiny, thereby improving the validity of findings. To address this concern, future research involving C. elegans paralysis should be performed in triplicate and subject to data analysis through ANOVA.

Similar to paralysis, results of the population assay (Fig. 4 & Fig. 7) also notably differed between trials. Likewise, Trial 1 (Fig. 4) was subject to similar oversights as Trial 1 of paralysis, such as the delivery method of ginseng treatment (Groups D & E). Further exacerbating these limitations, nematodes lacked a consistent food source throughout the experimental duration. Although C. elegans was transferred from agar spotted with OP50, this limited food source may have been depleted through the trial. Considering that both Omega-3 and ginseng are extensively researched to increase metabolism in both human and animal models, OP50 may have been depleted sooner in treatment groups, thereby inducing a starved state (Yu et al., 2021; Yarizadeh et al., 2021). Additionally, WT became contaminated at around day 4, which resulted in a final population lower than that of CL -G/-O (Cl2355 without treatment). As extensive research shows Cl2355 to possess highly impaired reproductive capabilities, this result ultimately invalidates the outcomes of Trial 1 (Tangrodchanapong et al., 2020). Trial 2 addressed these limitations by changing ginseng administration to spreading and implementing more rigorous sterilization. To that end, the results of Trial 2 (Fig. 6) suggest interventions to have marginal, insignificant effects on restoring reproductive capability and preventing embryonic lethality of Cl2355. Moreover, results regarding groups A & B (Fig. 6) were now consistent with prior research (Tangrodchanapong et al., 2020).

A chemotaxis index assay was performed to further assess the validity of the paralysis assay and, by extension, the neuroprotective properties of ginseng & ω-3FAs. The C. elegans strain CL2355 produces Aβ in the neurons through the synaptobrevin orthologous (snb-1) promoter, leading to flaws in 5-Hydroxytryptamine (5-HT) sensitivity, neuronal manners, and chemotaxis. Figure 5 shows that the nematodes treated with a combined application of ginseng and ω-3FAs had the greatest increase in chemotactic ability out of the groups containing nematodes expressing Aꞵ. The sole ginseng treatment showed similar effects when compared to CL -G/-O, the CL2355 group, with no external treatment. These findings indicate that the combined treatment of ginseng and ω-3FAs strengthens the proteostasis of transgenic C. elegans expressing Aꞵ, mitigating the neurodegenerative effects of excessive amyloid-ꞵ deposits.

Despite this, the chemotaxis assay possessed several limitations. Previous studies have suggested that C. elegans at different life stages can respond differently to certain stressors (Margie et al., 2013). Previous research recommends using C. elegans in the young adult stage when analyzing chemotactic ability. Although the nervous system is fully developed in C. elegans at the end of the L1 life stage, there are some intermediate changes within the chemosensory neurons in nematodes at the L4 life stage (Hart, 2006). Earlier studies have used bleach to synchronize C. elegans' life stages; however, bleach synchronization was implemented in this study due to constraints of laboratory equipment available to student researchers. However, variance in nematode life stages was minimized by having a relatively high sample size in each quadrant . Additionally, most studies performing chemotaxis assay apply an anesthetic (i.e. sodium azide) to paralyze C. elegans upon entering the quadrant. However, most, if not all, anesthetics, such as sodium azide, are highly volatile and are quickly absorbed by the skin within a few seconds of exposure, leading to potential poisoning. To mimic the results of anesthetics, a 60-minute video was taken of the chemotaxis plates where the first quadrant that each C. elegans entered was recorded as their final quadrant, and the recorded nematodes were transferred to refrain from rescoring.

C. elegans, as a model of AD, possesses several advantages and disadvantages. Firstly, C elegans lack many hallmark AD factors such as the APOE gene, complex mammalian neurophysiology affected by AD pathogenesis (namely, hippocampus, BBB, cortex, and secretion/utilization of molecules such as BDNF), tau phosphorylation (in models expressing Aβ) and microbiota profile similar to humans (Alvarez et al., 2022). For instance, a recent study from the Journal of Ginseng Research found that restored lactobacillus dominance (a probiotic essential to human health that is not present in C. elegans) plays a critical role in ginseng's ability to ameliorate cognitive defects in mice model of AD (Lee et al., 2022). As mentioned previously, one of the main mechanisms of ω-3FAs' unique ability to prevent AD is its role in strengthening the BBB through mitigating age-related loss of the DHA transporter, MFSD2a. In C. elegans, this mechanism is absent as nematodes lack a BBB.

Nevertheless, C. elegans still possesses valuable features, making it a valuable model of AD. For instance, researchers generally agree that the short lifespan and versatility (ex, temperature-induced Aβ secretion) of C. elegans make it a perfect candidate for rapid testing of novel treatments (Alvarez et al., 2022). When sequenced, 83% of the C. elegans proteome has human homologous genes (Lai et al., 2000). Although C. elegans lacks mammalian-specific genes such as BDNF, it still processes similar evolutionary conserved functions such as klotho production, whose expression is upregulated by ginseng intervention (lim et al., 2019; Château et al., 2010). Additionally, the present study is the first to evaluate the efficacy of ω-3FAs' in the C. elegans model of AD, thereby establishing viability and educating future research. Together, these strengths and weaknesses of C. elegans as a model for Alzheimer's are important considerations when determining the potential of a specific treatment and underscore the importance of further research in higher-order organisms in the case of promising results. To make use of the versatility of C. elegans, future research should explore the effect of combined treatment (Panax ginseng extract and ω-3FAs) on AD models expressing tau protein and gene knockout C. elegans to uncover underlying mechanisms. Additionally, western blot assessing Anti-Ab 6E10 & anti-oligomer A11 antibodies should be performed to confirm the ability of combined treatment to specifically target Aβ oligomers. Conclusion

The results of this study suggest promising preliminary evidence that combined treatments consisting of Panax ginseng extract and ω-3FAs may enhance individual effects and optimize benefits. Together, findings suggest that the synergistic or at least additive effects of the treatments can potentially lead to better managing of Aβ toxicity, damage, and the harmful effects of Aβ oligomers. The present study also was the first to confirmed the efficacy of ω-3FAs' in the mitigating Aβ toxicity in the C. elegans model of AD, however mechanisms behind its effects has yet to be uncovered. As millions of worldwide cases of AD are expected to grow; with expensive drugs only effective at the early stages of AD, exploring treatments' potential as viable therapeutic strategies are of utmost importance. Future research should focus on performing similar studies on additional C. elegans strains, and the use of these treatments on more complex models prior to widespread clinical adoption.

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