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== Abstract ==
 
== Abstract ==
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This study aims to investigate the effects of GlyNAC on planarian tissue regeneration. Planarian and organism regeneration are needed to maintain a healthy body. However, as one ages, their regeneration declines. There has been a focus on anti-aging in the older population due to the adverse effects that come with it, such as muscle fatigue, a decrease in tissue regeneration, etc. As a result, there was an increase in research to find an effective supplement to combat health issues and aging. GlyNAC, a combination of Glycine and N-acetylcysteine, has been shown to reverse and improve health by increasing glutathione levels. Glutathione is an antioxidant with multiple health benefits, such as protecting against oxidative stress, a main factor in causing cell death. To conduct this experiment, planarians are going to be used because they have a similar central nervous system, tissue regeneration, and glutathione production to humans. There will be 1 control group and 3 experimental groups. The control group will be fed regular ground beef, while the experimental group will get ground beef with GlyNAC mixed into it. For tissue regeneration, each group would be weighed and measured before and after the ground beef or GlyNAC-mixed ground beef. The behaviors that are going to be observed are the positive behavior, the negative behavior, and the planarians’ motility by recording them for 30 minutes with grid paper underneath their petri dishes.
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This study investigates the effects of GlyNAC—a combination of glycine and N-acetylcysteine (NAC)—on planarian tissue regeneration, locomotor behavior, and eyespot reformation. As organisms age, tissue regeneration declines, partly due to a deficiency of glutathione (GSH), a key antioxidant synthesized from glycine and cysteine. GlyNAC has been shown to restore GSH levels and reverse aging-associated deficits in humans; however, its effect on in vivo tissue regeneration in a whole-organism model has not been evaluated. Dugesia tigrina planarians were used as the model organism due to their pluripotent neoblast-driven regeneration, similar central nervous system organization to vertebrates, and endogenous GSH redox system. Forty planarians were assigned to four groups (n = 10 per group) receiving ground beef mixed with 0%, 1%, 5%, or 10% GlyNAC (g/g) for seven days prior to transverse amputation. Following amputation, eyespot regeneration and fragment body length were recorded every Monday, Wednesday, and Friday for 11 days. Locomotor activity was assessed by counting grid-line crossings during 30-minute video recordings before and after treatment. Statistical analysis used one-way analysis of variance (ANOVA) followed by Tukey's honest significant difference (HSD) post hoc test (significance threshold: α = 0.05). Results indicated that GlyNAC increased eyespot regeneration and fragment length relative to controls, supporting the hypothesis that GlyNAC promotes tissue regeneration. Limitations, including manual movement scoring, scheduling gaps, and planarian mortality at 10% GlyNAC in Trial 1 are discussed. Future research should employ automated tracking, longer treatment durations, and larger sample sizes.
  
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Introduction
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Keywords: Dugesia tigrina, GlyNAC, glutathione, tissue regeneration, oxidative stress, locomotor activity
  
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GlyNAC
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== 1. Introduction ==
  
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There has been an interest in anti-aging in modern-day society due to one’s fear of aging and the many symptoms that come with it, such as muscle fatigue, inflammation, a decrease in tissue regeneration, and oxidative damage(Kumar, et al. 2023). Oxidative stress occurs when there's an imbalance between the production of reactive oxygen species (ROS) and the body's ability to detoxify them (Pizzino et al,.2017). Excessive oxidative stress leads to aging and cell death, while low oxidative stress supports cell regeneration (Lee et al.,2018). 
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=== 1.1 GlyNAC and Aging ===
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<nowiki> </nowiki>  There is limited research on why older people face these symptoms and the lack of effective treatments to treat these symptoms. Despite this, many try to preserve their health and prevent these symptoms from happening through various means such as dieting, supplements, exercise, and antioxidants. Previous experiments have shown that both glycine and N-acetylcysteine, are antioxidants that can protect against oxidative stress (Ruiz-Ramírez, 2014; Kerksick, et al. 2005). These components are precursors to glutathione(GSH), an important antioxidant that protects against oxidative stress, increased energy, cell proliferation, etc. Therefore, GlyNAC is expected to boost glutathione production by combining Glycine and N-acetylcysteine (Kumar, et al. 2023).
+
There has been growing interest in anti-aging research due to the widespread symptoms associated with aging, including muscle fatigue, chronic inflammation, decreased tissue regeneration, and oxidative damage [1]. Oxidative stress occurs when there is an imbalance between the production of reactive oxygen species (ROS) and the body's ability to detoxify them [2]. Excessive oxidative stress accelerates aging and cell death, whereas low oxidative stress supports cell regeneration [3].
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<nowiki> </nowiki> As one age, there is a deficiency of GSH due to the lack of glycine and cysteine needed to synthesize the GSH. With this deficiency of GSH, fewer antioxidant defenses are protecting against oxidative stress that can damage cells that promote a healthy body. Therefore, GlyNAC demonstrates that it has the potential and ability to benefit those who are aging.
+
  
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Planarian
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Despite limited mechanistic understanding of why older individuals experience these symptoms, many attempt to preserve health through dietary strategies, supplements, and antioxidants. Previous research has demonstrated that both glycine and N-acetylcysteine (NAC) independently protect against oxidative stress [4,5]. Crucially, these compounds serve as direct precursors to glutathione (GSH), a master intracellular antioxidant that defends against oxidative damage, supports energy metabolism, and promotes cell proliferation [6]. GlyNAC—a combined supplement of glycine and NAC—has been shown to restore GSH levels and reverse multiple aging-associated health deficits in older adults [1].
  
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Planaria, specifically Dugesia tigrina, are flatworms known for their remarkable tissue regeneration abilities and nervous system. This makes them ideal for studying tissue regeneration and the nervous system, which helps provide insights into human tissue regeneration and the nervous system since both planaria and humans use stem cells for this process and have similar nervous systems. Planaria are famously used to research tissue regeneration, toxicology, and other pharmacology studies. Planarians can regenerate their tissue by using stem cells which are unspecialized cells that can develop into various types of cells, such as skin and nerve cells (Reddien, et al 2018). Additionally, planaria have glutathione in their redox system and glutathione is an essential component in balancing the redox system (Bijnens, et al.2021). With this planaria can create glutathione in balancing their redox system.
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With aging, GSH deficiency arises from insufficient availability of glycine and cysteine required for GSH synthesis. The resulting decline in antioxidant defenses allows oxidative stress to accumulate, impairing cells that sustain tissue homeostasis. GlyNAC supplementation thus represents a rational strategy for restoring antioxidant capacity and supporting regenerative function in aging organisms.
  
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Materials and Method:
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=== 1.2 Dugesia tigrina as a Model Organism ===
 +
Planarians, specifically Dugesia tigrina, are freshwater flatworms renowned for their exceptional tissue regeneration capacity. When bisected, each fragment activates a population of pluripotent adult stem cells called neoblasts, which proliferate and differentiate to reconstitute the entire organism within days [7]. This process relies on stem cell mechanisms that parallel vertebrate tissue repair, making planarians a broadly informative model for regenerative biology.
  
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Preparation of GlyNAC treatment
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Planarians also possess an endogenous GSH-based redox system that is essential for balancing oxidative status during regeneration [8]. This makes D. tigrina particularly well-suited to investigate whether GlyNAC-driven GSH augmentation can enhance regenerative outcomes. Additionally, the species exhibits quantifiable behavioral outputs—including locomotor activity and stereotyped aversive responses—that provide indirect readouts of nervous system integrity and overall physiological condition.
  
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Glycine and N-acetylcysteine) were purchased on Amazon (Nutricost). Glycine and N-acetylcysteine were mixed with a 1:1 ratio to make GlyNAC. The GlyNAC was blended into the ground beef with their desired mass concentrations of 0%, 1%, 5%, and 10% (Table 1.)
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The present study was designed to test whether dietary GlyNAC supplementation at graded concentrations (0–10% g/g in ground beef) would enhance tissue regeneration, eyespot reformation, and locomotor activity in D. tigrina. A positive result would support further investigation of GlyNAC in vertebrate regeneration models and strengthen the translational case for its use in aging populations.
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{| class="wikitable"
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 +
== 2. Materials and Methods ==
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 +
=== 2.1 Preparation of GlyNAC Treatment ===
 +
Glycine and N-acetylcysteine were purchased from a commercial supplier (Nutricost, via Amazon). The two compounds were combined in a 1:1 mass ratio to produce GlyNAC, which was then blended into ground beef at target concentrations of 0%, 1%, 5%, and 10% (g/g) for groups A–D, respectively (Table 1). In Trial 2, the Group D concentration was reduced from 10% to 5% following substantial planarian mortality observed at the 10% concentration in Trial 1.
 +
 
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==== Table 1. Composition of GlyNAC treatment diets by group. ====
 +
{|
 +
|Group
 +
|GlyNAC in Beef (% g/g)
 +
|GlyNAC (g)
 +
|Glycine (g)
 +
|NAC (g)
 +
|Ground Beef (g)
 +
|n
 
|-
 
|-
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! Group !! Percentage of GlyNAC in Beef (g/g) !! Amount of GlyNAC used (g) !! Amount of Glycine (g) !! Amount of N-acetylcysteine (g) !! Amount of Ground Beef !! Number of Planarians
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|A
 +
|0
 +
|0
 +
|0
 +
|0
 +
|20
 +
|10
 
|-
 
|-
−
| A || 0 || 0 || 0 || 0 || 20 || 10
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|B
 +
|1%
 +
|0.2
 +
|0.1
 +
|0.1
 +
|19.8
 +
|10
 
|-
 
|-
−
| B || 1% || 0.2 || 0.1 || 0.1|| 19.8 || 10
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|C
 +
|5%
 +
|1.0
 +
|0.5
 +
|0.5
 +
|19
 +
|10
 
|-
 
|-
−
| C || 5% || 1.0 || 0.5 || 0.5 || 19 || 10
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|D*
−
|-
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|10% (T1); 5% (T2)
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| D || 10% || 2.0 || 1.0 || 1.0 || 18 || 10
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|2.0 / 1.0
 +
|1.0 / 0.5
 +
|1.0 / 0.5
 +
|18 / 19
 +
|10
 
|}
 
|}
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★ The treatments were changed since in trial 1 majority of group D’s planarians had died. So, Group D’s treatment was changed to 5% of GlyNAC.
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<nowiki>*</nowiki>Group D concentration was reduced to 5% in Trial 2 following mortality observed at 10% in Trial 1.
  
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Preparation of Planarians
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=== 2.2 Organism Husbandry ===
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Forty Dugesia tigrina planarians were obtained from a commercial supplier (Carolina Biological Supply, catalog no. 132954) and randomly assigned to four groups of ten. Animals were housed individually in loosely sealed glass jars containing Poland Spring water and stored in a darkened cabinet at room temperature. Planarians were acclimated for seven days on plain ground beef (three feedings per week: Monday, Wednesday, Friday) prior to experimental treatment. Following each feeding, leftover beef was removed and fouled water was replaced with fresh Poland Spring water using a pipette, taking care not to aspirate any organisms. All handling was performed with appropriate personal protective equipment (PPE), and work surfaces were disinfected with a 10% bleach solution before each session.
  
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40 planarians have been ordered from the website, Carolina.com, and the catalog number is 132954. The planarians were kept in loosely sealed jars of Poland spring water. They were then separated into 4 groups of 10. Which were each used for different concentrations of GlyNAC. Afterward, the planarian jars were stored in the cabinet with a gap. The planarians were fed ground beef for a week.
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=== 2.3 Treatment Administration ===
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Following acclimation, each group received 20 g of the appropriate GlyNAC-supplemented (or control) ground beef preparation up to three times per week for seven days prior to amputation. Water was replaced and residual food was removed after each feeding as described in Section 2.2.
  
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Preparation of Treatment Solutions
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2.4 Locomotor Activity Assessment
  
−
All preparation procedures were conducted under sterile conditions. Work surfaces were disinfected using a 10% bleach solution, and hands were washed with soap and water prior to beginning. Standard personal protective equipment (PPE), including laboratory coats, gloves, and safety goggles, was worn throughout the process. Materials required for treatment preparation included beakers, graduated cylinders, pipettes, an analytical balance, weighing boats, and Poland Spring® water. The volume of Poland Spring® water specified in Table 1 was measured using a graduated cylinder. The appropriate volume of Trichosanthes root extract, also specified in Table 1, was then added to the water to create a stock solution, which was mixed thoroughly in a beaker. To prepare the working concentrations, a measured volume of the stock solution (e.g., 10 mL for Group B) was combined with Poland Spring® water (e.g., 90 mL for Group B) to reach a total volume of 100 mL. The solution was mixed by gently swirling the beaker. This procedure was repeated for each treatment group (A–D), based on the concentrations indicated in Table 1. Upon completion, all materials were cleaned, disinfected, and returned to their designated storage locations.
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Baseline locomotor activity was recorded before treatment initiation. For each session, 30 mL of Poland Spring water was added to four sterile Petri dishes placed over graph paper grids (Fig. 1). Planarians from each group were transferred to their respective dishes, allowed a brief acclimation period, and then video-recorded for 30 minutes. The number of grid lines crossed by each individual was counted from the recordings as a proxy for locomotion. This procedure was repeated after the treatment period to assess any treatment-related changes in motility.
  
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Treating Planarian
 
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−
Before the planarians are used, they must be nurtured. When working with them, safety equipment was worn and materials used in the experiment, as well as the workspace, were disinfected beforehand. The groups were treated with 20g of correspondent food according to Table 1. This was done a maximum of 3 times a week. After feeding, leftover beef was removed from the container using a pipette. Additionally, dirtied water was replaced with clean Poland spring water using a pipette, avoiding taking any planarians along with the dirtied water. The planarians were treated for 7 days before beginning the experiment.
 
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Mobility Measurement
 
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Before adding the treatment, the behaviors of planarians were measured. To do this, safety gear was worn, and all materials, gear, and workspace were disinfected. Next, 30 mL of Poland spring water was poured into 4 petri dishes. Then, the planarians were put into the petri dishes based on their respective groups. Afterwards, graph paper “(Figure 1) was placed below the petri dishes, and a camera was used to record the planarian groups for 30 minutes.
 
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Figure 1. Experimental setup for movement
 
 
[[File:Review_337222142936_7562_Grid.png]]
 
[[File:Review_337222142936_7562_Grid.png]]
  
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Planarian Cutting
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==== Fig. 1. Experimental setup for locomotor activity assessment. Petri dishes placed over graph paper grids to enable quantification of grid-line crossings as a locomotion proxy.Planarian Cutting ====
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+
−
Before cutting the planarians, the scalpel was disinfected, which was done by wetting a paper towel with ethanol. Next, group A planarians were put onto ice for one minute and all the planarians were cut individually with the disinfected scalpel before the pharynx. The scalpel was disinfected every 3 cuts and repeated the process for groups B, C,  and D. Then, a small jar was used and labeled as “AH” and another jar was labeled as “AT”. The tails of group A planarian were put into the “AT” jar and the heads of group A planarian were put into the “AH”. This process was repeated for the other groups but the letter of the labels was changed to represent the appropriate group.  After that, the planarians were measured for the average mass of the planaria groups, AH, BH, CH, DH, AT, BT, CT, and DT to record the measurements.
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−
 
+
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Tissue regeneration
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−
 
+
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Every Monday, Wednesday, and Friday, the planarians’ eyespot and length would be recorded and observed. The amputated planarian tail groups were observed under a microscope to see the time when the planarian's eye spot appeared; this process was repeated for other groups. Then, the planarians’ lengths were measured in millimeters and the lengths were recorded in an Excel sheet of all planarians in group A and then the process was repeated for other groups. After the trials, the planarians were given to the supervisor to be autoclaved.
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−
 
+
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Data analysis
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−
 
+
−
The planarian motility was collected by observing the number of line tiles crossed by each planarian under a grid paper and was recorded on an Excel sheet before and after the treatment for 2 weeks. The means and standard deviation were done by Excel standard functions. Then, created a scatter plot line graph. The data’s significance was calculated by using ANOVA (Analysis of Variance) followed by Tukey HSD (from https://www.socscistatistics.com/tests/anova/default2.aspx). P-values under 0.05 were considered significant. The graphs were a scatter plot of each concentration tile crossed per day.
+
  
−
The planarian regeneration was done by comparing how fast the eye spots grow between the tail groups. The length was measured between both the heads and tails. The means and standard deviation were done by Excel standard functions. The data’s significance was calculated by using ANOVA (Analysis of Variance) followed by Tukey HSD (from https://www.socscistatistics.com/tests/anova/default2.aspx) P-values under 0.05 were considered significant. The graphs made were bar graphs showing the difference in growth from day 0 to day 11 and the mean scoring of the eyespot regeneration plotted as a line graph.
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==== 2.5 Transverse Amputation ====
 +
Prior to amputation, planarians were cold-anesthetized on ice for one minute. Each animal was transversely bisected anterior to the pharynx using a scalpel sterilized with 70% ethanol; the scalpel was re-sterilized after every three cuts to prevent cross-contamination. Head and tail fragments from each group were placed into separately labeled containers (e.g., AH = Group A heads; AT = Group A tails) containing the appropriate treatment solution. The average mass of each fragment group (AH, BH, CH, DH, AT, BT, CT, DT) was recorded immediately following amputation.
  
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Result
+
=== 2.6 Regeneration Assays ===
 +
Fragment body length (mm) and eyespot regeneration were assessed every Monday, Wednesday, and Friday for 11 days following amputation. Eyespot appearance in tail fragments was observed under a dissecting microscope using a three-point ordinal scale: 0 = absent; 0.5 = partial; 1 = fully formed bilaterally symmetric eyespots. Fragment lengths were measured with a ruler and recorded in Microsoft Excel. After the completion of both trials, all planarians were submitted to a faculty supervisor for ethical euthanasia by autoclaving at 121°C.
  
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[[File:Review_337222142936_6553_FIG 2.png]]
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=== 2.7 Statistical Analysis ===
 +
<nowiki>Group means and standard deviations were calculated using standard Excel functions. Between-group differences were assessed by one-way ANOVA followed by Tukey's HSD post hoc test using an online statistics platform (https://www.socscistatistics.com/tests/anova/default2.aspx). A significance threshold of α = 0.05 was applied. Locomotor data were graphed as scatter plots of grid-line crossings per day; regeneration data were presented as bar graphs of growth from Day 0 to Day 11 and line graphs of mean eyespot scores over time.</nowiki>
  
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[[File:Review_337222142936_2328_FIG 3.png]]
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== Result ==
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[[File:Review_337222142936_6283_FIG 4.png]]
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[[File:Review_337222142936_6553_FIG 2.png|1100x1100px]]
  
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[[File:Review_337222142936_4460_FIG 6.png]]
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[[File:Review_337222142936_2328_FIG 3.png|1100x1100px]]
 +
[[File:Review_337222142936_6283_FIG 4.png|1154x1154px]]
  
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[[File:Review_337222142936_7516_FIG 8.png]]
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[[File:Review_337222142936_4460_FIG 6.png|1100x1100px]]
  
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[[File:Review_337222142936_5995_FIG 9.png]]
+
[[File:Review_337222142936_7516_FIG 8.png|1100x1100px]]
  
−
Discussion/Conclusion
+
[[File:Review_337222142936_5995_FIG 9.png|1100x1100px]]
  
−
The data reveals that the GlyNAC had increased the regeneration of eyespots and length. This supports the hypothesis that GlyNAC has a positive effect on the regeneration of a planarian. This suggests that GlyNAC may also have a positive effect on tissue regeneration in humans. This is because since there is increased regeneration in planarians then these effects will show up in the human regeneration of cells.  
+
== 4. Discussion ==
 +
The data indicate that GlyNAC supplementation increased eyespot regeneration rate and fragment body length relative to the control group, supporting the hypothesis that GlyNAC exerts a positive effect on tissue regeneration in D. tigrina. Given the mechanistic parallels between planarian neoblast-driven regeneration and vertebrate stem cell repair [7], these findings suggest GlyNAC may similarly promote tissue regeneration in humans, potentially through restoration of GSH levels and reduction of oxidative stress.
  
−
The limitation of this experiment is that it can cause errors within this experiment. During the data collection, the researchers were manually counting the number of lines crossed by the planarians; this can lead to human error and time-consuming. This issue was solved by having the video recording shortened. This allows the researchers to be more time-efficient. On the contrary, this did not solve the problem of human error. Another limitation is holidays, where data cannot be collected. This can make comparing data from different trials hard. Trials were also not long enough to see the full extent of GlyNAC’s impact. Due to the time constraints, this was not resolved. In trial 1 the planarians treated with 10% had died. The researchers had resolved the issue by changing the Group D treatment to be 5% of GlyNAC. This resolution had guided the researchers to seeing the benefits of GlyNAC.
+
Several methodological limitations may have affected the results. Locomotor activity was quantified by manual counting of grid-line crossings from video recordings, which is susceptible to inter-observer variability and fatigue-related errors. Shortening the recording duration from 30 minutes was adopted as a partial mitigation, but did not eliminate observer-related error. Scheduling constraints and holidays created gaps in the data collection timeline, complicating comparison across trials and reducing the ability to detect gradual trends. Trial duration was insufficient to capture the full temporal extent of GlyNAC's effects. In Trial 1, all planarians in Group D (10% GlyNAC) died, indicating that this concentration is toxic to D. tigrina; the concentration was reduced to 5% in Trial 2, which resolved the mortality issue and yielded informative data.
  
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Future Research
+
Despite these limitations, this study provides preliminary in vivo evidence that dietary GlyNAC supplementation can enhance planarian tissue regeneration. The finding that GlyNAC is most likely to benefit aging organisms—due to their pronounced GSH deficiency—has direct relevance to translational aging research.
  
−
This is because our substance (GlyNAC) is most effective in older patients because of their deficiency of glutathione. Future research should use a thermal camera or an artificial intelligence to track the movements of the planarian better because when counting it manually it can be more inaccurate. Additionally, future research should focus on a better way of treating the planarians. Such as soaking the planarian in a GlyNAC solution. Lastly, future researchers should extend the time giving planarians GlyNAC and increase the sample size.
+
== 5. Conclusions ==
 +
This study investigated whether dietary GlyNAC supplementation at graded concentrations (0–10% g/g) enhances tissue regeneration, eyespot reformation, and locomotor activity in Dugesia tigrina across two independent trials. GlyNAC-treated groups showed increased eyespot regeneration and fragment length relative to controls, consistent with the proposed mechanism of GSH restoration reducing oxidative barriers to neoblast activity. These results contribute to a growing evidence base for GlyNAC as a pro-regenerative intervention and support further investigation in vertebrate models.
  
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Acknowledgments
+
Future work should employ automated tracking systems (e.g., thermal cameras or machine learning-based motion analysis) to reduce observer error in locomotor assessments. Alternative delivery routes, such as immersion in GlyNAC solution rather than dietary administration, should be evaluated to improve bioavailability. Extended treatment durations and larger sample sizes would strengthen statistical power and allow the full temporal trajectory of GlyNAC's effects to be characterized.
  
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Dr. D. Marmor​, Mrs. N. Jaipershad​, Dr. L. Wang, Ms. Zhu, Ms. Khemlani, Dr. J. Cohen​, Dr. S. Lin​, Mr. Z. Liang, Ms. DePietro
+
Acknowledgements. 
  
−
References:
+
The authors thank Dr. D. Marmor, Mrs. N. Jaipershad, Dr. L. Wang, Ms. Zhu, Ms. Khemlani, Dr. J. Cohen, Dr. S. Lin, Mr. Z. Liang, and Ms. DePietro for their support and guidance throughout this research.
  
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Bijnens, K., Jaenen, V., Wouters, A., Leynen, N., Pirotte, N., Artois, T., &    Smeets, K. (2021). A spatiotemporal characterization of redox molecules in planarians, with a focus on the role of glutathione during regeneration. Biomolecules, 11(5), 714.
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== References. ==
 +
<nowiki>[1] P. Kumar, C. Liu, J. W. Hsu, S. Chacko, C. Minard, F. Jahoor, and R. V. Sekhar, "Glycine and N-acetylcysteine (GlyNAC) supplementation in older adults improves glutathione deficiency, oxidative stress, mitochondrial dysfunction, inflammation, insulin resistance, endothelial dysfunction, genotoxicity, muscle strength, and cognition: results of a pilot clinical trial," Clinical and Translational Medicine, vol. 11, no. 3, p. e372, 2021. https://doi.org/10.1002/ctm2.372</nowiki>
  
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Jeong, E. M., Yoon, J. H., Lim, J., Shin, J. W., Cho, A. Y., Heo, J., ... & Kim, I. G. (2018). Real-time monitoring of glutathione in living cells reveals that high glutathione levels are required to maintain stem cell function. Stem cell reports, 10(2), 600-614.
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[2] G. Pizzino, N. Irrera, M. Cucinotta, G. Pallio, F. Mannino, V. Arcoraci, and A. Bitto, "Oxidative stress: harms and benefits for human health," Oxidative Medicine and Cellular Longevity, vol. 2017, 2017.
  
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Pawelec, Graham, David Goldeck, and Evelyna Derhovanessian. "Inflammation, aging, and
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[3] J. Lee, Y. S. Cho, H. Jung, and I. Choi, "Pharmacological regulation of oxidative stress in stem cells," Oxidative Medicine and Cellular Longevity, vol. 2018, 2018.
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chronic disease." Current opinion in immunology 29 (2014): 23-28.
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Kawai, Nobuhiro, et al. "The sleep-promoting and hypothermic effects of glycine are mediated by NMDA receptors in the suprachiasmatic nucleus." Neuropsychopharmacology 40.6 (2015): 1405-1416.’
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[4] A. Ruiz-Ramírez, E. Ortiz-Balderas, G. Cardozo-Saldaña, E. Diaz-Diaz, and M. El-Hafidi, "Glycine restores glutathione and protects against oxidative stress in vascular tissue from sucrose-fed rats," Clinical Science, vol. 126, no. 1, pp. 19–29, 2014.
  
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Kumar, P., Liu, C., Hsu, J. W., Chacko, S., Minard, C., Jahoor, F., & Sekhar, R. V. (2021). Glycine and N‐acetylcysteine (GlyNAC) supplementation in older adults improves glutathione deficiency, oxidative stress, mitochondrial dysfunction, inflammation, insulin resistance, endothelial dysfunction, genotoxicity, muscle strength, and cognition: results of a pilot clinical trial. Clinical and translational medicine, 11(3), e372.
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[5] C. Kerksick and D. Willoughby, "The antioxidant role of glutathione and N-acetyl-cysteine supplements and exercise-induced oxidative stress," Journal of the International Society of Sports Nutrition, vol. 2, no. 2, pp. 38–44, 2005.
  
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Lee, J., Cho, Y. S., Jung, H., & Choi, I. (2018). Pharmacological regulation of oxidative stress in stem cells. Oxidative Medicine and Cellular Longevity, 2018.
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[6] A. Pompella, A. Visvikis, A. Paolicchi, V. De Tata, and A. F. Casini, "The changing faces of glutathione, a cellular protagonist," Biochemical Pharmacology, vol. 66, no. 8, pp. 1499–1503, 2003.
  
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Pizzino, G., Irrera, N., Cucinotta, M., Pallio, G., Mannino, F., Arcoraci, V., ... & Bitto, A. (2017). Oxidative stress: harms and benefits for human health. Oxidative medicine and cellular longevity, 2017.
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[7] P. W. Reddien, "The cellular and molecular basis for planarian regeneration," Cell, vol. 175, no. 2, pp. 327–345, 2018.
  
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Pompella, A., Visvikis, A., Paolicchi, A., De Tata, V., & Casini, A. F. (2003). The changing faces of glutathione, a cellular protagonist. Biochemical pharmacology, 66(8), 1499-1503.
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[8] K. Bijnens, V. Jaenen, A. Wouters, N. Leynen, N. Pirotte, T. Artois, and K. Smeets, "A spatiotemporal characterization of redox molecules in planarians, with a focus on the role of glutathione during regeneration," Biomolecules, vol. 11, no. 5, p. 714, 2021.
  
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Reddien, P. W. (2018). The cellular and molecular basis for planarian regeneration. Cell, 175(2), 327-345.
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[9] E. M. Jeong, J. H. Yoon, J. Lim, J. W. Shin, A. Y. Cho, J. Heo, and I. G. Kim, "Real-time monitoring of glutathione in living cells reveals that high glutathione levels are required to maintain stem cell function," Stem Cell Reports, vol. 10, no. 2, pp. 600–614, 2018.
  
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Robaczewska, J., Kędziora-Kornatowska, K., Kozakiewicz, M., Żary-Sikorska, E., Pawluk, H., Pawliszak, W., & Kędziora, J. (2016). Role of glutathione metabolism and glutathione-related antioxidant defense systems in hypertension. J Physiol Pharmacol, 67(3), 331-337.
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[10] J. Robaczewska, K. Kędziora-Kornatowska, M. Kozakiewicz, E. Żary-Sikorska, H. Pawluk, W. Pawliszak, and J. Kędziora, "Role of glutathione metabolism and glutathione-related antioxidant defense systems in hypertension," Journal of Physiology and Pharmacology, vol. 67, no. 3, pp. 331–337, 2016.
  
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Ruiz-Ramírez, A., Ortiz-Balderas, E., Cardozo-Saldaña, G., Diaz-Diaz, E., & El-Hafidi, M. (2014). Glycine restores glutathione and protects against oxidative stress in vascular tissue from sucrose-fed rats. Clinical Science, 126(1), 19-
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[11] N. Kawai, N. Sakai, M. Okuro, S. Karakawa, Y. Tsuneyoshi, N. Kawasaki, and S. Nishino, "The sleep-promoting and hypothermic effects of glycine are mediated by NMDA receptors in the suprachiasmatic nucleus," Neuropsychopharmacology, vol. 40, no. 6, pp. 1405–1416, 2015.

Revision as of 18:26, 30 May 2026

Abstract

This study investigates the effects of GlyNAC—a combination of glycine and N-acetylcysteine (NAC)—on planarian tissue regeneration, locomotor behavior, and eyespot reformation. As organisms age, tissue regeneration declines, partly due to a deficiency of glutathione (GSH), a key antioxidant synthesized from glycine and cysteine. GlyNAC has been shown to restore GSH levels and reverse aging-associated deficits in humans; however, its effect on in vivo tissue regeneration in a whole-organism model has not been evaluated. Dugesia tigrina planarians were used as the model organism due to their pluripotent neoblast-driven regeneration, similar central nervous system organization to vertebrates, and endogenous GSH redox system. Forty planarians were assigned to four groups (n = 10 per group) receiving ground beef mixed with 0%, 1%, 5%, or 10% GlyNAC (g/g) for seven days prior to transverse amputation. Following amputation, eyespot regeneration and fragment body length were recorded every Monday, Wednesday, and Friday for 11 days. Locomotor activity was assessed by counting grid-line crossings during 30-minute video recordings before and after treatment. Statistical analysis used one-way analysis of variance (ANOVA) followed by Tukey's honest significant difference (HSD) post hoc test (significance threshold: α = 0.05). Results indicated that GlyNAC increased eyespot regeneration and fragment length relative to controls, supporting the hypothesis that GlyNAC promotes tissue regeneration. Limitations, including manual movement scoring, scheduling gaps, and planarian mortality at 10% GlyNAC in Trial 1 are discussed. Future research should employ automated tracking, longer treatment durations, and larger sample sizes.

Keywords: Dugesia tigrina, GlyNAC, glutathione, tissue regeneration, oxidative stress, locomotor activity

1. Introduction

1.1 GlyNAC and Aging

There has been growing interest in anti-aging research due to the widespread symptoms associated with aging, including muscle fatigue, chronic inflammation, decreased tissue regeneration, and oxidative damage [1]. Oxidative stress occurs when there is an imbalance between the production of reactive oxygen species (ROS) and the body's ability to detoxify them [2]. Excessive oxidative stress accelerates aging and cell death, whereas low oxidative stress supports cell regeneration [3].

Despite limited mechanistic understanding of why older individuals experience these symptoms, many attempt to preserve health through dietary strategies, supplements, and antioxidants. Previous research has demonstrated that both glycine and N-acetylcysteine (NAC) independently protect against oxidative stress [4,5]. Crucially, these compounds serve as direct precursors to glutathione (GSH), a master intracellular antioxidant that defends against oxidative damage, supports energy metabolism, and promotes cell proliferation [6]. GlyNAC—a combined supplement of glycine and NAC—has been shown to restore GSH levels and reverse multiple aging-associated health deficits in older adults [1].

With aging, GSH deficiency arises from insufficient availability of glycine and cysteine required for GSH synthesis. The resulting decline in antioxidant defenses allows oxidative stress to accumulate, impairing cells that sustain tissue homeostasis. GlyNAC supplementation thus represents a rational strategy for restoring antioxidant capacity and supporting regenerative function in aging organisms.

1.2 Dugesia tigrina as a Model Organism

Planarians, specifically Dugesia tigrina, are freshwater flatworms renowned for their exceptional tissue regeneration capacity. When bisected, each fragment activates a population of pluripotent adult stem cells called neoblasts, which proliferate and differentiate to reconstitute the entire organism within days [7]. This process relies on stem cell mechanisms that parallel vertebrate tissue repair, making planarians a broadly informative model for regenerative biology.

Planarians also possess an endogenous GSH-based redox system that is essential for balancing oxidative status during regeneration [8]. This makes D. tigrina particularly well-suited to investigate whether GlyNAC-driven GSH augmentation can enhance regenerative outcomes. Additionally, the species exhibits quantifiable behavioral outputs—including locomotor activity and stereotyped aversive responses—that provide indirect readouts of nervous system integrity and overall physiological condition.

The present study was designed to test whether dietary GlyNAC supplementation at graded concentrations (0–10% g/g in ground beef) would enhance tissue regeneration, eyespot reformation, and locomotor activity in D. tigrina. A positive result would support further investigation of GlyNAC in vertebrate regeneration models and strengthen the translational case for its use in aging populations.

2. Materials and Methods

2.1 Preparation of GlyNAC Treatment

Glycine and N-acetylcysteine were purchased from a commercial supplier (Nutricost, via Amazon). The two compounds were combined in a 1:1 mass ratio to produce GlyNAC, which was then blended into ground beef at target concentrations of 0%, 1%, 5%, and 10% (g/g) for groups A–D, respectively (Table 1). In Trial 2, the Group D concentration was reduced from 10% to 5% following substantial planarian mortality observed at the 10% concentration in Trial 1.

Table 1. Composition of GlyNAC treatment diets by group.

Group GlyNAC in Beef (% g/g) GlyNAC (g) Glycine (g) NAC (g) Ground Beef (g) n
A 0 0 0 0 20 10
B 1% 0.2 0.1 0.1 19.8 10
C 5% 1.0 0.5 0.5 19 10
D* 10% (T1); 5% (T2) 2.0 / 1.0 1.0 / 0.5 1.0 / 0.5 18 / 19 10

*Group D concentration was reduced to 5% in Trial 2 following mortality observed at 10% in Trial 1.

2.2 Organism Husbandry

Forty Dugesia tigrina planarians were obtained from a commercial supplier (Carolina Biological Supply, catalog no. 132954) and randomly assigned to four groups of ten. Animals were housed individually in loosely sealed glass jars containing Poland Spring water and stored in a darkened cabinet at room temperature. Planarians were acclimated for seven days on plain ground beef (three feedings per week: Monday, Wednesday, Friday) prior to experimental treatment. Following each feeding, leftover beef was removed and fouled water was replaced with fresh Poland Spring water using a pipette, taking care not to aspirate any organisms. All handling was performed with appropriate personal protective equipment (PPE), and work surfaces were disinfected with a 10% bleach solution before each session.

2.3 Treatment Administration

Following acclimation, each group received 20 g of the appropriate GlyNAC-supplemented (or control) ground beef preparation up to three times per week for seven days prior to amputation. Water was replaced and residual food was removed after each feeding as described in Section 2.2.

2.4 Locomotor Activity Assessment

Baseline locomotor activity was recorded before treatment initiation. For each session, 30 mL of Poland Spring water was added to four sterile Petri dishes placed over graph paper grids (Fig. 1). Planarians from each group were transferred to their respective dishes, allowed a brief acclimation period, and then video-recorded for 30 minutes. The number of grid lines crossed by each individual was counted from the recordings as a proxy for locomotion. This procedure was repeated after the treatment period to assess any treatment-related changes in motility.

Review 337222142936 7562 Grid.png

Fig. 1. Experimental setup for locomotor activity assessment. Petri dishes placed over graph paper grids to enable quantification of grid-line crossings as a locomotion proxy.Planarian Cutting

2.5 Transverse Amputation

Prior to amputation, planarians were cold-anesthetized on ice for one minute. Each animal was transversely bisected anterior to the pharynx using a scalpel sterilized with 70% ethanol; the scalpel was re-sterilized after every three cuts to prevent cross-contamination. Head and tail fragments from each group were placed into separately labeled containers (e.g., AH = Group A heads; AT = Group A tails) containing the appropriate treatment solution. The average mass of each fragment group (AH, BH, CH, DH, AT, BT, CT, DT) was recorded immediately following amputation.

2.6 Regeneration Assays

Fragment body length (mm) and eyespot regeneration were assessed every Monday, Wednesday, and Friday for 11 days following amputation. Eyespot appearance in tail fragments was observed under a dissecting microscope using a three-point ordinal scale: 0 = absent; 0.5 = partial; 1 = fully formed bilaterally symmetric eyespots. Fragment lengths were measured with a ruler and recorded in Microsoft Excel. After the completion of both trials, all planarians were submitted to a faculty supervisor for ethical euthanasia by autoclaving at 121°C.

2.7 Statistical Analysis

Group means and standard deviations were calculated using standard Excel functions. Between-group differences were assessed by one-way ANOVA followed by Tukey's HSD post hoc test using an online statistics platform (https://www.socscistatistics.com/tests/anova/default2.aspx). A significance threshold of α = 0.05 was applied. Locomotor data were graphed as scatter plots of grid-line crossings per day; regeneration data were presented as bar graphs of growth from Day 0 to Day 11 and line graphs of mean eyespot scores over time.

Result

Review 337222142936 6553 FIG 2.png

Review 337222142936 2328 FIG 3.png Review 337222142936 6283 FIG 4.png

Review 337222142936 4460 FIG 6.png

Review 337222142936 7516 FIG 8.png

Review 337222142936 5995 FIG 9.png

4. Discussion

The data indicate that GlyNAC supplementation increased eyespot regeneration rate and fragment body length relative to the control group, supporting the hypothesis that GlyNAC exerts a positive effect on tissue regeneration in D. tigrina. Given the mechanistic parallels between planarian neoblast-driven regeneration and vertebrate stem cell repair [7], these findings suggest GlyNAC may similarly promote tissue regeneration in humans, potentially through restoration of GSH levels and reduction of oxidative stress.

Several methodological limitations may have affected the results. Locomotor activity was quantified by manual counting of grid-line crossings from video recordings, which is susceptible to inter-observer variability and fatigue-related errors. Shortening the recording duration from 30 minutes was adopted as a partial mitigation, but did not eliminate observer-related error. Scheduling constraints and holidays created gaps in the data collection timeline, complicating comparison across trials and reducing the ability to detect gradual trends. Trial duration was insufficient to capture the full temporal extent of GlyNAC's effects. In Trial 1, all planarians in Group D (10% GlyNAC) died, indicating that this concentration is toxic to D. tigrina; the concentration was reduced to 5% in Trial 2, which resolved the mortality issue and yielded informative data.

Despite these limitations, this study provides preliminary in vivo evidence that dietary GlyNAC supplementation can enhance planarian tissue regeneration. The finding that GlyNAC is most likely to benefit aging organisms—due to their pronounced GSH deficiency—has direct relevance to translational aging research.

5. Conclusions

This study investigated whether dietary GlyNAC supplementation at graded concentrations (0–10% g/g) enhances tissue regeneration, eyespot reformation, and locomotor activity in Dugesia tigrina across two independent trials. GlyNAC-treated groups showed increased eyespot regeneration and fragment length relative to controls, consistent with the proposed mechanism of GSH restoration reducing oxidative barriers to neoblast activity. These results contribute to a growing evidence base for GlyNAC as a pro-regenerative intervention and support further investigation in vertebrate models.

Future work should employ automated tracking systems (e.g., thermal cameras or machine learning-based motion analysis) to reduce observer error in locomotor assessments. Alternative delivery routes, such as immersion in GlyNAC solution rather than dietary administration, should be evaluated to improve bioavailability. Extended treatment durations and larger sample sizes would strengthen statistical power and allow the full temporal trajectory of GlyNAC's effects to be characterized.

Acknowledgements. 

The authors thank Dr. D. Marmor, Mrs. N. Jaipershad, Dr. L. Wang, Ms. Zhu, Ms. Khemlani, Dr. J. Cohen, Dr. S. Lin, Mr. Z. Liang, and Ms. DePietro for their support and guidance throughout this research.

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