Effects of GlyNAC on Tissue Regeneration, Behavior, and Locomotor Activity in Dugesia tigrina

Abstract

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 like 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, negative behavior, and the planarians’ motility by recording them for 30 minutes with grid paper underneath their petri dishes.

Introduction GlyNAC

   There has been an interest in anti-aging in the modern day 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).  
   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).
  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.

Planarian

  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.

Materials and Method:

Preparation of GlyNAC treatment

  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.)  

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
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%	2.0	1.0	1.0	18	10

★ 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.

Preparation of Planarians

  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.

Preparation of Treatment Solutions

 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.

Treating Planarian 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.

Mobility Measurement 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.

Figure 1. Experimental setup for movement

Planarian Cutting

  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. 

Tissue regeneration

  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.

Data analysis

  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.

Result

Discussion/Conclusion 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. 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.

Future 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.

Acknowledgments

  Dr. D. Marmor​, Mrs. N. Jaipershad​, Dr. L. Wang, Ms. Zhu, Ms. Khemlani, Dr. J. Cohen​, Dr. S. Lin​, Mr. Z. Liang, Ms. DePietro 

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