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Epigallocatechin-3-gallate as a modulator of motor coordination, repetitive behavior, and sensory function in autism spectrum disorder
*Corresponding author: Irwanto Irwanto, Department of Child Health, Faculty of Medicine, Universitas Airlangga, Surabaya, East Java, Indonesia. irwanto@fk.unair.ac.id
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Received: ,
Accepted: ,
How to cite this article: Fithriyah I, Irwanto I, Setiawati Y, Chuanardi W. Epigallocatechin-3-gallate as a modulator of motor coordination, repetitive behavior, and sensory function in autism spectrum disorder. J Neurosci Rural Pract. doi: 10.25259/JNRP_359_2025
Abstract
Objectives:
This study aims to determine the effect of epigallocatechin-3-gallate (EGCG) on motor coordination, repetitive behavior, and sensory function in an animal model of autism.
Materials and Methods:
This study is a true experimental post-test control group design in pregnant mice injected with 600 mg/kg body weight (BW) valproic acid intraperitoneally. The research subjects were divided into 5 groups, namely a negative control group without valproic acid induction, a positive control group with valproic acid induction, and 3 treatment groups, which were given EGCG at doses of 2 mg/kg BW, 20 mg/kg BW, and 200 mg/kg BW.
Results:
There is a significant difference in the geotropism test on day 19 between groups (P < 0.05). Results from the negative control group were not different from the treatment group given EGCG at doses of 20 mg/kg BW and 200 mg/kg BW. Self-grooming test on day 21 showed improvement in repetitive self-grooming symptoms and was similar to the negative control in groups given EGCG doses of 20 mg/kg BW and 200 mg/kg BW. The results of the hot plate test on day 39 showed significant improvements occurred in the group given EGCG at a dose of 200 mg/kg BW.
Conclusion:
Administration of EGCG at doses of 20 mg/kg BW and 200 mg/kg BW during mouse pregnancy can improve motor coordination, repetitive behavior, and sensory function in an animal model of autism.
Keywords
Epigallocatechin-3-gallate
Mental health
Motor coordination
Repetitive behavior
Sensory function
INTRODUCTION
Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder characterized by impairments in behavior, social interaction, communication, special interests, and sensory processing.[1] Autism is a heterogeneous, multifactorial developmental disability, where unusual developmental patterns occur in infancy and toddlerhood.[2] The average age of first diagnosis is usually before 4 years old.[3]
The prevalence of ASD is expected to increase over time.[4] From a meta-analysis study, a total of 74 studies with 30,212,757 participants showed that the global prevalence of ASD is 0.6% (95% confidence interval: 0.4–1%).[5] The percentage of 8-year-old children with severe autism among those with autism is 26.7%.[6]
The pathogenesis of ASD is currently unclear. Immune dysregulation may be involved in the pathogenesis of ASD, including biochemical markers and inflammatory cytokines.[7] Maternal immune activation (MIA) induces microglia activation, oxidative stress, and mitochondrial dysfunction, which may damage the brain, leading to neuroinflammation and neurodevelopmental pathology in the offspring.[8] Increased inflammatory cytokines were also associated with poorer behavioral scores and increased stereotypy, which was significantly associated with Interleukin-6 (IL-6) concentrations.[9] Further research may help to develop more cost-effective ways of preventing ASD and thereby reduce the burden.[10]
Clinically effective treatment has not been established, but the use of natural antioxidants and anti-inflammatories may have significant benefits.[11] Epigallocatechin-3-gallate (EGCG) is one of the polyphenols.[12] It reduces oxidative stress through free radical scavenging, antioxidant enzyme activation, and mitochondrial function stabilization.[13] EGCG is also said to be effective and beneficial for neurodegenerative diseases, such as Alzheimer’s Disease.[14] It functions as an antioxidant and anti-inflammatory.[15] The molecule is emerging as a natural compound to combat chronic neuroinflammation and oxidative stress, offering a novel alternative for neuroprotective strategies in the treatment of neurological disorders.[16] Autism, as a neurodevelopmental disorder, has a basis in neuroinflammation and oxidative stress.[17] EGCG is predicted to be able to reduce behavioral disorders of autism. This study aims to determine the effect of EGCG on motor coordination, repetitive behavior, and sensory function in an animal model of autism.
MATERIALS AND METHODS
Materials
This study used valproic acid sodium salt P4543 produced by Sigma Aldrich (St. Louis, Missouri, United States) to induce autism in mice. Brain exposure to valproic acid induces granular cell death and cerebellar hypoplasia, which further causes changes to Purkinje cell structure and dendritic abnormalities. These mechanisms could disrupt motoric activity and coordination, cognition, sensory function, and social interaction capacity.[18,19] EGCG used has received a certificate for analysis from Xi’an Rongsheng Biotechnology Co., Ltd (Xi’an, Shaanxi, China). This study used syringes, mice cages, a gastric tube for mice, a hot plate testing device, standard food, a board to evaluate negative geotropism test, and a stopwatch. The mice used were healthy adult Mus musculus, weighing between 25-40g, from a licensed institution, aged 8–10 weeks.
Methods
This study used a randomized control trial group design. The mice were randomly divided into five groups, with each group containing six mice. Before the experiment, the mice were acclimated for 2 weeks in experimental cages. The cages were regulated by a light-dark cycle. The room temperature was set at 22 ± 2°C, and the humidity was set at 65 ± 5%. Food and water were freely regulated. After 2 weeks of acclimatization, male and female mice were mated at a ratio of one male to two female mice per cage. Vaginal plug examinations were performed every morning. Mice that had vaginal plugs were considered to have gone through the fertilization process and counted as day 1/embryo 1 (E1). On embryonic day 12.5 (E12.5), mice in the negative control group (C−) received an intraperitoneal saline injection, while the positive control group (C+) was injected with 600 mg/kg body weight (BW) of valproic acid intraperitoneally. There were three treatment groups, which were also given intraperitoneal injection of 600 mg/kg BW of valproic acid and later given intragastric EGCG until delivery at doses of 2 mg/kg BW (group P1), 20 mg/kg BW (group P2), and 200 mg/kg BW (group P3). Mice were allowed free access to food and waited until delivery.
Mice were allowed to care for their offspring until the time of evaluation of autistic behaviors. Autistic symptoms in animals manifest in repetitive symptoms, impaired motor coordination, and impaired sensory stimulation. Repetitive symptoms in this study are tested with the self-grooming test, impaired motor coordination is tested with the geotropism test, and impaired sensory stimulation is tested with the hot plate test.[20]
The geotropism test involved placing mice face down on a 45° incline in a temperature-controlled setting. The time it took for each mouse to rotate 180° so that its head faced upward along the incline was recorded, with a maximum limit of 30 s per trial. This test of negative geotropism reflects the development of motor skills, which rely on the effective coordination of sensory input and motor control.[20,21]
Self-grooming test was examined by counting stereotypic and repetitive behaviors in autism-model mice. Each mouse was placed individually into a standard mouse cage illuminated at 40 lx. Mice were habituated to the test cage for 5 min, and trained observers counted the number of self-grooming episodes within 10 min.[20]
To assess sensory function, mice were placed individually on a hotplate set to 55.0 ± 0.3°C, and the time until the first hind paw response was recorded. A response was defined as either a paw shake or a paw lick. A 30-s cutoff was applied for each trial. The nociceptive index, which measures the pain threshold to thermal stimuli, was assessed using this hotplate test.[20,21]
The geotropism test was conducted on day 19, the self-grooming test was done on day 21, and the hot plate test was done on day 39. On day 40, all the mice used for the research were sacrificed. This study hypothesizes that there will be a significant difference indicating improvement of autistic symptoms in the intervention groups (administered with EGCG), compared to the control group. The statistical package for the social sciences version 25 (International Business Machines Corporation (IBM Corp.) Armonk, New York, United States) was used for data analysis. For data with normal distribution, an analysis of variance test was conducted for multi-group comparison, while Fisher’s least significant difference test was conducted for pairwise comparison between group means. Kruskal–Wallis test was used in non-normal distribution to test the difference between multiple groups, while Mann–Whitney test was used to compare results between each group in the experiment.
RESULTS
Geotropism test results
Geotropism test results that indicate motor coordination, as shown in Table 1, show a significant difference between the negative control group (C− group) and the positive control group (C+ group) with P < 0.001. This indicates that the C+ group injected with valproic acid showed a longer time in the negative geotropism test compared to the C− group. Prolonged time to reorient on an inclined plane may represent autistic behaviors in an animal model. Group P1, which received valproic acid injections followed by oral intragastric EGCG at a dose of 2 mg/kg BW, showed a decrease in the time needed to return to an upright position compared to the C+ group. Group P2, which received valproic acid and EGCG at a dose of 20 mg/kg BW, showed improvement as indicated by a decrease in the time needed to return to an upright position compared to group P1, and was not significantly different to the C− group. Group P3, which received valproic acid injections and oral intragastric EGCG at a dose of 200 mg/kg BW, showed more decrease in the time needed to return from an incline position, and was not significantly different from the time needed C− group.
| Groups | n | Median in seconds (min-max) | P-value |
|---|---|---|---|
| C- | 6 | 3.55 (1.76–3.90)a | |
| C+ | 6 | 11.19 (10.28–12.87)b | |
| P1 | 6 | 7.75 (5.97–9.83)bc | <0.001 |
| P2 | 6 | 4.51 (3.00–5.89)ac | |
| P3 | 6 | 2.93 (1.00–3.91)ab |
Different superscripts indicate significant differences from Mann Whitney test, P-value significant at 0.05.
Self-grooming test results
This study showed significant differences in the self-grooming test that indicate repetitive behavior, as shown in Table 2. The C− group showed a significant difference (P < 0.001) compared to the C+ group. The result of the self-grooming test of P1 group was not significantly different from C+ group (P = 0.216), while the two other intervention groups administered with EGCG showed significant decreases (P = 0.009 for P2 and P < 0.001 for P3) compared to the C+ group. Groups P2 and P3, administered with EGCG at doses of 20 mg/kg BW and 200 mg/kg BW, respectively, showed the most significant improvements, with no significant difference from the C− group.
| Groups | n | Median (min-max) | P-value |
|---|---|---|---|
| C- | 6 | 1.0 (1–2)a | |
| C+ | 6 | 4.5 (3–8)b | |
| P1 | 6 | 3.0 (2–3)bc | <0.001 |
| P2 | 6 | 2.0 (1–3)bc | |
| P3 | 6 | 1.0 (1–2)a |
Different superscripts indicate significant differences from Mann–Whitney test, P-value significant at 0.05.
Hot plate test results
The hot plate test results that indicate sensory function, as shown in Table 3, show significant differences between groups (P < 0.001). The C− group showed a significant difference in the time required to respond to heat compared to the C+ group. The C+ group showed a longer response time to temperature stimuli. The intervention groups receiving EGCG therapy showed a significantly shorter response time to temperature stimuli compared to the C+ group. Group P3, which received EGCG intervention of 200 mg/kg BW intragastric orally, showed results close to the time recorded by C− group in response to temperature stimuli.
| Groups | n | Mean in seconds±standard deviation | P-value |
|---|---|---|---|
| C− | 6 | 11.78±2.079a | |
| C+ | 6 | 26.90±1.979d | |
| P1 | 6 | 22.87±2.230c | <0.001 |
| P2 | 6 | 14.63±2.658b | |
| P3 | 6 | 12.18±1.566ab |
Different superscripts indicate significant differences from the Least Significant Difference test, P-value significant at 0.05.
DISCUSSION
This study shows that EGCG administration to an animal model of autism injected with valproic acid showed promising results. EGCG could decrease the time to reorient from an inclined plane in autistic mice. Administration to P2 and P3 groups resulted in the same reorientation response time as the negative control group, which is considered normal mice. Administration of EGCG at doses of 20 mg/kg BW and 200 mg/kg BW improved reflex development and motor coordination, which are markers of autism in animals. The geotropism test is an unlearned automatic response that directs movement against gravitational cues, which helps sensory and proprioceptive functions. This is one of the initial behavioral tests conducted to evaluate motoric development (reflexes) and vestibular function.[22] An abnormal geotropism developmental pattern may serve as a marker for detecting ASD in animals.[23]
In this study, the positive control group mice showed a high frequency of self-grooming, a repetitive behavioral symptom in the autism model of mice. Mice given oral intragastric EGCG intervention at doses of 20 mg/kg BW and 200 mg/kg BW had decreased frequency of self-grooming, meaning the repetitive symptoms in mice were reduced and were found equal to the negative control group. Administration of EGCG at a dose of 2 mg/kg BW provided some improvements, but was not optimal and did not match the negative control group. Self-grooming is a core repetitive symptom in the autism model of mice.[24] Excessive grooming in these mice is associated with altered gene expression in the striatum in Shank3-deficient mice.[25]
In this study, administration of EGCG at doses of 2 mg/kg, 20 mg/kg, and 200 mg/kg showed a reduction in the time required for pain response. The most optimal reduction occurred in group P3 mice given EGCG at a dose of 200 mg/kg. The hot plate test in this study demonstrated sensory function in response to painful stimuli, specifically thermal ones. This test was conducted to identify abnormal reactions in the autism model of mice.[26] In addition, differences in sensory reactivity are estimated to occur in up to 94% of adults with autism.[27]
Valproic acid induces activation in multiple brain regions, with evidence of persistent glial activation in the hippocampus and cerebellum. Several studies have suggested that changes in social behavior in adult mice are caused by cerebellar inflammation, as the cerebellum is thought to be involved in executive and cognitive functions.[28] Studies have shown the important involvement of IL-6 in triggering core symptoms associated with proinflammatory responses in the MIA model of autism.[29] Recent research has shown evidence of increased oxidative stress in autism.[30] Postmortem basal ganglia (BG) studies of individuals with ASD compared to neurotypical controls have shown significant increases in dopamine 2 receptors mRNA in medium spiny neurons in the caudate and putamen of individuals with ASD, implicating the indirect BG pathway. Disruption of this pathway leads to motor dysfunction, stereotypy, and other repetitive behaviors in individuals with ASD.[31]
EGCG is the most abundant polyphenol in green tea and functions as an antioxidant that can scavenge superoxide and hydroxyl radicals.[28] The molecule also possesses metal-chelating properties. Two structures that give this compound its metal-chelating properties include an ortho-3’,4’-dihydroxy group and a 4-keto, 3-hydroxyl or 4-keto and 5-hydroxyl group. Catechins prevent the formation of potentially damaging free radicals by chelating metal ions. EGCG can inactivate iron ions, thereby suppressing the superoxide-induced Fenton reaction, which is considered the most important pathway in the formation of reactive oxygen species.[32] Supplementation with EGCG has the potential to reduce oxidative stress found in the brains of individuals with autism and decrease neuroinflammation, as indicated by a reduction in pro-inflammatory cytokine markers. This process is believed to help alleviate symptoms of motor coordination disorders, repetitive behaviors, and preserve sensory function in autism.[33]
The doses of EGCG in this study that were effective were 20 mg/kg BW and 200 mg/kg BW. This is in accordance with research by Machin et al., which used EGCG doses of 10 mg/kg BW, 20 mg/kg BW, and 30 mg/kg BW, with the 20 mg/kg BW dose providing the most optimal effect.[34] In contrast to research by Kumaravel et al., which showed data on the use of EGCG at doses of 1, 2, and 5 mg/kg BW, the dose that has the most potential to influence neurotransmitter changes in animal models of autism with valproic acid was 2 mg/kg BW.[33] Our study has the limitation of not including the social interaction and communication aspects of autism as variables, which may be difficult to assess in animal research. Future research could evaluate ECGC supplementation in humans with ASD. Further research is needed to determine the most appropriate dosage for preventing autism.
CONCLUSION
Administration of EGCG at doses of 2 mg/kg BW, 20 mg/kg BW, and 200 mg/kg BW resulted in improvements in behavioral symptoms of autism in animal models, including improved motor coordination, repetitive symptoms, and sensory function. Significant improvements approaching normal conditions (negative control) were seen at doses of 20 mg/kg BW and 200 mg/kg BW. Further research is needed to examine the biomolecular effects of EGCG, determine the optimal dose for significant improvement of autism symptoms, and establish therapeutic protocols in humans.
Acknowledgment:
The author would like to thank the research group that has collaborated and made this research successful.
Ethical approval:
The research/study was approved by the Institutional Review Board at Animals Ethics Committee, Faculty of Veterinary Medicine, Universitas Airlangga, Animal Care and Use Committee, approval number 2. KEH.021.02.2024, dated 7th February, 2024.
Declaration of patient consent:
Patient’s consent not required as there are no patients in this study.
Conflicts of interest:
There are no conflicts of interest.
Use of artificial intelligence (AI)-assisted technology for manuscript preparation:
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript, and no images were manipulated using AI.
Financial support and sponsorship: Nil.
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