Effects of ergothioneine on oxidative DNA damage and immune response induced by circadian rhythm disturbance in mice

Abstract

Ergothioneine has antioxidant, anti-inflammatory, and cell-protective properties. Circadian rhythm disruption can lead to health issues, such as insomnia, mental illness, chronic diseases, and cancer. However, the impact of ergothioneine, as an antioxidant, on oxidative DNA damage and immune variations caused by circadian rhythm disruptions remains unclear. To investigate the effect of ergothioneine on oxidative DNA damage and immune responses caused by circadian rhythm disruption, 8-week-old mice were subjected to night time feeding and exercise restrictions for 14 days. Body weight, daytime running wheel activity, and anxiety-like behavior showed no significant differences between the night-restricted groups, regardless of ergothioneine administration. Serum interleukin-6 levels, 8-hydroxy-2'-deoxyguanosine levels in urine and nuclear DNA of the liver, testes, lungs, and pancreas were significantly reduced in the night-restricted group receiving ergothioneine compared with that of the group without ergothioneine, with no significant differences observed when compared to the control group. Ergothioneine can mitigate immune function changes and oxidative DNA damage induced by circadian rhythm disruption caused by abnormal dietary timing in mice. However, it did not alleviate obesity or mental state dysregulation. These findings have important implications for improving night-shift workers health and developing therapies for diseases associated with circadian rhythm disturbances.

Introduction

Ergothioneine (EGT) is a distinct low-molecular-weight compound derived from histidine. It occurs naturally in substantial quantities in fermented foods and mushrooms, and is known for its strong antioxidant and cell-protective properties.⁽¹⁾ Animals and humans are unable to synthesize EGT and can only obtain it through their diet.⁽²⁾ EGT is found in various tissues, where it is believed to protect against oxidative damage and cellular stress, and accumulates in these tissues after ingestion.^(3,4) EGT possesses potent biochemical and biological activities, including cytoprotective, antioxidant, anti-inflammatory, and anti-apoptotic effects.^(5–7) Consequently, it is regarded as an essential dietary nutrient for the prevention of age-related inflammatory and neurodegenerative diseases.^(5,8,9)

Circadian rhythms are 24-h cycles that regulate behavior and physiological processes, essential for homeostasis and mental and physical health. Circadian rhythm disruptions are linked to various disorders, including psychiatric, cardiometabolic, and immune disorders.⁽¹⁰⁾ Nutrition considerably influences circadian rhythms, and the quality, timing, and quantity of food intake profoundly impact the circadian system. Circadian rhythms, in turn, influence nutrient metabolism.^(11,12) This interaction plays a role in metabolic disorders and obesity, and in optimizing exercise performance. Shifting feeding times in mice from night to day disrupts circadian gene expression and rhythms.^(13,14) Oxidative stress plays an important role in the onset of metabolic disorders, neurological diseases, and cancer.⁽¹⁵⁾ Several studies have highlighted the intricate relationship between circadian rhythms and oxidative stress. Circadian rhythms can affect ROS production and detoxification, whereas oxidative stress has the potential to disrupt these rhythms at the molecular level.^(16,17) Circadian disruption can alter the daily immune rhythms, impairing the ability to combat pathogens or heal tissue injuries. It may also disturb the balance between the anti-inflammatory and pro-inflammatory mechanisms, leading to immunosuppression or a pro-inflammatory state that promotes the development of chronic inflammatory diseases.⁽¹⁸⁾ Interleukin-6 (IL-6) is a cytokine produced by immune and non-immune cells that plays a key role in immune responses and inflammation. Elevated IL-6 levels are linked to chronic diseases, such as cardiovascular diseases, diabetes, and cancer. Sleep loss and stress-related disorders are correlated with increased IL-6 levels.^(19,20) Understanding the links among circadian disruption, oxidative stress, and immune function is vital for the development of strategies to prevent or treat related diseases.

Recently, we used a mouse model to study the effects of circadian rhythm disruption on oxidative stress and immunity. The characteristics of mice subjected to a daytime-restricted diet and exercise (12-h access from 7:00 p.m. to 7:00 a.m., restricted from 7:00 a.m. to 7:00 p.m.) showed no significant differences compared to those of mice on an unrestricted diet and exercise regimen throughout the day. However, mice with daytime food intake and exercise exhibited increased body weight, elevated IL-6 levels, and higher levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage, which may contribute to health risks associated with circadian rhythm disorders.⁽²¹⁾ In this study, we used a mouse model of circadian rhythm dysregulation induced by time-restricted feeding and spontaneous exercise. We aimed to evaluate the potential of EGT to improve oxidative DNA damage and immune system disruption caused by circadian rhythm disruption in this model.

Materials and Methods

Animal experiments

Seven-week-old male BALB/c mice were purchased from SLC Japan, Inc. (Shizuoka, Japan). Starting at 8 weeks of age, the mice were provided daily with EGT solution (2 ‍mg/L, sourced from Cayman Chemical Co., Ann Arbor, MI) or tap water as their drinking water for 14 days. Twenty mice were divided into four groups (n = 5 per group). In the day-restricted groups [CC (without EGT) and CE (with 2 ‍mg/L EGT)], mice were supplied with a running wheel and a commercially available 8-OHdG-free diet (Dyet no. 110952; Dyets Inc., Bethlehem, PA) only during the night (12-h access from 7:00 p.m. to 7:00 a.m., with restriction from 7:00 a.m. to 7:00 p.m.). The CC group received tap water, whereas the CE group was administered a 2 ‍mg/L EGT solution. In the night-restricted groups [SC (without EGT) and SE (with 2 ‍mg/L EGT)], mice had access to the running wheel and 8-OHdG-free diet only during the day (12-h access from 7:00 a.m. to 7:00 p.m., with restriction from 7:00 p.m. to 7:00 a.m.). The SC group received tap water, and the SE group was administered 2 ‍mg/L EGT solution. The running wheel activity was determined using custom-made cages (Bio Research Center Co., Ltd., Nagoya, Japan) equipped with running wheels. The running wheel activity was continuously recorded every 4 ‍s for 14 days using LabVIEW 13.0 software (National Instruments, Tokyo, Japan). On days 0, 7, and 14 following the dietary and rotational restriction regimens, the mice were individually housed in metabolic cages for 24 ‍h to measure their daily body weight, food intake, water consumption, fecal and urine volumes, and urine samples. Urine samples were stored at −20°C for subsequent measurement of 8-OHdG levels. After 14 days of the experiment, the mice were euthanized between 1:00 p.m. and 3:00 p.m. Serum and selected organs were collected and stored at −80°C for further analysis.

Throughout the experiment, the mice were kept in a temperature-controlled room maintained at 25°C with a 12-h light/dark cycle. The drink (water or 2 ‍mg/L EGT solution) and diet were fed ad libitum. All animal experiments complied with the guidelines established in the Japanese Guide for the Care and Use of Laboratory Animals. This study was reviewed and approved by the Animal Care and Use Committee of University of Occupational and Environmental Health, Japan (AE 21-009).

Open field test

The open field test was conducted on days 0, 7, and 14 after the start of the experiment to assess anxiety-like behavior. To minimize the effects of circadian rhythms, the test was performed between 1:30 p.m. and 5:00 p.m. A square chamber measuring 40 × 40 × 32 ‍cm³ was used as the open field apparatus, and a central area of 20 × 20 ‍cm² was marked on the floor of the arena. To acclimatize the mice to the experimental environment, they were moved to the testing room 3 ‍h before the test. Each mouse was lightly placed in a corner of the chamber, and its behavior was video-recorded for 15 ‍min using the Ethovision^® XT 15.0 software (BrainScience idea. Co., Ltd., Osaka, Japan). The total distance moved, number of entries into the central area, and time spent in the center were observed and recorded. The chamber was cleaned with 70% ethanol after each test.

Analysis of serum IL-6

A mouse IL-6 enzyme-linked immunosorbent assay (ELISA) kit (Proteintech Group Inc., Rosemont, IL) was used to assess serum IL-6 levels, following the manufacturer’s instructions.

Analysis of urinary 8-OHdG

A previously described high-performance liquid chromatography system with electrochemical detection was used to analyze the urinary 8-OHdG levels.^(22,23) The 8-OHdG concentration was normalized to the urinary creatinine concentration.

Analysis of 8-OHdG in tissue DNA

A DNA Extraction WB Kit (FUJIFILM Wako Pure Chemical Co., Ltd., Osaka, Japan) was used to isolate nuclear DNA from 100 ‍mg of tissue. Nuclear DNA 8-OHdG levels in tissues were measured using a previously described method.⁽²⁴⁾ The results are expressed as the number of 8-OHdG molecules per 10⁶ deoxyguanosine molecules.

Data analysis and statistics

One-way analysis of variance (ANOVA) was used to evaluate individual differences using IBM SPSS ver. 29.0 (SPSS Inc., Chicago, IL). Data are expressed as the mean ± SD. Statistical significance was set at *p<0.05 and **p<0.01.

Results

Characteristics of the animals

The body weights of the mice in the SC group were significantly higher than those in the CC group on days 7 and 14 (p = 0.001 and p = 0.018, respectively; Table 1). Mice in the SE group also showed a significantly higher body weight than those in the CC group (p = 0.001 and p = 0.006, respectively), with no significant difference compared with the SC group. This suggests that EGT administration had no effect on the body weight of the mice. There were no significant differences in water consumption, dietary intake, urine output, or fecal output among the four groups. Liver and spleen weights in the SC group were notably lower than those in the CC group on day 14 (Table 2). However, liver weight in the SE group was not significantly different from that in the CC group. In contrast, spleen weights were significantly lower in the SC and SE groups than in the CC group but were similar between SC and SE groups. Testis weights in the CE group were significantly higher than those in the CC group.

Table 1.

Characteristics of the animals

Characteristics	Group	Administration period (day)
		0	7	14
Body weight (24 ‍h)	CC	1	0.99 ± 0.02	1.01 ± 0.05
	CE	1	0.99 ± 0.02	1.02 ± 0.03
	SC	1	1.05 ± 0.02**	1.07 ± 0.04*
	SE	1	1.05 ± 0.03**	1.09 ± 0.03**
Water consumption (24 ‍h)	CC	1	0.98 ± 0.18	1.05 ± 0.30
	CE	1	1.27 ± 0.13	1.31 ± 1.27
	SC	1	1.14 ± 0.24	1.01 ± 0.24
	SE	1	1.09 ± 0.32	1.13 ± 0.38
Diet intake (24 ‍h)	CC	1	1.18 ± 0.59	1.22 ± 0.70
	CE	1	1.45 ± 0.67	1.28 ± 0.95
	SC	1	1.36 ± 0.32	1.34 ± 0.43
	SE	1	0.98 ± 0.10	0.90 ± 0.23
Urine (24 ‍h)	CC	1	1.26 ± 0.71	1.41 ± 0.67
	CE	1	1.45 ± 0.74	1.56 ± 1.06
	SC	1	1.78 ± 1.08	1.40 ± 0.63
	SE	1	1.43 ± 0.68	1.81 ± 1.19
Feces (24 ‍h)	CC	1	0.86 ± 0.24	0.84 ± 0.31
	CE	1	0.98 ± 0.59	1.13 ± 0.69
	SC	1	0.95 ± 0.29	1.03 ± 0.28
	SE	1	0.81 ± 0.19	0.85 ± 0.19

CC, day-restricted diet and exercise group, drinking water; CE, day-restricted diet and exercise group, drinking 2 ‍mg/L ergothioneine solution; SC, night-restricted diet and exercise group, drinking water; SE, night-restricted diet and exercise group, drinking 2 ‍mg/L ergothioneine solution. Day 0 values were measured just before the initiation start of the experiments. Day 7 and 14 values are expressed as ratios to the day 0 values. Compared to the CC group: *p<0.05, **p<0.01.

Table 2.

Tissue weight of the animals on day 14 after dietary and exercise restriction

Group	Tissue
	Liver/BW (%)	Kidney/BW (%)	Spleen/BW (%)	Lung/BW (%)	Testes/BW (%)
CC	6.80 ± 0.78	1.74 ± 0.10	0.41 ± 0.04	0.65 ± 0.03	0.74 ± 0.06
CE	6.91 ± 0.44	1.65 ± 0.03	0.39 ± 0.07	0.65 ± 0.12	0.82 ± 0.04*
SC	5.85 ± 0.78**	1.77 ± 0.14	0.37 ± 0.03*	0.63 ± 0.02	0.73 ± 0.08
SE	6.91 ± 0.72	1.80 ± 0.04	0.35 ± 0.04**	0.62 ± 0.01	0.73 ± 0.05

CC, day-restricted diet and exercise group, administrated with tap water; CE, day-restricted diet and exercise group, administrated with 2 ‍mg/L ergothioneine solution; SC, night-restricted diet and exercise group, administrated with tap water; SE, night-restricted diet and exercise group, administrated with 2 ‍mg/L ergothioneine solution. Compared to the CC group: *p<0.05, **p<0.01.

Running wheel activity

As expected for nocturnal animals, the nighttime (12-h) running wheel activities of the CC and CE groups were significantly higher than that of the daytime (12-h) activities of the SC and SE groups (Fig. 1A). Notably, the CE group displayed significantly higher nighttime running wheel activity compared with that of the CC group throughout the experiment (Fig. 1B). In contrast, the SE group did not show a significant difference in daytime wheel running activity compared with the SC group during the experimental period (Fig. 1C). However, a more rapid decline in running wheel activity was observed in the SE group by day 14, with reductions of 55% and 79% in the SC and SE groups, respectively, compared with that at day 1.

Fig. 1.

Ergothioneine affected the running wheel activity. (A) Total running wheel activity of BALB/C mice on days 1, 7, and 14 under dietary and exercise restriction conditions for the following groups: day-restricted (CC), day-restricted with 2 ‍mg/L ergothioneine (CE), night-restricted (SC), and night-restricted with 2 ‍mg/L ergothioneine (SE). (B) Running wheel activity measured from 7:00 p.m. to 7:00 a.m. (C) Running wheel activity measured from 7:00 a.m. to 7:00 p.m. Values are expressed as mean ± SD (n = 5). Statistical significance is indicated by *p<0.05 and **p<0.01, determined using one-way analysis of variance (ANOVA).

Anxiety-like behavior

The open field test was used to evaluate spontaneous activity levels and anxiety-like behaviors. Although the SC group spent significantly more time in the central area of the chamber on day 7 than did the CC group, this increase was significantly reduced in the SE group (Fig. 2A). Specifically, the time spent in the central area increased by 46% in the SC group compared with that of the CC group. In contrast, the time spent in the central area decreased by 52% in the SE group compared with that in the SC group. By day 14, no significant differences were observed between the CC, CE, and SC groups. However, a significant reduction in the time spent in the central area was observed in the SE group compared with that in the SC group. There was no significant difference in the number of fecal pellets between the SC and SE groups throughout the experimental period (Fig. 2B). However, the number of fecal pellets in the SC and SE groups was significantly higher than that in the CC group. No significant difference was observed in the total distance moved among the four groups during the experimental period (Fig. 2C). These findings suggest a trend towards increased anxiety-like behavior in the SC and SE groups, although no differences in the time spent in the center area were observed compared with that in the CC group on day 14. This indicates that EGT intake did not significantly improve anxiety-like behavior induced by circadian rhythm disruptions.

Fig. 2.

Behavior in the open field test. (A) Time spent in the center of the chamber. (B) Number of fecal pellets. (C) Total distance moved. Values are expressed as the mean ± SD (n = 5). Statistical significance is indicated by *p<0.05 and **p<0.01 (ANOVA).

Serum IL-6 levels

IL-6 levels were significantly higher in the SC group than that in the CC, CE, and SE groups on day 14 (Fig. 3). IL-6 levels in the SC group were 1.52 times those in the CC group. This increase was significantly suppressed in the SE group after EGT administration (p<0.001). No significant differences were observed among the CC, CE, and SE groups.

Fig. 3.

Effect of ergothioneine on serum interleukin-6 (IL-6) levels on day 14 after dietary and exercise restriction in the four groups (CC, CE, SC, and SE). Values are expressed as the mean ± SD (n = 5). **p<0.01 (ANOVA).

8-OHdG levels

As a biomarker of oxidative DNA damage, 8-OHdG levels were measured in the urine and tissue DNA. Urinary 8-OHdG levels in the SC group significantly increased throughout the experiment (Fig. 4A). The levels in the SC group were 1.29 and 1.20 times those of the CC group on days 7 and 14, respectively. Urinary 8-OHdG levels were significantly lower in the SE group than those in the SC group (p<0.001 and p = 0.005, respectively), with no significant differences observed among the CC, CE, and SE groups.

Fig. 4.

Effect of ergothioneine on 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in the four groups (CC, CE, SC, and SE). A: Urinary 8-OHdG levels, normalized to urinary creatinine concentration. B: 8-OHdG levels in tissue DNA on day 14 after dietary and exercise restriction. Tissue DNA values were calculated as the number of 8-OHdG molecules per 106 deoxyguanosine (dG) molecules. Values are expressed as the mean ± SD (n = 5). **p<0.01 (ANOVA).

Nuclear DNA 8-OHdG levels in the liver, testes, lungs, and pancreas of the SC group significantly increased 14 days after dietary and exercise restriction during the dark period (Fig. 4B). These levels in the SC group were 1.37 to 1.51 times those in the CC group. In contrast, the 8-OHdG levels in the SE group were significantly lower than those in the SC group, with no significant differences observed among the CC, CE, and SE groups. However, nuclear 8-OHdG levels in the brain, kidney, and spleen did not differ significantly among the four groups (data not shown).

Discussion

In this study, our findings demonstrated the protective effects of EGT, a potent antioxidant, against oxidative DNA damage and immune dysfunction caused by circadian rhythm disruption. Some studies have found that clock-gene-mutant mice or mice fed only during the day exhibited circadian rhythm disruption and higher body weights, despite having similar food intake as the control mice.^(25,26) Circadian rhythm disruption may be a risk factor for weight gain and obesity. It has also been linked to reduced levels of leptin, satiety hormones, and leptin resistance, all of which can contribute to obesity.^(27,28) In this study, we obtained similar results; the body weights of mice in the SC group were significantly higher than those of mice in the CC group (Table 1). However, this effect was not altered by EGT administration in the SE group. Calvo et al.⁽²⁹⁾ also found that EGT did not affect leptin levels in adults with metabolic syndrome. These results indicated that EGT did not significantly inhibit weight gain.

Increased time spent in the central area during open field tests, without changes in total movement or vertical exploration, is an indicator of anxiety-like behavior.^(30,31) Additionally, the number of excreted fecal pellets is recognized as a marker of anxiety during open field tests. Although the fecal output in the SC group was significantly higher than that in the CC group, no difference was observed in the number of fecal pellets excreted during the test between the SC and SE groups (Fig. 2B). These results indicate that night-feeding-restricted mice exhibited significant anxiety-like behaviors, and that EGT intake did not improve these behaviors. One study found that night-time workers with both daytime and night-time eating patterns had increased depression and anxiety symptoms, unlike daytime-only eating or daytime workers.⁽³²⁾ Altering the timing of food intake to misalign it with regular circadian rhythms may increase the risk of mental health disorders owing to disruptions in the circadian system. Nakamichi et al.⁽³³⁾ found that when EGT is orally ingested through diet, it crosses the blood-brain barrier and produces an antidepressant-like effect in mice, as demonstrated by the forced swimming test. However, it did not exhibit anxiolytic effects in the open field test. Piriyaprasath et al.⁽³⁴⁾ reported that in mice treated with EGT, the total movement distance and time spent in the center area were significantly greater than those in the saline-treated group on day 3 after EGT administration in an open field test. However, in the present study, no significant differences in the total movement distance or time spent in the center area were observed among the CC, CE, and SE groups (Fig. 2). The SE group did not show a significant difference in daytime wheel running activity compared with the SC group during the experimental period (Fig. 1C). This discrepancy may be due to differences in EGT administration concentrations, methods, or observation times. These results indicate that EGT does not alleviate anxiety-like behaviors induced by circadian rhythm disturbances caused by daytime-restricted feeding and exercise in mice. Some studies have indicated that circadian rhythm disruption not only alters the hippocampal protein levels of clock-related genes and induces depression-like behavior, but also increases IL-6 levels in animal experiments.^(21,35,36) Hemalathaa et al.⁽³⁷⁾ also suggested that IL-6 levels in the serum and tears of night-shift workers were significantly higher than those of day-shift workers. In this study, the serum IL-6 levels in the SC group were significantly higher than those in the CC group. However, IL-6 levels were significantly reduced in the EGT-treated SE group (Fig. 3). No significant differences were observed among the CC, CE, and SE groups. These results indicated that EGT significantly suppressed IL-6 levels induced by circadian rhythm disruption. Studies have suggested that EGT significantly inhibits the increase in IL-6 levels caused by exposure to certain chemicals or diseases.^(38,39) Wang et al.⁽³⁸⁾ suggested that EGT can inhibit IL-1β-induced IL-6 production in osteoarthritis chondrocytes by activating the SIRT6 pathway. However, the mechanism by which EGT inhibits IL-6 production in circadian rhythm disorders remains unclear.

Circadian rhythm disruption leads to oxidative stress accumulation, which contributes to various diseases associated with circadian rhythm disturbances.^(40,41) Our previous study showed that oxidative DNA damage can be induced by circadian rhythm disturbances from nighttime-restricted feeding and exercise in mice.⁽²¹⁾ Our study revealed that, on day 14 following dietary and exercise restrictions, the liver and spleen weights were significantly lower in the SC group compared to the CC group (Table 2). Furthermore, the levels of 8-OHdG in urine and DNA from the liver, testes, lungs, and pancreas were markedly higher in the SC group than in the CC group (Fig. 4). However, 8-OHdG levels significantly decreased in the SE group after EGT administration. No significant differences were observed among the CC, CE, and SE groups. EGT, a potent antioxidant with cytoprotective properties, reduces oxidative stress by preventing the formation of free radicals and scavenging existing free radicals, including O₂^•− and ^•OH.⁽⁴²⁾ EGT is widely present in the blood and tissues and accumulates through dietary intake, including in the liver, pancreas, testes, and lungs.^(3,4) Its metabolic turnover rate is relatively low, suggesting a prolonged antioxidant effect in various tissues.^(43,44) EGT concentrations are notably higher in oxidative tissues, underscoring its protective role against oxidative damage.⁽⁴⁵⁾ Some studies have shown that disruptions in the circadian rhythm are associated with an elevated risk of cancers, including liver, pancreatic, and lung cancers.^(46–48) Circadian regulation of glutathione and oxidative stress-related enzymes is well established.^(49–51) Shift work can impair semen quality in men without affecting testosterone levels.⁽⁵²⁾ Dare et al.⁽⁵³⁾ reported that EGT supplementation significantly reduced malondialdehyde levels in damaged testes of cisplatin-treated Wistar rats, increased superoxide dismutase (SOD) and catalase (CAT) activity, and decreased the percentage of dead sperm cells. Some studies have shown that EGT administration can decrease liver and lung injury by increasing the activity of enzymes or the concentration of antioxidants that combat oxidative stress, including glutathione, SOD, CAT, and glutathione peroxidase.^(7,54,55) Dare et al.⁽⁵⁵⁾ reported that EGT does not significantly decrease blood glucose levels or insulin resistance, although it significantly reduces oxidative stress in the liver. However, the combination of EGT and metformin significantly reduced blood glucose levels and insulin resistance. These results indicate that EGT can protect the pancreas by suppressing oxidative DNA damage, but does not influence blood glucose levels and insulin resistance. However, the underlying mechanisms require further investigation.

In the present study, we used a mouse model to investigate the effects of EGT on oxidative stress and immunity in the context of circadian rhythm disruption. EGT administration significantly inhibited the increase in IL-6 levels and oxidative DNA damage induced by circadian rhythm disruption in mice subjected to daytime food intake and exercise activity. However, EGT administration did not affect body weight, running wheel activity, or anxiety-like behavior in mice with circadian rhythm disruption. Supplementation with EGT can reduce the health risks associated with circadian rhythm disorders by mitigating the accumulation of oxidative DNA damage and IL-6. However, further studies are needed to understand the precise mechanisms by which EGT improves oxidative DNA damage and immunity induced by circadian rhythm disruptions. In addition, it is necessary to assess a broader range of oxidative stress biomarkers, immune markers, and behavioral changes. A better understanding of this relationship could provide a theoretical basis for the potential benefits of EGT supplementation in improving the health status and reducing disease risks associated with circadian rhythm disruption in shift workers. The results of this experiment are based on studies involving mice. Although the circadian rhythm in mice is evolutionarily conserved, these findings need to be validated in animals with sleep patterns more similar to those of humans, or through epidemiological studies.

Author Contributions

Y-SL, HF, KF, and KK designed and critically discussed the study. Animal experiment, Y-SL and HF; Project administration and data analysis, Y-SL and HF; Methodology, Y-SL and HF; Supervision, KF; Validation, KK and KF; Writing, Y-SL; Obtained funding, Y-SL. All authors have read and approved the final manuscript.

Abbreviations

CAT

catalase

EGT

ergothioneine

IL-6

interleukin-6

8-OHdG

8-hydroxy-2'-deoxyguanosine

ROS

reactive oxygen species

SOD

superoxide dismutase

Conflict of Interest

No potential conflicts of interest were disclosed.