Effects of Resveratrol on Muscle Inflammation, Energy Utilisation, and Exercise Performance in an Eccentric Contraction Exercise Mouse Model

Eccentric contraction can easily cause muscle damage and an inflammatory response, which reduces the efficiency of muscle contraction. Resveratrol causes anti-inflammatory effects in muscles, accelerates muscle repair, and promotes exercise performance after contusion recovery. However, whether resveratrol provides the same benefits for sports injuries caused by eccentric contraction is unknown. Thus, we explored the effects of resveratrol on inflammation and energy metabolism. In this study, mice were divided into four groups: a control group, an exercise group (EX), an exercise with low-dose resveratrol group (EX + RES25), and an exercise with high-dose resveratrol group (EX + RES150). The results of an exhaustion test showed that the time before exhaustion of the EX + RES150 group was greater than that of the EX group. Tumour necrosis factor-α (Tnfα) mRNA expression was lower in the EX + RES150 group than in the EX group. The energy utilisation of the EX + RES150 group was greater than that of the EX + RES25 group in different muscles. High-dose resveratrol intervention decreased Tnfα mRNA expression and enhanced the mRNA expressions of sirtuin 1, glucose transporter 4, AMP-activated protein kinase α1, and AMP-activated protein kinase α2 in muscles. These results revealed that high-dose resveratrol supplementation can reduce inflammation and oxidation and improve energy utilisation during short-duration high-intensity exercise.


Introduction
A habit of exercise contributes to effective metabolism, strong muscles, and low fatigue [1] and protects against metabolic disorders and even cognitive impairment in older adults [2,3]. However, poor exercise habits can cause muscle damage. Mice were made to execute eccentric contraction that caused the overextension of muscle fibres [4]. When such muscle damage occurs, macrophages are polarised as M1 macrophages by tumour necrosis factor-α (TNF-α) and interleukin 6 (IL-6) to prevent damage to the muscle cells [4,5]. During a rest period, these M1 macrophages are converted to M2 macrophages by anti-inflammatory factors (i.e., insulin-like growth factor 1 and interleukin 10) for muscle reconstruction [5,6]. Increased levels of IL-6 and TNF-α in adipose tissue have been observed in individuals with obesity during long-duration eccentric contraction exercise [7]; however, the related cytokine mechanism is not understood.
the Taipei Medical University Laboratory Animal Center. All animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Taipei Medical University (LAC-2021-0310).

Experiment Design
C57BL/6J mice were housed in the animal facility for 1 week for them to adapt to the environment before the study. Twenty-four mice were randomly assigned into four equal groups: control (NC), exercise (EX), exercise with low-dose resveratrol (25 mg/kg body weight; EX + RES25), and exercise with high-dose resveratrol (150 mg/kg body weight; EX + RES150). The mice in the EX, EX + RES25, and EX + RES150 groups underwent 3 days of acclimation running before undertaking the incremental load test (ILT). Acclimation involved running 10 m/min for 15 min with an incline of 0° each day for 3 days. All the mice underwent an exhaustion test the day after the ILT, and the downhill running section of the study started the following week. Resveratrol was dissolved in distilled water. EXtreated mice were gavaged with various amounts (25 mg/kg/d [RES25] and 150 mg/kg/d [RES150] in 0.5 mL of normal saline) of resveratrol or the vehicle (0.5 mL of normal saline) for C and EX groups for 4 weeks (Figure 1).

ILT, Exhaustion Test, and Downhill Running
Mice undertook the ILT on a motorised treadmill (Rodent Treadmill 47300, Ugo Basile, Italy). The intensity of exercise was programmed to increase by 3 m/min (initial speed: 12 m/min) in the first 3 min and mice ran on a 0% gradient until exhaustion [24]. Individual exhaustion velocity (EV) was determined from the results. The same exercise protocol was used in the exhaustion test. After a 3-day acclimation to running and the ILT, the mice of groups EX, EX + RES25 and EX + RES150 ran on a motorised treadmill (−15° slope) at 22 m/min (60% EV), 2 h/day, 5 days/week for 2 weeks [25]. Training interventions were conducted between 10 am and 6 pm.

ILT, Exhaustion Test, and Downhill Running
Mice undertook the ILT on a motorised treadmill (Rodent Treadmill 47300, Ugo Basile, Italy). The intensity of exercise was programmed to increase by 3 m/min (initial speed: 12 m/min) in the first 3 min and mice ran on a 0% gradient until exhaustion [24]. Individual exhaustion velocity (EV) was determined from the results. The same exercise protocol was used in the exhaustion test. After a 3-day acclimation to running and the ILT, the mice of groups EX, EX + RES25 and EX + RES150 ran on a motorised treadmill (−15 • slope) at 22 m/min (60% EV), 2 h/day, 5 days/week for 2 weeks [25]. Training interventions were conducted between 10 am and 6 pm.

Sample Collection
After the downhill running test, mice were euthanized by a CO 2 inhalation overdose and were sacrificed for biopsy, including biopsies of the liver, epididymal adipose tissue (eWAT), gastrocnemius muscle, soleus muscle, and tibialis anterior muscle, which were collected and stored at −80 • C for biochemical analyses. Skin irritation was also evaluated in all animals at the end of the study. RNA was extracted using RNAzol RT (Molecular Research Center, Cincinnati, OH, USA) according to manufacturer guidelines. 1 µg of RNA was reverse transcribed using a TOOLSQuant II fast RT kit (BIOTOOLS, Taiwan). Genes were quantified using specific primers with SYBR green using the Applied Biosystems QuantStudio 1 Real-Time PCR System. The final quantification was executed using the 2−∆∆CT method with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primer as a control. The sequences of primers are shown in Table 1.

Next Generation Sequencing Analysis
First, cDNA libraries were collected from four independent samples in each group. The cDNA libraries were assessed using the Agilent 2100 Bioanalyzer system and the Real-Time PCR system. β-Actin served as an internal control to verify the quality and quantity of cDNA in the PCR system. The sequenced libraries underwent fragment size detection using the Agilent Bioanalyzer 2100 system and library concentration determination using the Real-Time PCR system; the quality-confirmed libraries underwent 150-bp paired-end sequencing on the Illumina NovaSeq 6000 sequencer (Genomics, BioSci & Tech Company, New Taipei City, Taiwan). Raw sequencing reads were filtered using Trimmomatic (version 0.36) [26]. Reads were aligned using Bowtie2 (version 2.3.5) [27]. Raw gene counts were extracted using RSEM (version 1.3.3) [28]. The R package edgeR (v3.16.5) was used for the differential gene expression analysis of two sample groups. Furthermore, the log 2 fold change was calculated as log 2 (sample count 1/sample count 2). The t-test was used to identify significant differences between samples from the two groups (p < 0.05). Gene ontology (GO) enrichment analysis was conducted on the differential genes obtained through screening. When p < 0.05, GO terms were significantly enriched [29]. The Kyoto Encyclopedia of Genes and Genomes was used for the gene enrichment of differentially expressed genes.

Statistical Analysis
All results are expressed in terms of the mean ± SEM. The significance of the difference was examined using GraphPad Prism version 9.0 (GraphPad Software; San Diego, CA, USA). All data were normally distributed. Significant differences were analyzed using a student's t-test or one-way analysis of variance with Tukey's test and Bonferroni's test for multiple comparisons. A p-value less than 0.05 was considered statistically significant.

Average Body Weight, Food Intake, and Tissue Relative Weight
Average body weight, food intake, and tissue relative weight are shown in Table 2. No significant differences in the body weight, food and water intake, and selected biopsy results were found. However, the relative weights of the eWAT in the EX, EX + RES25, and EX + RES150 groups were significantly lower than that of the NC group (p < 0.05). 1.00 ± 0.05 a 0.61 ± 0.05 b 0.60 ± 0.03 b 0.68 ± 0.04 b Data displayed as mean ± S.E.M. Data were analysed using one-way analysis of variance and a t-test followed by the Bonferroni multiple comparison test. Different superscripts (a and b) in each row indicate significant differences among groups (p < 0.05).

Exercise Performance in Exhaustion Test
The time before exhaustion in the EX group was shorter than in the NC group (p < 0.05); however, the time before exhaustion of the EX and EX + RES25 groups showed no difference. A significant difference was found between the results of the EX and EX + RES150 groups (p < 0.05; Figure 2). CA, USA). All data were normally distributed. Significant differences were analyzed using a student's t-test or one-way analysis of variance with Tukey's test and Bonferroni's test for multiple comparisons. A p-value less than 0.05 was considered statistically significant.

Average Body Weight, Food Intake, and Tissue Relative Weight
Average body weight, food intake, and tissue relative weight are shown in Table 2. No significant differences in the body weight, food and water intake, and selected biopsy results were found. However, the relative weights of the eWAT in the EX, EX + RES25, and EX + RES150 groups were significantly lower than that of the NC group (p < 0.05). Table 2. Body weight, food and water intake and tissue relative weight in mice.

Exercise Performance in Exhaustion Test
The time before exhaustion in the EX group was shorter than in the NC group (p < 0.05); however, the time before exhaustion of the EX and EX + RES25 groups showed no difference. A significant difference was found between the results of the EX and EX + RES150 groups (p < 0.05; Figure 2).

Lactate Dehydrogenase (LDH) and Creatine Kinase (CK)
The blood biochemical values of the mice after the exhaustion test showed that the levels of LDH and CK in the EX group were significantly higher than those in the NC group (p < 0.05). Compared with the NC group, LDH and CK in the EX + RES25 and EX + RES150 groups were also significantly increased (p < 0.05 for both), but LDH and CK in the EX + RES150 group were significantly lower than those in the EX group (p < 0.05; Figure 3). The blood biochemical values of the mice after the exhaustion test showed that the levels of LDH and CK in the EX group were significantly higher than those in the NC group (p < 0.05). Compared with the NC group, LDH and CK in the EX + RES25 and EX + RES150 groups were also significantly increased (p < 0.05 for both), but LDH and CK in the EX + RES150 group were significantly lower than those in the EX group (p < 0.05; Fig  . Data are presented as mean ± SEM. Data were analysed using one-way analysis of variance and a t-test followed by the Bonferroni multiple comparison test. Superscript letters (a, b and c) in columns denote a significant difference (p < 0.05).

Gastrocnemius Muscle
Compared with that of the NC group and the EX + RES150 group, the mRNA expression of TNF-α in the gastrocnemius muscle was increased higher in the EX group (p < 0.05; Figure 4A-1). Il6 mRNA expression was significantly lower in the EX group than in the other groups (p < 0.05; Figure 4B-1).

Tibialis Anterior Muscle
Compared with the NC group, the mRNA expression of TNF-α and IL-6 in the tibialis anterior muscle were significantly higher in the EX, EX + RES25, and EX + RES150 groups (p < 0.05; Figure 4A-2, 4B-2). Tnfα mRNA expression in the EX + RES25 and EX + RES150 groups was significantly higher than that in the NC group (p < 0.05; Figure 4A-2). The mRNA expression of TNF-α in the EX + RES25 and EX + RES150 groups was significantly lower than that in the EX group ( Figure 4A-2). No difference in Il6 mRNA expression among the four groups was found ( Figure 4B-2).

Soleus Muscle
Compared with that of the NC group, the mRNA expression of TNF-α in the soleus muscle was significantly higher in the EX group. After resveratrol was administered, soleus-muscle Tnfα mRNA expression in the EX + RES25 and EX + RES150 groups was significantly lower than that in the EX group (p < 0.05; Figure 4A-3). No difference in soleus muscle Il6 mRNA expression between the groups was found ( Figure 4B-3). . Data are presented as mean ± SEM. Data were analysed using one-way analysis of variance and a t-test followed by the Bonferroni multiple comparison test. Superscript letters (a, b and c) in columns denote a significant difference (p < 0.05).

Gastrocnemius Muscle
Compared with that of the NC group and the EX + RES150 group, the mRNA expression of TNF-α in the gastrocnemius muscle was increased higher in the EX group (p < 0.05; Figure 4A-1). Il6 mRNA expression was significantly lower in the EX group than in the other groups (p < 0.05; Figure 4B-1).

Tibialis Anterior Muscle
Compared with the NC group, the mRNA expression of TNF-α and IL-6 in the tibialis anterior muscle were significantly higher in the EX, EX + RES25, and EX + RES150 groups (p < 0.05; Figure 4A-2,B-2). Tnfα mRNA expression in the EX + RES25 and EX + RES150 groups was significantly higher than that in the NC group (p < 0.05; Figure 4A-2). The mRNA expression of TNF-α in the EX + RES25 and EX + RES150 groups was significantly lower than that in the EX group ( Figure 4A-2). No difference in Il6 mRNA expression among the four groups was found ( Figure 4B-2).

Soleus Muscle
Compared with that of the NC group, the mRNA expression of TNF-α in the soleus muscle was significantly higher in the EX group. After resveratrol was administered, soleus-muscle Tnfα mRNA expression in the EX + RES25 and EX + RES150 groups was significantly lower than that in the EX group (p < 0.05; Figure 4A-3). No difference in soleus muscle Il6 mRNA expression between the groups was found ( Figure 4B-3).

Gastrocnemius Muscle
The results showed that the mRNA expression of SIRT1, AMPK α1, and AMPK α2 in the gastrocnemius muscle in the EX + RES25 group was significantly higher than that in the EX group (p < 0.05; Figure 5A,C,D). The mRNA expression levels of GLUT4, AMPK α1, and PGC-1α in the gastrocnemius muscle in the EX + RES150 group were significantly higher than those in the EX group (p < 0.05; Figure 5B,C,E). The mRNA expression of SIRT1 and PGC-1α significantly differed between the EX + RES25 and EX + RES150 groups (p < 0.05; Figure 5A,E).   (3) soleus muscle during short-d downhill running. mRNA expression was normalized to GAPDH and expressed as fold cha ative to the control group. NC: control (n = 6); EX: exercise (n = 6); EX + RES25: exercise with trol 25 mg/kg (n = 6); EX + RES150: exercise with resveratrol 150 mg/kg (n = 6). Data are p as mean ± SEM. Data were analysed using one-way analysis of variance and a t-test followe Bonferroni multiple comparison test. Superscript letters (a and b) in columns denote a sig difference (p < 0.05).

Gastrocnemius Muscle
The results showed that the mRNA expression of SIRT1, AMPK α1, and AM in the gastrocnemius muscle in the EX + RES25 group was significantly higher th in the EX group (p < 0.05; Figure 5A,C,D). The mRNA expression levels of GLUT4, α1, and PGC-1α in the gastrocnemius muscle in the EX + RES150 group were signi higher than those in the EX group (p < 0.05; Figure 5B,C,E). The mRNA expres SIRT1 and PGC-1α significantly differed between the EX + RES25 and EX + RES150  (3) soleus muscle during short-duration downhill running. mRNA expression was normalized to GAPDH and expressed as fold change relative to the control group. NC: control (n = 6); EX: exercise (n = 6); EX + RES25: exercise with resveratrol 25 mg/kg (n = 6); EX + RES150: exercise with resveratrol 150 mg/kg (n = 6). Data are presented as mean ± SEM. Data were analysed using one-way analysis of variance and a t-test followed by the Bonferroni multiple comparison test. Superscript letters (a and b) in columns denote a significant difference (p < 0.05). . Data are presented as mean ± SEM. Data were analysed us one-way analysis of variance and a t-test followed by the Bonferroni multiple comparison test. perscript letters (a, b and c) in columns denote a significant difference (p < 0.05).

Tibialis Anterior Muscle
The mRNA expression levels of SIRT1 and AMPK α1 in the tibialis anterior mus in the EX + RES150 group were significantly higher compared with those in the NC a EX groups (p < 0.05; Figure 6A,C). The mRNA expression of AMPK α1 increased sign cantly with resveratrol dosage, exhibiting a dose-dependent effect in the two RES grou (p < 0.05; Figure 6C). The mRNA expression of PGC-1α was significantly higher in the E EX + RES25, and EX + RES150 groups than in the NC group (p < 0.05; Figure 6E). No d ference between the groups in the expression of GLUT4 and AMPK α2 was found (Figu 6B,D). Glut4 mRNA (E) Pgc1α mRNA. mRNA expression was normalized to GAPDH and is expressed in terms of fold change relative to the control group. NC: control (n = 6); EX: exercise (n = 6); EX + RES25: exercise with resveratrol 25 mg/kg (n = 6); EX + RES150: exercise with resveratrol 150 mg/kg (n = 6). Data are presented as mean ± SEM. Data were analysed using one-way analysis of variance and a t-test followed by the Bonferroni multiple comparison test. Superscript letters (a, b and c) in columns denote a significant difference (p < 0.05).

Tibialis Anterior Muscle
The mRNA expression levels of SIRT1 and AMPK α1 in the tibialis anterior muscle in the EX + RES150 group were significantly higher compared with those in the NC and EX groups (p < 0.05; Figure 6A,C). The mRNA expression of AMPK α1 increased significantly with resveratrol dosage, exhibiting a dose-dependent effect in the two RES groups (p < 0.05; Figure 6C). The mRNA expression of PGC-1α was significantly higher in the EX, EX + RES25, and EX + RES150 groups than in the NC group (p < 0.05; Figure 6E). No difference between the groups in the expression of GLUT4 and AMPK α2 was found ( Figure 6B,D).

Soleus Muscle
The soleus muscle mRNA expression levels of SIRT1, GLUT4, AMPK α1, AMPK α2, and PGC-1α in the EX + RES150 group were significantly higher than in the NC group (p < 0.05; Figure 6). Sirt1, Glut4, Ampkα2, and Pgc1α mRNA expression increased with resveratrol dosage significantly (p < 0.05; Figure 7A,B,D,E). The mRNA expression level of GLUT4 in the EX group was significantly lower than in the NC group ( Figure 7B). to GAPDH and is expressed in terms of fold change relative to the control group. NC: control (n = 6); EX: exercise (n = 6); EX + RES25: exercise with resveratrol 25 mg/kg (n = 6); EX + RES150: exercise with resveratrol 150 mg/kg (n = 6). Data are presented as mean ± SEM. Data were analysed using one-way analysis of variance and a t-test followed by the Bonferroni multiple comparison test. Superscript letters (a, b and c) in columns denote a significant difference (p < 0.05).

Soleus Muscle
The soleus muscle mRNA expression levels of SIRT1, GLUT4, AMPK α1, AMPK α2, and PGC-1α in the EX + RES150 group were significantly higher than in the NC group (p < 0.05; Figure 6). Sirt1, Glut4, Ampkα2, and Pgc1α mRNA expression increased with resveratrol dosage significantly (p < 0.05; Figure 7A,B,D,E). The mRNA expression level of GLUT4 in the EX group was significantly lower than in the NC group ( Figure 7B). Glut4 mRNA (E) Pgc1α mRNA. mRNA expression was normalized to GAPDH and is expressed in terms of fold change relative to the control group. NC: control (n = 6); EX: exercise (n = 6); EX + RES25: exercise with resveratrol 25 mg/kg (n = 6); EX + RES150: exercise with resveratrol 150 mg/kg (n = 6). Data are presented as mean ± SEM. Data were analysed using one-way analysis of variance and a t-test followed by the Bonferroni multiple comparison test. Superscript letters (a, b and c) in columns denote a significant difference (p < 0.05).

MA Plot, Volcano Plot, and Bar Charts for EX vs. EX + RES150 Groups
In the EX and EX + RES150 groups, 37 genes were more significantly upregulated, and 77 genes were more significantly downregulated in the high-dose group than in the EX group ( Figure 8).
As shown in the biological process plot, pathway regulation occurred due to the intervention of high-dose resveratrol, such as the positive regulation of cytokine production ( Figure 9A). Analysis of molecular function showed that the regulation of pathways related to energy hydrolysis enzymes also occurred, indicated, for example, by nucleosidetriphosphatase regulator activity and GTPase regulator activity ( Figure 9B). In addition, as shown by the cellular component bar chart, most regulated pathways were found to be related to immunity, such as MHC class II proteins or lysosomal membrane proteins ( Figure 9C). Higher doses of resveratrol have a greater effect on facial features during exercise and may have greater anti-inflammatory, energy utilisation-related, and immuneboosting effects.

MA Plot, Volcano Plot, and Bar Charts for EX vs. EX + RES150 Groups
In the EX and EX + RES150 groups, 37 genes were more significantly upregulate and 77 genes were more significantly downregulated in the high-dose group than in t EX group (Figure 8).  Glut4 mRNA (E) Pgc1α mRNA. mRNA expression was normalized to GAPDH and is expressed in terms of fold change relative to the control group. NC: control (n = 6); EX: exercise (n = 6); EX + RES25: exercise with resveratrol 25 mg/kg (n = 6); EX + RES150: exercise with resveratrol 150 mg/kg (n = 6). Data are presented as mean ± SEM. Data were analysed using one-way analysis of variance and a t-test followed by the Bonferroni multiple comparison test. Superscript letters (a, b and c) in columns denote a significant difference (p < 0.05). . Data are presented as mean ± SEM. Data were analysed using one way analysis of variance and a t-test followed by the Bonferroni multiple comparison test. Supe script letters (a, b and c) in columns denote a significant difference (p < 0.05).

MA Plot, Volcano Plot, and Bar Charts for EX vs. EX + RES150 Groups
In the EX and EX + RES150 groups, 37 genes were more significantly upregulated and 77 genes were more significantly downregulated in the high-dose group than in th EX group (Figure 8).  triphosphatase regulator activity and GTPase regulator activity ( Figure 9B). In addition, as shown by the cellular component bar chart, most regulated pathways were found to be related to immunity, such as MHC class II proteins or lysosomal membrane proteins (Figure 9C). Higher doses of resveratrol have a greater effect on facial features during exercise and may have greater anti-inflammatory, energy utilisation-related, and immune-boosting effects.

Discussion
In this experiment, we used short-duration downhill running and different dosages of resveratrol to evaluate inflammation and energy utilisation in the different muscles of mice. The main findings were: (1) the time before exhaustion decreased due to exercise injury; this was counteracted by the intervention of resveratrol; (2) inflammation in the

Discussion
In this experiment, we used short-duration downhill running and different dosages of resveratrol to evaluate inflammation and energy utilisation in the different muscles of mice. The main findings were: (1) the time before exhaustion decreased due to exercise injury; this was counteracted by the intervention of resveratrol; (2) inflammation in the muscles was reduced by the intervention of resveratrol; (3) the expression of genes related to energy utilisation in the EX + RES150 group was significantly higher than in the EX group, indicating that the intervention of resveratrol increases muscle energy utilisation.
The time to exhaustion in the EX group was significantly lower than that in the NC, EX + RES25, and EX + RES150 groups. The results of this experiment demonstrate that resveratrol supplementation is beneficial for endurance exercise. Our study found no difference in endurance performance between the low-dose group and the high-dose group. This difference between this study and ours may have been due to differences in exercise mode (weight-loaded swimming in the previous study).
TNF-α and IL-6 levels are often used to indicate inflammation. When an inflammatory response occurs, the production of cytokines in injured tissues increases significantly. Tnfα mRNA expression in the EX group was significantly higher than that in the NC group, confirming that exercise injury elevated the production of TNF-α [30]. Resveratrol effectively reduced inflammation; high doses of resveratrol exerted anti-inflammatory effects in different muscles. In long-duration exercises, reaching exhaustion always results in high levels of oxidative stress. A high-intensity cycling-based clinical trial demonstrated that resveratrol supplementation attenuated exercise-induced serum interleukin-6 levels but not oxidative stress [31]. Such physical phenomena imply that resveratrol affects antiinflammation and antioxidation processes independently. IL-6 production is positively correlated with glucose uptake by GLUT4 in muscles [32][33][34]. In our study, the mRNA expression of IL-6 did not differ between different muscle tissues, but the repression of Glut4 mRNA was observed in the gastrocnemius and soleus muscles. This suggests that increased mRNA levels of IL-6 and GLUT4 occur synchronously; thus, IL-6 may trigger GLUT4-induced glucose utilisation in muscle.
Many studies have found that during endurance exercise, due to the reduction in available glycogen in the body, more AMPK is activated to facilitate the production of downstream products, providing the body with energy [35]. During exercise, the intervention of resveratrol resulted in the activation not only of AMPK but also SIRT1 and its downstream proteins GLUT4 and PGC-1α and in miR-22-3p in a muscle cell model [36]. Resveratrol also exhibited dual bioactive effects in our study; however, the effects of resveratrol remain unclear.
Type I muscle fibres support long-duration exercise, type II fibres support explosive short-duration exercise, and type IIx fibres transform into different types of fibres in relation to the intensity of the physical activity at hand [37]. The mRNA expression of AMPK α1 in the gastrocnemius and tibialis anterior muscles in the EX group did not differ from that in the NC group. We hypothesise that the type IIx muscle fibres in these muscles were not converted into type I muscle fibres because the exercise period was not long enough for the fibres to adapt to endurance exercise. The effect of resveratrol on AMPK activation in the gastrocnemius and tibialis anterior muscles was greater than that caused by exercise. However, no differences in Ampkα1 and Ampkα2 mRNA expression in the soleus muscle between the EX and the NC groups were found. These apparently conflicting results were also observed in a 12-week study [38], and we speculate that the two-week duration of this experiment was insufficient for a long-term exercise study. However, the soleus muscle has a high proportion of red muscle, which supports aerobic exercise. Thus, the significant differences between the NC and EX groups can be attributed to resveratrol intervention.
The mRNA expression of GLUT4 in the gastrocnemius muscle and soleus muscle in the EX + RES150 group was significantly higher than that in the EX group. This result is consistent with the findings of a previous study that the intervention of resveratrol in muscle cells effectively increases the amount of GLUT4 translocated to the plasma membrane [39]. The tibialis anterior muscle does not exhibit similar effects; the ratio of red to white muscle in the tibialis anterior muscle may contribute to this [38]. Studies have demonstrated that white muscle fibres can activate GLUT4 translocation during exercise [40]; the gastrocnemius muscle has a higher proportion of white muscle and exhibits higher Glut4 mRNA expression. Pereira BC et al. reported that excessive eccentric contraction training impairs insulin signal transduction in mice's skeletal muscles and reduces the rate of GLUT4 translocation [41]. Thus, muscle injury may have caused the mRNA expression of GLUT4 in the soleus muscle in the EX group to be significantly lower than in the other groups.
As an oxidoreductase, SIRT1 regulates various cellular events, including apoptosis, cell survival, endocrine signalling, and gene transcription [42]. SIRT1 activates the downstream product PGC-1α, and PGC-1α regulates mitochondrial biosynthesis to facilitate the regulation of oxidative metabolism [43]. The mRNA expression of SIRT1 and PGC-1α in the gastrocnemius muscle, tibialis anterior muscle, and soleus muscle increased sig-nificantly with the intervention of high-dose resveratrol compared with the NC group, which is consistent with the results of previous studies [44], indicating resveratrol intervention for short-duration exercise can effectively increase energy utilisation and cause an antioxidising effect.
In this study, we assessed genome expression under resveratrol intervention in shortduration eccentric contraction exercises. We focused on muscle inflammation and energy utilisation-related gene expression. One gene that was intensively activated after high-dose resveratrol intervention was myosin light chain kinase 2 (ENSMUSG00000027470), which is intensively expressed in cardiac and skeletal muscle [45] and regulates the interaction of myosin and actin through the concentration of calcium ions protein cross bridge [46]. In this experiment, high-dose resveratrol intensively activated this gene, thus we hypothesise that resveratrol increases muscle contraction ability during exercise. NADH dehydrogenase subunit 5 (ENSMUSG00000064367) is part of mitochondrial complex I, a large enzyme complex that is active in mitochondria [47]. We found that this gene was upregulated in the presence of intervening resveratrol. Therefore, we hypothesise that due to the increase in the expression of this gene, oxidative phosphorylation on the inner mitochondrial membrane and the production of ATP increased, providing the cell with more energy and thereby increasing the utilisation of energy during exercise. Gene ontology enrichment table of EX vs. EX + RES150 included biological process, molec-ular function and cellular component is shown in Supplementary Table S1.

Conclusions
High-dose resveratrol intervention prolonged the time before exhaustion for shortduration downhill running. High-dose resveratrol intervention decreased Tnfα mRNA expression and enhanced the mRNA expressions of SIRT1, GLUT4, AMPK α1, and AMPK α2 in some muscles. These results indicate that high-dose resveratrol supplementation can reduce inflammation and oxidation and improve the utilisation of energy during short-duration high-intensity exercise.