Muscular TOR knockdown and endurance exercise ameliorate high salt and age-related skeletal muscle degradation by activating the MTOR-mediated pathway

Abstract

The target of rapamycin(TOR)gene is closely related to metabolism and cellular aging, but it is unclear whether the TOR pathways mediate endurance exercise against the accelerated aging of skeletal muscle induced by high salt intake. In this study, muscular TOR gene overexpression and RNAi were constructed by constructing MhcGAL4/TOR-overexpression and MhcGAL4/TORUAS-RNAi systems in Drosophila. The results showed that muscle TOR knockdown and endurance exercise significantly increased the climbing speed, climbing endurance, the expression of autophagy related gene 2(ATG2), silent information regulator 2(SIR2), and pparγ coactivator 1(PGC-1α) genes, and superoxide dismutases(SOD) activity, but it decreased the expression of the TOR gene and reactive oxygen species(ROS) level, and it protected the myofibrillar fibers and mitochondria of skeletal muscle in Drosophila on a high-salt diet. TOR overexpression yielded similar results to the high salt diet(HSD) alone, with the opposite effect of TOR knockout found in regard to endurance exercise and HSD-induced age-related skeletal muscle degradation. Therefore, the current findings confirm that the muscle TOR gene plays an important role in endurance exercise against HSD-induced age-related skeletal muscle degeneration, as it determines the activity of the mammalian target of rapamycin(MTOR)/SIR2/PGC-1α and MTOR/ATG2/PGC-1α pathways in skeletal muscle.

Fig 1. Muscle TOR gene, endurance exercise activates the MTOR/ SIR2/PGC-1α and MTOR/ATG2/PGC-1α pathway and regulates age-related decline in skeletal muscle.

Fig 2. Comparison of MTOR gene expression levels in overexpressed/knock-down Drosophila aged 3 weeks.

Data were expressed by mean ±SEM. * P < 0.05; * * P < 0.01; * * * P < 0.001; n indicates no significant difference. The sample size was muscle of 20 flies per group, measured 3 times.

Fig 3. Effects of exercise on body weight, climbing ability and skeletal muscle physiological structure of TOR^UAS-OE and TOR^UAS-RNAi flies.

(A) Fatigue time of 1-week-old flies in TOR^UAS-OE group. (B) Fatigue time of 3-week-old flies in TOR^UAS-OE group. (C) Fatigue time of flies aged 4 weeks in TOR^UAS-OE group. (D) Fatigue time of 5-week-old flies in TOR^UAS-OE group. (E) Body weight of 5-week-old flies in TOR^UAS-OE group. (F) Climb height of 1,3,4,5 week-old flies in TOR^UAS-OE group within 3 seconds. (G) Skeletal muscle ROS levels in TOR^UAS-OE group. (H) SOD level in skeletal muscle of TOR^UAS-OE group. (I) Relative expression of TOR gene in skeletal muscle of TOR^UAS-OE group. (J) Relative expression of ATG2 gene in skeletal muscle of TOR^UAS-OE group. (K) Relative expression of PGC-1α gene in skeletal muscle of TOR^UAS-OE group. (L) Relative expression of SIR2 gene in skeletal muscle of TOR^UAS-OE group. (M) Fatigue time of 1-week-old flies in TOR^UAS-RNAi group. (N) Fatigue time of 3-week-old flies in TOR^UAS-RNAi group. (O) Fatigue time of flies aged 4 weeks in TOR^UAS-RNAi group. (P) Fatigue time of 5-week-old flies in TOR^UAS-RNAi group. (Q) Body weight of 5-week-old flies in TOR^UAS-RNAi group. (R) Climb height of 1,3,4,5 week-old flies in TOR^UAS-RNAi group within 3 seconds. (S) Skeletal muscle ROS levels in TOR^UAS-RNAi group. (T) SOD level in skeletal muscle of TOR^UAS-RNAi group. (U) Relative expression of TOR gene in skeletal muscle of TOR^UAS-RNAi group. (V) Relative expression of ATG2 gene in skeletal muscle of TOR^UAS-RNAi group. (W) Relative expression of PGC-1α gene in skeletal muscle of TOR^UAS-RNAi group. (X) Relative expression of SIR2 gene in skeletal muscle of TOR^UAS-RNAi group. (Y) myosin heavy chain immunofluorescence in TORUAS-OE group, with blue dots as nucleus and red fluorescent bands as myofibril. HSD reduces fluorescence expression (scale: white line is 5 microns). (Z) TOR^UAS-RNAi group myosin heavy chain immunofluorescence. (α) Skeletal muscle transmission electron microscopy (scale: small mesh represents 0.2 microns). The white arrow points to the myofibril. The red arrow points to the mitochondria. Climbing height measurements, the sample size of each group is about 150–170 animals. Univariate analysis of variance (ANOVA) and minimum significance difference (LSD) tests were used to determine differences between groups. The P-values of climbing endurance curve and survival curve were calculated by log-rank test. Data were expressed by mean ±SEM. * P < 0.05; * * P < 0.01; * * * P < 0.001; n indicates no significant difference. The sample size of proteins and ROS was muscle of 20 flies per group, measured 3 times. The sample size for RT-PCR and ELISA was muscle of 20 flies per group, measured 3 times. Univariate analysis of variance (ANOVA) and minimum significance difference (LSD) tests were used to determine differences between groups. (A) Data are represented by mean ±SEM. * P < 0.05; * * P < 0.01; n indicates no significant difference.

Fig 4. Effects of HSD on body weight, climbing ability and skeletal muscle physiological structure of TOR^UAS-OE and TOR^UAS-RNAi flies.

(A) Fatigue time of 1-week-old flies in TOR^UAS-OE group. (B) Fatigue time of 3-week-old flies in TOR^UAS-OE group. (C) Fatigue time of flies aged 4 weeks in TOR^UAS-OE group. (D) Fatigue time of 5-week-old flies in TOR^UAS-OE group. (E) Body weight of 5-week-old flies in TOR^UAS-OE group. (F) Climb height of 1,3,4,5 week-old flies in TOR^UAS-OE group within 3 seconds. (G) Skeletal muscle ROS levels in TOR^UAS-OE group. (H) SOD level in skeletal muscle of TOR^UAS-OE group. (I) Climb height of 1,3,4,5 week-old flies in TOR^UAS-RNAi group within 3 seconds. (J) Body weight of 5-week-old flies in TOR^UAS-RNAi group. (K) Skeletal muscle ROS levels in TOR^UAS-RNAi group. (L) SOD level in skeletal muscle of TOR^UAS-RNAi group. (M) Fatigue time of 1-week-old flies in TOR^UAS-RNAi group. (N) Fatigue time of 3-week-old flies in TOR^UAS-RNAi group. (O) Fatigue time of flies aged 4 weeks in TOR^UAS-RNAi group. (P) Fatigue time of 5-week-old flies in TOR^UAS-RNAi group. (Q) Relative expression of TOR gene in skeletal muscle of TOR^UAS-RNAi group. (R) Relative expression of ATG2 gene in skeletal muscle of TOR^UAS-RNAi group. (S) Relative expression of PGC-1α gene in skeletal muscle of TOR^UAS-RNAi group. (T) Relative expression of SIR2 gene in skeletal muscle of TOR^UAS-RNAi group. (U) myosin heavy chain immunofluorescence in TOR^UAS-OE group, with blue dots as nucleus and red fluorescent bands as myofibril. HSD reduces fluorescence expression (scale: white line is 5 microns). (V) TOR^UAS-RNAi group myosin heavy chain immunofluorescence. (W) Skeletal muscle transmission electron microscopy (scale: small mesh represents 0.2 microns). The white arrow points to the myofibril. The red arrow points to the mitochondria. Climbing height measurements, the sample size of each group is about 150–170 animals. Univariate analysis of variance (ANOVA) and minimum significance difference (LSD) tests were used to determine differences between groups. The P-values of climbing endurance curve and survival curve were calculated by log-rank test. Data were expressed by mean ±SEM. * P < 0.05; * * P < 0.01; * * * P < 0.001; n indicates no significant difference. The sample size of proteins and ROS was muscle of 20 flies per group, measured 3 times. The sample size for RT-PCR and ELISA was muscle of 20 flies per group, measured 3 times. Univariate analysis of variance (ANOVA) and minimum significance difference (LSD) tests were used to determine differences between groups. (A) Data are represented by mean ±SEM. * P < 0.05; * * P < 0.01; n indicates no significant difference.

Fig 5. Effects of exercise and HSD on body weight, climbing ability and skeletal muscle physiological structure of TOR^UAS-OE and TOR^UAS-RNAi flies.

(A) Fatigue time of 1-week-old flies in TOR^UAS-OE group. (B) Fatigue time of 3-week-old flies in TOR^UAS-OE group. (C) Fatigue time of flies aged 4 weeks in TOR^UAS-OE group. (D) Fatigue time of 5-week-old flies in TOR^UAS-OE group. (E) Body weight of 5-week-old flies in TOR^UAS-OE group. (F) Climb height of 1,3,4,5 week-old flies in TOR^UAS-OE group within 3 seconds. (G) Skeletal muscle ROS levels in TOR^UAS-OE group. (H) SOD level in skeletal muscle of TOR^UAS-OE group. (I) Relative expression of TOR gene in skeletal muscle of TOR^UAS-OE group. (J) Relative expression of ATG2 gene in skeletal muscle of TOR^UAS-OE group. (K) Relative expression of PGC-1α gene in skeletal muscle of TOR^UAS-OE group. (L) Relative expression of SIR2 gene in skeletal muscle of TOR^UAS-OE group. (M) Fatigue time of 1-week-old flies in TOR^UAS-RNAi group. (N) Fatigue time of 3-week-old flies in TOR^UAS-RNAi group. (O) Fatigue time of flies aged 4 weeks in TOR^UAS-RNAi group. (P) Fatigue time of 5-week-old flies in TOR^UAS-RNAi group. (Q) Body weight of 5-week-old flies in TOR^UAS-RNAi group. (R) Climb height of 1,3,4,5 week-old flies in TOR^UAS-RNAi group within 3 seconds. (S) Skeletal muscle ROS levels in TOR^UAS-RNAi group. (T) SOD level in skeletal muscle of TOR^UAS-RNAi group. (U) Relative expression of TOR gene in skeletal muscle of TOR^UAS-RNAi group. (V) Relative expression of ATG2 gene in skeletal muscle of TOR^UAS-RNAi group. (W) Relative expression of PGC-1α gene in skeletal muscle of TOR^UAS-RNAi group. (X) Relative expression of SIR2 gene in skeletal muscle of TOR^UAS-RNAi group. (Y) myosin heavy chain immunofluorescence in TOR^UAS-OE group, with blue dots as nucleus and red fluorescent bands as myofibril. HSD reduces fluorescence expression (scale: white line is 5 microns). (Z) TOR^UAS-RNAi group myosin heavy chain immunofluorescence. (α) Skeletal muscle transmission electron microscopy (scale: small mesh represents 0.2 microns). The white arrow points to the myofibril. The red arrow points to the mitochondria. Climbing height measurements, the sample size of each group is about 150–170 animals. Univariate analysis of variance (ANOVA) and minimum significance difference (LSD) tests were used to determine differences between groups. The P-values of climbing endurance curve and survival curve were calculated by log-rank test. Data were expressed by mean ±SEM. * P < 0.05; * * P < 0.01; * * * P < 0.001; n indicates no significant difference. The sample size of proteins and ROS was muscle of 20 flies per group, measured 3 times. The sample size for RT-PCR and ELISA was muscle of 20 flies per group, measured 3 times. Univariate analysis of variance (ANOVA) and minimum significance difference (LSD) tests were used to determine differences between groups. Data were expressed by mean ±SEM. * P < 0.05; * * P < 0.01; n indicates no significant difference.

Fig 6. Effects of TOR overexpression on body weight, climbing ability and skeletal muscle physiological structure of TOR^UAS-OE flies’.

(A) Fatigue time of 1-week-old flies. (B) Fatigue time of flies at 3 weeks of age. (C) Fatigue time of flies at 4 weeks of age. (D) Fatigue time of 5 week-old flies. (E) Body weight of flies at 5 weeks of age. (F) Climb height of 1,3,4,5 week-old flies within 3 seconds. (G) Skeletal muscle ROS levels. (H) SOD level in skeletal muscle. (I) Relative expression of TOR gene in skeletal muscle. (J) Relative expression of skeletal muscle ATG2 gene. (K) Relative expression of PGC-1α gene in skeletal muscle. (L) Relative expression of SIR2 gene in skeletal muscle. (M) myosin heavy chain immunofluorescence in TOR^UAS-OE group, with blue dots as nucleus and red fluorescent bands as myofibril. HSD reduces fluorescence expression (scale: white line is 5 microns). (N) Skeletal muscle transmission electron microscopy (scale: small mesh represents 0.2 microns). The white arrow points to the myofibril. The red arrow points to the mitochondria. Climbing height measurements, the sample size of each group is about 150–170 animals. Univariate analysis of variance (ANOVA) and minimum significance difference (LSD) tests were used to determine differences between groups. The P-values of climbing endurance curve and survival curve were calculated by log-rank test. Data were expressed by mean ±SEM. * P < 0.05; * * P < 0.01; * * * P < 0.001; n indicates no significant difference. The sample size of proteins and ROS was muscle of 20 flies per group, measured 3 times. The sample size for RT-PCR and ELISA was muscle of 20 flies per group, measured 3 times. (A) Univariate analysis of variance (ANOVA) and minimum significance Difference (LSD) tests were used to determine inter-group differences. Data were expressed by mean ±SEM. * P < 0.05; * * P < 0.01; n indicates no significant difference.

Fig 7. Effects of exercise on body weight, climbing ability and skeletal muscle physiological structure of TOR^OE flies.

(A) Fatigue time of 1-week-old flies. (B) Fatigue time of flies at 3 weeks of age. (C) Fatigue time of flies at 4 weeks of age. (D) Fatigue time of 5 week-old flies. (E) Body weight of flies at 5 weeks of age. (F) Climb height of 1,3,4,5 week-old flies within 3 seconds. (G) Skeletal muscle ROS levels. (H) SOD level in skeletal muscle. (I) Relative expression of TOR gene in skeletal muscle. (J) Relative expression of skeletal muscle ATG2 gene. (K) Relative expression of PGC-1α gene in skeletal muscle. (L) Relative expression of SIR2 gene in skeletal muscle. (M) myosin heavy chain immunofluorescence in TOR^UAS-OE group, with blue dots as nucleus and red fluorescent bands as myofibril. HSD reduces fluorescence expression (scale: white line is 5 microns). Climbing height measurements, the sample size of each group is about 150–170 animals. Univariate analysis of variance (ANOVA) and minimum significance difference (LSD) tests were used to determine differences between groups. The P-values of climbing endurance curve and survival curve were calculated by log-rank test. Data were expressed by mean ±SEM. * P < 0.05; * * P < 0.01; * * * P < 0.001; n indicates no significant difference. The sample size of proteins and ROS was muscle of 20 flies per group, measured 3 times. The sample size for RT-PCR and ELISA was muscle of 20 flies per group, measured 3 times. (A) Univariate analysis of variance (ANOVA) and minimum significance Difference (LSD) tests were used to determine inter-group differences. Data were expressed by mean ±SEM. * P < 0.05; * * P < 0.01; n indicates no significant difference.

Fig 8. Effects of HSD on body weight, climbing ability and skeletal muscle physiological structure of TOR^OE flies.

(A) Fatigue time of 1-week-old flies. (B) Fatigue time of flies at 3 weeks of age. (C) Fatigue time of flies at 4 weeks of age. (D) Fatigue time of 5 week-old flies. (E) Body weight of flies at 5 weeks of age. (F) Climb height of 1,3,4,5 week-old flies within 3 seconds. (G) Skeletal muscle ROS levels. (H) SOD level in skeletal muscle. (I) Relative expression of TOR gene in skeletal muscle. (J) Relative expression of skeletal muscle ATG2 gene. (K) Relative expression of PGC-1α gene in skeletal muscle. (L) Relative expression of SIR2 gene in skeletal muscle. (M) myosin heavy chain immunofluorescence in TOR^UAS-OE group, with blue dots as nucleus and red fluorescent bands as myofibril. HSD reduces fluorescence expression (scale: white line is 5 microns). Climbing height measurements, the sample size of each group is about 150–170 animals. Univariate analysis of variance (ANOVA) and minimum significance difference (LSD) tests were used to determine differences between groups. The P-values of climbing endurance curve and survival curve were calculated by log-rank test. Data were expressed by mean ±SEM.* P < 0.05; * * P < 0.01; * * * P < 0.001; n indicates no significant difference. The sample size of proteins and ROS was muscle of 20 flies per group, measured 3 times. The sample size for RT-PCR and ELISA was muscle of 20 flies per group, measured 3 times. (A) Univariate analysis of variance (ANOVA) and minimum significance Difference (LSD) tests were used to determine inter-group differences. Data were expressed by mean ±SEM. * P < 0.05; * * P < 0.01; n indicates no significant difference.

Fig 9. Effects of exercise and HSD on body weight, climbing ability and skeletal muscle physiological structure of TOR^OE flies.

(A) Fatigue time of 1-week-old flies. (B) Fatigue time of flies at 3 weeks of age. (C) Fatigue time of flies at 4 weeks of age. (D) Fatigue time of 5 week-old flies. (E) Body weight of flies at 5 weeks of age. (F) Climb height of 1,3,4,5 week-old flies within 3 seconds. (G) Skeletal muscle ROS levels. (H) SOD level in skeletal muscle. (I) Relative expression of TOR gene in skeletal muscle. (J) Relative expression of skeletal muscle ATG2 gene. (K) Relative expression of PGC-1α gene in skeletal muscle. (L) Relative expression of SIR2 gene in skeletal muscle. (M) myosin heavy chain immunofluorescence in TOR^UAS-OE group, with blue dots as nucleus and red fluorescent bands as myofibril. HSD reduces fluorescence expression (scale: white line is 5 microns). Climbing height measurements, the sample size of each group is about 150–170 animals. Univariate analysis of variance (ANOVA) and minimum significance difference (LSD) tests were used to determine differences between groups. The P-values of climbing endurance curve and survival curve were calculated by log-rank test. Data were expressed by mean ±SEM. * P < 0.05; * * P < 0.01; * * * P < 0.001; n indicates no significant difference. The sample size of proteins and ROS was muscle of 20 flies per group, measured 3 times. The sample size for RT-PCR and ELISA was muscle of 20 flies per group, measured 3 times. (A) Univariate analysis of variance (ANOVA) and minimum significance Difference (LSD) tests were used to determine inter-group differences. Data were expressed by mean ±SEM. * P < 0.05; * * P < 0.01; n indicates no significant difference.

Fig 10. Effects of TOR knockdown on body weight, climbing ability and skeletal muscle physiological structure of TOR^UAS-RNAi flies.

(A) Fatigue time of 1-week-old flies. (B) Fatigue time of flies at 3 weeks of age. (C) Fatigue time of flies at 4 weeks of age. (D) Fatigue time of 5 week-old flies.(E) Body weight of flies at 5 weeks of age. (F) Climb height of 1,3,4,5 week-old flies within 3 seconds. (G) Skeletal muscle ROS levels. (H) SOD level in skeletal muscle. (I) Relative expression of TOR gene in skeletal muscle. (J) Relative expression of skeletal muscle ATG2 gene. (K) Relative expression of PGC-1α gene in skeletal muscle. (L) Relative expression of SIR2 gene in skeletal muscle. (M) myosin heavy chain immunofluorescence in TOR^UAS-OE group, with blue dots as nucleus and red fluorescent bands as myofibril. HSD reduces fluorescence expression (scale: white line is 5 microns). Climbing height measurements, the sample size of each group is about 150–170 animals. Univariate analysis of variance (ANOVA) and minimum significance difference (LSD) tests were used to determine differences between groups. The P-values of climbing endurance curve and survival curve were calculated by log-rank test. Data were expressed by mean ±SEM. * P < 0.05; * * P < 0.01; * * * P < 0.001; n indicates no significant difference. The sample size of proteins and ROS was muscle of 20 flies per group, measured 3 times. The sample size for RT-PCR and ELISA was muscle of 20 flies per group, measured 3 times. (A) Univariate analysis of variance (ANOVA) and minimum significance Difference (LSD) tests were used to determine inter-group differences. Data were expressed by mean ±SEM. * P < 0.05; * * P < 0.01; n indicates no significant difference.

Fig 11. Effects of exercise on body weight, climbing ability and skeletal muscle physiological structure of TOR^RNAi flies.

(A) Fatigue time of 1-week-old flies. (B) Fatigue time of flies at 3 weeks of age. (C) Fatigue time of flies at 4 weeks of age. (D) Fatigue time of 5 week-old flies. (E) Body weight of flies at 5 weeks of age. (F) Climb height of 1,3,4,5 week-old flies within 3 seconds. (G) Skeletal muscle ROS levels. (H) SOD level in skeletal muscle. (I) Relative expression of TOR gene in skeletal muscle. (J) Relative expression of skeletal muscle ATG2 gene. (K) Relative expression of PGC-1α gene in skeletal muscle. (L) Relative expression of SIR2 gene in skeletal muscle. (M) myosin heavy chain immunofluorescence in TOR^UAS-OE group, with blue dots as nucleus and red fluorescent bands as myofibril. HSD reduces fluorescence expression (scale: white line is 5 microns). Climbing height measurements, the sample size of each group is about 150–170 animals. Univariate analysis of variance (ANOVA) and minimum significance difference (LSD) tests were used to determine differences between groups. The P-values of climbing endurance curve and survival curve were calculated by log-rank test. Data were expressed by mean ±SEM.* P < 0.05; * * P < 0.01; * * * P < 0.001; n indicates no significant difference. The sample size of proteins and ROS was muscle of 20 flies per group, measured 3 times. The sample size for RT-PCR and ELISA was muscle of 20 flies per group, measured 3 times. (A) Univariate analysis of variance (ANOVA) and minimum significance Difference (LSD) tests were used to determine inter-group differences. Data were expressed by mean ±SEM. * P < 0.05; * * P < 0.01; n indicates no significant difference.

Fig 12. Effects of HSD on body weight, climbing ability and skeletal muscle physiological structure of TOR^RNAi flies.

(A) Fatigue time of 1-week-old flies. (B) Fatigue time of flies at 3 weeks of age. (C) Fatigue time of flies at 4 weeks of age. (D) Fatigue time of 5 week-old flies. (E) Body weight of flies at 5 weeks of age. (F) Climb height of 1,3,4,5 week-old flies within 3 seconds. (G) Skeletal muscle ROS levels. (H) SOD level in skeletal muscle. (I) Relative expression of TOR gene in skeletal muscle. (J) Relative expression of skeletal muscle ATG2 gene. (K) Relative expression of PGC-1α gene in skeletal muscle. (L) Relative expression of SIR2 gene in skeletal muscle. (M) myosin heavy chain immunofluorescence in TOR^UAS-OE group, with blue dots as nucleus and red fluorescent bands as myofibril.HSD reduces fluorescence expression (scale: white line is 5 microns). Climbing height measurements, the sample size of each group is about 150–170 animals. Univariate analysis of variance (ANOVA) and minimum significance difference (LSD) tests were used to determine differences between groups. The P-values of climbing endurance curve and survival curve were calculated by log-rank test. Data were expressed by mean ±SEM.* P < 0.05; * * P < 0.01; * * * P < 0.001; n indicates no significant difference. The sample size of proteins and ROS was muscle of 20 flies per group, measured 3 times. The sample size for RT-PCR and ELISA was muscle of 20 flies per group, measured 3 times. (A) Univariate analysis of variance (ANOVA) and minimum significance Difference (LSD) tests were used to determine inter-group differences. Data were expressed by mean ±SEM. * P < 0.05; * * P < 0.01; n indicates no significant difference.

Fig 13. Effects of exercise and HSD on body weight, climbing ability and skeletal muscle physiological structure of TOR^RNAi flies.

(A) Fatigue time of 1-week-old flies. (B) Fatigue time of flies at 3 weeks of age. (C) Fatigue time of flies at 4 weeks of age. (D) Fatigue time of 5 week-old flies. (E) Body weight of flies at 5 weeks of age. (F) Climb height of 1,3,4,5 week-old flies within 3 seconds. (G) Skeletal muscle ROS levels. (H) SOD level in skeletal muscle. (I) Relative expression of TOR gene in skeletal muscle. (J) Relative expression of skeletal muscle ATG2 gene. (K) Relative expression of PGC-1α gene in skeletal muscle. (L) Relative expression of SIR2 gene in skeletal muscle. (M) myosin heavy chain immunofluorescence in TOR^UAS-OE group, with blue dots as nucleus and red fluorescent bands as myofibril. HSD reduces fluorescence expression (scale: white line is 5 microns). Climbing height measurements, the sample size of each group is about 150–170 animals. Univariate analysis of variance (ANOVA) and minimum significance difference (LSD) tests were used to determine differences between groups. The P-values of climbing endurance curve and survival curve were calculated by log-rank test. Data were expressed by mean ±SEM.* P < 0.05; * * P < 0.01; * * * P < 0.001; n indicates no significant difference. The sample size of proteins and ROS was muscle of 20 flies per group, measured 3 times. The sample size for RT-PCR and ELISA was muscle of 20 flies per group, measured 3 times. (A) Univariate analysis of variance (ANOVA) and minimum significance Difference (LSD) tests were used to determine inter-group differences. Data were expressed by mean ±SEM. * P < 0.05; * * P < 0.01; n indicates no significant difference.