Rhodiola Crenulata ameliorates exhaustive exercise-induced fatigue in mice by suppressing mitophagy in skeletal muscle

Abstract

The aim of present study was to evaluate the potential effects of Rhodiola crenulata oral liquid (RCOL) on exhaustive exercise (EE)-induced fatigue in mice. Male Institute of Cancer Research mice from five treatment groups (n=10 per group) were orally administered with sterilized water for the Control and EE groups and/or RCOL at doses of 1.02, 3.03 and 6.06 ml/kg/day, once daily for 2 weeks. Anti-fatigue activity was subsequently evaluated by measuring the levels of creatine kinase (CK), lactic acid (LA), lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and total anti-oxidative capability (T-AOC). Histopathology was assessed using hematoxylin and eosin staining. Ultrastructures of mitochondria were observed by transmission electron microscopy. Energy supply capacity was assessed using citrate synthase (CS), succinate dehydrogenase (SDH), Na⁺-K⁺-ATPase, and liver and quadriceps glycogen content assays. Expression levels of mRNA and protein associated with mitophagy in the skeletal muscle were measured by reverse transcription-quantitative PCR and western blotting, respectively. RCOL was observed to markedly inhibit fatigue-induced oxidative stress by increasing the activities of SOD, CAT and T-AOC, whilst reducing the accumulation of LA, CK, LDH and MDA. Histological analysis of the quadriceps femoris tissue suggested increased numbers of muscle fibers in the RCOL groups compared with those in the EE group. RCOL administration was found to reverse EE-induced mitochondrial structural damage and alleviated defects inflicted onto the energy supply mechanism by increasing CS, SDH, Na⁺-K⁺-ATPase and glycogen levels. Additionally, RCOL reduced the protein expression of PTEN-induced kinase 1 (PINK1), Parkin, microtubule-associated proteins 1A/1B light chain 3, sequestosome 1 and ubiquitin, whilst lowering the gene expression of PINK1 and Parkin. Taken together, results from the present study clarified the anti-fatigue effect of RCOL, where the underlying mechanism may be associated with increased antioxidant activity, enhanced energy production and the inhibition of mitophagy by suppressing the PINK1/Parkin signaling pathway.

Keywords: Rhodiola crenulata oral liquid; anti-fatigue; loaded swimming; mitophagy; quadriceps femoris.

Figure 1

Experimental workflow of the present study. RCOL, Rhodiola crenulata oral liquid; EE, exhaustive exercise; ICR, Institute of Cancer research; W, week.

Figure 2

High performance liquid chromatography determination of the six major components contained within RCOL. (A) Chemical structures of the six mixed reference substances. (B) Determination of the relative content of the six main active components of RCOL. 1, gallic acid; 2, salidroside; 3, tyrosol; 4, catechin; 5, caffeic acid; 6, p-coumaric acid; RCOL, Rhodiola crenulata oral liquid.

Figure 3

Effects of RCOL administration on the histology and ultrastructure of quadriceps femoris tissue of mice subjected to EE. (A) Schematic diagram of quadriceps femoris of mice tissues. (B) Hematoxylin and eosin staining observed by optical light microscopy. The black arrow indicates areas of reduced muscle fibers. Scale bar, 50 µm. (C) Ultrastructural analysis of mitochondria by TEM. (C₁) Compared with the control group, (C₂) mice in the EE group exhibited more areas of damaged mitochondria. (C_3-5) Mice in the RCOL groups appeared to have less damaged ultrastructures with a comparable level of uniformity as that observed in the control group. Yellow arrows indicate the location of mitochondria. Red boxes indicate bright zones. The dark zone is located between the Z and M lines. Yellow boxes indicate H zones. Scale bar, 2 µm. n=5. RCOL, Rhodiola crenulata oral liquid; EE, exhaustive exercise; TEM, transmission electron microscopy; L, low dose; M, medium dose; H, high dose.

Figure 4

Effects of RCOL administration on energy production in mice. (A) Liver and (B) muscle glycogen content. (C) CS protein expression was measured in quadriceps femoris tissue and (D) quantified by semi-quantitative analysis. (E) SDH activity in quadriceps femoris tissue. (F) Na⁺-K⁺-ATPase activity in quadriceps femoris tissue. Data are presented as the mean ± SD. n=6. ^**P<0.01 vs. Control; ^#P<0.05 and ^##P<0.01 vs. EE. RCOL, Rhodiola crenulata oral liquid; EE, exhaustive exercise; L, low dose; M, medium dose; H, high dose; CS, citrate synthase; SDH, succinate dehydrogenase; ATP, adenosine triphosphate; prot, protein.

Figure 5

Effects of RCOL administration on the protein and mRNA expression of key PINK1/Parkin signaling pathway components. (A) Representative western blotting images showing the protein expression of PINK1, Parkin, LC3-II/LC3-I, p62 and ubiquitin in mice quadriceps femoris tissues. Semi-quantitative densitometric analysis of (B) PINK1, (C) p62, (D) Parkin, (E) LC3I and (F) ubiquitin. Relative mRNA expression of (G) PINK1 and (H) Parkin, two key proteins associated with mitophagy. Data are presented as the mean ± SD. n=6. ^**P<0.01 vs. Control; ^#P<0.05 and ^##P<0.01 vs. EE. RCOL, Rhodiola crenulata oral liquid; EE, exhaustive exercise; L, low dose; M, medium dose; H, high dose; PINK1, PTEN-induced kinase 1; LC3, microtubule associated protein 1 light chain 3.

Figure 6

Proposed molecular mechanism for the effect of RCOL pre-administration on improved exercise performance in fatigued mice. RCOL, Rhodiola crenulata oral liquid; EE, exhaustive exercise; PINK1, PTEN-induced kinase 1.