Protective Role And Potential Mechanism Of Dihydromyricetin And Myricetin On Physical Performance Under Acute Hypoxic Conditions

Backgrounds:When people go to high altitudes in a short time, reduced oxygen availability at decreased atmospheric pressures will lead to a sinificantly reduced physical performance. Pre-arranged altitudinal adaptation which presented a stairs type to continuously rising is one of the best preventive strategies. However, the troops need to march rapidly forward to the high plateau and race to the battle in the future military warfare, thus the aforementioned acclimatization method makes little sense in wartime. Recently studies on exogenous nutrition intervention for troops carrying out a mission at high altitude, so as to acclimate hypobaric hypoxia, were becoming the cross-interdisciplinary hot research area including sport medicine, military medicine and nutriology.Based on the literature review and our pilot study, we noted that polyphenolic compounds have been increasingly addressed according to their multiple biological activities including antioxidation, anti-inflammation and free radicals scavenging, et al. Several studies have demonstrated that quercetin, a representative polyphenolic compound, could increase mitochondrial biogenesis and improve physical performance in normoxic conditions. Therefore we proposed the research hypothese: hypoxia-induced mitochondrial dysfunction contributes to a impaired energy production in skeletal muscle and then results in the exercise intolerance in hypoxia.Natural polyphenolic compounds can effectively improve exercise performance under acute hypoxia exposure by alleviating mitochondrial impairment and promoting mitochondrial repair.Objective:1. To investigate the effects of polyphenolic compounds on the physical performance in hypobaric hypoxia conditions in rats, and screen the candidates with effective protection, by the classical run-to-fatigue animal models under simulated high altitude conditions.2. To clarify the relationship between mitochondrial dysfunction of skeletal muscle and reduced physical performance under the plateau hypoxia, then explore the potential mechanism of the protective role of the selected compounds on physical performance in acute hypoxia conditions.Methods:Part I: the effect of common polyphenolic compounds on rat’s physical performance under hypobaric hypoxia conditions1. Rats were supplied with the corresponding reagents for consecutive 7 days after being divided into the following groups: a normoxia control group, a hypoxia control group, a rhodiola rosea positive control group, a quercetin experiment group, a myricetin experiment group, a dihydromyricetin(DHM) experiment group and a resveratrol experiment group. Each compound group was divided into several subgroups according to the predetermined dose;2. Rats were placed in a specially designed hypobaric chamber to expose to a simulated high altitude conditions, and then their physical performance was assessed with the run-to-fatigue procedure on a motorized treadmill. We observed the effect of the representative four compounds including quercetin, myricetin, DHM and resveratrol on rat’s run-to-fatigue time under the plateau hypoxia;3. Blood was collected immediately following the run-to-fatigue assessment test, then serum urea nitrogen(BUN), creatine kinase(CK), and lactate dehydrogenase(LDH) were measured in an automatic analyzer;4. Fresh muscle specimens were collected to observe ultrastructure of skeletal muscle through transmission electron microscopy(TEM).Part II: The effects and potential mechanisms of DHM on exercise-induced skeletal muscle mirco-damage.First quarter: DHM effectively reduces the ultrastructural damage of skeletal muscle by attenuating mitochondrial metabolic disturbance1. The muscle specimens of three dose subgroup in DHM and the corresponding control group were selected to analyze the enzymatic activities of electron transfer complexesⅠ,Ⅱ,Ⅳ and Ⅴ using an enzyme-linked immuno-sorbent assay(ELISA);2. The mt DNA was isolated from gastrocnemius muscle tissues, and quantification of mt DNA copy number was performed by real-time PCR;3. Proteins expression of mitochondrial fusion and fission markers was detected by western blotting.Second quarter: The role and mechanism of SIRT3 in DHM’s protective effect on exercise-induced muscle mirco-damage1. WT and SIRT3 KO 129Sv male mice were divided into five groups: a rested control group, an exercise control group, a DHM-exercise control group(100mg/kg.bw/d), an exercise SIRT3 KO group and a DHM-exercise SIRT3 KO group(100mg/kg.bw/d). Dosages were administered intragastrically through all groups(4 weeks length);2. All animals(except rested control group) were running on a motorized treadmill for 4 weeks’ exhaustive exercise(5 days/week). The next day of the experiment, all exercise mice performed the run-to-fatigue procedure. Then blood serum and muscle specimens were collected;3. serum creatine kinase(CK), lactate dehydrogenase(LDH), total superoxide dismutase(T-SOD) and tissue malondialdehyde(MDA) activities were measured using related kit;4. Ultrastructure of muscle was observed through transmission electron microscopy(TEM);5. Proteins expression of SIRT3 and mitochondrial functional markers in gastrocnemius muscle tissues was detected by western blotting and immunohistochemistry;6. Co-immunoprecipitation of SIRT3-OPA1 in gastrocnemius muscle tissues was detected.Part III: The effects and potential mechanisms of myricetin on mitochondrial biogenesis in skeletal muscle cells under hypoxia conditions1. The rat L6 myogenic cell line was cultured and induced differentiation. The extent of differentiation was established by assaying the gene expression of myogenin and myosin heavy chain(MHC) by real-time PCR. Differentiated L6 cells were divided into five groups: a normoxia control group, a hypoxia control group, a myricetin group, a SIRT1-inhibited myricetin group and an AMPK-inhibited myricetin group, the latter four groups were then remained in the hypoxic incubator for 16 h incubation periods;2. Mitochondrial membrane potential of the rat L6 myogenic cell was measured by JC-1 staining;3. The muscle specimens of three dose subgroup in myricetin group and the corresponding control groups were selected to detect the activities of electron transfer complexes I,II,IV and V in skeletal muscle using an enzyme-linked immuno-sorbent assay(ELISA);4. Ultrastructure of mitochondria was observed through transmission electron microscopy(TEM);5. The mtDNA was isolated from gastrocnemius muscle tissues and L6 cells and quantification of mt DNA copy number was performed by real-time PCR;6. Proteins expression of mitochondrial biogenesis markers in both gastrocnemius muscle tissues and L6 cells was detected by western blotting;7. Proteins expression of mitochondrial biogenesis markers in gastrocnemius muscle tissues and L6 cells was detected by Immunohistochemistry;8. Proteins expression of mitochondrial biogenesis markers in L6 cells was detected by Immunofluorescence staining.Results:I: The polyphenolic compounds including quercetin, DHM and myricetin protect exercise performance of rat in acute hypobaric hypoxia exposure.1. The average run-to-fatigue time in the hypoxia control group dramatically decreased, and was only approximately one fifth compared with that in normoxia control group;2. With the exception of resveratrol, pretreatment of each of the other three representative polyphenolic compounds including quercetin, DHM and myricetin increased run-to-fatigue time compared to their respective hypoxia control group, however, with different dose-effect curves. The protevtive efficiency on run-to-fatigue time of DHM and myricetin was similar and more prominent. At 75 mg/kg.bw/d dose, both DHM and myricetin could significantly increase run-to-fatigue time, whereas quercetin could play such role at 150mg/kg.bw/d dose;3. After the run-to-fatigue procedure under simulated high altitude conditions, the plasma LDH, BUN, and CK in the hypoxia control group were significantly higher than those in the normoxia control group;4. DHM and myricetin pretreatment had significant impact on BUN, both of them caused lower BUN levels than the hypoxia control group. However, quercetin and resveratrol had little effect on BUN levels, even at the highest dose of 200mg/kg.bw/d;5. With the exception of resveratrol, pretreatment of each of the other three representative polyphenolic compounds could cause lower plasma LDH levels after the run-to-fatigue procedure under the plateau hypoxia, however, with different dose-effect curves, and myricetin had the strongest effect;6. Quercetin, DHM and myricetin could decrease lower plasma CK levels after the run-to-fatigue procedure under the plateau hypoxia, and DHM had the strongest effect;7. Acute hypobaric hypoxia exposure resulted in the ultrastructural damage of skeletal muscle, which manifested as obvious disorder of myofibril, Blurred Z line and dissolution of sarcoplasmic reticulum. DHM pretreatment could significantly improve the above injury, whereas the effect of the other three compounds were not really obvious.II: DHM administration effectively reduces the ultrastructural damage of skeletal muscle, maintains mitochondrial dynamics of fusion/fission, protects the activity of mitochondrial respiratory chain complexes and promotes mitochondrial proliferation in an acute hypobaric hypoxia exposure.1. Acute hypobaric hypoxia exposure caused a significant decrease in the activities of mitochondrial complexes I, II, IV, and V in skeletal muscle of rats. Compared to the hypoxia control group, the DHM group had higher complex I, II, but not V, activities in muscle cells at the dose of 100mg/kg.bw/d;2. The relative amount of mtDNA in muscle cells significantly decreased in the hypoxia control group, and was only half compared with that in normoxia control group. DHM pretreatment could effectively increased the mtDNA content and in a dose-dependent manner;3. Mitochondrial fusion-related proteins(MFN1 and MFN2) expressions were significantly decreased and the fission-related proteins(DRP1 and FIS1) expressions were increased in the hypoxia control group compared to normoxia control group, and DHM pretreatment could effectively restrain the above trend of mitochondrial fission and fusion at the dose of 100mg/kg.bw/d.III: SIRT3 plays a critical role in DHM-induced protections on excessive exercise-caused muscle mirco-damage1. DHM could protect skeletal muscle in the wild-type mice from ultrastructural damage;2. DHM showed no significant effects on skeletal muscle in SIRT3 KO mice with ultrastructural damage;3. Excessive exercise resulted in the decrease of the protein expression of PGC-1α and ERRα in skeletal muscle in the wild-type mice, which were significantly attenuated by DHM administration, and this effect was not influenced by the SIRT3 knockout;4. Co-immunoprecipitation results showed that SIRT3 and OPA1 were closely related by deacetylation. Western blotting results indicated that mitochondrial morphodynamic process was facilitated towards catabolism by excessive exercise, which were significantly attenuated by DHM treatment. However, such effect was not observed in SIRT3 KO mouse.IV: Myricetin could promote mitochondrial biogenesis of skeletal muscle in both acute hypoxia exposure and normoxic conditions, and attenuate acute hypoxia-induced mitochondrial dysfunction via protecting the activities of respiratory chain complexes and mitochondrial membrane potential(ΔΨm)1. Acute hypobaric hypoxia exposure resulted in mitochondrial structural impairments, and myricetin could effectively protect mitochondria from the impairment in a dose-dependent manner;2. Acute hypobaric hypoxia exposure and exhausted exercise caused a significant decrease in the activities of mitochondrial complexes I, II, IV, and V in skeletal muscle of rats, while treatment with myricetin significantly improved the activities of mitochondrial complexes(I, II, IV) compared with the hypoxia control group;3. Acute hypoxia resulted in dissipation of ΔΨm. However, Myricetin treatment could significantly prevent hypoxia-induced dissipation of ΔΨm in L6 cells;4. Pretreatment with myricetin effectively prevented the decline of the mt DNA content caused by acute hypobaric hypoxia exposure, and maintained protein expression of mitochondrial biogenesis parameters including PGC-1a, NRF-1 and TFAM in acute plateau hypoxia both in gastrocnemius muscle and L6 cells;5. Myricetin supplementation could increase mt DNA amount and promote mitochondrial biogenesis parameters(PGC-1a, NRF-1 and TFAM) of skeletal muscle under normoxic conditions;6. Acute hypobaric hypoxia exposure and exhausted exercise resulted in the decrease of the protein expression of SIRT1 and AMPK in gastrocnemius muscle and L6 cells, which were significantly attenuated by myricetin treatment;7. The AMPK inhibitor significantly compromised the effects of myricetin on the expression of mitochondrial biogenesis markers and mt DNA content, while the SIRT1 inhibitor showed no apparent influence.Conclusions:1. Supplementation of quercetin, DHM or myricetin before acute hypoxic exposure could be beneficial to maintain exercise capacity. The efficiency of DHM and myricetin was similar and more prominent at the dose of 100mg/kg.bw/d.2. DHM effectively alleviates the ultrastructural damage of skeletal muscle in acute hypobaric hypoxia exposure by maintaining mitochondrial dynamics of fusion/fission, moreover it protects activity of mitochondrial respiratory chain complexes and promotes mitochondrial proliferation, which contribute to the significant preventions on the impairment of exercise capacity.3. SIRT3 plays a key role in DHM-induced protective effects on excessive exercise-caused muscle mirco-damage.The protection of DHM is achieved partailly by regulating the mitochondrial morphodynamics(fussion and fission) via PGC-1α-SIRT3-OPA1 pathway.4. Myricetin protects exercise performance in acute hypobaric hypoxia exposure by counteracting mitochondrial dysfunction and promoting mitochondrial biogenesis, in which AMPK-PGC-1a and AMPK-SIRT1-PGC-1a pathways play a crucial role in this process.In a word, the present study firstly found the protective effect of DHM and myricetin on exercise performance under acute hypobaric hypoxia conditions, by alleviating ultrastructural damages of skeletal muscle and promoting mitochondrial biogenesis, respectively. The action targets are clearly different from that of classic anti-altitude sickness drugs. Thus the present study may expand researchers’ idea on anti-altitude sickness drugs and provide new solutions for rapid acclimatization under hypobaric hypoxia conditions.