Effects Of Exercises And Huangqidanshen On Mitochondrial Biogenesis In Rat Skeletal Muscle

Mitochondria play a central role not only in a variety of metabolic pathways including the supply of the bulk of cellular ATP through oxidative phosphorylation (OXPHOS), but also in complex processes such as development, apoptosis, and aging. Mitochondrial biogenesis can be defined as an increase in mitochondrial numbers and/or mass, formation of new mitochondria in cells and their phylogenesis. The human mtDNA is a circle of only 16.6kb double stranded DNA encoding just 13 proteins and 22 tRNA and 2 rRNA. It is not feasible to construct so complex an organelle from so few proteins, and a total of about 850 proteins are in fact required, the majority encoded in the nuclear DNA (nDNA), thus the mitochondrial biogenesis needs the coexpression of both mtDNA and nDNA. ROS, maily is hydrogen peroxide can act as a second messenger, regulates the signal transduction and the involved factors. As a result, the construction, function and mitochondrial biogenesis are regulated. Recent research revealed exercises increase the biogenesis of mitochondria, but the mechanisms still remain unclear. Based on the above reasons, there will be practical significance in the research on the effects of exercise and Huangqidanshen on mitochondrial biogenesis in order to understand the adaptation mechanisms of exercise and pharmacology action.The research aims to: (1) explore the hypothesis that phasic changes of mitochondrial biogenesis related genes in PGC-1α, NRF-1, mtTFA and mtTFB2 are involved in acute exercise and endurance training. And Complex IV (COX IV), Nitric Oxide Synthase (NOS), Superoxide Dismutase (SOD) and phospho-AKt are related to the exercises. (2) find out the hypothesis that increases in PGC-1α, NRF-1, mtTFA and mtTFB2 are involved in the primary skeletal muscle cells treated with H₂O₂. To know whether ROS affected the mitochondrial biogenesis related factors. (3) understand that if changes in mitochondrial biogenesis related genes: mtTFA, PGC-1α, NRF-1 and mtTFB2 are involved in the adaptive response of muscle to endurance training and/or with Huangqidanshen. 1. Effects of acute and endurance exercise on mitochondrial biogenesis in rat skeletal muscleIn this research, the Sprange-Dawley male rats are the subjects. We used two kinds of exercises: acute exercise (AE) and endurance training (ET). 42 three-month-old rats of AE were randomly divided into 7 groups: at rest (C1) , Oh group (tested immediately after swimming), 3h, 12h, 6h, 18h, 24h after one swimming bout. The rats were accustomed to swimming for 10 min/day for 2 days. They were then exercised by a modification of the procedure of Ploug et al, which involves swimming for two 3h long bouts separated by a 45 min rest, during which the rats were kept warm and given food and water. The rats swam in group of six in water tank filled to depth of 60cm, with water maintained at 35℃. All rats performed this exercise program once and then were anesthetized for removal of gastrocnemius muscles 0, 3, 6, 12, 18, 24h after cessation of exercise. 42 male SD rats of ET, weighed averagely 170±10g, were randomly divided into 7 groups : control group(C2) , immediate group (OH) ,and 3H, 6H, 12H, 18H, 24H after the last swimming exercise. The rats were accustomed to swimming for 10 min/day for 2 days and then swam for 40min for first four weeks and swam for 60min in next two weeks. After 6 weeks of swimming, gastrocnemius muscles were isolated from all seven groups. Mitochondria were isolated and then the activity of enzymes were measured. mRNA expression of PGC-1α, NRF-1, mtTFA and mtTFB2 were measured with Real Time PCR. Phospho-Akt (Ser473) were detected by Western-blotting. Skeletal muscle mitochondrial structure were assayed by using transmission electron microscope.Results: PGC-1αall increased at 6h and 12h postexercise in two exercise types. NRF-1 increased at 3h and 6h after AE, and increased at 6h and 12h after ET. AE increased mtTFA at 12,18 and 24h, but in ET it increased at 6h and 12h postexercise. mtTFB2 only increased at 18h AE and at 12h ET. In AE the activity of NOS increased firstly and then decreased whereas the activity of NOS increased at all time courses in ET, it is the same of the activity of COX IV. The activity of SOD increased at Oh, 3h, 6h, 12h in AE but only increased at 18h, 24h in ET. The number of cristaes decreased and the matrix expanded in mitochondria of AE, but it still normal in ET.Conclusion: (1) Both acute exercise and endurance training can induce the mitochondrial biogenesis mainly through the PGC-1α/ mtTFA pathway. (2) Exercises can increase NOS, SOD and COX IV activity, which inverse modulate the mitochondrial biogenesis and the function of respiratory chain. (3) Exercises induced mitochondrial biogenesis partly through activating the AKt pathway. (4) PGC-1αmay not involved in the beginning of mitochondrial biogenesis.2. Effects of H₂O₂ on mitochondrial biogenesis in primary skeletal muscle cellsPrimary skeletal muscle cell cultures were obtained by trypsinization of small muscle pieces of 1 day-old rats (Sprague-Dawley) hindlimbs. Survival of cells seeded in 96-well microtiter plates with different concentration (0, 25, 50,100, 200, 400μM) of H₂O₂. Cells were seeded and treated with up to 100μM H₂O₂ (THO), and then collected at different time point (0, 1h, 6h, 18h) for RNA extraction, MTT assay and transmission electron microscope detection.Results: With cells THO 1h increased in NRF-1 mRNA level, 3h THO increased in PGC-1α, mtTFA and mtTFB2, at time course of 6 and 18h, the mtTFB2 also increased significantly. The activity of NOS increased in cells THO 1h and 3h. COXIV activity increased at 1h and 3h. The number of mitochondria were increased and swelled like a ball at 1h. The cristaes were almost disappeared, and the membrane of mitochondria are damaged at 3h and 6h. At 18h, the matrix of mitochondria changed into floccule and most form a big vacuole.Conclusion: (1) Concentration of H₂O₂ is more important than time course affect the cell survival. 100μM H₂O₂ may be the proper concentration of exogenous addition. (2) ROS may be an important second molecular messenger which intermediates in primary metabolism and regulates the specific signal transduction pathways and gene expression involved the biogenesis of mitochondria. (3) The effect of proper amount H₂O₂ on cells is the activation of Akt, which preserves the mitochondrial integrity. 3. Effects of Compound Huangqidanshen with swimming training on mitochondrial biogenesis in Rat Skeletal MuscleIn this research, 36 male SD rats, about 2 months were randomly divided into 6 groups, which including: the normal control group(C2); injection every three days control group (C3); injection eyery five days control group (C5); swimming for six weeks group (OH); injection every three days with swimming group (SD); injection every five days with swimming group (SI5). The activity of gastrocnemius mitochondrial COX IV, NOS and SOD were detected. PGC-1α, mtTFA, mtTFB2 and NRF-1 mRNA expression were detected by SYBRGreen I Quantitave reverse transcription-PCR. Phospho-Akt (Ser473) were detected by Western-blotting. Skeletal muscle mitochondrial structure were assayed by using transmission electron microscope.Results As compared to C2 group , COX IV activity increased in all groups(p<0.01), NOS activity only decreased in OH group, SOD activity only increased in SD group, while the levels of PGC-1αmRNA expression were significantly increased respectively in SD and SI5 group, but decreased in OH group after six weeks of swimming training(p<0.01). mtTFA mRNA expression were significantly increased in SD group, but decreased in C5 group. mtTFB2 mRNA expression were significantly increased in SD group, but decreased in OH group. NRF-1 mRNA level were only increased in SD group. As compared to OH group, COX IV activity increase in SD and SI5 group, SOD activity only increased in SD group. NOS activity and PGC-1αmRNA level is all the same, wherease the mtTFA mRNA level is only increase in SD group (p<0.01). Both mtTFB2 and NRF-1 mRNA level increased in SD and SI5 group. For Phospho-Akt, compared with C2 group, all the groups were increased. SD and SI5 groups were decreased respectively when compared with OH, whereas C3 and C5 group were increased (p<0.01). Both the quantity and volume of mitochondria were increased and mitochondrial structure was still in good condition. Conclusion (1) Both swimming and huangqidanshen injection can induce the biogenesis of mitochondria, and keep the mitochondrial function in good condition. Mainly through the Akt—NRF-1—mtTFA pathway; Akt—NOS mitochondria pathway and the MAPK—PGC-1α—NRF-1—mtTFA pathway. (2) Combination is better than single stimulation since the mRNA level is highest in combination group. There may exist some new factors or pathways besides PI3K/Akt triggering mitochondrial biogenesis, but it must involved the downstream factor: PGC-1α, NRF-1, mtTFA, mtTFB2. (3) New insights should be provided into possible therapeutic interventions to care for those who have mitochondrially-based diseases but unable to withstand regular physical activity or for those who want to improve the capacity of exercise.Summary: 1. Phasic changes of mitochondrial biogenesis related genes and enzymes are involved in acute exercise and endurance training. 2. H₂O₂ act as the second signal induced the mitochondrial biogenesis decided on its density and effect time. 3. Also huangqidanshen could affect mitochondrial biogenesis through AKt pathway.