The amplification was performed in a total volume of 20 μL, which included 2 μL of cDNA, 1 μL of each primer (10 pmol/μL), and 16 μL of DEPC (Diethyl pyrocarbonate) water. The array of forward and reverse primer is the same as shown in Table 3. If the RNA pellet was completely dried, the RNA pellet was dissolved by adding the 0.01% DEPC-treated distilled water of 30 μL. 70% ethanol of 1 mL was added on RNA pellet formed on the bottom of tube by centrifugation, and the pellet was washed twice (4,500 rpm, 4☌, 5 minutes). The only clear supernatant solution was separated from the centrifuged solution into a new tube, and then was allowed to stand at room temperature for 10 minutes after adding the same amount of isopropanol (Sigma, USA) into this supernatant solution, and it was centrifuged (13,000 rpm, 4☌, 10 minutes).
Chloroform (Sigma, Missouri, USA) of 200 μL was added into this solution, and then the solution was allowed to stand for 3 minutes at room temperature after mixing for 15 seconds so that the chloroform was wholly mixed well, and was centrifuged (13,000 rpm, 4☌, 15 minutes). In order to extract total RNA, 40 μg of tissue was put into Trizol (Qiagen, Hilden, Germany) and the tissue was grinded for 20 seconds using a homogenizer, and the homogenized solution prepared from the homogenizer was allowed to stand at room temperature for 5 minutes. Nonetheless, there are only a few studies comparing the effects of exercise training and chrysin supplementation.
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Also, chrysin shows promising hepatoprotective and anti-hyperlipidemic effects, which are evidenced by the decreased levels of triglycerides, free fatty acids, total cholesterol, phospholipids, low-density lipoprotein-C, and very low-density lipoprotein-C and increased levels of high-density lipoprotein-C in the plasma and tissues of hepatotoxicity in rats.
Multiple studies have indicated antioxidant, antihypertensive, antidiabetogenic, and anxiolytic functions. Chrysin (5,7dihydroxyflavone) is a natural flavonoid contained in many plant extracts, honey and propolis. Flavonoids were reported to enhance insulin secretion, promote pancreatic β-cells proliferation, glucose uptake, and reduce insulin resistance, inflammation and oxidative stress. Flavonoids, one of these treat, have been reported for anti-inflammatory, antidiabetic, anticancer, neuro-protective and cardio-protective activities. Recently, many scientists have used a variety way to treat obesity induced metabolic complication such as caloric restriction and functional nutrition diet. Also, COX-IV is used as an indicator of aerobic exercise performance because it is controlled by PGC-1α that regulate mitochondrial biogenesis and function. COX-IV protein plays an important role in aerobic exercise capacity by regulation of mitochondrial oxiative phosphorylation.
And it has been known that the trained individuals showed a higher number of SIRT-3 proteins and mitochondria compared to sedentary individuals. showed that treadmill running increased the amount of SIRT-3 protein and mitochondria density in the skeletal muscle of the rats. showed that the expression of SIRT-3 was increased in 7-week-old FVB/NJ rats after 6 weeks of wheel-cage exercise. SIRT-1 (silent mating type information regulation 2, homolog 1) is activated by the PGC-1α and recent studies have shown that SIRT-3 (silent mating type information regulation 2, homolog 2) exists in the mitochondria and increases PGC-1α that induce mitochondrial biogenesis by regulating mitochondrial number and substrate utilization.
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