![]() ![]() Treatment of muscle atrophy through nutritional supplements from plants is becoming more and more popular. Muscle atrophy is a feature of sarcopenia that occurs often in elderly patients with chronic diseases such as type II diabetes mellitus (T2DM), chronic liver and kidney diseases, and chronic obstructive pulmonary disease (COPD). Effect of MON on the AKT/mTOR/FOXO3a Signaling Pathway in DEX-Treated Mice These results indicate that MON prevents the induction of atrophy in muscle by the down-regulation of the negative regulators of muscle growth thus increasing MyHC expression. The expression of Myostatin was significantly decreased in atrophied muscle tissues ( Figure 4E) after the administration of MON at levels of 40 mg/kg ( p < 0.05) and 80 mg/kg ( p < 0.001). As expected, a significant decrease in the expression of Atrogin1 ( p < 0.001, Figure 4C) and MuRF1 ( p < 0.001, Figure 4D) was observed in gastrocnemius tissues by the administration of MON at a level of 80 mg/kg to mice with DEX-induced muscle atrophy. The administration of MON to mice at levels of 40 mg/kg ( p < 0.05) and 80 mg/kg ( p < 0.01) significantly increased the expression of MyHC in their muscle tissues ( Figure 4B). The expression of MyHC in the gastrocnemius tissues of mice with DEX-induced muscle atrophy was significantly decreased ( p < 0.001) compared with the normal group. In the in vivo study, DEX stimulation was shown to significantly up-regulate the expression of Atrogin1, MuRF1, and Myostatin compared to the normal group ( Figure 4A). The expression of each target protein was normalized to β-actin (Sigma-Aldrich) as an internal control. The protein signals were measured under a ChemiDoc MP Imaging System (Bio-Rad) and the band density was quantified by densitometry using the ImageJ programming software (ImageJ, NIH, Bethesda, MD, USA). The membranes were incubated with the secondary antibodies, including anti-mouse IgG or anti-rabbit IgG (Bio-Rad, Hercules, CA, USA), for 2 h at room temperature and washed three times with 1× TBST buffer. The membranes were then washed three times with 1× Tris-buffered saline (pH 7.4) containing 0.1% Tween-20 (TBST) buffer for 15 min. PA5-91959, Thermo Fisher Scientific), Myostatin (Cat. 2972s, Cell Signaling, Danvers, MA, USA), p-mTOR (Cat. bs-2539R, Bioss Antibodies, Woburn, MA, USA), mTOR (Cat. After transferring the membranes and blocking with 5% skim milk, the membranes were incubated with the primary antibodies against MyHC (sc-376157, Santa Cruz, Dallas, CA, USA), MuRF1 (Cat. Equal amounts of each protein were separated into 8% acrylamide gels, and SDS-polyacrylamide gel electrophoresis (PAGE) was performed. The protein concentrations of the supernatants were measured using a protein assay solution. 78510, Thermo Fisher Scientific) containing protease/phosphatase inhibitors, put on ice for 20 min, and centrifuged at 14,000 rpm at 4 ☌ for 20 min. For the in vivo study, gastrocnemius muscle tissues were homogenized using a homogenizer (T10 basic, IKA, Staufenim Breisgau, Germany) with tissue lysis buffer (Cat. 89901, Thermo Fisher Scientific), which included protease/phosphatase inhibitors, and then total proteins were isolated by centrifugation at 14,000 rpm for 20 min at 4 ☌. Measurement of Grip Strengthįor the in vitro study, the C2C12 myotubes were added to the RIPA lysis and extraction buffer (Cat. During treatment with drugs, the body weights of mice were measured once three days. Two different concentrations of MON (40 and 80 mg/kg) were administered once a day. (III) One group was injected DEX and administered 40 mg/kg MON (MON40 group) and (IV) one group was injected DEX and administered 80 mg/kg MON (MON80 group). Louis, MO, USA) as DEX group subsequently (Nor group) (II) the dexamethasone was dissolved into PEG400 and intraperitoneally injected DEX 10 mg/kg, then administrated 0.9% saline once a day for 10 days (DEX group) MON was dissolved into 0.9% saline. ![]() After adaption for one week, all the mice were randomly divided into four groups ( n = 8 animals per group) as follows: (I) the normal group was administered 0.9% saline, intraperitoneal injection of the same volume of PEG400 (Cat. They were given free access to food and water. The mice were maintained under controlled conditions at 22 ± 3 ☌ and a 12 h/12 h light/dark cycle. All animal studies were approved by the Use Committee of the Dongguk University (IACUC-2021-10) and performed in accordance with the guidelines for Institutional Animal Care. Eight-week-old male C57BL/6N mice were obtained from Koatech (Pyeongtaek, Korea).
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