Icariin (ICA) is a pharmacologically active flavonoid glycoside that shows promise in the treatment and prevention of osteoporosis (OP). However, the mechanisms underlying the anti-osteoporotic effects of ICA remain largely unclear. The present study used quantitative polymerase chain reaction, western blot and immunohistochemical analysis to examine the effects of ICA on several key targets in the Notch signaling pathway in bone tissue in ovariectomized rats. It was observed that ICA has a pronounced beneficial effect on OP rats and inhibits the expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα) and fatty acid-binding protein 4 (FABP4) mRNA. In addition, it was identified that ICA downregulates the expression of notch1 intracellular domain (N1ICD) and Jagged1 proteins in bone tissue, and suppresses the effect of N1ICD on Notch2 mRNA expression. It is proposed that ICA inhibits the differentiation of mesenchymal stem cells into adipocytes by inhibiting the expression of PPARγ, C/EBPα and FABP4 mRNA via the Notch signaling pathway. In addition, it is proposed that ICA inhibits the expression of Notch2 mRNA by suppressing the effect of N1ICD. In conclusion, the results provide further mechanistic evidence for the clinical efficacy of ICA in the treatment of OP.

Icariin (ICA), the main active flavonoid glucoside isolated from Herba Epimedii, has been shown to prevent postmenopausal bone loss in vitro. However, the mechanisms by which ICA prevents bone loss in vivo remain poorly understood. In the present study, the effect of ICA in an ovariectomized (OVX) rat model of osteoporosis was evaluated. Sprague-Dawley rats were divided into sham-operated and OVX groups. The OVX rats were randomly divided into five groups: OVX group (water only), Fosamax (positive) group (5.04 mg/kg, weekly, administered orally), and OVX-ICA groups (125, 250 or 500 mg/kg, daily, administered orally) and treated for 12 weeks. The 125, 250 and 500 mg/kg doses of ICA were designated as low (L-ICA), medium (M-ICA) and high (H-ICA), respectively. Compared with the sham-operated group, the OVX rats had significantly decreased bone mineral density (BMD), reduced serum osteoprotegerin (OPG) and increased serum bone gla protein (BGP) concentrations. ICA significantly increased BMD, biomechanical strength, trabecular bone number and trabecular bone thickness, and reduced lumbar trabecular bone separation. Treatment with ICA also completely normalized the expression of osteoblast markers by increasing serum concentrations of OPG and BGP. Enhanced mineralization was demonstrated by increased expression of differentiation markers. Although further in vivo studies are required to investigate the efficacy of ICA in improving bone mass, this study demonstrates that ICA has strong osteogenic activity, inducing osteogenic differentiation and inhibiting resorption by osteoclasts. It also demonstrates an antiosteoporotic effect for ICA on the basis of BMD, biochemical markers, biomechanical tests and histopathological parameters. Compared with L-ICA and H-ICA, M-ICA was more effective and caused no liver or kidney damage.

A previous study demonstrated that leucine upregulates the slow myosin heavy chain mRNA expression in C2C12 cells. However, the role of leucine in slow-twitch muscle fibers expression and mitochondrial function of porcine skeletal muscle satellite cells as well as its mechanism remain unclear. In this study, porcine skeletal muscle satellite cells cultured in differentiation medium were treated with 2 mM leucine for 3 days. Sirt1 inhibitor EX527, AMPK inhibitor compound C, and AMPKα1 siRNA were used to examine its underlying mechanism. Here we showed that leucine increased slow-twitch muscle fibers and mitochondrial function-related gene expression, as well as increased succinic dehydrogenase (SDH) and malate dehydrogenase (MDH) activities. Moreover, leucine increased the protein levels of Sirt1 and phospho-AMPK. We also found that AMPKα1 siRNA, AMPK inhibitor compound C, or Sirt1 inhibitor EX527 attenuated the positive effect of leucine on slow-twitch muscle fibers and mitochondrial function-related gene expression. Finally, we showed that Sirt1 was required for leucine-induced AMPK activation. Our results provide, for the first time, evidence that leucine induces slow-twitch muscle fibers expression and improves mitochondrial function through Sirt1/AMPK signaling pathway in porcine skeletal muscle satellite cells. K E Y W O R D Sleucine, mitochondrial function, porcine skeletal muscle satellite cells, Sirt1/AMPK, slowtwitch muscle fibers expression

The study was conducted to investigate the effects of dietary leucine on antioxidant activity and expression of antioxidant-and mitochondrial-related genes in longissimus dorsi muscle and liver of piglets. Three diets were formulated with different levels of supplemented leucine (0%, 0.25%, 0.5%). Results showed that supplementation of 0.25% leucine significantly increased antisuperoxide anion (ASA) and antihydroxyl radical (AHR) levels and activities of total superoxide dismutade (T-SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST), and total antioxidant capacity (T-AOC) in serum, longissimus dorsi muscle and liver of piglets as compared with the control group. The SOD2, catalase (CAT), GPx, GST, glutathione reductase (GR), and nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA levels in longissimus dorsi muscle and liver were significantly increased by 0.25% leucine supplementation. However, the malondialdehyde (MDA) content and the mRNA level of Kelchlike ECH-associated protein 1 (Keap1) exhibited an opposite tendency. Additionally, supplementation of 0.25% leucine significantly increased the mRNA levels of mitochondrial-related genes in longissimus dorsi muscle and liver of piglets. Results suggested that supplementation of 0.25% leucine improved antioxidant activity and mitochondrial biogenesis and function of piglets, which was related to the increase in antioxidant enzymes activities and upregulation of expression of antioxidant-and mitochondrial-related genes. K E Y W O R D S antioxidant activity, leucine, mitochondrial, piglets | 991 CHEN Et al.

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