Eurycomanone stimulates bone mineralization in zebrafish larvae and promotes osteogenic differentiation of mesenchymal stem cells by upregulating AKT/GSK-3ß/ß-catenin signaling.

Background: Eurycomanone (EN) is a diterpenoid compound isolated from the roots of Eurycoma longifolia (E. longifolia). Previous studies have confirmed that E. longifolia can enhance bone regeneration and bone strength. We previously isolated and identified ten quassinoids from E. longifolia, and the result displayed that five aqueous extracts have the effects on promotion of bone formation, among whom EN showed the strongest activity. However, the molecular mechanism of EN on bone formation was unknown, and we further investigated in this study. Methods: After the verification of purity of extracted EN, following experiments were conducted. Firstly, the pharmacologic action of EN on normal bone mineralization and the therapeutic effect of EN on Dex-induced bone loss using zebrafish larvae. The mineralization area and integral optical density (IOD) were evaluated using alizarin red staining. Then the vital signaling pathways of EN relevant to OP was identified through network pharmacology analysis. Eventually in vitro, the effect of EN on cell viability, osteogenesis activities were investigated in human bone marrow mesenchymal stem cells (hMSCs) and C3H10 cells, and the molecular mechanisms by which applying AKT inhibitor A-443654 in hMSCs. Results: In zebrafish larvae, the administration in medium of EN (0.2, 1, and 5 µM) dramatically enhanced the skull mineralization area and integral optical density (IOD), and increased mRNA expressions of osteoblast formation genes (ALP, RUNX2a, SP7, OCN). Meanwhile, exposure of EN remarkably alleviated the inhibition of bone formation induced by dexamethasone (Dex), prominently improved the mineralization, up-regulated osteoblast-specific genes and down-regulated osteoclast-related genes (CTSK, RANKL, NFATc1, TRAF6) in Dex-treated bone loss zebrafish larvae. Network pharmacology outcomes showed the MAPK and PI3K-AKT signaling pathways are closely associated with 10 hub genes (especially AKT1), and AKT/GSK-3ß/ß-catenin was selected as the candidate analysis pathway. In hMSCs and C3H10 cells, results showed that EN at appropriate concentrations of 0.008-5 µM effectively increased the cell proliferation. In addition, EN (0.04, 0.2, and 1 µM) significantly stimulated osteogenic differentiation and mineralization as well as significantly increased the protein phosphorylation of AKT and GSK-3ß, and expression of ß-catenin, evidencing by the results of ALP and ARS staining, qPCR and western blotting. Whereas opposite results were presented in hMSCs when treated with AKT inhibitor A-443654, which effectively inhibited the pro-osteogenesis effect induced by EN, suggesting EN represent powerful potential in promoting osteogenesis of hMSCs, which may be closely related to the AKT/GSK-3ß/ß-catenin signaling pathway. Conclusions: Altogether, our findings indicate that EN possesses remarkable effect on bone formation via activating AKT/GSK-3ß/ß-catenin signaling pathway in most tested concentrations. The translational potential of this article: This study demonstrates EN is a new effective monomer in promoting bone formation, which may be a promising anabolic agent for osteoporosis (OP) treatment.

Musculoskeletal Disorders Symptoms among Taiwanese Bakery Workers.

In this study, the Nordic Musculoskeletal Questionnaire (NMQ) was administered to a valid sample of 81 Taiwanese bakery workers to explore their discomfort or symptoms of work-related musculoskeletal disorders and identify the risk factors. Wrist postures were also examined during 3 typical dough operations (kneading, rolling, and rounding) by using an electrogoniometer. The prevalence of musculoskeletal discomfort in any part of the body in the past year among the respondents was 93.0%, with the highest prevalence of 66.3% and 51.8% in the hands/wrists (right and left), followed by the prevalence of 50.6% and 45.8% in the shoulders (right and left) and the lower back (48.2%), respectively. The results also revealed that during the 3 dough processing operations, the workers' wrist movements in specific operations were close to the recommended limits suggested in previous studies, especially the ulnar deviation and palm flexion of the right wrist during dough kneading and the radial deviation of the left wrist during dough rolling and rounding. The study findings can be used to explain why the bakers self-report a high proportion of wrist and shoulder disorders and can also serve as a reference for task rearrangement and redesign.

Spraying Brassinolide improves Sigma Broad tolerance in foxtail millet (Setaria italica L.) through modulation of antioxidant activity and photosynthetic capacity.

To explore the role of Brassinolide (BR) in improving the tolerance of Sigma Broad in foxtail millet (Setaria italica L.), effects of 0.1 mg/L of BR foliar application 24 h before 3.37 g/ha of Sigma Broad treatment at five-leaf stage of foxtail millet on growth parameters, antioxidant enzymes, malondialdehyde (MDA), chlorophyll, net photosynthetic rate (P N), chlorophyll fluorescence and P700 parameters were studied 7 and 15 d after herbicide treatment, respectively. Results showed that Sigma Broad significantly decreased plant height, activities of superoxide dismutase (SOD), chlorophyll content, P N, PS II effective quantum yield (Y (II)), PS II electron transport rate (ETR (II)), photochemical quantum yield of PSI(Y (I)) and PS I electron transport rate ETR (I), but significantly increased MDA. Compared to herbicide treatment, BR dramatically increased plant height, activities of SOD, Y (II), ETR (II), Y (I) and ETR (I). This study showed BR pretreatment could improve the tolerance of Sigma Broad in foxtail millet through improving the activity of antioxidant enzymes, keeping electron transport smooth, and enhancing actual photochemical efficiency of PS II and PSI.