Abstract
Background and PurposeThe polyphenol resveratrol (RSV) exists in high quantities in certain foods (e.g. grapes and nuts). However, the capacity of RSV to confer physiological health benefits and a biological mechanism through which this might occur remains unclear.Experimental ApproachAged, RSV‐treated (300 mg·kg−1·day−1) and genetically modified [endothelial NOS (eNOS−/−)] female mice were assessed using histomorphometric and μCT analysis. Alongside in vivo analysis, molecular siRNA knockdown and pharmacological manipulation of eNOS, BMP2 and sirtuin 1 (SIRT1) and functional cellular assays in an osteoblast cell line panel, explored the mechanism through which RSV might impact overall bone volume.Key ResultsRSV promoted osteoblast activity and bone growth in vivo. RSV dose‐dependently and simultaneously increased alkaline phosphatase (ALP) and eNOS levels. Similarly, NO‐donor treatment increased ALP, runt homology transcription factor 2, BMP2 and stimulated bone formation, whilst eNOS‐deficient mice displayed a bone loss phenotype. Moreover, RSV‐induced increase in ALP and BMP2 expression was blocked in eNOS−/− osteoblasts and by BMP‐inhibitor noggin. The longevity‐linked SIRT1 enzyme was positively regulated by RSV and SIRT1 deletion reduced eNOS, BMP2 and ALP. Like eNOS deletion, loss of SIRT1 blocked RSV‐induced osteoblast activity; however, SIRT1 levels remained unchanged in eNOS−/− mice, indicating RSV activation of SIRT1 stimulates BMP2 release via eNOS. This signalling axis is supported by decreased SIRT1, eNOS and BMP2 confirmed in old versus young bone.Conclusions and ImplicationsThese findings suggest a new mechanism of action in bone remodelling and the ageing skeleton, where RSV positively impacts bone homeostasis via SIRT1 activation of BMP2.
Highlights
In MC3T3 (Figure 2A) and 2T3 (Figure 2B) osteoblast cell lines treated with RSV, endothelial NOS (eNOS) gene expression was dose-dependently upregulated, concomitantly with soluble levels of nitrite product (Figure 2C) and total eNOS protein levels (Figure 2D)
Given the positive effects of RSV treatment on eNOS and NO expression, combined with our observations indicating loss of eNOS reduced bone whilst NO generation stimulates osteoblast activity, we investigated whether eNOS deficiency impairs RSV effects in bone-forming osteoblasts
FoxO1 and FoxO3a can be activated by RSVSIRT1 and regulate eNOS activity, where deletion of FoxO1 and FoxO3a abolished the effect of RSV on eNOS expression in endothelial cells (Xia et al, 2013) and deletion eNOS and NO-donors stimulate bone growth. eNOS mRNA expression in MC3T3 (A) and 2T3 (B) cells treated with RSV (1–100 μM) or vehicle for 24 h (n = 5) determined by real time PCR
Summary
Diet impacts growth and ageing in all species, yet investigations exploring various dietary constituents as experimental medicines are limited in mechanism and translational impact, despite the potential for significant beneficial effects matching those offered by current pharmaceuticals.Resveratrol (RSV; 3,40,5-trihydroxy-transstilbene) is a plant polyphenol found in foods such as nuts, grapes and chocolate, and like other polyphenolic compounds (e.g. catechins in green tea extract) demonstrates various beneficial effects in preclinical and clinical studies for disorders such as cardiovascular disease (Smoliga et al, 2011; Sin et al, 2015a), diabetes (Szkudelski and Szkudelska, 2015) and inflammation (Huang et al, 2014; Conte et al, 2015).Within the skeleton, dietary components are important in regulating cellular metabolism and providing crucial constituents within bone (Sahni et al, 2015) calcium. Bone is formed through the activities of osteoblasts These mesenchymal-derived, matrix-producing cells lay down unmineralized osteoid in a highly regulated process, whilst simultaneously directing local mineralization through the release of matrix-modifying enzymes, primarily alkaline phosphatase (ALP; de Gorter and ten Dijke, 2013). RSV promotes osteoblast differentiation from mesenchymal stem cells in vitro (Zhou et al, 2009; Kupisiewicz et al, 2010; Shakibaei et al, 2012), reduces the formation of boneresorbing osteoclasts (Shakibaei et al, 2011) and may be protective in an experimental model of accelerated bone disease (Wang et al, 2017) suggesting that RSV supplementation may be beneficial for bone health to prevent the age-related decline in functional integrity or as an experimental medicine in disorders of excessive bone destruction. Molecular siRNA knockdown and pharmacological manipulation of eNOS, BMP2 and sirtuin 1 (SIRT1) and functional cellular assays in an osteoblast cell line panel, explored the mechanism through which RSV might impact overall bone volume
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