Antioxidant avenanthramides prevent osteoblast and osteocyte apoptosis and induce osteoclast apoptosis by Nrf2-independent mechanisms

PELLEGRINI GG; MORALES CC; WALLACE TC; PLOTKIN L; BELLIDO T

Congreso; ASBMR Annual Meeting; 2016

ASBMR

Oats contain different types of phytochemicals with antioxidant properties and are gaining increasing scientific interest for their nutritional benefits. Avenanthramides (AVAs), uniquely found in oats, have been shown to enhance the endogenous antioxidant response in human kidney cells through activation of the transcription factor Nrf2. Here, we examined the ability of the three major AVAs 2f, 2c and 2p, which differ in the type of hydroxylcinnamic acid component (ferulic, caffeic or p-coumaric acid), to regulate bone cell apoptosis and the potential Nrf2 involvement. To examine the effect of AVAs in basal and induced apoptosis, apoptosis was quantified by trypan blue uptake after 6 h of treatment in OB-6 osteoblastic, MLO-Y4 osteocytic and primary Nrf2 WT or KO osteoblastic cells. Bone marrow precursors derived from WT and KO mice were differentiated to osteoclasts (RANKL 80 ng/ml and M-CSF 20 ng/ml). Osteoclasts were enumerated after staining with TRAPase and hematoxylin (TRAPase + cells > 3 nuclei, were considered osteoclasts). Osteoclast gene expression was also assessed. Treatment with 1-100 µM AVAs did not affect basal levels of apoptosis of OB-6 or MLO-Y4 cells. However, treatment with AVAs prevented apoptosis induced by the DNA topoisomerase etoposide (50 µM), the glucocorticoid dexamethasone (10 µM) and the reactive oxygen species (ROS) H2O2 (50 µM). AVAs (1 µM) also prevented apoptosis of primary WT as well as KO osteoblastic cells, demonstrating that survival by AVAs does not require Nrf2 expression in these cells. Bone marrow precursors derived from KO mice produced 30.21±2.25% more osteoclasts than WT precursors. Furthermore, KO cultures exhibited lower number of apoptotic osteoclasts compared to WT cultures (13.25±2.47 % vs 22.97±1.84 % for KO and WT, respectively). AVAs did not affect either number or apoptosis of WT osteoclasts, whereas in KO osteoclasts, AVA 2p reversed the decreased apoptosis to WT levels. Consistent with this, the expression of the osteoclast markers Cat K, Cal R and TRAP was higher in the KO cultures compared to WT and AVA 2p significantly decreased the expression of CAL R in KO osteoclasts. AVAs did not affect the expression of Nrf2 or the phase II antioxidant enzymes NQO1 and HMOX1, known to depend on Nrf2. These results demonstrate that AVAs prevent osteoblast/osteocyte apoptosis and increase osteoclast apoptosis; and that these regulatory actions are Nrf2 independent.