Accumulation of reactive oxygen species (ROS) is an important pathogenic mechanism underling the loss of bone mass and strength with aging and other conditions leading to osteoporosis. The transcription factor erythroid 2-related factor2 (Nrf2) plays a central role in activating the cellular response to ROS. Here, we examined the endogenous response of bone regulated by Nrf2, and its relationship with bone mass and architecture in the male and female murine skeleton. Young (3 month-old) and old (15 month-old) Nrf2 knockout (KO) mice of either sex exhibited the expected reduction in Nrf2 mRNA expression compared to wild type (WT) littermates. Nrf2 deletion did not lead to compensatory increase in Nrf1 or Nrf3, other members of this transcription factor family; and instead, Nrf1 expression was lower in KO mice. Compared to the respective WT littermate controls, female KO mice, young and old, exhibited lower expression of both detoxifying and antioxidant enzymes; young male KO mice, displayed lower expression of detoxifying enzymes but not antioxidant enzymes; and old male KO mice showed no differences in either detoxifying or antioxidant enzymes. Moreover, old male WT mice exhibited lower Nrf2 levels, and consequently lower expression of both detoxifying and antioxidant enzymes, compared to old female WT mice. These endogenous antioxidant responses lead to delayed rate of bone acquisition in female KO mice and higher bone acquisition in male KO mice as quantified by DXA and μCT, demonstrating that Nrf2 is required for full bone accrual in the female skeleton but unnecessary and even detrimental in the male skeleton. Therefore, Nrf2 regulates the antioxidant endogenous response and bone accrual differently depending on sex and age. These findings suggest that therapeutic interventions that target Nrf2 could be developed to enhance the endogenous antioxidant response in a sex- and age-selective manner.
Oats contain unique bioactive compounds known as avenanthramides (AVAs) with antioxidant properties. AVAs might enhance the endogenous antioxidant cellular response by activation of the transcription factor Nrf2. Accumulation of reactive oxygen species plays a critical role in many chronic and degenerative diseases, including osteoporosis. In this disease, there is an imbalance between bone formation by osteoblasts and bone resorption by osteoclasts, which is accompanied by increased osteoblast/osteocyte apoptosis and decreased osteoclast apoptosis. We investigated the ability of the synthethic AVAs 2c, 2f and 2p, to 1-regulate gene expression in bone cells, 2-affect the viability of osteoblasts, osteocytes and osteoclasts, and the generation of osteoclasts from their precursors, and 3-examine the potential involvement of the transcription factor Nrf2 in these actions. All doses of AVA 2c and 1 and 5 µM dose of 2p up-regulated collagen 1A expression. Lower doses of AVAs up-regulated OPG (osteoprotegerin) in OB-6 osteoblastic cells, whereas 100 μM dose of 2f and all concentrations of 2c down-regulated RANKL gene expression in MLO-Y4 osteocytic cells. AVAs did not affect apoptosis of OB-6 osteoblastic cells or MLO-Y4 osteocytic cells; however, they prevented apoptosis induced by the DNA topoisomerase inhibitor etoposide, the glucocorticoid dexamethasone, and hydrogen peroxide. AVAs prevented apoptosis of both wild type (WT) and Nrf2 Knockout (KO) osteoblasts, demonstrating that AVAs-induced survival does not require Nrf2 expression. Further, KO osteoclast precursors produced more mature osteoclasts than WT; and KO cultures exhibited less apoptotic osteoclasts than WT cultures. Although AVAs did not affect WT osteoclasts, AVA 2p reversed the low apoptosis of KO osteoclasts. These in vitro results demonstrate that AVAs regulate, in part, the function of osteoblasts and osteocytes and prevent osteoblast/osteocyte apoptosis and increase osteoclast apoptosis; further, these regulatory actions are independent of Nrf2.
Catalase activity was evaluated in Long Evans rat retina after ischemia and reperfusion. Ischemia was induced by ligation of the optic nerve and vessels. Rats were sacrificed after 15 and 120 min of reperfusion, respectively. Catalase activity was assessed by Claiborne’s method and was expressed as U/mg of protein. In the first group, retinas of each animal were pooled. In the second group, ischemia was induced in the right eye with the left eye serving as control. In the first group, enzyme activity was 7.39 ± 0.26 (n= 11), 7.67 ± 0.27 (n = 9) and 9.15 ± 0.45 (n = 7) for the sham-operated, 15- and 120-min reperfusion groups, respectively. There was a significant difference between the control and 120-min reperfusion groups (p < 0.001). In the second group, there was a significant (p < 0.01) increase in catalase activity in the ischemic eye compared to the non-ischemic eye after 15 (n = 7) and 120 min (n = 9) of reperfusion. These findings may suggest a rapid activation of catalase activity during the ischemia-reperfusion sequence.
Increasing evidence suggests that long term consumption of some whole grain cereals, promotes good health. Oat is a commonly consumed whole‐grain cereal and contains different types of phytochemicals with antioxidant properties, such as tocotrienols, phenolic acids, flavonoids, sterols, and phytic acid. Oats are gaining increasing scientific and public interest for their antioxidant‐associated health benefits. Avenanthramides, a group of alkaloids uniquely found in oats that contain a phenolic group, have been reported to exhibit strong antioxidant activity both in vitro and in vivo in different cell types. To date, there are no studies addressing the effect of avenanthramides in bone in vivo or in bone cells in vitro, on bone homeostasis or the function of the cells of bone. Here, we investigated in vitro the ability of avenanthramides 2c, 2f and 2p to regulate the function of osteoblasts by determining their effects on gene expression and on basal and induced apoptosis in osteoblastic cells. In order to determine in vitro the ability of avenanthramides to regulate the function of osteoblasts, a 24‐hour dose response for osteoblast mRNA gene expression was conducted. OB‐6 osteoblastic cells were cultured in αMEM + 1% PSG + 10% FBS media for one day. Medium was changed and cells were cultured overnight. The next day, medium was removed and cells were cultured for 24‐h with fresh αMEM + PSG + 2% FBS (500 μl/well) with control (DMSO) and 0.01, 0.1, 1, 10 and 100 μM of avenantramide 2c, 2f and 2p. RNA was extracted and gene expression was determined by quantitative PCR and quantified by the delta CT method. Although the expression of osteoblasts markers osteocalcin, runx2, collagen 1, osterix and alkaline phosphatase was not affected by avenanthramides 2c, 2f and 2p, and RANKL was not detected, there was an increase in OPG (osteoprotegerin) gene expression with the lowest doses (0.01 and 0.1 μM) of avenanthramides 2f and 2c. To examine the effect of avenanthramide 2f on basal and induced apoptosis in OB‐6 cells, cells were culture for 20‐h in αMEM + PSG + 10% FBS. Medium was changed to αMEM + PSG + 2% FBS+ control (DMSO) or avenanthramide 2f at 1, 10 and 100 μM (300 μl/well) and cultured for 1 h. The pro‐apoptotic agent etoposide (50 μM) was added to the corresponding wells for 6 h. Cell viability was determined by trypan blue uptake. The presence of avenanthramide 2f did not affect basal levels of apoptosis of osteoblastic OB‐6 cells. However, avenanthramide 2f prevented etoposide‐induced apoptosis in a dose dependent manner. These results demonstrate that avenanthramides may directly affect gene expression and survival of osteoblasts upregulating the expression of the anti‐osteoclastogenic cytokine OPG and preventing osteoblast apoptosis. Thus, the beneficial effects of avenanthramides, antioxidant compounds found in oats, might be related to regulation of bone cell function and life span.Support or Funding InformationThis study was supported by the National Osteoporosis Foundation through unrestricted educational grants from the Dairy Management Inc. and the Egg Nutrition Center and PepsiCo, Inc., Quaker Oats Center of Excellence, PepsiCo R & D, Inc, Barrington, IL, USA
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