Progress 12/15/15 to 12/14/16
Target Audience:The ultimate goal of this project is to elevate clinical or experimental evidence of the health benefits of berries to the public health arena and contribute to expanding the recommended dietary intake of anthocyanin-rich berries by reducing the risk of aging-associated bone loss. Under this goal, the evaluation of the inhibitory effect of dietary blackcurrant on the formation of advanced oxidation protein products (AOPPs) and aging-associated bone loss in a mouse model of aging can provide a scientific basis for the future studies planned. The potential target audience of the study will include researchers in nutrition and health science areas, food and neutraceutical industry, public health policy makers, nutrition educators, any gender, especially postmenopausal women, and anyone interested in increasing antioxidant consumption. Changes/Problems:For the proposed study, we planned to use the 18 month-old mouse colonies from the National Institute of Aging (NIA) at no cost. However, NIA recently changed their policy and now we must purchase the animals. In addition, to acquire 18 month old mice, we would have to wait at least 9-12 months. Thus we decided to use 12-month old female mice for the study without OVX surgical treatment since this surgery increases death rates by 30% resulting in huge increase in cost for the animal purchase. The change of age of the study animal will not affect the study results significantly since 12 month old female mouse model without OVX surgery is still appropriate since middle aged females have much greater risk of AAO due to the cumulative impacts of aging. As initially planned, we will feed the animals for up to 4 months. Unlike most dietary intervention animal studies, we propose this long term dietary intervention in this project since a dietary strategy for reducing aging-associated bone deterioration can be more effective when it is based on a long-term lifestyle modification. What opportunities for training and professional development has the project provided?While conducting the project, our graduate students who participated in this project gained knowledge and learned the science relevant to the study and also acquired technical skills needed to conduct the compositional analytical studies. How have the results been disseminated to communities of interest?Findings were disseminated to scientific communities via journal articles, professional society conferences, and invited seminars. To date, we published 1 paper and gave 1 invited seminar with the study findings. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue mechanistic studies to investigate how anthocyanin-rich black currant extract attenuates OVX bone loss in mice. By adding gene expression data, we will update and submit one manuscript to a peer-reviewed journal. In addition, we will start an animal feeding study to expend our research questions in an in vivo model.
What was accomplished under these goals?
1. Major goals of the project The goal of this project is to investigate the inhibitory effect of dietary blackcurrant on the formation of advanced oxidation protein products (AOPPs) and aging-associated bone loss in a mouse model of aging. Evidence indicates that AOPPs play a crucial role in aging-associated bone loss and suggests that the consumption of antioxidant-rich foods may lower the risk of aging-associated osteoporosis by reducing AOPPs. Our preliminary studies with cell and animal models have shown that blackcurrant or its anthocyanin-rich extract is effective in reducing bone loss in ovariectomized (OVX) mice. However, the animal model used might not adequately mimic the conditions of aging-associated bone loss coupled with menopause that exist in middle-aged and older women. Furthermore, the mechanisms underlying the inhibitory effect of antioxidants on the formation of AOPPs in vivo still remain unknown. Thus, the main objectives of our proposed study are to determine the effects of blackcurrant in attenuating aging-associated bone loss and to define the underlying mechanisms in a mouse model of bone aging. 2. Majoraccomplishments Under these major goals, during the 1st year of the project, we have conducted in vitro and ex vivo studies. 1)In Vitro Studies: Since we have trouble acquiring aged mice, we performed the majority of osteoclast assay preliminary results using young female mice at 8 weeks of age. We found blackcurrant (0.5 - 5 ug/ml) downregulated osteoclast formation in vitro in a dose dependent manner and this was not due to the alteration in cell proliferation or apoptosis of the cells that received blackcurrant treatment. We also found that the treatment with blackcurrant was the most effective as soon as cells were plated when compared to the first half or second half of culture period. In terms of osteoclast specific gene expression in response to blackcurrant treatment, we cultured bone marrow macrophage (BMM) cells with or without blackcurrant at 5 ug/ml for 2 or 4 days in the presence of M-CSF and RANKL. Osteoclast specific genes (TRAP, Cathepsin K, MMP-9, DC-STAMP, OC-STAMP and Atp6v0d2) were downregulated by blackcurrant treatment with the exception of MFR. We also examined the effect of blackcurrant in TNF-induced osteoclastogenesis. We found that blackcurrant downregulated TNF-induced osteoclast formation in vitro. These experiments were performed to determine if blackcurrant has an effect on the inflammation induced osteoclast formation. Subsequently, we examined the signaling pathways that blackcurrant may modulate. NFATc1, a master regulator of osteoclast differentiation, was reduced in response to blackcurrant treatment along with the downregulation of c-fos and JNK expression indicating that blackcurrant directly modulates RANKL mediated signaling to downregulate osteoclastogenesis. These preliminary experiments will be the foundation for the subsequent experiments using aged mice. 2)Ex Vivo studies: To determine whether blackcurrant anthocyanins affect osteoclast precursor cells in vivo, bone marrow (BM) cells from Sham, OVX, OVX+BC mice were collected and allowed to differentiate into OCL cells ex vivo in the presence of M-CSF and RANKL for 5 days. BM cells from ovariectomized mice significantly increased osteoclast formation 1.5-fold compared with Sham controls (P < .05). Furthermore, BM cells from blackcurrant supplemented OVX mice downregulated TRAP(+) osteoclast formation compared to OVX at eighth week (P < .05) indicating that in vivo supplementation of blackcurrant significantly affected osteoclastic potential of precursor cells to differentiate. We next examined whether blackcurrant supplementation affected bone resorption. BM cells from Sham, OVX, and OVX+BC were cultured on bone slices in the presence of M-CSF and RANKL as described and bone resorption area by individual osteoclast was evaluated. Interestingly, BM cells from ovariectomized mice did not affect bone resorption compared to those from Sham controls (Fig. 6b). However, BM cells from in vivo blackcurrant supplementation significantly decreased bone resorption. With the findings, we have published one paper to a peer-reviewed journal: Xi Zheng, Se Hwan Mun, Sang Gil Lee, Terrence Vance, Patrice Hubert, Sung I. Koo, Sun Kyeong Lee, Ock K. Chun. Anthocyanin-rich black currant extract attenuates ovariectomy-induced bone loss in mice. J Medicinal Food 19(4):390-7, 2016. (Support by the USDA/National Institute of Food and Agriculture Award (#2016-67018-24492) was acknowledged in the paper)
Xi Zheng, Se Hwan Mun, Sang Gil Lee, Terrence Vance, Patrice Hubert, Sung I. Koo, Sun Kyeong Lee, Ock K. Chun. Anthocyanin-rich black currant extract attenuates ovariectomy-induced bone loss in mice. J Medicinal Food 19(4):390-7, 2016.