Source: UNIV OF CONNECTICUT submitted to
BLACKCURRANT PREVENTS OSTEOPOROSIS ASSOCIATED WITH AGING BY INHIBITING FORMATION OF ADVANCED OXIDATION PROTEIN PRODUCTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1007778
Grant No.
2016-67018-24492
Project No.
CONS2015-05474
Proposal No.
2015-05474
Multistate No.
(N/A)
Program Code
A1341
Project Start Date
Dec 15, 2015
Project End Date
Dec 14, 2017
Grant Year
2016
Project Director
Chun, O. K.
Recipient Organization
UNIV OF CONNECTICUT
(N/A)
STORRS,CT 06269
Performing Department
Nutritional Sciences
Non Technical Summary
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. 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. The proposed research is designed to answer an important question of whether, and if so how, long-term berry consumption reduces aging-associated bone loss in this mouse model of aging. Thus, this proposed study is an important step toward discovering potential dietary agents that may be effective in preventing bone loss. Findings from the proposed research may serve as a basis for future clinical trials to evaluate the bone-protective effect of blackcurrant in high-risk older women. The proposed study is consistent with the USDA NIFA Program Area Priority of Function and Efficacy of Nutrients (A1341).
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
70211291010100%
Goals / Objectives
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. 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.
Project Methods
The overall goal of this two-year project is to investigate the inhibitory effect of a blackcurrant consumption on the formation of AOPPs and aging-associated bone loss. For this purpose, we will use an animal model of female OVX C57BL/6 mice aged 18 months, which is equivalent to 60 years in human age based on musculoskeletal maturity. Our team has extensive expertise and experience with mouse aging models.Study Protocols for Aim 1:Assay of AOPP levels: AOPP levels in plasma and left femur will be spectrophotometrically determined according to the previously described method. AOPP concentrations will be expressed as μmol/L (plasma) or nmol/mg protein (femur) of chloramine-T equivalents.Assay of MDA levels: Malondialdehyde (MDA) levels will be measured according to the previously described method. Absorbance at 532 nm will be expressed as μmol/L (plasma) or nmol/mg protein (femur).NO measurements: Nitric oxide (NO) concentration will be determined by the enzymatic conversion of nitrate to nitrite using nitrate reductase.Determination of AGEs: AGE determination will be based on spectrofluorometric detection. Fluorescence intensity is expressed in arbitrary units per gram of protein (AU/g protein).Determination of SOD activity: Superoxide dismutase (SOD) activity will be analyzed according to the method of Sun et al. SOD activities in the plasma and femur will be expressed as U/mL and U/mg protein, respectively.Mononuclear cell isolation: Mononuclear cells will be isolated from whole blood.Total RNA extraction and quantitative realtime PCR (qRT-PCR): Total RNA will be extracted from mononuclear cells using Trizol reagent (Life Technology, Rockville, MD) following the manufacturer's protocol. Reverse transcription for cDNA synthesis and qRT-PCR analysis for genes related to AOPP formation, including NOX1, NOX2, NOX4, Nrf2, NFκB, and RAGE will be performed. Whole mouse femurs will be homogenized using Polytron and total RNA extracted using Trizol reagent.Study Protocols for Aim 2:Biomarkers for bone metabolism Plasma P1NP activity will be quantified by immunoassay in a microtiter format (Immunodiagnotic, Inc, Scottsdale, AZ). Plasma CTX and TRAP will be determined colorimetrically using commercially available kits (Immunodiagnostic Inc, Scottsdale, AZ).DXA & micro-computed tomography and histomorphometric analyses: The right femur and 1st lumbar vertebrae (L1) will be isolated and fixed in 70% ethanol at 4ºC for bone density analysis by using Dual-energy X-ray absorptionmetry (DXA) to determine BMD and BMC followed by high-resolution X-ray computed microtomography (µCT40-Scanco Medical, AG, Bassersdorf, Switzerland). Trabecular and cortical morphometry will be quantified within the metaphyseal region of the distal femur. Cortical morphometry will be analyzed by assessing average thickness and cross-section area within a 600 µm long section at mid-diaphysis of the femur. The left femur will be dissected out and fixed in 4% paraformaldehyde in PBS before tissues are decalcified to determine the number of osteoblasts and osteoclasts in vivo by histomorphometric analysis.To determine the effect of blackcurrant in vivo on the differentiation and activity of osteoblasts and osteoclasts, we will culture BM cells from the mice fed the diets with or without blackcurrant. Both osteoblasts and osteoclasts differentiation experiments will be performed. BM stromal cells and BM macrophages will be prepared from the mice fed blackcurrant for 2 or 4 months. To further define the mechanisms underlying the effect of blackcurrant, we will culture BM-derived stromal cells obtained from mice at 18 months of age in the presence or absence of blackcurrant anthocyanin extract. The extract will be prepared and added at 20 μg/ml which was found to be effective in inhibiting NOX1 and NOX2 mRNA expression. The cells will be cultured for up to 21 days in osteogenic medium to facilitate osteoblast differentiation. Alkaline phosphatase (ALP) and von Kossa staining will be conducted to determine the nodule formation as well as ALP activity using cell lysates. In addition, RNA samples will be prepared to examine if blackcurrant extract alters osteoblast specific gene expression (ALP, collagen, and bone sialoprotein) in these cultures by real time RT-PCR. To determine the effect of blackcurrant on the production of AOPPs in osteoblasts and osteoclasts, we will culture BM cells from the mice in the presence or absence of blackcurrant extract. To determine the effect on osteoblasts, BM stromal cells will be cultured in osteogenic medium and mRNA and protein will be prepared at 7, 14, and 21 days. To evaluate the anti-osteoclastogenic effect of blackcurrant, we will prepare BM macrophages to differentiate into osteoclasts. The macrophages will be prepared from mice at 18 month of age not fed blackcurrant by using Ficoll-Hypaque gradient centrifugation and will be cultured for 4, 5 and 6 days in the presence of M-CSF and RANKL (both at 30 ng/ml, R&D Systems, Minneapolis, MN). At the conclusion of culture, the osteoclasts formed will be fixed and stained for TRAP using a commercial kit (Leukocyte acid phosphatase, Sigma). TRAP(+) osteoclasts (more than 3 nuclei) will be counted. We will also determine the bone resorptive activity of osteoclasts as previously described.Statistical Analysis: Data will be reported as means ± SEM if normally distributed and as geometric means if strongly skewed as expected in serum and plasma data. Statistical software SAS 9.2 will be employed to conduct the analyses. Differences between the baseline and each dietary treatment group will be analyzed by one-way analysis of variance (ANOVA) to determine age effect. For Aim 1, the measurements will be compared between Control and BC Groups. For Aim 2, treatment effects will be analyzed by ANOVA followed by post hoc analysis using the Tukey's least squares means separation test. Two-way ANOVA will be used to determine significant differences between treatments and time points among groups. Differences will be considered to be significant at P<.05.Limitations, Potential Problems, and Alternative Strategies: We anticipate no difficulty in conducting the proposed work, since we have all of the proposed techniques and protocols in place between the project director (PD)'s and Co-PD' laboratories. For the proposed experiment ex-vivo, the co-PD (Dr. Lee) has the expertise and experience with all of the proposed techniques and is currently engaged in a collaborative project with the PD.

Progress 12/15/15 to 12/14/16

Outputs
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.

Impacts
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)

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: 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.