Tart Cherry Prevents Bone Loss through Inhibition of RANKL in TNF-Overexpressing Mice

Abstract

Current drugs for the treatment of rheumatoid arthritis-associated bone loss come with concerns about their continued use. Thus, it is necessary to identify natural products with similar effects, but with fewer or no side effects. We determined whether tart cherry (TC) could be used as a supplement to prevent inflammation-mediated bone loss in tumor necrosis factor (TNF)-overexpressing transgenic (TG) mice. TG mice were assigned to a 0%, 5%, or 10% TC diet, with a group receiving infliximab as a positive control. Age-matched wild-type (WT) littermates fed a 0% TC diet were used as a normal control. Mice were monitored by measurement of body weight. Bone health was evaluated via serum biomarkers, microcomputed tomography (µCT), molecular assessments, and mechanical testing. TC prevented TNF-mediated weight loss, while it did not suppress elevated levels of interleukin (IL)-1β and IL-6. TC also protected bone structure from inflammation-induced bone loss with a reduced ratio of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) to a degree comparable to infliximab. Furthermore, unlike with infliximab, TC exhibited a moderate improvement in TNF-mediated decline in bone stiffness. Thus, TC could be used as a prophylactic regimen against future fragility fractures in the context of highly chronic inflammation.

Keywords: OPG; RANKL; Runx2; TNF; bone mass; infliximab; osteoblast; osteoclast; rheumatoid arthritis; tart cherry.

Figure 1

High-doses of TC recovered a TNF-mediated loss of body weight, but it did not suppress TNF-mediated elevation of proinflammatory cytokines in the blood. After 4 weeks of treatment, body weight was measured in wild-type (WT), transgenic (TG), TG with 5% TC, TG with 10% TC, and TG with infliximab mice. Collected serum after sacrifice was also used to measure protein levels of human TNF (hTNF), mouse interleukin (mIL)-1β, and mIL-6. Values are mean ± SEM (n = 4–8 per group). *, p < 0.05 vs. WT; #, p < 0.05 vs. TG; $, p < 0.05 vs. TG + 5% TC; %, p < 0.05 vs. TG + 10% TC.

Figure 2

TC diet dose-dependently inhibited TNF-induced decline of trabecular bone mass. (A) Microcomputed tomography (µCT) scans representing metaphyseal trabecular bone were taken from the femurs of WT, TG, TG + 5% TC, TG + 10% TC, and TG + infliximab mice. (B) Trabecular number (Tb.N), thickness (Tb.Th), space (Tb.Sp), and bone mass (Tb.BV/TV) were quantified using µCT analysis. Values are mean ± SEM (n = 4–8 per group). *, p < 0.05 vs. WT; #, p < 0.05 vs. TG; $, p < 0.05 vs. TG + 5% TC; %, p < 0.05 vs. TG + 10% TC.

Figure 3

TC diet improved modification of cortical bone structure by inflammation. (A) Microcomputed tomography (µCT) scans representing sections of cortical bone were taken for femurs of WT, TG, TG + 5% TC, TG + 10% TC, and TG+infliximab mice. (B) Cortical thickness (Ct.Th), porosity (Ct.Po), and bone mass (Ct.BV/TV) were quantified using µCT analysis. Values are mean ± SEM (n = 4–8 per group). *, p < 0.05 vs. WT; #, p < 0.05 vs. TG.

Figure 4

TC downregulated gene expressions of RANKL and TRAP. Total RNA was extracted from histological sections of metaphyseal area in each femur of WT, TG, TG + 5% TC, TG + 10% TC, and TG + infliximab mice. cDNA converted from total RNA was applied to measure relative transcript levels of TNF, IL-1β, Runx2, COL I, RANKL, and TRAP that were normalized to GAPDH using RT-PCR. Values are means (n = 4–8) ± SEM. *, p < 0.05 vs. WT; #, p < 0.05 vs. TG; $, p < 0.05 vs. TG + 5% TC; %, p < 0.05 vs. TG + 10% TC.

Figure 5

TC exhibited a modest improvement of bone stiffness devastated by TNF overexpression. Right femurs were placed on the machine with a three-point bending mechanical system to measure and determine failure load, stiffness, and modulus. The 5% and 10% TC mice were combined for these parameters to make a total of four groups: WT, TG, TG + TC, and TG + infliximab. Values are means (n = 4–9) ± SEM. *, p < 0.05 vs WT.