Anti-osteoporotic effects of Boswellia serrata gum resin extract in vitro and in vivo

Abstract

Background/objectives: This study evaluated the beneficial effects of an ethanol extract of Boswellia serrata gum resin (FJH-UBS) in osteoporosis.

Materials/methods: MC3T3-E1 osteoblastic cells and RAW 264.7 osteoclastic cells were treated with FJH-UBS. The alkaline phosphatase (ALP) activity, mineralization, collagen synthesis, osteocalcin content, and Runt-related transcription factor 2 (RUNX2) and Osterix expression were measured in MC3T3-E1 cells. The actin ring structures, tartrate-resistant acid phosphatase (TRAP) activity, and the nuclear factor of activator T-cells, cytoplasm 1 (NFATc1) expression were evaluated in RAW 264.7 cells. Ovariectomized ICR mice were orally administered FJH-UBS for eight weeks. The bone mineral density (BMD) and the serum levels of osteocalcin, procollagen 1 N-terminal propeptide (P1NP), osteoprotegerin, and TRAP 5b were analyzed.

Results: FJH-UBS increased the ALP activity, collagen, osteocalcin, mineralization, and RUNX2 and osterix expression in MC3T3-E1 osteoblastic cells, whereas it decreased the TRAP activity, actin ring structures, and NFATc1 expression in RAW 264.7 osteoclastic cells. In ovariectomy-induced osteoporosis mice, FJH-UBS positively restored all of the changes in the bone metabolism biomarkers (BMD, osteocalcin, P1NP, osteoprotegerin, and TRAP 5b) caused by the ovariectomy.

Conclusion: FJH-UBS has anti-osteoporotic activity by promoting osteoblast activity and inhibiting osteoclast activity in vitro and in vivo, suggesting that FJH-UBS is a potential functional food ingredient for osteoporosis.

Keywords: Boswellia; osteoblasts; osteoclast; osteoporosis; ovariectomy.

Fig. 1. Effects of FJH-UBS on the viability of MC3T3-E1 cells and RAW 264.7 cells.

MC3T3-E1 cells (A) and RAW 264.7 cells (B) were plated in 24-well plates. After incubation for 24 h, the cells were incubated for 72 h in media containing 0–200 µg/mL of FJH-UBS. The cell viability was measured using the MTT assay. Each bar represents the mean ± SE (n = 4). Means without a common letter are significantly different at P < 0.05.

Fig. 2. Effects of FJH-UBS on the ALP activity, collagen synthesis, mineralization, and osteocalcin production in MC3T3-E1 cells.

MC3T3-E1 cells were plated in multi-well plates. After incubation for 24 h, the cells were incubated in ObDIM containing various concentrations of FJH-UBS for five days (ALP activity), eight days (collagen synthesis), or 18 days (mineralization and osteocalcin production). (A) ALP activity was measured using TRACP and ALP assay kits. (B) Collagen synthesis was measured by Sirius Red staining. (C) Mineralization was measured by Alizarin Red S staining. (D) Twenty-four hour-conditioned media were collected. The contents of osteocalcin in the 24 h-conditioned media were measured using an osteocalcin enzyme-linked immunosorbent assay kit. Each bar represents the mean ± SE (n = 6). ObDIM, osteoblast differentiation-inducing medium; ALP, alkaline phosphatase. ^***P < 0.001 significantly different from the ObDIM-untreated group. Means without a common letter are significantly different at P < 0.05.

Fig. 3. Effects of FJH-UBS on mRNA expression of Runx2 and Osterix in MC3T3-E1 cells.

MC3T3-E1 cells were plated in multi-well plates. After incubation for 24 h, the cells were incubated in ObDIM containing various concentrations of FJH-UBS for three days. The total RNA in cells was extracted, reverse transcribed, and real-time PCR was performed. The relative mRNA expression of Runx2 (A) and Osterix (B) were analyzed. Targeted mRNA expression was normalized to that of Gapdh and is represented relative to the ObDIM-treated group. Each bar represents the mean ± SE (n = 6). ObDIM, osteoblast differentiation-inducing medium; Runx2, Runt-related transcription factor 2; Gapdh, glyceraldehyde 3-phosphate dehydrogenase. ^***P < 0.001 significantly different from the ObDIM-untreated group. Means without a common letter are significantly different at P < 0.05.

Fig. 4. Effects of FJH-UBS on TRAP activity and actin ring formation in RAW 264.7 cells.

RAW 264.7 cells were plated in multi-well plates. After incubation for 24 h, the cells were incubated in OcDIM containing various concentrations of FJH-UBS for five days. (A) TRAP activity was measured using TRACP and ALP assay kits. Each bar represents the mean ± SE (n = 6). (B) The cells were fixed with 4% paraformaldehyde containing 0.1% Triton X-100 and stained with Alexa Fluor 594 Phalloidin and DAPI to stain actin and nuclei, respectively. Morphological changes of actin ring in cells were observed under a microscope, Magnification, 200 x. Representative staining images are shown (n = 3). OcDIM, osteoclast differentiation-inducing medium; TRAP, tartrate-resistant acid phosphatase; DAPI, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride. ^***P < 0.001 significantly different from the OcDIM-untreated group. Means without a common letter are significantly different at P < 0.05.

Fig. 5. Effects of FJH-UBS on Nfatc1 mRNA expression in RAW 264.7 cells.

RAW 264.7 cells were plated in multi-well plates. After incubation for 24 h, the cells were incubated in OcDIM containing various concentrations of FJH-UBS for three days. The total RNA in cells was extracted, reverse transcribed, and real-time polymerase chain reaction was performed. The relative mRNA expression of Nfatc1 was analyzed. Targeted mRNA expression was normalized to that of Gapdh and is represented relative to the OcDIM-treated group. Each bar represents the mean ± SE (n = 6). OcDIM, osteoclast differentiation-inducing medium; Nfatc1, the nuclear factor of activator T-cells, cytoplasm 1; Gapdh, glyceraldehyde 3-phosphate dehydrogenase. ^***P < 0.001 significantly different from the OcDIM-untreated group. Means without a common letter are significantly different at P < 0.05.

Fig. 6. Effect of FJH-UBS administration on the serum levels of bone turnover markers in ovariectomized mice.

Ovariectomized mice were orally administered either FJH-UBS (80 mg/kg body weight (BW)/day) or soy isoflavones (5 mg/kg BW/day) for eight weeks. Serum levels of osteocalcin (A), P1NP (B), osteoprotegerin (C), and TRAP 5b (D) were measured with the relevant ELISA kit. Each bar represents the mean ± SE (n = 10). P1NP, procollagen 1 N-terminal propeptide; TRAP 5b, tartrate-resistant acid phosphatase 5b; Sham, sham-operated group; OVX, ovariectomized group; OVX+FU, ovariectomized and 80 mg/kg body weight FJH-UBS-treated group; OVX+SI, ovariectomized and 5 mg/kg body weight soy isoflavones (a positive control)-treated group. ^*P < 0.01, ^**P < 0.05, ^***P < 0.001 significantly different from the Sham group. Mean without a common letter differs among OVX, OVX + FU, and OVX + SI groups at P < 0.05.