Anti‑osteoclastogenic effect of fermented mealworm extract by inhibiting RANKL‑induced NFATc1 action

Abstract

Augmented osteoclast activity and differentiation can lead to destructive bone diseases, such as arthritis and osteoporosis. Therefore, modulating osteoclastogenesis and differentiation may serve to be a possible strategy for treating such diseases. Tenebrio molitor larvae, also known as mealworms, are considered a good source of protein with nutritional value, digestibility, flavor and functional properties, such as antioxidant, anti-diabetic and anti-obesity effects. However, the role of mealworms in osteoclastogenesis remains poorly understood. The present study therefore investigated the effects of fermented mealworm extract (FME) on receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in bone marrow-derived macrophages (BMMs) whilst also attempting to understand the underlying mechanism, if any. The cells treated with RANKL were used as the negative control. To prepare FME, defatted mealworm powder was fermented with a Saccharomyces cerevisiae strain, and then extracted with fermented alcohol. Cell viability of BMMs isolated from 5-week-old Institute of Cancer Research mice was measured using Cell Counting Kit-8 assay. Subsequently, the effects of FME on osteoclast differentiation were measured using tartrate-resistant acid phosphatase (TRAP) staining. In addition, expression of markers associated with osteoclast differentiation was assessed by reverse transcription-quantitative PCR. Expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) was assessed by western blotting. TRAP staining revealed that FME inhibited osteoclast differentiation in a dose-dependent manner (10-100 µg/ml) without causing cytotoxicity. Specifically, the formation of osteoclasts appear to have been suppressed by FME as indicated by the reduction in the number of TRAP-positive multinucleated cells observed. Furthermore, FME treatment significantly decreased the mRNA expression of c-Fos, whilst also significantly decreasing the expression of NFATc1 on both protein and mRNA levels. c-Fos and NFATc1 are transcription factors that can regulate osteoclast differentiation. FME treatment also reduced the expression of genes associated with osteoclast differentiation and function, including dendritic cell-specific transmembrane protein, osteoclast associated Ig-like receptor, Cathepsin K and TRAP, compared with that in the control group. Subsequently, FME was found to effectively suppress RANKL-induced osteoclast differentiation compared with that by the non-fermented mealworm extract. These findings suggest that FME may confer anti-osteoclastogenic effects, providing insights into its potential application in treatment of osteoporosis.

Keywords: cytoplasmic 1; fermentation; mealworm; nuclear factor of activated T-cells; osteoclastogenesis; receptor activator of NF-κB ligand.

Figure 1

Preparation of non-fermented and fermented mealworm extracts. DMP, defatted mealworm powder.

Figure 2

Comparison of the effects of ME and FME (1, 3, 10 and 30 µg/ml) on (A) RANKL-induced osteoclast differentiation and (B) TRAP-positive multinucleated cells with three or more nuclei. The results of three independent experiments are presented as the mean ± SEM values and analyzed by two-way ANOVA followed by Holm-Sidak post hoc analysis. Scale bars, 100 µm. ^***P<0.001 vs. RANKL and ^###P<0.001 vs. RANKL + ME. FME, fermented mealworm extract; ME, non-fermented mealworm extract; RANKL, receptor activator of nuclear factor κB ligand; TRAP, tartrate-resistant acid phosphatase.

Figure 3

FME suppresses RANKL-induced osteoclast differentiation. (A) FME dose-dependently inhibited osteoclast differentiation and TRAP-positive multinucleated cells with three or more nuclei. Scale bars, 100 µm. BMMs were cultured with 30 ng/ml macrophage colony-stimulating factor for 24 h, before the indicated concentrations of FME were added and incubated for 3 days. (B) The effects of FME on the viability of BMMs were measured using a Cell Counting Kit-8 assay. The results of three independent experiments are presented as the mean ± SEM values and analyzed by one-way ANOVA followed by Holm-Sidak post hoc analysis. ^***P<0.001 vs. 0 µg/ml FME, ^###P<0.001 vs. 10 µg/ml FME and ^†††P<0.001 vs. 30 µg/ml FME. BMM, bone marrow-derived macrophages; FME, fermented mealworm extract; receptor activator of nuclear factor κB ligand; TRAP, tartrate-resistant acid phosphatase.

Figure 4

Effects of FME on the mRNA expression levels of cFos and NFATc1 and NFATc1 protein expression in RANKL-induced cells. (A) cFos and NFATc1 mRNA expression was measured by reverse transcription-quantitative PCR. GAPDH was used as an internal control. (B) Effects of FME on NFATc1 protein expression in RANKL-induced cells were measured by western blotting. Day 0 represents a 2-h reaction period. The results of three independent experiments are presented as the mean ± SEM values and analyzed by one-way ANOVA followed by Holm-Sidak post hoc analysis. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. RANKL. ^†P<0.05, ^††P<0.01 and ^†††P<0.001 vs. 30 µg/ml FME. FME, fermented mealworm extract; RANKL, receptor activator of nuclear factor κB ligand, NFATc1, nuclear factor of activated T cells, cytoplasmic 1.

Figure 5

Effects of FME on the expression of osteoclastogenic-associated marker genes TRAP, CTSK, OSCAR and DC-STAMP. Reverse transcription-quantitative PCR was performed. Day 0 represents a 2-h reaction period. The results of three independent experiments are presented as the mean ± SEM values and analyzed using an unpaired Student's t-test. ^***P<0.001 vs. RANKL. FME, fermented mealworm extract; RANKL, receptor activator of nuclear factor κB ligand; TRAP, tartrate-resistant acid phosphatase; CTSK, cathepsin K; OSCAR, osteoclast associated Ig-like receptor; DC-STAMP, dendritic cell-specific transmembrane protein.