Non-polar lipid from greenshell mussel (Perna canaliculus) inhibits osteoclast differentiation

Abstract

The osteoclast-dependent bone resorption process is a crucial part of the bone regulatory system. The excessive function of osteoclasts can cause diseases of bone, joint, and other tissues such as osteoporosis and osteoarthritis. Greenshell mussel oil (GSM), a good source of long chain omega-3 polyunsaturated fatty acids (LCn-3PUFAs), was fractionated into total lipid, polar lipid, and non-polar lipid components and their anti-osteoclastogenic activity tested in RAW 264.7 cell cultures. Osteoclast differentiation process was achieved after 5 days of incubation with RANKL in 24-well culture plates. Introducing the non-polar lipid fraction into the culture caused a lack of cell differentiation, and a reduction in tartrate-resistant acid phosphatase (TRAP) activity and TRAP cell numbers in a dose-dependent manner (50% reduction at the concentration of 20 μg/mL, p < 0.001). Moreover, actin ring formation was significantly diminished by non-polar lipids at 10-20 μg/mL. The bone digestive enzymes released by osteoclasts into the pit formation were also compromised by downregulating gene expression of cathepsin K, carbonic anhydrase II (CA II), matrix metalloproteinase 9 (MMP-9), and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1). This study revealed that the non-polar lipid fraction of GSM oil contains bioactive substances which possess potent anti-osteoclastogenic activity.

Keywords: AA, Arachidonic acid; ALA, Alpha linolenic acid; CAII, Carbonic anhydrase II; DHA, Docosahexaenoic acid; DMSO, dimethyl sulfoxide; DPA, Docosapentaenoic acid; EPA, Eicosapentaenoic acid; FFAR, Free fatty acid receptor; GSM, Greenshell mussel; Greenshell mussel; LA, Linoleic acid; LPS, Lipopolysaccharide; MMP-9, Matrix metalloproteinase 9; MUFA, Monounsaturated fatty acid; NF-κB, Nuclear factor κB; NFATc1, Nuclear factor of activated T-cells, cytoplasmic 1; OA, Osteoarthritis; Omega 3 fatty acid; Osteoarthritis; Osteoclasts; Osteoporosis; PA, Palmitic acid; PPAR, Peroxisome proliferator activated receptor; PUFA, Polyunsaturated fatty acid; RANKL, Receptor activator of nuclear factor κB ligand; SFA, Saturated fatty acid; TRAP, Tartrate-resistant acid phosphatase.

Graphical abstract

Fig. 1

Cell viability RAW 264.7 cells were incubated with different fractions of GSM lipids at concentration of 5-80 μg/mL or docosahexaenoic acid (DHA) at concentrations of 5-20 μg/mL in 96-well plates for 48 h. The viability of the cells was measured by MTT assay. Bar chart represents mean ± SD to demonstrate intra-and inter-experimental variability of percent cell viability from three independent experiments each conducted in duplicate. 10% DMSO was used as a cytotoxic reference. Individual fractions were compared with the vehicle control (VC) using one-way ANOVA followed by LSD post hoc test. Asterisk * indicates significantly different when compared to vehicle control at p < 0.05.

Fig. 2

Effect of greenshell mussel lipid on TRAP enzymatic activity The three fractions of GSM lipids at the concentration range from 5 to 20 μg/mL were tested in osteoclast differentiation assay and 10 μg/mL of docosahexaenoic acid (DHA) was used as a positive control for anti-osteoclastogenesis. The amount of TRAP was measured in supernatants by enzymatic activity. Bar chart shows mean ± SD of percentage of differences between the test substances and the vehicle control (n = 3 per condition). Individual fractions were compared with the vehicle control using one-way ANOVA followed by LSD post hoc test. Asterisk * and *** indicate significant different when compared to vehicle control at p < 0.05 and 0.001 respectively.

Fig. 3

Effect of greenshell mussel lipid on TRAP-positive cell counting The three fractions of GSM lipids at the concentration ranging from 5 to 20 μg/mL were tested in osteoclast differentiation assay. All cultures but “No RANKL” were treated with 15 ng/mL RANKL to induce osteoclastogenesis; vehicle control represents the negative control with RANKL only while 10 μg/mL of docosahexaenoic acid (DHA) was used as a positive control for anti-osteoclastogenesis. All treatments, including No RANKL, contained the vehicle (0.1% DMSO). TRAP positive cells with more than four nuclei were visually counted in each well under a microscope and expressed as percentage. Bar chart shows mean ± SD of percentage of differences between the test substances and the vehicle control (n = 3 per condition). Individual fractions were compared with the vehicle control using one-way ANOVA followed by LSD post hoc test. Asterisk * and *** indicate significant differences when compared to vehicle control at p < 0.05 and 0.001 respectively. Representative images show the effect of non-polar lipid GSM on anti-osteoclastogenesis in dose dependent manner. The red arrows identify the border of fusion osteoclasts (Vehicle control) or an osteoclast (the other images).

Fig. 4

Effect of non-polar lipid GSM on actin ring formation RAW 264.7 cells were seeded on 24-well culture plates at a density of 1.5 × 10⁴ cells per well with various concentrations of non-polar lipid GSM and 15 ng/mL RANKL. After 5 days of incubation, the cultures were stained with fluorescent dyes phalloidin (green) for actin and Hoechst 3324 (blue) for nuclei and then actin rings were counted in each well. Bar chart shows mean ± SD of percentage of differences between the test substances and the vehicle control (n = 3 per condition). Individual fractions were compared with the vehicle control using one-way ANOVA followed by LSD post hoc test. Asterisk *** indicate significant differences when compared to the vehicle control at p < 0.001. A demonstration of one actin ring is enclosed with a circle in the vehicle control and red arrows indicate actin ring formation. GSM = greenshell mussel, DHA = docosahexaenoic acid. Scale bars = 100 μm.

Fig. 5

Effect of non-polar lipid GSM on gene expression RAW 264.7 cells were seeded on 24-well culture plates at a density of 1.5 × 10⁴ cells per well with various concentrations of non-polar lipid GSM and 15 ng/mL RANKL for 5 days. The specific mRNA expression of gene relevant for osteoclast function was determined by quantitative RT-PCR. Relative quantification to non-RANKL vehicle-only control (0.1% DMSO) was calculated in fold change. The data are shown as mean ± SD (n = 3 per condition). Asterisk * and ** indicate significant differences of each concentration when compared to the vehicle control (VC) using one-way ANOVA followed by LSD post hoc test at p < 0.01 and 0.001 respectively. MMP-9 = Matrix metallopeptidase 9, Cat K = Cathepsin K, CAII = Carbonic anhydrase, NFATc1 = Nuclear Factor of Activated T Cells 1.