Suppressive effects of an apoptotic mimicry prepared from jumbo-flying squid-skin phospholipids on the osteoclastogenesis in receptor activator of nuclear factor kappa B ligand/macrophage colony-stimulating factor-induced RAW 264.7 cells

Abstract

Background:

Liposomes containing docosahexaenoic acid (DHA) and phosphatidylserine were claimed to inhibit osteoclast formation and bone resorption in the inflammatory status. Herein, we proposed that an apoptotic mimicry (SQ liposome) prepared from squid-skin phospholipids can explore the suppressive osteoclastogenesis.

Methods:

The intermolecular fatty-acid composition in the phospholipid of squid-skin extract was analyzed by GC-FID. The SQ liposome structure was characterized by size distribution and zeta potential (ζ). RAW 264.7 cell is used to study the effect of SQ liposomes on osteoclast differentiation. Secretion of prostaglandin E2 (PGE2) and transforming growth factor-β (TGF-β) from RAW 264.7 cells were assayed. Antiosteoclastogenesis effects were performed via the tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell (MNC) counting, bone resorption pit assay, and TRAP activity analysis. The specific gene expressions related to antiosteoclastogenesis were also detected.

Results:

An apoptotic mimicry through the use of a single-layer liposome (SQ liposome) with phosphatidylserine exposure contains DHA (28.7%) and eicosapentaenoic acid (EPA, 11.8%). Co-treatment with receptor activator of nuclear factor kappa B ligand (RANKL)/macrophage colony-stimulating factor induced RAW 264.7-cell differentiation into mature osteoclasts, thus enhancing PGE2 and TGF-β secretion. However, cotreatment with 1 mg/mL of SQ liposome restored (p < 0.05) the cell viabilities under the RANKL stress. Increased PGE2 levels was downregulated (p < 0.05) in cotreatments with 0.11 and 0.33 mg/mL of SQ liposome, but on the TGF-β levels were not (p > 0.05) influenced in SQ liposome cotreatments. Cotreatments with 0.33–1 mg/mL of SQ liposome suppressed (p < 0.05) the osteoclast maturation (such as decreased MNCs and bone pit formation), inhibited TRAP activities, and downregulated the osteoclastogenesis-related gene expressions.

Conclusion:

In summary, current data support that a possible prevention of our prepared SQ liposomes which are rich in DHA and EPA on bone loss is through the suppression of osteoclastogenesis. Moreover, based on the results from this study an in vivo study warrants a further investigation.

1. Introduction

It is critical to regulate osteoclastogenesis and osteogenesis in bone remodeling. However, bone homeostasis after adulthood often becomes unbalanced and tends to lead to overactive osteoclastogenesis, thus resulting in increased bone resorption and a decline in bone mineral density that may cause bone fracture risk. Some bone-related inflammatory responses were also characterized as a high risk in terms of bone metabolic disorders, such as periodontitis in the alveolus and arthritis in the ankle joints. Therefore, the question of how to alleviate inflammation and overactive osteoclastogenesis is pivotal in retaining bone mass, especially in the elderly.

An osteoclast, also called an osteal macrophage, in bony tissues is derived from hematopoietic progenitors of the monocyte-macrophage lineage and is known as the protagonist in osteoclastogenesis.¹ The RAW 264.7 cells, also known as monocyte/macrophage-like cells, originating from the Abelson leukemia virus, transformed the cell line derived from BALB/c mice.² Owing to the fact that primary osteoclast cultures are difficult to maintain and isolate, the RAW 264.7 cell is, therefore, the most commonly used to study osteoclast differentiation and its signaling pathways.^2,3 Docosahexaenoic acid (DHA), one of the n-3 polyunsaturated fatty acids (PUFAs), has been claimed to have an in vitro antiosteoclastogenic effect through suppression of the proliferation and differentiation of the bone-marrow–derived macrophages or the enhancement of the apoptosis of mature osteoclasts.^3,4 It was also reported that DHA supplementation can inhibit osteoclast formation and bone resorption in LPS-induced inflammatory mice.⁵ The cause of alveolar bone loss is usually addressed in chronic periodontitis, and a massive investigation of the prevalence of periodontitis indicated that a higher dietary intake of DHA is associated with a lower prevalence of periodontitis in adult American and elder Japanese.^6,7 It has been indicated that DHA supplementation, combined with aspirin treatment, shows significantly improved periodontal outcomes in adults.⁶ Furthermore, a series of studies focused on the inhibition of osteoclastogenesis by using either phosphatidylserine (PS) liposome or the apoptotic mimicry. A further study prepared a liposome using egg phosphatidylcholine (PC) and bovine brain PS that mimicked the apoptotic cell which characterized PS exposure on the cell surface.⁸ They investigated the effects of the PS liposome on osteoclastogenesis by receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast cells and adjuvant-induced ankle joint arthritic (AA) rats, and then indicated that the engulfed PS liposome of a phagocyte can inhibit osteoclastogenesis by generating transforming growth factor beta (TGF-β) and prostaglandin E2 (PGE2), and then alleviating AA-induced trabecular bone loss. Furthermore, it was also demonstrated that PS liposomes suppress inflammatory bone loss in AA rats via the regulation of cytokine secretion provoked by infiltrated macrophages.⁹ Though both of the n-3 PUFA or PS liposomes’ effect on bone health function had been mentioned, the phospholipid extracts in soybean or livestock materials have low intermolecular PUFA content.

The integument, brain, arm, and gonad of the Humboldt squid (Dosidicus gigas) are enriched by the PUFA in the form of phospholipids and are usually discarded or used only as feed additives for livestock.¹⁰ In response to the Asia-Pacific Economic Cooperation policy on food loss and waste reduction throughout the food processing chain, the processing of marine byproducts should be fully carried out. In our previous studies, a squid-skin (SQ) liposome was prepared by extracting phospholipids from squid integument, which is a byproduct of the squid processing stream.¹¹ SQ liposomes can engage in apoptotic mimicry to elicit the resolution of inflammation in LPS-induced RAW 264.7 cells and ameliorated carrageenan-induced paw edema in mice.¹¹ In this study, we would like to rearrange the phospholipid in SQ liposomes into a single-layer lipid droplet and then evaluate whether PS exposure on the liposomal surface can elicit phagocyte engulfment. Herein, the suppressive effects of SQ liposome cotreatment on osteoclastogenesis were also evaluated by RANKL-induced osteoclast differentiation of RAW 264.7 cells.

2. METHODS

2.1. Materials

The followings were purchased from Sigma-Aldrich Co. (St. Louis, MO): β-estradiol (Cat#: E8875) and pamidronate disodium (Cat#: P2371), as the reference compounds for detecting osteoclastogenesis levels in the cell-based assay; dimethyl sulfoxide (DMSO) (Cat#: D5879) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Cat#: M2128), which were used in analyzing cell viability and cytotoxicity, respectively; and an acid phosphatase leukocyte (tartrate-resistant acid phosphatase [TRAP]) kit (Cat#: 387A), which was used for measuring mature osteoclast enzyme activities. The following were supplied by GIBCO, Life Technologies (Grand Island, NY): the cell culture medium and serum including Dulbecco’s Eagle medium (DMEM) (Cat#: 21063-29); α-MEM (Cat#: 41061-029); and fetal bovine serum and penicillin streptomycin solution, Pen-Strep (10 000 U/mL) (Cat#: 15140). Formaldehyde solution, 10% in aqueous phosphate buffer (Cat#: H121-08, Macron Fine Chemicals), which was used for cell fixation, was obtained from Avantor Performance Materials Taiwan Co., Ltd. (Hsinchu County, Taiwan, ROC). A bone resorption assay kit (Cat#: 485453479, Cosmo Bio Co., Ltd., Carlsbad, CA), PGE2 ELISA kits (Cat#: 514010, Cayman Chemical Co., Ann Arbor, MI), and RNeasy mini kit (Cat#: 74104 and 74106, Qiagen Inc., Valencia, CA) were purchased from local suppliers in Taiwan. Recombinant mouse TRANCE/RANKL/TNFSF11 (Cat#: 462-TEC, R&D System, Inc., Minneapolis, MN) and recombinant mouse M-CSF protein (Cat#: 416-ML, R&D System, Inc.) for stimulating macrophage proliferation, as well as a TGF-β kit (Cat#: DY1679) for cytokine detection, were acquired from R&D System, Inc.

2.2. Phospholipid extraction and SQ liposome preparation

Phospholipid extraction and SQ liposomes from squid skin were prepared according to our previous study.¹¹ In brief, the dried squid-skin extract pellet was further suspended in α-MEM and sonicated (UP50H Ultrasonic homogenizer, Hielscher Ultrasonics GmbH, Teltow, Germany) for 10 minutes on ice. All the SQ liposomes were prepared 1 day before use in the in vitro experiments and stored at −20°C.

2.3. Fatty acid composition in squid-skin phospholipid extracts

After the phospholipid fraction was isolated from the squid skin, the solvent was removed under a nitrogen gas flow. The intramolecular fatty acid (FA) methyl ester and the composition analysis were commissioned to the Food Industry Research and Development Institute (Hsinchu City, Taiwan, ROC) and performed according to AOAC official method 969.06 by using gas chromatography with flame-ionization detection (GC-FID). Briefly, 100–200 mg of phospholipid extract was added to 1 mL of 1 N NaOH-methanol in a Pyrex screw-cap tube, shaken for 30 seconds, and then kept in the heater at 80°C for 15 minutes. One milliliter of 14% BF3-methanol was further added to the tube and boiled to 110°C for another 15 minutes. After the sample tube was cooled, 1 mL of hexane and 6 mL of saturated NaCl solution were added to the tube, shaken, and separated. The up-layer hexane phase was recovered and stocked in brown vials with a bit of sodium sulphate. The composition of methyl-esterified FAs was carried out through use of a CP-Sil 88 capillary column (CP7489, Agilent Technologies, Santa Clara, CA) with a 100 m × 0.25 mm inner diameter and a 0.20-μm film thickness. Chromatographic peaks were detected by a flame ionization detector. The carrier gas (helium) flow rate was 0.75 mL/min. The oven temperature was programmed from 170°C hold for 40 minutes as well as then increased to 200°C at a ramp rate of 3°C/min and maintained at 200°C for 50 minutes. Chromatographic peaks were identified by comparison of the retention times of a 17 component FAME standard mix (Sigma-Aldrich Co.). The amount of each identified FA was determined using standard curves of authentic compounds. The amount of each identified FA in the phospholipids extracted from the squid skin was symbolized as %.

2.4. Measurement of the size distribution and zeta potential of SQ liposomes

After the SQ liposome suspension (1 mg/mL) was loaded into the disposable folded capillary cells (Cat#: DTS1070, Zetasizer Nano Series, Malvern Instruments Ltd., Worcestershire, UK), the average size distribution and zeta potential (ζ) of SQ liposomes in α-MEM were further determined by a Zetasizer Nano-Zs (Malvern Instruments Ltd. and Zetasizer software 6.30). The zeta potentials of SQ liposomes were measured five times and then averaged. During measurements, the temperature was maintained at 37°C, and all measurements were made in triplicate.

2.5. RAW 264.7 cell culture and stimulated osteoclast differentiation

RAW 264.7 cells were purchased from the Bioresource Collection and Research Center (BCRC 60001; Food Industry Research and Development Institute) and cultured in DMEM media supplemented with 10% heat-inactivated fetal bovine serum, 100 unit/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere of 5% CO₂ at 37°C. To induce RAW 264.7 cell differentiation into mature osteoclasts, the media were replaced with α-MEM in the presence or absence of a combination of both RANKL (40 ng/mL) and M-CSF (40 ng/mL) and then coincubated with various concentrations of the SQ liposomes or reference compounds including 100 nM β-estradiol and 100 μM pamidronate for 4–5 days without changing the medium.

2.6. Cytotoxicity and cell viability

MTT assays were performed using serial threefold dilutions of the SQ liposomes (0.04, 0.11, 0.33, and 1 mg/mL) in α-MEM cultural media with or without a combination of both 40 ng/mL RANKL and 40 ng/mL M-CSF at 37°C for 5 days. After careful removal of the supernatant, 100 μL of 0.5 mg/mL MTT reagent was added to each well. The plates were subsequently incubated at 37°C for 1 hour. The supernatant was discarded, and 100 μL of DMSO was added. The plates were then gently shaken to dissolve the formed formazan. The absorbance of each well was measured at 570 nm through use of a microplate reader (ELx800 ELISA Reader, BioTek Instruments Inc., Winooski, VT), and the cell viability of control cells was assumed to be 100%.

2.7. TGF-β and PGE2 quantification

The supernatant from each culture well was collected on day 5 of the experiments. According to the manufacturer’s instructions, TGF-β and PGE2 levels in the supernatant were measured by using commercial TGF-β and PGE2 ELISA kits, respectively.

2.8. TRAP-positive cell multinucleated counting

For the counting of TRAP-positive multinucleated cells, the RAW 264.7 cells (5 × 10³ cells/mL) were seeded into an FBS coated 8-well chamber slide (Cat#: C86024, Millicell@ EZ slides, Merck KGaA, Darmstadt, Germany). After the cell adhesion, a threefold serial dilution of the SQ liposomes or reference compounds (100 μM pamidronate or 100 nM β-estradiol) was added in the presence or absence of a combination of both 40 ng/mL RANKL and 40 ng/mL M-CSF at 37°C for 4–5 days. TRAP staining was conducted according to the manufacturer’s instructions. The cells were fixed and stained through use of the TRAP staining kit according to the manufacturer’s instructions. The TRAP-positive multinucleated cells were identified as more than three nuclei by a light microscope observation (CKX41, Olympus, Tokyo, Japan).

2.9. Bone resorption pit assays

A bone resorption assay kit was used to evaluate the suppressive effects of SQ liposomes on RANKL-induced osteoclast differentiation. In a typical assay procedure, the murine macrophage cell line RAW 264.7 cell (2.5 × 10³ cells/well) was seeded in the 24-well calcium phosphate (CaP)-coated plate (Cosmo Bio Co., Ltd.) for 24 h. Then, the medium was changed into each indicated condition medium including pamidronate, β-estradiol, or SQ liposome with or without a RANKL (40 ng/mL)/M-CSF (40 ng/mL) stimulation. After a 5-day incubation period, the cells were removed with 5% sodium hypochlorite for 5 minutes and the plate was washed and rinsed with ddH₂O. The photograph of resorption pits in each well was taken through use of a microscope observation (CKX41, Olympus) and quantified through use of Adobe Photoshop CS3 software. Then, the results of the bone pit area were expressed as pixels of each well pit area.

2.10. TRAP activity analysis

In addition, RAW 264.7 cells were suspended in phenol-free α-MEM containing 10% FBS and seeded overnight in a 96-well culture plate at a concentration of 5 × 10⁴ cells/mL. After the cell adhesion, threefold serial dilutions (0.04, 0.11, 0.33, and 1 mg/mL) of the SQ liposomes or reference compounds were added in the presence or absence of a combination of 40 ng/mL RANKL and 40 ng/mL M-CSF for 4–5 days. Then, the medium was removed and the cell was gently washed twice with DPBS. The cells were then lysed with 200 mL of 0.2% Triton X-100.TRAP activity in cell lysate was determined through use of a TRAP kit. A total of 100 μL of TRAP buffer was added to each well of cell lysate and followed by incubation for 1 hour. The reaction was stopped by the addition of 0.1 N NaOH and absorbance was measured at 405 nm through use of a microplate reader (ELx800 ELISA Reader, BioTek Instruments Inc.).

2.11. Osteoclastogenesis-specific gene expression

For the specific gene expression of osteoclastogenesis analysis, RAW 264.7 cells (5 × 105 cells/mL) were seeded into a Nunc 6-well cell culture plate (Cat#: 140675, Thermo Fisher Scientific Taiwan Co. Ltd., Taipei, Taiwan, ROC). After the cell adhesion, a twofold serial dilution of the SQ liposomes (0.125, 0.25, 0.5, and 1 mg/mL) was added in the presence or absence of a combination of both 40 ng/mL RANKL and 40 ng/mL M-CSF for 6 hours. After the supernatant was discarded, the total RNA of RAW 264.7 cells was isolated through use of the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) followed by the DNase on column digestion (RNase-Free Dnase Set). For real-time PCR (RT-PCR) analyses, single-strand complementary deoxyribonucleic acid (cDNA) was synthesized from 1 μg of total RNA through the use of reverse transcriptase (SuperScript III First-Stand; Invitrogen, Carlsbad, CA). The RT-PCR analysis of each target gene was performed through use of SYBR Green qPCR Master Mixes (Cat#: 3003230, Thermo Fisher Scientific Inc., Waltham, MA). The results were detected through use of a CFX Real-Time PCR System (Bio-Rad Laboratories, Inc., Hercules, CA). PCR amplification was performed through use of the following program: 95°C for 10 minutes and then 40 cycles of 95°C for 10 seconds and 65°C for 5 seconds. The primer sequences of osteoclastogenesis used in this study were shown as following mouse sequences: nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1, forward: 5′-GGGTCAGTGTGACCGAAGAT-3′, reverse: 5′-GGAAGTCAGAAGTGGGTGGA-3′, GenBank no.: AF239169.1), proto-oncogene tyrosine-protein kinase Src (c-Src, forward: 5′-CCAGGCTGAGGAGTGGTACT-3′, reverse: 5′-CAGCTTGCGGATCTTGTAGT-3′, GenBank no.: NM_009271.3), receptor activator of nuclear factor-kappa B (RANK, forward: 5′-GGCTGCCATCATCTTCGGCGTT-3′, reverse: 5′-TTGCCGAGTGGGAACCAGCA-3′, GenBank no.: AF019046.1), matrix metallopeptidase 9 (MMP-9, forward: 5′-AGTTTGG TGTCGCGGAGCAC-3′, reverse: 5′-TACATGAGCGCTTCCGGCAC-3′, GenBank no.: NM 013599.4), cathepsin K (forward: 5′-GGCCAACTCAAGAAGAAAAC-3′, reverse: 5′-GTGCTTGCTTCCCTTCTGG-3′, GenBank no.: NM 007802.4), TRAP (forward: 5′-CGCTGACTTCATCATGTCTCTG-3′, reverse: 5′-ATAAAGATGGCCACAGTTATGTTTG-3′, GenBank no.: BC012911.1), β3-Integrin (forward: 5′-TGACATCGAGCAGGTGAAAG-3′, reverse: 5′-GAGTAGCAAGGCCAATGAGC-3′, GenBank no.: NM 016780.2), dendrocyte-expressed seven transmembrane protein (DC-STAMP, forward: 5′-TCCTCCATGAACAAACAGTTCCAA-3′, reverse: 5′-AGACGTGGTTTAGGAATGCAGCTC-3′, GenBank no.: NM_029422.4), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, forward: 5′-AACTTTGGCATTGTGGAAGG-3′, reverse: 5′-ACACATTGGGGGTAGGAACA-3′, GenBank no.: NM_001289726.1). The sizes of the reaction products are as follows: NFATc1, 225 bp; c-Src, 193 bp; RANK, 146 bp; MMP-9, 754 bp; cathepsin K, 426 bp; TRAP, 276 bp; β3-Integrin, 225 bp; DC-STAMP, 149 bp; and GAPDH, 221 bp. Data were analyzed using the 2^−ΔΔCT method, and all values were normalized to the level of the housekeeping gene, GAPDH.

2.12. Statistical analysis

The experimental data were analyzed through use of the SAS 9.2 software (SAS Institute Inc., Cary, NC). All data were given as mean and SD. The experimental data were analyzed through use of one-way analysis of variance (ANOVA). If a significant difference (p < 0.05) was detected among the groups, Duncan’s multiple range test was further applied to distinguish the differences between the groups.

3. RESULTS

3.1. Characterization of SQ liposomes

As shown in Fig. 1A, the major FAs from the isolation of SQ phospholipids were DHA (28.7%), palmitic acid (27.2%), and eicosapentaenoic acid (EPA, 11.8%). Over 40% of the omega-3 PUFA (n-3 PUFA)-primarily EPA and DHA—in SQ phospholipids were received. The particle size with the diameter and the surface charge (ζ, Zeta potential) of the SQ liposome in α-MEM were 146.4 ± 73.4 nm and −24.5 ± 12.7 mV, respectively, determined through use of a Zetasizer Nano-Zs (Fig. 1B).

Fig. 1.

The characterization of phospholipids in squid skin and SQ liposomes: (A) intermolecular fatty acid composition in phospholipids and (B) zeta potential. The zeta potential of SQ liposomes was measured by Zetasizer Nano-Zs (Malvern Instruments, Worcestershire, UK and Zetasizer software 6.30). The intermolecular fatty acid composition of squid skin phospholipid extraction was analyzed according to AOAC official methods 969.33 by GC-FID. GC-FID = gas chromatography with flame-ionization detection; SQ = squid skin.

3.2. Effects of SQ liposomes on RANKL-induced TGF-β and PGE2 secretion

The cell viability effects of co-treatment with SQ liposomes were evaluated on RANKL/M-CSF (40 ng/mL of RANKL and 40 ng/mL of M-CSF)-stimulated RAW 264.7 cells (Fig. 2A). In comparison to the control group (no RNAKL/M-CSF stimulation), though the cell viabilities decreased (p < 0.05) in the RNAKL/M-CSF stimulation group and the further cotreated with low-dose SQ liposome groups (0.012, 0.04, 0.11, and 0.33 mg/mL of SQ liposome), the entire cell viabilities of all groups were higher than 80%. Interestingly, the decreased cell viability of the RANKL/M-CSF stimulation was restored (p < 0.05) after a cotreatment with 1 mg/mL of SQ liposome. In addition, the TGF-β and PGE2 levels measured from the cell suspension of groups were further examined. As shown in Fig. 2B, C, in comparison to the control group (no RANKL/M-CSF stimulation) on day 5, the TGF-β and PGE2 secretion levels significantly increased from 76 to 358 pg/mL and from 97.8 to 171.5 pg/mL, respectively, after the RANKL/M-CSF stimulation. However, all of the groups including cotreatments with 100 μM pamidronate, 100 nM β-estradiol, or 0.04–1 mg/mL of SQ liposomes did not affect the TGF-β secretion levels. Meanwhile, an enhancement of PGE2 levels by RANKL/M-CSF stimulation was suppressed (p < 0.05) during cotreatment with 100 μM pamidronate, 100 nM β-estradiol, or 0.11–0.33 mg/mL of SQ liposomes, though not during cotreatment with 1 mg/mL.

Fig. 2.

The effects of cotreated SQ liposomes on the cell viability, and TGF-β and PGE2 secretion levels in the RANKL/M-CSF stimulated RAW 264.7 cells for 120h: (A) Cell viability, (B) TGF-β, level, and (C) PGE2 level. The cell viability analysis was measured by MTT assay. Quantitative analyses of TGF-β and PGE2 were determined using an ELISA kit. Data are given as mean ± SD (n = 3). Data bars in each tested parameter without a common letter indicate a significance between groups (p < 0.05). M-CSF = macrophage colony-stimulating factor; PGE2 = prostaglandin E2; RANKL = receptor activator of nuclear factor kappa B ligand; SQ = squid skin; TGF-β = transforming growth factor-β.

3.3. Effects of SQ liposomes on TRAP staining and multinuclear formation

TRAP-positive staining and multinuclear giant cell formation are critical features of mature osteoclasts by RANKL/M-CSF-stimulated RAW 264.7 cells. As shown in Fig. 3A, after the RAW 264.7 cells were treated with RANKL/M-CSF for 120 hours, the cell morphologies of the positive control group (RANKL/M-CSF+) had significantly changed and the TRAP staining images showed that the area with purple-red around the cytoplasm of osteoclasts was markedly increased. This was accompanied by cell-cell fused phenomena and the formation of a multinuclear giant cell. Here, pamidronate and β-estradiol, the generic drugs for osteoporosis, had been used as a reference to inhibit osteoclastogenesis. As a result, the addition of both generic drugs significantly suppressed TRAP-positive multinuclear (nuclei > 3) cell formation. Moreover, in the experimental groups, depending on the increased addition of SO-liposome doses, the purple-red stained with a multinuclear cell number illustrated a decreased trend. The quantitative data of the multinuclear cell counting in each group with average in triplicate experiments was also conducted, as shown in Fig. 3B. The experimental groups with over 0.33 mg/mL of SQ liposomes illustrated a potent inhibition (p < 0.05) of the TRAP-positive multinuclear cell formation.

Fig. 3.

The effects ofaco-treated SQ liposome on the RANKL/M-CSF-induced RAW 264.7-cell differentiation to become osteoclasts. The 5 × 10³ cells/mL of RAW 264.7 cells were seeded into 8-well chamber slides.After cell attachment, the indicated SQ liposome condition medium or osteoporosis drug (pamidronate or β-estradiol) was coincubated with 40 ng/mL of RANKL and 40 ng/mL of M-CSF at 37°C, 5% CO₂. After 120 h of culture, the cells were fixed and stained with a TRAP staining kit. A, The illustrations are represented in triplicate by inverted microscope photography. B, The numbers of TRAP-positive MCFs in each group were counted on the microphotograph (Adobe Photoshop CS3) by 3 representative fields. Data are given as Mean ± SD (n = 3). Data bars without a common letter indicate a significance between groups (p < 0.05). M-CSF = macrophage colony-stimulating factor; RANKL = receptor activator of nuclear factor kappa B ligand; SQ = squid skin; TRAP = tartrate-resistant acid phosphatase.

3.4. Effects of SQ liposomes on RANKL/M-CSF-stimulated bone pit formation

Following the above outcomes, similar cell-based experiments were performed on the calcium-phosphate-coated cell culture plate for 120 hours. After the cells were removed, washed, and rinsed with ddH₂O, the bone resorption pit areas in each well were taken and analyzed. It was observed that not only the osteoporosis drugs (100 μM of pamidronate or 100 nM of β-estradiol), but also SQ liposome treatment significantly reduced the bone resorption pit area (Fig. 4A, B). In addition, a dose-dependent suppression of the pit area formation on RANKL/M-CSF-stimulated RAW 264.7 cells was shown in an addition of 0.1–1 mg/mL of SQ liposome.

Fig. 4.

The effects of co-treated SQ liposomes on RANKL/M-CSF-stimulated bone pit formation. RAW 264.7 cells were seeded in calcium phosphate (CaP)-coated plates overnight and treated with the indicated dosage of SQ liposome or osteoporosis drugs in the presence or absence of RANKL/M-CSF medium for 120 h. A, After the cell was removed, the images of the resorption pits on the plates were taken using an optical inverted microscope. B, Resorption areas were quantified using Adobe Photoshop CS3 software and the results were expressed as pixels of each well pit area. Data are given as mean ± SD (n = 3). Data bars without a common letter indicate a significance between groups (p < 0.05). M-CSF = macrophage colony-stimulating factor; RANKL = receptor activator of nuclear factor kappa B ligand; SQ = squid skin.

3.5. Effects of SQ liposomes on TRAP activity and gene expression related to RANKL/M-CSF-stimulated osteoclastogenesis

In addition, Fig. 5A shows that RANKL/M-CSF treatment significantly increases (p < 0.05) TRAP activity in RAW 264.7 cells, while TRAP activity was dose-dependently downregulated by the SQ liposome. After TRAP-positive multinuclear cell counting, the results of the TRAP activity assay reconfirmed the inhibitory ability of the TRAP activity by SQ liposomes. Consequently, to understand which role involved in the osteoclastogenesis inhibitory effect of our prepared SQ liposomes, we screened the osteoclast differentiation-related gene expressions, including NFATc1, RANK, c-Src, β3-integrin, cathepsin K, MMP-9, TRAP, and DC-STAMP. As presented in Fig. 5B, all of the mRNA levels showed a significant upregulation of at least two folds when stimulated with RANKL/M-CSF (p < 0.05) for 24 hours; the exception was MMP-9 (only 1.2-fold). However, the NFATc1, RANK, c-Src, cathepsin K, TRAP, and DC-STAMP mRNA levels were noticeably downregulated (p < 0.05) when the RANKL/M-CSF-stimulated RAW 264.7 cells were cotreated with 0.1–1 mg/mL of SQ liposomes. Increased MMP-9 gene expression by RANKL/M-CSF stimulation was downregulated in SQ liposome added levels higher than 0.25 mg/mL (p < 0.05).

Fig. 5.

The effect of an SQ liposome on TRAP activity and osteoclastogenesis gene expressions in RANKL/M-CSF induced RAW 264.7 cells: (A) TRAP activity and (B) the gene expressions including NFATc1, c-Src, RANK, MMP-9, Cathepsin K, TRAP, β3-Integrin, and DC-STAMP. Data are given as mean ± SD (n = 3). Data bars in each tested parameter without a common letter indicate a significance between groups (p < 0.05). DC-STAMP = dendrocyte-expressed seven transmembrane protein; M-CSF = macrophage colony-stimulating factor; MMP-9 = matrix metallopeptidase 9; NFATc1 = nuclear factor of activated T-cells, cytoplasmic 1; RANKL = receptor activator of nuclear factor kappa B ligand; SQ = squid skin; TRAP = tartrate-resistant acid phosphatase.

4. DISSCUSSION

Our previous study had indicated that approximately 100 nm of SQ liposome can be formed as a unilamellar lipid droplet; meanwhile, the major phospholipid classes of the squid skin extract, including phosphatidylinositol, PS, phosphatidylethanolamine, PC, and Lyso-PC, had been claimed.¹¹ As we know, most of the cellular phospholipids are from the lipid bilayer of the plasma membrane or intracellular membrane. PS is a phospholipid that is conjugated with a negatively charged serine group. During cell apoptosis, one of the critical characterizations is PS translocation from the inner to the outer membrane leaflets to elicit the recognition and clearance of macrophage-like cells. Here, we propose a hypothesis that our prepared SQ liposomes should appear to have negative zeta potential when the phospholipids were re-assembling into a single-layer liposome in a polar solution. Rodriguez-Fernandez et al¹² characterized an empty PS liposome showing the negative charge (−38.9 ± 2.52 mV) at the liposome surface in comparison to a PC liposome (−7.6 ± 2.68 mV). Our results also had a similar observation. Moreover, PS liposomes suppressed osteoclastogenesis, while trabecular bone loss in an adjuvant-induced-arthritis rat model had continuously been proposed.^8,9 The amphipathic phospholipid compounds also contain two intramolecular FA chains. Saito et al¹⁰ examined the phospholipids and FAs from the Humboldt squid (D. gigas) tissue, including the mantle, skin, arm, and gonad, while the main FAs in those tissue phospholipids are rich in both EPA and DHA. Those omega-3 FAs have been well-known as the precursors to the resolved inflammatory regulators, including E- and D-series resolvins, as well as protectins. It has been demonstrated that DHA and PS improve the in vitro and in vivo antioxidant activities of rat studies.¹³ Moreover, it was also indicated that long-chain n-3 PUFAs are beneficial to bone health.¹⁴ Additionally, it has been reported that DHA has an antiosteoclastogenic effect through suppression of the proliferation and differentiation of bone marrow-derived macrophages or enhancement of the apoptosis of mature osteoclasts.^3,4 Kishikawa et al⁵ investigated the effects of DHA on inflammation-induced osteoclast formation in in vitro and in vivo studies and suggested that DHA inhibits LPS-induced osteoclast formation and bone resorption through G protein-coupled receptor 120 (GPR120), a functional receptor for n-3 FAs. Our results also showed that rich n-3 PUFAs had been measured in SQ phospholipid extracts. The above literature related both PS liposome and n-3 PUFAs supported the notion that those elements have bone-protective effects.

After the RANKL binging to the receptor (RANK) of osteoclast precursors, the active downstream of RANK signaling cascades is initiated mainly by phosphorylation of TRAF complexes that belong to the TNF receptor superfamily, which responds to TNF members. RANKL, as other members of the TNF superfamily, has the ability to inhibit cell growth and induce cell apoptosis.¹⁵ Therefore, the cell viabilities of RANKL treatments on our experiments were slightly decreased, and we also observed the prevention of RANKL-induced RAW 264.7-cell apoptosis by cotreating with one mg/mL of the SQ liposome group. Iwasaki et al⁷ observed that PS liposome could be engulfed by the osteoclast precursor cells and then increase the plasma levels of TGF-β and PGE2 on the adjuvant-induced-arthritis rat model. They further inferred that osteoclastogenesis inhibition may act in an autocrine manner. Furthermore, the secretion of TGF-β by osteoblasts and osteoclasts was observed in the bone matrix during bone remodeling. TGF-β acts as a central component of the coupling of bone formation to resorption.¹⁶ Meanwhile, it was also observed that osteoclastic activity contributes to the enhancement of TGF-β for further stimulation of osteoblast maturation at sites of bone resorption. Our in vitro cell-based results also supported the notion that TGF-β increases after RANKL-stimulated RAW 264.7-cell osteoclastogenesis, though no changes in TGF-β levels were found after cotreatment with PS liposome or the reference compounds. On the other hand, PGE2 secreted by osteoblasts increased when bone density decreased, as demonstrated in osteoporotic animal models.¹⁷ Results of these articles illustrated the differences between the in vitro and in vivo studies.

Pereira et al¹⁸ indicated that multinuclear cell formation is one of the cell signatures in chronic inflammatory bone diseases. Mature multinuclear osteoclasts cells were fusion by monocyte or macrophage lineage through recognize each other of fusion-mediating molecules on the cell surfaces.¹⁹ For example, the DC-STAMP, CD44, and the tetraspanins CD9 and CD81 are involved in the fusion-mediating molecules; however, some of the underlying mechanisms are not clear.¹⁸ On the other hand, TRAP, which is a kind of enzyme involved in bone matrix degradation during the resorption process, is also one of the pivotal histochemical markers on mature osteoclasts.²⁰ A previous study indicated that a cotreatment of PS liposomes for 96 hours can suppress the formation of TRAP-positive multinuclear cells in the bone marrow and osteoclast precursor.⁷ In the RANK/M-CFS-induced RAW 264.7 cell-based assay, DHA has the strongest inhibitory effect on TRAP-positive osteoclast formation.³ Kasonga et al²¹ indicated that long-chain n-3 PUFAs potentially inhibit TRAP activity in a RANK/M-CFS-triggered human CD14+ monocyte differentiation model. Those articles provided convincing data supporting the suppression of RANKL/M-CSF-stimulated osteoclast maturation by using either the PS liposome or n-3 PUFA. Our prepared SQ liposome shares similar characteristics with the PS molecular properties that appeared on the liposomal surface and contained n-3 PUFA-rich phospholipids. Our pit suppression outcomes also corresponded to the previous experiment involving TRAP staining and multinuclear formation (Figs. 3 and 4). SQ liposomes attenuated the bone resorptive activity of an osteoclast in vitro cell-based assay. Disodium pamidronate to a class of drugs known as bisphosphonates, the pharmacological mechanism in bones, including the adsorption of calcium phosphate (hydroxyapatite) crystals in bone, may directly block the dissolution of this mineral component of bone.²² In vitro studies also reported that inhibition of osteoclast activity contributes to the inhibition of bone resorption.²¹ However, long-term use of pamidronate had been correlated with numerous side effects, including hypocalcemia, secondary hyperparathyroidism, musculoskeletal pain, various ocular events, and osteonecrosis of the jaw.²³ It was also reported the β-estradiol is able to inhibit osteoclast bone resorption by directly inducing osteoclast apoptosis in a dose- and time-dependent manner.²⁴ Adverse effects of estrogen on the long-term use also exist including increased risk of breast cancer, cardiovascular disease, and dementia.²⁵ Although, there was not experiments of PS liposome effects on bone resorption pit assays have been proven, inhibition of trabecular bone loss in the AA rat model treated with PS liposomes has been proposed.^7,8 Hence, the inhibition of the osteoclast differentiation and bone resorption pit formed by our prepared SQ liposomes and obtained from squid skin is reasonable and reliable.

González-Galván et al²⁶ reported that the RANK responding to the RANK ligand (RANKL) exists on the osteoclast precursor cell (OP cell) surface that initiates OP cell differentiation into mature osteoclasts. NFATc1 is a pivotal transcription factor that responds to RANKL-RANK-induced signal transduction, and then regulates downstream osteoclastogenesis and differentiation.^27,28 A collaboration between cathepsin K and MMP-9 contributes to the degradation of type I collagen in the bone matrix.²⁹ DC-STAMP is an essential molecule for mononuclear osteoclast fusion³⁰ and giant cell formation that increases the resorption activity of osteoclasts.^19,31 The adhesion molecules β3-Integrin and c-Src play important roles in the cytoskeleton organization to form the sealing zone by mediating their migration and adhesion activities.^32,33 Those reports, integrated into our studies, implied that SQ liposomes play a role in the regulation of osteoclastogenesis, including affecting the cell signal transcription factor through downregulation of NFATc1 mRNA, suppressed the bone degradation enzymes through the downregulation of TRAP and cathepsin K, affected osteoclast adhesion and sealing on the bone surface through a decrease in c-Scr mRNA expression, and influenced the cell-cell fusion into a multinuclear cell through the downregulation of DC-STAMP mRNA levels.

In conclusion, the results showed that our prepared SQ liposomes potentially inhibited the RANKL/M-CSF-induced osteoclastogenesis of RAW 264.7 cells by suppressing multinuclear cell formation, matured osteoclast sealing, bone material degradation enzyme expression, and central nuclear signal activation via osteoclast gene expression (Fig. 6). Herein, we propose a rational application to design a liposomal that revealed PS on the single-layer liposome surface as apoptotic mimicry through use of the phospholipid extract of squid integument. Moreover, the inhibitory effects of our prepared SQ liposomes on the RANKL/M-CSF-induced osteoclastogenesis of RAW 264.7 cells were suppressed by a suppression of cell-cell fusion via the downregulation of DC-STAMP gene expression, the inhibition of osteoclast sealing formation via decreased c-Src gene expression (not β3-integrin) which involves a conjunction with αv-subunit as a vitronectin receptor response to bone surface adhesion, and suppression of bone excavation enzymes via a decrease in gene expressions of TRAP, MMP-9, and cathepsin K, as well as TRAP enzyme activity, finally leading to decreased bone pit formation as the resulting phenomena.

Fig. 6.

An illustration showing that our prepared SQ liposomes characterized phosphatidylserine exposure to mimic an apoptotic body may down-regulate osteoclastogenesis on RANKL-induced RAW 264.7-cell differentiations. At least, SQ liposomes show that negative feedback of NFATc1 transcription thus suppressed gene expressions of bone excavation enzymes, such as TRAP, MMP-9, and Cathepsin K, also affecting the cell-cell fusion-related mRNA expression (dendritic cell-specific transmembrane protein, DC-STAMP) and down-regulating the receptor that mainly induced osteoclastogenesis (RANK). DC-STAMP = dendrocyte-expressed seven transmembrane protein; MMMP-9 = matrix metallopeptidase 9; NFATc1 = nuclear factor of activated T-cells, cytoplasmic 1; RANKL = receptor activator of nuclear factor kappa B ligand; SQ = squid skin; TRAP = tartrate-resistant acid phosphatase.

ACKNOWLEDGMENTS

We acknowledge the funding of this research from the Council of Agriculture, Executive Yuan, Taiwan (Project: 106AS-3.5.3-AI-A1) and the Ministry of Science and Technology, Taiwan (Project: MOST108-2221-E-002-116-MY2).