| BackgroudWith the increasing of population of elderly in our country, osteoporosis along its complication has become a more and more common disease. Osteoporosis lead to fragile fractures such as spine, hip and waist fractures affecting the life quality of patients as well as bringing burdensome to our society and country. It is as far as concerned that the potential mechanism of osteoporosis may be excess osteoclastic activity, causing by osteoclasts and osteoblasts, an imbalance of bone remodeling processes mediated. Thus with decreased bone mass and impaired microarchitecture, skeleton is prone to fractures. The effect of pharmacological intervention used for osteoporosis in clinic is barely satisfactory due to its undesirable side effects and long time to take some effects. In order to find an effective method to treat osteoporosis, researchers should pay attention to the key process of osteoporosis and force on how to inhibit osteoclastic differentiation and reduce the numbers of mature osteoclast.As we know, osteoporosis Osteoclast is unique, multinucleated and highly differentiated giant cells responsible for the decalcificationand resorption of the bone matrix, differentiating from the monocytemacrophage lineage. Tartrate resistant acid phosphatase and cathepsin K have been seen as the typical character of osteoclast. At this moment, a variety of signaling pathways involved in the differentiation of osteoclast. Among them, RANKL/RANK signaling pathway acts as one of the major roles for regulation of osteoclastic differentiation. RANK, combined with its ligand RANKL, making active of cascade reactions and the transcription factor nuclear factor of activated T cells (NFATcl), which is required for osteoclastic differentiation, finally induces osteoclast differentiation. NFATcl is a kind of transcription factors which is closely related with the progress of differentiation and maturity of osteoclasts. Papers in recent years have claimed that the Ca2+-calcineurin-NFATcl signaling pathway is one of the major mechanisms for regulating osteoclastic differentiation in the RANKL/RANK/OPG signaling system, and is activated and regulated by intracellular calcium. Calcineurin is major Ca2+-binding protein in the signaling pathway and the vitality is dependent of intracellular calcium:NFATcl is mainly regulated by the serine/threonine phosphatase calcineurin, which is activated by intracellular Ca2+. When the calcium is uploaded, serine residues in NFATs became dephosphorylation underthrough calcineurin activation, which leads to exposure of the nuclear-localization position and being translocatde into the cell nucleus. In osteoclasts, NFATcl undergoes efficient nuclear translocation in response to RANKL stimulation, inducing the development of osteoclastic differentiation. What this reaction suggests that Ca2+-calcineurin-NFATcl signaling pathway palys a vital role for the course of osteoclastic differentiation.Pulsed electromagnetic field, with the mechanism of affecting cellular electricity activity through electromagnetic induction, has longtimely been used for treatment. PEMF is divided for various kinds with respect to different frequency and intensity, for example static electromagnetic field (SEMF), low-frequency pulsed electromagnetic field (LPEMF), high-frequency pulsed electromagnetic field (HPEMF) and rotated electromagnetic field (REMF). SEMF can provide a static magnetic environment while lack of variety of bioelectricity. HPEMF is used to treat diseases for instance tumors and calculus because of its powerful ability to clear diseased tissue. In comparison with SEMF and HPEMF, LPEMF, also called PEMF, contains moderate electromagnetic effect and can lead a change of cellular biological function. A lot of evidence has showed that PEMF, one of the noninvasive and effective physiotherapies for improving bone healing, can increase bone mineral density (BMD) in patients as well as prevent loss of bone mass in ovariectomized (OVX) rats, causing hardly pain in human and animals. In 2013, Pall ML et al have came up with the idea that the biological effect on cell palyed by PEMF was mainly affected by intracellular calcium. Consequently, Lu XW et al have found that SEMF may lead to the discharge of calcium either from intracellular calcium store or extracelluar through calcium channels located on the membrane, and then result in calcium oscillation in cells in 2014. Furthermore, data from other experiments have demonstrated that osteogenic differentiation of adipose-derived stem cells could be accelerated by EMF. All of these findings make us think of whether PEMF can inhibit osteoclastic differentiation through regulating intracellular calcium.Therefore, according to above theoretical infinite, a hypothesis we bring up is that PEMF inhibit osteoclastic differentiation through regulating intracellular calcium and Ca2+-calcineurin-NFATc1 signaling pathway. Despite many studies have reported some mechanisms of PEMF about its biological effects, few studies have investigated the results of PEMF on the Ca2+-calcineurin-NFATc1 signaling pathway. In this study, we planned to confirm the effect of PEMF on RANKL-induced osteoclastic differentiation of RAW264.7 cells in vitro, meanwhile investigate the molecular mechanism underlying this effect. If the hypothesis is correct, it’s inspiring for providing a new aspect for treatment of osteoporosis.ObjectiveIn this study, we will use RANKL-dependent RAW264.7 cells in vitro to investigate the molecular mechanism of PEMF, and the three parts experiments: Firstly, to test out whether PEMF can inhibit osteoclastic differentiation of RANKL-dependent RAW264.7 cells; Secondly, to investigate the effect of PEMF on the calcium signal in RAW264.7 cells; Finally, to discuss whether PEMF attention osteoclastic differentiation through regulating Ca2+-calcineurin-NFATc1 signaling pathway.Methods1. The experiment groupsIn vitro, the experiment groups are set as follow:Control group, RANKL alone group, PEMF along group, RANKL+PEMF group, RANKL+Nifedipin group, RANKL+FK506 group, RANKL+SKF-96365 group, RANKL+PEMF+Nife group and RANKL+PEMF+FK506 group.2. The contents of experiment2.1 Effects of PEMF on RANKL-dependent RAW264.7 cells osteoclastic differentiation.2.1.1 Cell viability assayWe used a Cell Counting Kit-8 assay to quantify the cell viability, all steps were based on the manufacturer’s instructions. Briefly, RAW264.7 cells, with a density of 1x103 cells/well, were seeded in a 96-well plate and stimulated by RANKL (50 ng/ml). At the set time points,100 μl of fresh medium containing 1/10 (v/v) CCK-8 reagent was added to each well before incubation for 4 hours at 37℃. The OD value for each well was measured at an absorbance wavelength of 450 nm.2.1.2 The examination of osteoclastic related genes, proteins and landmarksTo generate multinucleated osteoclasts, RAW264.7 cells were cultured in a 96-well plate and 50ng/ml RANKL was added to the culture systems. We changed the culture medium every other day. After a 4-day induction, cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP), western blot and RT-PCR. After 7days, cells were test for pit formation.2.2 Measurement of intracellular Ca2+RAW264.7 cells cultured in confocal dishes and activated for 24 hours with RANKL were first loaded with 10 μM Fluo 4-AM in physiological salt solution (final concentration of DMSO,0.1%) at room temperature for 30 min protected from light, Fluorescence was measured every 0.6 s for 2 minutes. We analysze image and data by software, and fluorescence of every cell in each field was measured.2.3 Effects of PEMF on Ca2+-calcineurin-NFATcl signaling pathway.Calcineurin activity was measured from RAW264.7 cell extracts using a colorimetric Calcineurin Cellular Activity Assay Kit. For the experiments, cells of all four groups were lysed in the buffer supplied by the manufacturer, and calcineurin activity was determined, Cytotic and neucleus NFATc1 protein was measured by western blot.3. Statistical analysisAll experiments were repeated three times and the data are performed in triplicate. The results are expressed as the means± S.D. One-way ANOVA and Student’s t-test from SPSS 19.0 software were used for statistical evaluation. P-values<0.05 were considered significant.Results1. Effects of PEMF on RANKL-dependent RAW264.7 cells osteoclastic differentiation.1.1. PEMF did not alter the cell viability of RAW264.7 cellsThe result of CCK-8 assay has evaluated the viability of RAW264.7 cells after PEMF stimulation. Cells were treated with or without RANKL (50 ng/ml) under PEMF (50 Hz,1 mT,3 h/day) for 4 days. There is no significant difference in cell viability observing between the groups. (different time of F are 2.961、0.521、0.560、 0.604/0.590, P value are 0.127、0.642、0.697、0.675、0.681)1.2. The measure of osteoclastic related genes and proteinsThe expression of osteoclastic differentiation genes was appraised. The levels of NFATc1 mRNA, an osteoclast-specific transcription factor, were significantly reduced with PEMF, as well as other levels of three incluging TRAP, CTSK and MMP-9. Then, western blotting was conducted to investigated differentiation markers under PEMF for more convincing results. Expression levels of CTSK, TRAP, MMP-9 and NFATc1 were notably reduced in the PEMF-treated group. TRAP staining showed that the numbers of TRAP-positive cells were reduced in the PEMF-treated group compared with the RANKL-treated group. TRAP activity was significantly decreased by PEMF treatment. (F=83.69, P<0.0001) Pit formation about mature osteoclasts on bovine bone slices was observed and calculated. The area of bone resorption in the PEMF treated-group was significantly lower than that in the RANKL-treated group. (F=55.06, P<0.0001)2. PEMF reduced RANKL-dependent intracellular Ca2+ oscillationsRAW264.7 cells were preincubated with Fluo-4AM, and the fluctuation of intracellular Ca2+ levels was observed using confocal microscopy. After RANKL stimulation, Ca2+ oscillations are triggered and maintained from either store-operated Ca2+ entry (SOCE) or extracellular Ca2+. To determine the contribution of PEMF to the change of intracellular Ca+, RAW264.7 cells were pretreated in the presence of the blockers of membrane calcium channels or SOC channels, respectively. The results showed that compared to the control group, RANKL could significantly induce intracellular Ca2+ oscillations wherase intracellular Ca2+notably decreased in the PEMF group (F=40.62, P<0.0001), as well as in the nifedipine (L-type calcium channel blocker) and SKF-96365 (SOC channel blocker) treatments. We noted it was in line with the nifedipine group, whereas the SKF-96365 treatment suppressed most of the Ca2+ oscillations in response to RANKL. (F=27.38, P<0.001) Meanwhile, levels of protein expression of NFATcl and CTSK were observed to be consistent with the changes in the percentages of cells showing intracellular Ca2+ oscillations in vitro. (F=11.92, P=0.003) However, the levels of TRAP and MMP-9 did not show significant changes. The results demonstrated that with extracellular Ca2+ infux may contribute to intracellular Ca2+ oscillations, while blocking calcium channels may attuned this phenomenon.3. PEMF suppressed the Ca2+-calcineurin-NFATc1 signaling pathway in RANKL-dependent osteoclastic differentiationRAW264.7 cells were treated with PEMF and nifedipine, calcium channel blocker and FK506, a specific inhibitor of calcineurin. Treatment with nifedipine or FK506 alone significantly inhibited RANKL-dependent NFATcl transcription and the levels of proteins specific for osteoclastic differentiation. Cotreating RANKL-stimulated cells with PEMF and nifedipine or FK506 only slightly increased the effect on the transcription of NFATc1 and protein levels. These results further indicate the causal relationship between inhibiting the Ca2+-calcineurin pathway and attenuating osteoclastic differentiation due to RANKL treatment. (FCTSK=3.184, P=0.031; FNFATC1=9.682, P<0.001)Conclusions:1.PEMF inhibited RANKL-induced RAW264.7 osteoclastic differentiation and suppressed activity of bone resportion in vitro.2. PEMF attenuated Ca2+ signal in RANKL-induced RAW264.7 cells.3. PEMF inhibited osteoclastic differentiation through the Ca2+-Calcineurin-NFATcl signaling pathway... |
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