| BackgroudOsteoporosis is a common disease in middle-aged and old people, characterized by inadequate bone mass and microarchitectural deterioration of bone tissue with a compromised bone strength and an increased risk of bone fragility fractures. The major consequence of osteoporosis was osteoporotic fracture, especially the fracture of hip, which had obviously decreased activity and a high mortality, was thought to the most dangerous consequence in osteoporosis. With the arrival of an aging society, the clinical and basic research of osteoporosis prevention and treatment has become a medical research focus.Exercise is an important factor in the prevention and treatment of osteoporosis.In recent years, physical factors characterized by a non-invasive method, less side effects, good compliance and other characteristics, have become an important way in the prevention and treatment of osteoporosis. As a physiotherapy method, whole-body vibration treatment produces a vibratory stimulation of the entire body for patients who stand on a vibrating platform through sinusoidal oscillations in a vertical or rotational mode. Recent studies have reported that low magnitude high frequency vibration (LMHFV) could increase bone formation and inhibit bone resorption in animal models and young adults and children with low bone mineral density or physical impairments, promote neuromuscular adaptation and increase muscle strength. As a new method for treating osteoporosis, whole body vibration exercise characterized by a non-invasive method, less side effects, good compliance, can effectively reduce the incidence of osteoporotic fractures, whole body vibration treatment increasingly gets more attention in the field of osteoporosis prevention and treatment.However, the mechanism of whole body vibration increases bone formation and inhibits bone resorption remains unclear.Studies have shown that vibration stress can affect the biological characteristics of osteoblasts, but the specific mechanisms of the vibration stress on the biological characteristics of osteoblasts remain unclear. Therefore, regarding the identification of the molecular mechanisms of vibration stress on the biological characteristics of osteoblasts as an entry point to further clarify the mechanism of whole body vibration in preventing and treating osteoporosis, which will provide a scientific basis for whole body vibration to prevent and cure osteoporosis.In the present study, MC3T3-E1 osteoblastic cells were subjected to low-magnitude (0.3g), various high-frequency (0,30,45,60,90 Hz) vibration (LMHFV) by using cell vibration stress loading system developed by ourselves and observed the effects of LMHFV on biological characteristics of osteoblasts in vitro. In addition, cyclooxygenase-2 protein synthesis and signal transduction pathways that mediated cellular responses to LMHFV were investigated using specific inhibitors and agonists. The research works are as follows:Objectives1. To explore the effects of low magnitude high frequency vibration (LMHFV) on the biological characteristics of osteoblasts in vitro.2. To investigate the roles played by cyclooxygenase-2 in effects of LMHFV on biological characteristics of osteoblasts in vitro.3. To investigate the preliminary mechanisms of LMHFV on expression of cyclooxygenase-2 protein in osteoblasts.MethodsI. Effects of low magnitude high frequency vibration (LMHFV) on the biological characteristics of osteoblasts in vitro.1. To investigate the effects of LMHFV on ratio of OPG/RANKL in MC3T3-E1 osteoblastic cells in vitro.(1) OPG/RANKL ratio assay:MC3T3-E1 cells were subjected to 0.3 g, and different high frequencies (0,30,45,60,90 Hz) LMHFV by using cell vibration stress loading system for 30 min, then soluble OPG, soluble RANKL levels in the cell medium collected at 0,0.5,1.5,3,6h post-LMHFV, respectively, were detected by ELISA Kit. By calculating the OPG/RANKL ratio, the frequency which showed a largest increase in OPG/RANKL ratio was selected as the one to used at subsequent experiments.(2) Effect of conditioned medium from the vibrated MC3T3-E1 cells on the regulation of osteoclasts:30Hz LMHFV-conditioned medium (CM) and OHz LMHFV-conditioned medium (CM) in MC3T3-E1 cells were collected at 6h post-LMHFV, respectively, then the CM (30Hz or OHz) was mixed (1:1,V/V) with the 1640 medium which contained 40ng/ml sRANKL,20ng/ml M-CSF.RAW264.7 cells were induced by 1640 medium which has been mixed with CM(30Hz) or CM(OHz) for 5,7 days,respectively. TRAP mRNA level of RAW264.7 cells in CM (30Hz) or CM (OHz) groups was analyzed by real-time quantitative PCR (qPCR) at 5 and 7 days, respectively. Samely, TRAP activity of RAW264.7 cells in CM (30Hz) or CM (OHz) groups was analyzed by TRAP Detection Kit at 5 and 7 days, respectively. At day 7, the RAW264.7 cells were stained by TRAP Staining Kit, the number of TRAP-positive multinuclear RAW-OCs in CM (30Hz) or CM (OHz) groups was recorded and analysed, respectively.2. To investigate the effect of LMHFV on osteogenic differentiation in MC3T3-E1 osteoblastic cells in vitro.(1) Effects of LMHFV on expression of alkaline phosphatase (ALP) mRNA and osteocalcin (OCN) mRNA in MC3T3-E1 osteoblastic cells in vitro.MC3T3-E1 cells were divided into two groups at random:Control group and LMHFV group, Control group was loaded with OHz,0.3g,30min/day vibration stress, LMHFV group was loaded with 30Hz,0,3g,30min/day vibration stress.Two groups were vibrated for 4,8 days, respectively. Cells were collected and total RNA was extracted using TaKaRa Kit according to the manufacturer's instruction. The mRNA expression of ALP, OCN were analyzed by qPCR. Relative expression of ALP,OCN were calculated using GAPDH mRNA expression as an internal control.(2) Effects of LMHFV on ALP activity and OCN expression in MC3T3-E1 osteoblastic cells in vitro.MC3T3-E1 cells were divide into two groups at random:Control group and LMHFV group, Control group was loaded with OHz,0.3g,30min/day vibration stress, LMHFV group was loaded with 30Hz,0,3g,30min/day vibration stress.Two groups were vibrated for 4,8 days, respectively. Cells were collected and their total protein was then prepared with RIPA lysis buffer. ALP commercial reagents and osteocalcin ELISA Kit were used to study the effects of LMHFV (OHz or 30Hz) on ALP activity and expression of OCN, respectively.The results of ALP activity, expression of OCN were normalised using the amount of total protein.(3) Effect of LMHFV on the mineralized nodule formation in MC3T3-E1 osteoblastic cells in vitro.MC3T3-E1 cells were divide into two groups at random:Control group and LMHFV group, Control group was loaded with OHz,0.3g,30min/day vibration stress, LMHFV group was loaded with 30Hz,0,3g,30min/day vibration stress.Two groups were vibrated for 14 days, respectively. Mineralized nodule formation was examined by the alizarin red staining.Ⅱ. To investigate the roles played by cyclooxygenase-2 (COX-2) in effects of LMHFV on biological characteristics of osteoblasts in vitro.1. Effect of LMHFV on expression of COX-2 mRNA, COX-2 protein in MC3T3-E1 osteoblastic cells in vitro.MC3T3-E1 cells were subjected to 0.3g,0 or 30 Hz LMHFV by using cell vibration stress loading system for 30 min.(1) Cells were collected and total RNA was extracted using TaKaRa Kit according to the manufacturer's instruction at 0,0.5,1.5,3,6h post-LMHFV, respectively. The mRNA expression of COX-2 was analyzed by qPCR. Relative expression of COX-2 mRNA were calculated using GAPDH mRNA expression as an internal control.(2) Cells were collected and their total protein was then prepared with RIPA lysis buffer at 0,0.5,1.5,3,6h post-LMHFV, respectively. The expression of COX-2 protein was analyzed by Western blot.2. Effect of LMHFV on prostaglandin E2 (PGE2) activity in MC3T3-E1 osteoblastic cells in vitro.MC3T3-E1 cells were subjected to 0.3g,0 or 30 Hz LMHFV by using cell vibration stress loading system for 30 min, then cell medium, cells were collected and their total protein was then prepared with RIPA lysis buffer at 0,0.5,1.5,3,6h post-LMHFV, respectively. Inhibition experiments were performed by 1h pretreatment with COX-2 inhibitor NS-398 before LMHFV loading. The PGE2 activity in cell medium was analyzed by PGE2 ELISA Kit, and the results of PGE2 activity were normalised using the amount of total protein.3. Effect of COX-2 inhibitor NS-398 on LMHFV-induced OPG secretion in MC3T3-E1 osteoblastic cells in vitro.MC3T3-E1 cells were subjected to 0.3g,0 or 30 Hz LMHFV by using cell vibration stress loading system for 30 min, then cell medium, cells were collected and their total protein was then prepared with RIPA lysis buffer at 6h post-LMHFV, respectively. Inhibition experiments were performed by 1h pretreatment with COX-2 inhibitor NS-398 before LMHFV loading. The OPG secretion in cell medium was analyzed by OPG ELISA Kit, and the results of OPG secretion were normalised using the amount of total protein.4. Effect of COX-2 inhibitor NS-398 on LMHFV-induced ALP activity and OCN expression in MC3T3-E1 osteoblastic cells in vitro.The MC3T3-E1 cells were exposed to 0.3g,0 or 30 Hz LMHFV for 8 days with or without COX-2 inhibitor NS-398, respectively. ALP commercial reagents and osteocalcin ELISA Kit were used to meastred ALP activity and expression of OCN, respectively.The results of ALP activity, expression of OCN were normalised using the amount of total protein.Ⅲ. To investigate the preliminary mechanisms of LMHFV on expression of cyclooxygenase-2 protein in osteoblasts. The expression of COX-2 protein was analyzed by Western blot.1. Preliminary investigation of signal transduction pathway that mediated LMHFV-induced expression of COX-2 protein in MC3T3-E1 cells.Inhibitors including NS-398,Staurosporine,H-89,U0126,SP600125 and SB203580 were used.After the MC3T3-E1 cells had been pre-incubated in the presence of each inhibitor for 60 min to permit these compounds to penetrate the cells and block their respective pathways, LMHFV at 0.3g, OHz or 30Hz was applied in culture for 30-min, and then cells were collected and their total protein was then prepared with RIPA lysis buffer at 6h post-LMHFV. The expression of COX-2 protein was analyzed by Western blot.2. Effect of cAMP/PKA signal pathway on LMHFV-induced expression of COX-2 protein in MC3T3-El cells.(1) Effect of LMHFV on the phosphorylation of PKA in MC3T3-E1 cells.MC3T3-E1 cells were subjected to 0.3g, OHz or 30 Hz LMHFV by using cell vibration stress loading system for 30 min, then cells were collected and their total protein was then prepared with RIPA lysis buffer at 0,0.5,1.5,3,6h post-LMHFV, respectively. The total protein and phosphorylated protein of PKA were analyzed by Western blot.(2) Effect of LMHFV on intracellular cAMP level in MC3T3-E1 cells.MC3T3-E1 cells were subjected to 0.3g, OHz or 30 Hz LMHFV by using cell vibration stress loading system for 30 min, then cells were collected at 0,0.5,1.5,3, 6h post-LMHFV, respectively and cAMP ELISA Kit was used to measure intracellular cAMP level in MC3T3-E1 cells according to the manufacturer's instruction.(3) Effect of LMHFV-conditioned medium (LMHFV-CM) on intracellular cAMP level in MC3T3-El cells.MC3T3-E1 cells were subjected to LMHFV-conditioned medium (LMHFV-CM) for 0,0.5,1.5,3 and 6h, CM from the 30Hz LMHFV loaded cells was regarded as Experiment group and CM from the OHz LMHFV loaded cells was regarded as Control group.Cells were collected at various time point, respectively and cAMP ELISA Kit was used to measure intracellular cAMP level in MC3T3-E1 cells according to the manufacturer's instruction.(4) Effect of adenylate cyclase activator forskolin (FSK) on intracellular cAMP level in MC3T3-E1 cells.The MC3T3-E1 cells were exposed to 15μM FSK for 0,0.5,1.5,3,6h or 0,5, 10,15,20μM FSK for 3h, respectively. Cells were collected at various time point, respectively and cAMP ELISA Kit was used to measure intracellular cAMP level in MC3T3-E1 cells according to the manufacturer's instruction.(5) Effect of PKA inhibitor H-89 on the stimulation of COX-2 protein in MC3T3-E1 cells by the LMHFV, FSK, LMHFV-conditioned medium (LMHFV-CM).After the MC3T3-E1 cells had been pre-incubated in the presence of 30μM H-89 for 60 min to permit H-89 to penetrate the cells and block its respective pathway, MC3T3-E1 cells were exposed to LMHFV for 30min and followed 3h post-LMHFV incubation, as well as exposed to 15μM FSK and LMHFV-CM for 3h, respectively. Cells were collected and their total protein was then prepared with RIPA lysis buffer at the corresponding detection time. The expression of COX-2 protein was analyzed by Western blot.ResultsI. Effects of low magnitude high frequency vibration (LMHFV) on the biological characteristics of osteoblasts in vitro.1. To investigate the effects of LMHFV on ratio of OPG/RANKL in MC3T3-E1 osteoblastic cells in vitro.(1) OPG/RANKL ratio assay:Compared to OHz-LMHFV loading, the secretion of OPG in MC3T3-E1 cells significantly increased at 6h after 30Hz-LMHFV loading (P<0.001), however, the secretion of RANKL in MC3T3-E1 cells showed little change at 6h after 30Hz-LMHFV loading.Correspondingly, the ratio of OPG/RANKL in MC3T3-E1 cells was highest at 6h after 30Hz-LMHFV loading (P<0.001).(2) Effect of conditioned medium from the vibrated MC3T3-E1 cells on the regulation of osteoclasts: ①After 5 days in culture, compared to 0Hz-LMHFV group; 30Hz-LMHFV group showed an insignificant increase in TRAP mRNA and TRAP activity, respectively (P=0.069, P=0.160).②After 7 days in culture, compared to 0Hz-LMHFV group,30Hz-LMHFV group showed an significant increase in TRAP mRNA and TRAP activity, respectively (P<0.001,P=0.043).③After 7 days in culture, compared to 0Hz-LMHFV group,30Hz-LMHFV group showed a light but in the number of RAW-OCs containing three to nine nuclei per cell, however, the population of RAW-OCs containing 10 or greater number of nuclei was found to be significantly lower in the cultures containing CM from the 30Hz-LMHFV loaded MC3T3-E1 cells (P<0.001).2. To investigate the effect of LMHFV on osteogenic differentiation in MC3T3-E1 osteoblastic cells in vitro.(1) Effects of LMHFV on expression of alkaline phosphatase (ALP) mRNA and osteocalcin (OCN) mRNA in MC3T3-E1 osteoblastic cells in vitro.After 4 days in culture, compared to 0Hz-LMHFV group,30Hz-LMHFV group showed a significant increase in ALP mRNA level (P=0.006). After 8 days in culture, compared to 0Hz-LMHFV group,30Hz-LMHFV group showed a significant increase in ALP mRNA level and OCN mRNA level, respectively (P=0.003, P=0.001)。(2) Effects of LMHFV on ALP activity and OCN expression in MC3T3-E1 osteoblastic cells in vitro.After 4 days in culture, compared to 0Hz-LMHFV group,30Hz-LMHFV group showed a significant increase in ALP activity (P=0.031). After 8 days in culture, compared to 0Hz-LMHFV group,30Hz-LMHFV group showed a significant increase in ALP activity and OCN expression, respectively (P=0.003, P=0.001)。(3) Effect of LMHFV on the mineralized nodule formation in MC3T3-E1 osteoblastic cells in vitro.After 14 days in culture, alizarin red staining showed that the mineralized nodule formation could be seen in OHz-LMHFV group and 30Hz-LMHFV group, however 30Hz-LMHFV group showed greater number and deeper color.Ⅱ. To investigate the roles played by cyclooxygenase-2 (COX-2) in effects of LMHFV on biological characteristics of osteoblasts in vitro.1. Effect of LMHFV on expression of COX-2 mRNA, COX-2 protein in MC3T3-E1 osteoblastic cells in vitro.(1) COX-2 mRNA level assay:Compared to OHz-LMHFV group, qPCR showed that 30Hz-LMHFV stimulation resulted in greater COX-2 mRNA level at 0,0.5,1.5, 3,6h post-LMHFV (allP<0.01), especially at 0.5 h post-LMHFV, 30Hz-LMHFV stimulation showed greatest COX-2 mRNA level (P<0.001).(2) COX-2 protein expression assay:Compared to OHz-LMHFV group, Western blot showed that 30Hz-LMHFV stimulation resulted in greater COX-2 protein expression at 0.5,1.5,3,6h post-LMHFV (allP<0.01), especially at 3 h post-LMHFV,30Hz-LMHFV stimulation showed greatest COX-2 protein expression (P<0.001).2. Effect of LMHFV on prostaglandin E2 (PGE2) activity in MC3T3-E1 osteoblastic cells in vitro.Compared to OHz-LMHFV group,30Hz-LMHFV stimulation resulted in greater PGE2 activity at0,0.5,1.5,3,6h post-LMHFV (all P<0.05), especially at 0.5h post-LMHFV,30Hz-LMHFV stimulation showed greatest PGE2 activity (P=0.032).Pretreatment of COX-2 inhibitor NS-398 could inhibit PGE2 activity induced by LMHFV at 3h post-LMHFV(P<0.001).3. Effect of COX-2 inhibitor NS-398 on LMHFV-induced OPG secretion in MC3T3-E1 osteoblastic cells in vitro. Pretreatment of COX-2 inhibitor NS-398 could inhibit the OPG secretion induced by LMHFV at 6h post-LMHFV(t=4.396,P=0.012) and NS-398 showed an inhibition effect on the increase of LMHFV-induced OPG secretion.4. Effect of COX-2 inhibitor NS-398 on LMHFV-induced ALP activity and OCN expression in MC3T3-E1 osteoblastic cells in vitro.After 8 days in culture, COX-2 inhibitor NS-398 could inhibit the increasing ALP activity and OCN expression induced by LMHFV, respectively (t=5.478,P=0.005;t=8.060,P=0.001)。Ⅲ. To investigate the preliminary mechanisms of LMHFV on expression of cyclooxygenase-2 protein in osteoblasts. The expression of COX-2 protein was analyzed by Western blot.1. Preliminary investigation of signal transduction pathway that mediated LMHFV-induced expression of COX-2 protein in MC3T3-E1 cells.The induction of COX-2 protein expression by 30Hz-LMHFV load was inhibited by COX-2 inhibitor NS-398, PKA inhibitor H-89, ERK1/2 inhibitor U0126, p38 inhibitor SB203580, respectively (all P<0.05).2. Effect of cAMP/PKA signal pathway on LMHFV-induced expression of COX-2 protein in MC3T3-E1 cells.(1) Effect of LMHFV on the phosphorylation of PKA in MC3T3-E1 cells.Compared to OHz-LMHFV group, Western blot showed that 30Hz-LMHFV stimulation resulted in increasing phosphorylation level of PKA at 0,0.5,1.5,3h post-LMHFV (all P<0.01), especially at 1.5 h post-LMHFV,30Hz-LMHFV stimulation showed greatest phosphorylation level of PKA (P< 0.001).(2) Effect of LMHFV on intracellular cAMP level in MC3T3-E1 cells.Compared to OHz-LMHFV group,30Hz-LMHFV stimulation resulted in greater intracellular cAMP level at 0,0.5,1.5h post-LMHFV (t=-10.85, P<0.001; t=-9.637,P=001;t=-4.941,P=008), especially at 0.5 h post-LMHFV, 30Hz-LMHFV stimulation showed greatest intracellular cAMP level (t=-4.941,P=008).(3) Effect of LMHFV-conditioned medium (LMHFV-CM) on intracellular cAMP level in MC3T3-E1 cells.Compared to 0Hz-LMHFV-CM group,30Hz-LMHFV-CM stimulation resulted in greater intracellular cAMP level at 0.5,1.5,3h (all P<0.05), especially at 1.5 h, 30Hz-LMHFV stimulation showed greatest intracellular cAMP level (P<0.05).(4) Effect of adenylate cyclase activator forskolin (FSK) on intracellular cAMP level in MC3T3-E1 cells.Compared to 0μM FSK,15μM FSK stimulation resulted in greater intracellular cAMP level at 0.5,1.5,3h (t=-6.175,P=0.003;t=-14.877, P<0.001;t=-20.895, P<0.001), especially at 3 h,15μM FSK stimulation showed greatest intracellular cAMP level.(5) Effect of PKA inhibitor H-89 on the stimulation of COX-2 protein in MC3T3-E1 cells by the LMHFV, FSK, LMHFV-conditioned medium (LMHFV-CM).Compared to the blank control group, Western blot analyses showed that the phosphorylation of PKA could be induced by 30Hz-LMHFV load,15μM FSK and 30Hz-LMHFV-CM, respectively (t=-13.294,P=000;t=-13.382,P=001; t=-13.275,P=008). Pretreatment of PKA inhibitor H-89 could inhibit the phosphorylation of PKA induced by 30Hz-LMHFV load,15μM FSK and 30Hz-LMHFV-CM, respectively (t=9.026,P=001;t=8.518,P=001; t=14.216,P=000).Conclusions1. Low-magnitude (0.3g), high-frequency (30Hz) vibration (LMHFV) showed positive influences on the biological characteristics of MC3T3-E1 cells:increasing the secretion of OPG and raising the concentration ratio of OPG/RANKL; promoting osteogenic differentiation in MC3T3-E1 cells.2. COX-2 signal involved in the effects of LMHFV on the biological characteristics of MC3T3-E1 cells. PKA signal, ERK1/2 signal and p38 signal involved in the induction of COX-2 protein by LMHFV in MC3T3-E1 cells.The autocrine effect of PGE2 production in MC3T3-E1 cells played an important role on the effect of LMHFV induce COX-2 protein expression in MC3T3-E1 cells through PGE2/cAMP/PKA signal pathways. |
