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Objective To study the influence of fluorine on signaling pathway of osteoprotegerin(OPG)/ receptor activator of NF-κB ligand(RANKL) in cultured rat osteoblasts.Methods Osteoblasts were isolated from skull of neonatal rats(＜ 24 hours) by enzyme digestion,and fluorine of different concentrations [0 (control),1 × 10-3,1 × 10-4,1 × 10-5,1 × l0-6 and 1 × 10-7 mol/L] were added into the culture medium of second generation of osteoblasts.The expressions of OPG and RANKL mRNA were determined using real-time PCR 24 and 48 hours after culturing.The expressions of OPG and RANKL protein were measured by Western blotting.Results ① After exposed to fluorine for 24 hours,the differences of RANKL and OPG mRNA expression had statistical significance between groups(F =30.95,22.62,all P ＜ 0.01),the expression of RANKL mRNA(5.99 ± 0.39) in the 1 × 10-5 mol/L group and the expressions of OPG mRNA(3.52 ± 0.09,4.81 ± 0.15,3.68 ± 0.04) in the 1 × 10-4,1 × 10-5 and 1 × 10-6 mol/L groups were higher than those of the control group(3.20 ± 0.19,3.09 ± 0.58,all P ＜ 0.05),but in the 1 × 10-3 mol/L group,RANKL mRNA(2.29 ± 0.18) was lower than that of the control group(P ＜ 0.05).After exposed to fluorine for 48 hours,the differences of RANKL and OPG mRNA expression had statistical significance between groups(F =26.62,5.72,all P ＜ 0.01),the expressions of RANKL and OPG mRNA(6.67 ± 0.49 and 5.05 ± 0.51) in the 1 × 10-5 mol/L group were higher than those of the control group(4.29 ± 0.07 and 4.34 ± 0.12,all P ＜ 0.05),and in the 1 × 10-3 mol/L group the expression of OPG mRNA(3.63 ± 0.49) was lower than that of the control group(P ＜ 0.05).② The expression of RANKL protein was not statistically significant between 24 hours and 48 hours groups (F =0.07,0.49,all P ＞ 0.05) ; the differences of OPG protein expression had statistical significance between groups(F =3.26,P ＜ 0.05),the expression of OPG protein in the 1 × 10-5 mol/L group(1.45 ± 0.10) was higher than that of the control group(1.05 ± 0.06,P ＜ 0.05) at the 24 hours.After 48 hours,the expression of OPG protein was not statistically significant(F =0.44,P ＞ 0.05).Conclusions At lower fluorine concentrations,bone formation is the main activity.But when fluorine concentration increased and time prolonged,the osteoclast differentiation and maturation are promoted,and the bone resorption is the main thing.
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Objective To explore the effects of daidzein (DA) on the expressions of estrogen receptors (ER) and peroxisome proliferator-activated recepor γ (PPARγ) in osteoblasts and the influence of estrogen on these effects.Methods A mouse osteoblastic cell line MC3T3-E1 cultured in α-MEM containing 2％ FBS was treated by 0.1 and 10 μmol/L DA.ER antagonist ICI182780 and PPARγ antagonist GW9662 in 0.1 μmol/L was added as required,and an equivalent amount of phosphate buffer solution (PBS) was used as control.For the study on estrogen effect,the cells were treated by DA in the serum-free medium with or without 10 nmol/L 17β-estradiol (E2).The expressions of ERa,ERβ and PPARγ were determined by real-time RT-PCR and Western blot analysis,respectively.Results DA inhibited ER,expression but stimulated PPARγ expression in the cells at the concentration of 0.1 and 10 μmol/L.The down-regulation of ERα by DA could be blocked by ICI182780,whereas the up-regulation of PPARγ could be repressed by GW9662 in transcription levels.Furthermore,the inhibitory effect of DA on ERβ expression was markedly enhanced,while its stimulatory effect on PPARγ expression was almost lost in serum-free medium with 10 nmol/L 17βestradiol as determined by real-time RT-PCR.Conclusions Besides its direct roles in ERs and PPARγ mediated gene transcriptions,DA could exert indirect effect on cellular pharmacological responses by altering ER and PPARγ expressions.The predominant influence on receptors expression probably involved in the time-related biphasic effects of DA on osteogenesis,which was supposedly influenced by estrogen level.
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Objective To compare the density of acetylcholine receptor (AchR) in orbicular muscle of mouth and gastrocnemius muscle and their affmity with rocuronium, trying to elucidate the mechanism for the difference in the sensitivity of the muscles innervated by facial and peripheral nerve respectively to muscle relaxant.Methods Eight pathogen-free adult male SD rats weighing 180-220 g were used in this study. Muscle strips were isolated from orbicular muscle of mouth and gastrocnemius muscle. Each muscle strip was further divided into 6 smaller and slender strips of same size using dissection microscope. One strip was stained with acetylcholinesterase to measure end-plate surface area (ESA). The other 5 strips were exposed to different concentrations of rocuronium (0, 2.5, 5.0, 7.5, 10.0μg/ml). The mean density of AchR at end-plate was obtained by AchR0/ESA. (AchR0 was defined as the number of AchR per end-plate without being exposed to rocuronium. AchRE was defined as the number of free AchR per end-plate after being exposed to different concentrations of rocuronium. ) The degree of saturation of AchR with different concentrations of rocuronium at each neuromuscular junction was calculated by (AchR0 - AchRE)/AchR0 which reflects the affinity of AchR with the rocuronium in orbicular muscle of mouth and gastrocnemius muscle. Results The density of AchR was significantly lower while the affinity with rocuronium was higher in gastrocnemius muscle than in orbicular muscle of mouth ( P ＜ 0.05). Conclusion The density of AchR is lower and the affinity of AchR at end-plate with rocuronium is significantly greater in gastrocnemius muscle innervated by sciatic nerve than in orbicular muscle of mouth innervated by facial nerve. This may explain the mechanism for different sensitivity of the muscles innervated by facial and peripheral nerves to rocuronium.
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To investigate the roles of daidzein in the expressions of steroid receptor coactivator-1 (SRC-1) and nuclear receptor corepressor (NcoR) in MC3T3-E1 osteoblastic cells.
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Objective To investigate the expression patterns of fibroblast growth factor 23 (FGF23) in osteoblast and its responses to calcium, phosphate, exogenous PTH and 1,25(OH)_2D~3. Methods The primary rat calvarial osteoblasts were cultured in MEM medium which containing 10% FBS, then were harvested when cells were in half-confluence, confluence, osteoid deposition and osteoid mineralization stages respectively. The procedure was monitored under microscopy. Total RNA was extracted from cells according to the Trizol procedure. FGF23 mRNA levels were determined by Real-time PCR. Further, the confluent osteoblasts were treated with 3.2 mmol/L CaCl_2, 4.4 mmol/L β-glycerophosphate, 10~(-9) mol/L rhPTH(1-34) and 10~(-8) mol/L 1,25(OH)_2D_3 respectively for 3 days, and same volume of the medium was added as the control. The gene expressions were determined by Real-time PCR. Results FGF23 expression was transiently up-regulated at cell confluent stage and down-regulated after that. The FGF23 mRNA levels were 7.5-fold higher in confluent cells compared with that in half-confluent cells (P<0.001). The markedlly stimulating effect (about 16 times) on FGF23 expression was stimulated by exogenous 1,25(OH)_2D_3 treatment while no significant effect was found on FGF23 mRNA levels by CaCl_2,β-glycerophosphate, and rhPTH(1-34) treatments when compared with the control. Conclusions The FGF23 expression in osteoblast is developmental stage-related and its powerful stimulator is 1,25(OH)_2D.
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BACKGROUND: Diphosphonate has a predominant therapeutic effect in the prevention and treatment of postmenopausal osteoporosis. Ibandronate, as a new-type diphosphonate preparation, is gradually becoming a study hotspot.OBJECTIVE: This study is to investigate the efficiency of ibandronate in interfering postmenopausal osteoposis by observing bone mass loss related indexes in ovariectomized rats, and made a comparison with nilestriol.DESIGN: A completely randomized grouping, and controlled animal experiment.SETTING: Study Room for Bone metabolism, Fudan University Medical College.MATERIALS: Forty SD female rats, aged 10-12 months, were involved in this study. Ibandronate was provided by the State Key Laboratory of Jiangsu Institute of Atomic Medicine. Nilestriol was produced in the Shanghai 12th Pharmaceutical Factory.METHODS: This experiment was carried out in the Study Room for Bone Metabolism, Institute of Radiation Medicine,Shanghai Medical University between August 1996 and June 1998. The rats were divided into 4 groups by a lot, 10 rats in each: sham-operation group, ovariectomized group, ovariectomized+ibandronate group and ovariectomized +nilestriol group. In the sham-operation group, only small pieces of adipose tissue around the ovary were resected from the rats.Three months after operation, each rat was intragastrically administrated with 1 mL normal saline; In the ovariectomized group, ovariectomized+ibandronate group and ovariectomized +nilestriol group, each rat was subjected to bilateral ovariectomy, and 3 months later, they were intragastrically administrated with normal saline, ibandronate water solution [0.5 mg/( kg·d)] and nilestriol suspension [1 mg/(kg· time)] respectively. Each rat in the latter three groups was administrated for 90 days, twice in the first week, and then once a week.MAIN OUTCOME MEASURES: Left femur was taken out, and its dry weight and ash weight were measured. Calcium content of bone was determined with an atomic absorption spectrophotometer, bone density of the whole body with a bone density apparatus, the bone density at the juncture of 1/2 right femoral bone length with a single photon bone density apparatus, and femoral anti-bending force was determined with a universal testing machine. Alkaline phosphatase were determined by dynamical method with an automatic biochemistry analyzer, urine calcium by EDTA titration method, urine creatinine by picric kinetic method, and urinary hydroxyproline by modified proline assay.Trabecular area was calculated.RESULTS: Forty rats were involved in the final analysis. ① Bone dry weight, bone ash weight and bone calcium content in the ovariectomized group were significantly lower than those in the other 3 groups, respectively (t =13.58-52.98, P ＜0.05). ② Femoral bone density and bone density of the whole body of rats in the ovariectomized group were significantly lower than those in the other 3 groups (t =3.31-5.61, P＜0.05), while anti-bending force was close between ovariectomized group and the other 3 groups (P＞0.05). ③ The ratio of urine calcium to urine creatinine was significantly lower in the ovariectomized+ibandronate group and ovariectomized +nilestriol group than in the ovariectomized group (t =4.04, 3.30, P＜0.05). No significant difference in the alkaline phosphatase and ratio of urinary hydroxyproline to urinary creatinine existed among the groups (P ＞ 0.05). ④Trabecular area of vertebrae in the ovariectomized group was significantly smaller than that in the other 3 groups (t =2.22,2.41,3.45,P ＜ 0.05), while the trabecular area of tibia in the ovariectomized group was only smaller than that in the ovariectomized +nilestriol group (t =2.45, P＜ 0.05).CONCLUSION: Osteoporosis apPears obviously in the SD rats 3 months after ovariectomy. Ibandronate has obviously inhibitory effects on the bone mass loss of rats with postmenopausal osteoposis, and it is equivalent to nilestriol in inhibitory effect.
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Objective To investigate the differences in function and collagen type I(COL-1) expression of cultured human ostoblastic cells between different age donors in vitro. Methods Human osteoblasts from different age donors(
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PurposeTo investigate the mechanism of BM210955 (ibandronate, a new bisphosphonate drug manufactured in China)in the prevention of bone loss in vitro. MethodsThe osteoclasts isolated from the long bone of 10-day-old Rabbit were cultured on glass and bone slices in different concentration of BM210955. TRAP(tartrate resistant acid phosphonase) staining for osteoclast and TB( toluidine blue)staining for resorption lacunae were used on bone slices and Fluorescence (orange acridine) staining for apoptosis cell on glass slices was used;Multinucleated( three and more nuclei) TRAP positive cell and Apoptosis cell and Pits were counted. ResultsThe BM210955 decreased the multinucleated cell number by 73% in 10-8 mol/ L;The inhibitory rate of pit formed was correlate to concentration as 31.58%, 76.32% and 87.99% to 10-12, 10-10 and 10-8 mol/L; The apoptosis induction was shown in above 10-8 mol/L, and the apoptosis rate was 62% in 10-4 mol/L. Conclusions Induction of osteoclast apoptosis and decrease of the cell number and inhibition of resorptive ability were the major mechanism for bone loss prevention effect of BM210955 in vitro.
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Human osteoblasts from donors of different age (≤5, 30-40, 50-60, ≥70) groups were isolated and cultured. Osteoprotegerin (OPG) and osteoclast differentiation factor (ODF) mRNA expressions were assayed by RT-PCR when the osteoblasts in the second passage were cultured for 14 days. Results showed that human osteoblasts from different age groups expressed OPG and ODF mRNA. Quantity of OPG mRNA and ODF mRNA expressions was correlated to donor′s age.