OBJECTIVE:To compare effects of low frequency pulsed electromagnetic fields on bone quality in growing rats between 1 h and 1.5 h.METHODS:Thirty male SPF SD rats aged 4 weeks selected, which with the average body weight (115.8± 1.5) g, were divided into three groups according to random number table, 10 rats in each group. Control groups put rats into electromagnetic field device with 1.5 h every day, the other two groups put rats in electromagnetic field for 1 h and 1.5 h with a 50 Hz 0.6 mT intensity pulsed. The body weight of rats was weighed every 2 weeks and detected bone mineral density. Rats were sacrificed after 6 weeks to measure bone mineral density and biomechanical value of the right femur and lumbar vertebrae. Serum osteocalcin (OC) and tartrate-resistant acid phosphatase 5b (TRACP 5b) concentrations were determined by ELISA methods. After the magenta-picric acid staining, the micro tissue structure of the right tibia was observed, and the parameters of trabecular bone were analyzed by IPP 6.0 software.RESULTS:There were no statistical difference in body weight and organ coefficient among each groups at different times. Bone mineral density results showed that the body thickness of the 1.5 h group was significantly increased compared with control group at 6 weeks, and bone mineral density of femoral and vertebra in 1.5 h group were higher than that of in 1 h group. The results of three point femoral bending and vertebral compression test showed that the maximum load value of femur and vertebrae in 1.5 h group increased significantly compared with control group, and the maximum femoral load value in 1.5 h group was significantly higher than that of 1 h group, while there was no difference in elastic modulus values among each groups. Results of serum biochemical indicators showed that level of OC in 1.5 h group was significantly increased compared with control group, and significantly higher than that of 1 h group, while no significant difference in TRACP 5b values among each groups. Bone histomorphometry analysis results showed that there was no statistical difference in trabecular thickness, number and resolution between 1 h group and control group, trabecular bone thickness and number in 1.5 h group were increased, and trabecular bone resolution was decreased; The thickness and number of trabecular bone in 1.5 h group were also significantly increased compared with 1 h group, and the degree of resolution was reduced, and had significant difference between two groups.CONCLUSION:Intervention of 50 Hz 0.6 mT low frequency pulsed electromagnetic field for 1.5 h could effectively increase peak bone mineral density and bone microstructure in young rats, enhance biomechanical properties of bone, promote concentration of bone formation markers in rat blood. The results indicating that pulsed electromagnetic field could be used as a good way to prevent and treat osteoporosis.

Objective To compare the effects of 50-Hz 0.6-mT low-frequency pulsed electromagnetic fields(PEMFs) and 50-Hz 1.8-mT sinusoidal alternating electromagnetic fields(SEMFs) in preventing bone loss in tail-suspended rats,with an attempt to improve the prevention and treatment of bone loss caused by weightlessness.Methods Tail-suspension rat models were used to simulate microgravity on the ground. Forty rats were randomly divided into four groups[control group,hindlimb-suspended(HLS) group,HLS+PEMFs group,and HLS+SEMFs group],with 10 rats in each group. In the PEMFs treatment group and SEMFs treatment group,the intervention was 90 min per day. Rats were sacrificed after four weeks. Bone mineral density(BMD) of femur and vertebra was measured by dual-energy X-ray absorptiometry and biomechanical strength by AG-IS biomechanical instrument. Serum osteocalcin(OC),tartrate-resistant acid phosphatase 5b(Tracp 5b),parathyroid hormone(PTH),and cyclic adenosine monophosphate(cAMP) were detected by ELISA. The microstructure of bone tissue was observed by Micro-CT and HE staining.Results The BMD of the femur(P=0.000) and vertebrae(P=0.001) in the HLS group was significantly lower than in the control group;the BMD of the femurs(P=0.001) and vertebrae(P=0.039) in the HLS+PEMFs group was significantly higher than in the HLS group;the BMD of the femurs in the HLS+SEMFs group was significantly higher than in the HLS group(P=0.003),but the BMD of the vertebrae showed no significant difference(P=0.130). There was no significant difference in the BMD of the femur(P=0.818) and vertebrae(P=0.614) between the HLS+PEMFs group and the HLS+SEMFs group. The maximum load(P=0.000,P=0.009) and elastic modulus(P=0.015,P=0.009) of the femurs and vertebrae in the HLS group were significantly lower than those in the control group;the maximum load of the femur(P=0.038) and vertebrae(P=0.087) in the HLS+PEMFs group was significantly higher than that in the HLS group,but the elastic modulus was not significantly different from that in the HLS group(P=0.324,P=0.091). The maximum load(P=0.190,P=0.222) and elastic modulus(P=0.512,P=0.437) of femurs and vertebrae in the HLS+SEMFs group were not significantly different from those in the HLS group. There were no significant differences in the maximum load and elastic modulus of femurs(P=0.585,P=0.948) and vertebrae(P=0.668,P=0.349) between the HLS+PEMFs group and the HLS+SEMFs group. The serum OC level in the HLS group was significantly lower than that in the control group(P=0.000),and the OC level in HLS+PEMFs group(P=0.000) and HLS+SEMFs group(P=0.006) were significantly higher than that in the HLS group. The serum Tracp 5b concentration in the HLS group was significantly higher than that in the control group(P=0.011). There was no significant difference between the HLS+PEMFs group(P=0.459) and the HLS+SEMFs group(P=0.469) compared with the control group.Serum Tracp 5b concentrations in the HLS+PEMFs group(P=0.056) and the HLS+SEMFs group(P=0.054) were not significantly different from those in the HLS group. The PTH(P=0.000) and cAMP concentrations(P=0.000) in the HLS group were significantly lower than those in the control group. The PTH(P=0.000,P=0.000) and cAMP concentrations(P=0.000,P=0.000) in the HLS+PEMFs group and the HLS+SEMFs group were significantly higher than in the HLS group. The femoral cancellous bone of the HLS group was very sparse and small compared with the control group. The density and volume of the cancellous bone were similar among the control group,HLS+PEMFs group,and HLS+SEMFs group. Compared with the control group,the HLS group had lower BMD(P=0.000),bone volume (BV)/tissue volume(TV)(P=0.000),number of trabecular bone (Tb.N)(P=0.000),and trabecular thickness(Tb.Th)(P=0.000) and higher trabecular bone dispersion(Tb.Sp)(P=0.000) and bone surface area(BS)/BV(P=0.000). Compared with the HLS group,the HLS+PEMFs group and the HLS+SEMFs group had significantly lower Tb.Sp(P=0.000,P=0.000) and BS/BV(P=0.000,P=0.000) and significantly increased BMD(P=0.000,P=0.000),BV/TV(P=0.001,P=0.004),Tb.Th(P=0.000,P=0.001),and Tb.N(P=0.000,P=0.001). The trabecular thickness significantly differed between the HLS+PEMFs group and the HLS+SEMFs group(P=0.024). The HLS group(P=0.000),HLS+PEMFs group(P=0.000),and HLS+SEMFs group(P=0.000) had the significantly lower osteoblast density on the trabecular bone surface than the control group;however,it was significantly higher in the HLS+SEMFs group(P=0.000) and the HLS+PEMFs group(P=0.000) than in the HLS group. The HLS group had significantly lower density of osteoblasts in the endothelium than the control group(P=0.000);however,the density of osteoblasts was significantly higher in HLS+PEMFs group(P=0.000) and HLS+SEMFs group(P=0.000) than HLS group and was significantly higher in HLS+PEMFs group than in HLS+SEMFs group(P=0.041). Compared with the control group,a large number of fatty cavities were produced in the bone marrow cavity in the HLS group,but the fat globules remarkably decreased in the treatment groups,showing no significant difference from the control group. The number of adipose cells per mm 2 bone marrow in the HLS group was 4 times that of the control group(P=0.000);it was significantly smaller in the HLS+PEMFs group(P=0.000) and HLS+SEMFs group(P=0.000) than in the HLS group,whereas the difference between the HLS+PEMFs group and the HLS+SEMFs group was not statistically significant(P=0.086). Conclusions 50-Hz 0.6-mT PEMFs and 50-Hz 1.8-mT SEMFs can effectively increase bone mineral density and biomechanical values in tail-suspended rats,increase the concentration of bone formation markers in rat blood,activate the cAMP pathway by affecting PTH levels,and thus further increase the content of osteoblasts to prevent the deterioration of bone micro-structure. In particular,PEMFs can prevent the reduction of bone mineral density and maximum load value by about 50% and increase the bone mass of tail-suspended rats by promoting bone formation.

Objective To investigate the signal transduction mechanisms of time-dependent effects of 50 Hz 1.8 mT sinusoidal electromagnetic fields(SEMFs)on osteoblastic activity.Methods Newborn rat calvarial osteoblasts(ROBs) were treated with 50 Hz 1.8mT SEMFs for 30,60,90,120,and 150 min,respectively.Intracellular alkaline phosphatase (ALP) activity was assayed,and protein expression of Runt-related transcription factor 2 (Runx2),Smad1/5/8 and mitogen-activated protein kinases(MAPKs)were examined by Western blot.The Dual-Luciferase Reporter Assay System was used to measure the activity of the Wnt pathway.Immunofluorescence staining and laser confocal microscopy were applied to examine the nuclear translocation of Smad1/5/8 and β-catenin.Changes in ALP activity were determined after inhibiting p38 MAPK using a specific inhibitor.Results ALP activity of ROBs increased after 30,60,90,120 and 150 min of SEMFs treatment,compared with the control group(51.41±5.21,59.47±4.02,67.56[4.68,63.69±3.92,and 58.16±3.61 vs.45.53± 3.24),and reached the highest value at 90 min and then started to decline.Protein expression of Runx-2,p-Smad1/5/8,p-p38 and β-catenin increased after SEMFs treatment,and reached the highest value at 90 min and then gradually returned to baseline levels.The values for Wnt pathway activity for the control group and with SEMFs treatment at 30,60,90,120 and 150 min were 0.49± 0.06,0.52 ± 0.09,0.75±0.05,0.77 ± 0.42,0.58 ± 0.08 and 0.42 ± 0.09,respectively.Wnt pathway activity,the nuclear translocation of β-catenin and Smad1/5/8 reached the highest level at 90 min.Values of ALP activity in the control group and the SEMFs group were 44.60±3.84 and 71.54±7.34,respectively,before specifically inhibiting p38 MAPK,and were 52.08±0.83 and 52.15±10.77,respectively,after p38 MAPK inhibition,indicating that ALP activity could not be increased with inhibition.Conclusions 50 Hz 1.8 mT SEMFs increase osteoblastic activity by activating the BMP-2/Smad1/5/8,Wnt/β-catenin and p38 MAPK signal pathways.The optimal duration of treatment is 90 min per day.

Objective To study the effects of the compound medicine of icariin and puerarin on peak bone mass in rats during growth period, and to explore its possible mechanism. Methods Forty female Sprague-Dawley rats aged 1 month were randomly divided into normal control group( C), icariin group( I), puerarin group( P), icariin and puerarin compound groupc(I+P), 10 in each group. The body weights were recorded once every two weeks, and the bone mineral density was measured by dual-energy X-ray absorptiometry every month. After the bone mineral density of the whole body was significantly different between the control group and drug groups the animals were sacrificed. The right femur and vertebrae were separated to measure the bone mineral density. The biomechanical properties of the femur and vertebra were detected by AG-IS series desktop electronic universal testing machine. The bone formation index osteocalcin, PINP and bone resorption index were determined by ELISA. Changes in the contents of tartrate-resistant acid phosphatase 5b(TRACP 5b) and CTX-1; and changes in trabecular bone related parameters were recorded after magenta-picric acid staining. Results There was no significant difference in body weight between the two groups (P＞0.05). There was no significant difference in whole body bone density after 1 month of treatment (P＞0.05). After 2 months of treatment, the body bone density of the drug-administered group was higher than that of the control group. Whole body bone density, femur and vertebral bone density, femur maximum load value, maximum vertebrae load value and trabecular bone number and area, serum OC and PINP levels increased, while TRACP 5b and CTX-1 levels decreased(P＜0.01) in drug group. The difference from the control group was statistically significant (P＜0. 05). There were significant differences in biochemical parameters and bone histomorphology between the compound drug group and the two-flavor monomer group ( P＜0. 01). There was no significant difference in bone mineral density and biomechanics, but the average value was higher than that of the monomer group. Conclusion The combination of icariin and puerarin can effectively increase the peak bone mass in rats.

Ethnopharmacological relevance: Epimedium sagittatum brevicornum Maxim. is an important traditional Chinese herb that has long been used to promote bone fracture healing and treat osteoporosis. Aim of the study: Achieving peak bone mass by adolescence has now been accepted to be fundamental for preventing osteoporosis in adulthood life. This study investigated the possibility of increasing peak bone mass in young rats using the total flavonoid extract of Epimedium herb (TFE). Materials and methods: TFE was intragastrically administered to one-month-old Wistar rats at a low (100 mg/kg), middle (200 mg/kg) or high dose (400 mg/kg). Whole body bone mineral density (BMD) was measured by dual energy X-ray absorptiometry every two weeks. When BMD of any one of TFE groups was found to be significantly higher than that of the control, all rats were sacrificed, serum samples were collected for bone turnover biochemical assays, and femurs, tibiae and vertebrae were isolated and used in BMD, mechanical, micro-structural, histomorphometric and mechanistic studies. Results: Administration of TFE at middle and high doses for two months significantly increased the whole body, femoral and vertebral BMDs, and improved the bone mechanical and micro-architectural properties. The serum turnover biochemical results and the enhanced expression levels of bone-formation regulatory genes (Runx-2, OSX, and BMP-2) demonstrated that TFE administration increased bone formation but had no effect on bone resorption. The increased phosphorylation levels in femurs of PICA and CREB and expression of AC10 (the only soluble form of adenylyl cyclase) and the increased serum CAMP level after 4 h of TFE administration indicated that TFE promoted bone formation by activating the AC10/cAMP/PKA/CREB pathway in vivo. Conclusions: Oral administration of TFE at 200 mg/kg for two months can increase the peak bone mass of growing rats, suggesting the possibility of using total flavonoid extract of Epimedium herb to increase the peak bone mass in adolescence which is important for preventing osteoporosis in adult life.

Objective To investigate whether Puerarin(PR)can improve the peak bone mass of young rats and play a role in the prevention of osteoporosis.Methods Thirty-six Wistar female healthy rats were randomly divided into four groups:control group(CON),Icariin(ICA,positive control group)and Puerarin group(PR).ICA group was administered with icariin 25 mg/(kg· d),rats in the PR group were treated with Puerarin 15.4 mg/(kg·d),and the control group was administered with equal volume of distilled water.All rats were sacrificed after significant differences in bone mineral density(after 2 months)were observed among the groups.The organ index of vital organs was calculated.Pathological analysis was performed by HE staining.Vertebrae and femur were assessed by dual energy X-ray absorptiometry for bone mineral density,universal testing machine was used for bone biomechanical testing,ELISA kits were used to assess serum bone metabolism indicators and VG staining was used for bone morphometric examination.Results Body weight increased in all three groups,but here were no significant differences in body weight and organ index of lung,kidney and uterus among the groups during the experiment.No pathological changes were observed in the pathology examination.At baseline,there were no significant differences in bone mineral density among the CON group,ICA group and the PR group,and the increase in bone mineral density in ICA group and PR group was not significantly different from that of the CON group at 4 weeks after the intragastric administration(both P＞0.05).However,compared with CON group,bone mineral density of ICA group and PR group significantly increased(both P＜0.01)at the end of the 8 weeks.Biomechanical result:Compared with CON group,the maximum load values of femur and vertebrae of ICA group and PR group significantly increased(P＜0.01,P＜0.05).Bone histomorphometry VG staining analysis:compared with CON group,the number of trabecular(Tb.N),trabecular thickness(Tb.Th)and bone volume / tissue volume(BV/TV)values significantly increased(all P＜0.05),and trabecular spacing(Tb.Sp)was significantly lower(P＜0.01).Compared with CON group,the levels of osteocalcin(OC)in ICA group and PR group were significantly higher(both P＜0.05),and of Tartrate-resistant acid phosphatase 5b(TRACP 5b)were significantly lower(both P＜0.05).Conclusion Puerarin can significantly improve the peak bone mineral density and bone mass of young rats,enhance the biomechanical properties of femur and vertebrae,and change the bone histomorphometry properties.The possible mechanism is that it has the dual activity of improving bone formation and inhibiting bone resorption.

ETHNOPHARMACOLOGICAL RELEVANCE:Epimedium sagittatum brevicornum Maxim. is an important traditional Chinese herb that has long been used to promote bone fracture healing and treat osteoporosis. AIM OF THE STUDY:Achieving peak bone mass by adolescence has now been accepted to be fundamental for preventing osteoporosis in adulthood life. This study investigated the possibility of increasing peak bone mass in young rats using the total flavonoid extract of Epimedium herb (TFE). MATERIALS AND METHODS:TFE was intragastrically administered to one-month-old Wistar rats at a low (100 mg/kg), middle (200 mg/kg) or high dose (400 mg/kg). Whole body bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry every two weeks. When BMD of any one of TFE groups was found to be significantly higher than that of the control, all rats were sacrificed, serum samples were collected for bone turnover biochemical assays, and femurs, tibiae and vertebrae were isolated and used in BMD, mechanical, micro-structural, histomorphometric and mechanistic studies. RESULTS:Administration of TFE at middle and high doses for two months significantly increased the whole body, femoral and vertebral BMDs, and improved the bone mechanical and micro-architectural properties. The serum turnover biochemical results and the enhanced expression levels of bone-formation regulatory genes (Runx-2, OSX, and BMP-2) demonstrated that TFE administration increased bone formation but had no effect on bone resorption. The increased phosphorylation levels in femurs of PKA and CREB and expression of AC10 (the only soluble form of adenylyl cyclase) and the increased serum cAMP level after 4 h of TFE administration indicated that TFE promoted bone formation by activating the AC10/cAMP/PKA/CREB pathway in vivo. CONCLUSIONS:Oral administration of TFE at 200 mg/kg for two months can increase the peak bone mass of growing rats, suggesting the possibility of using total flavonoid extract of Epimedium herb to increase the peak bone mass in adolescence which is important for preventing osteoporosis in adult life.

To investigate the effect of Xianling Gubao capsule in preventing postmenopausal osteoporosis, forty-eight female Wistar rats were randomly divided into four groups: sham group (Sham), ovariectomized group (OVX), ethinylestradiol group (EE) and Xianling Gubao capsule group (XLGB). Rats in each group received ovariectomy, except for sham group. The XLGB group received Xianling Gubao capsule at the dose of 378 mg·kg⁻¹·d⁻¹. The dosage of EE group was 200 μg·kg⁻¹·d⁻¹, and OVX and Sham groups were only fed with equal volume of distilled water. All of the rats were put to death two months later. Bone mineral density, bone biomechanics, bone histomorphometry Micro-CT scanning and organ index of vital organs were calculated and pathologically observed. There was no significant difference in the body weight of rats and organ indexes of lung, kidney, heart and spleen in the experimental groups. There was also no significant change in their pathological observation, but the uterine index of OVX group and XLGB group was significantly lower than that of Sham group. According to the results of BMD test, compared with the OVX group, femurs and vertebrae BMD of the other three groups were increased, with statistically significant differences. On the basis of the results of bone biomechanical test, compared with OVX group, the maximum load values of femur and vertebrae of the other three groups were increased, with statistically significant differences, while the change of elastic modulus was not statistically significant. According to the bone histomorphometry results of VG staining, compared with Sham group, the number of trabecular bone was significantly lower than that in OVX group. Compared with OVX group, the number of trabecular bone in EE group and XLGB group was increased, but with no significant difference between EE and XLGB groups. The results of serum biochemical indexes showed that compared with Sham group, osteocalcin (OC) decreased, while tartrate resistant acid phosphatase 5b (TRACP 5b) increased in OVX group, with statistically significant differences. Compared with OVX group, the OC content of XLGB group and EE group increased, while the content of TRACP 5b decreased, with statistically significant differences. On the basis of the results of Micro-CT scanning, the change trends of femur volume BMD, number of trabecular bone (Tb.N), trabecular bone thickness (Tb.Th), trabecular bone separation (Tb.Sp), bone volume/tissue volume (BV/TV) in the groups were consistent with those of bone histomorphometry. There was no significant change in femoral cortical bone between the two groups. Xianling Gubao capsule can prevent osteoporosis in ovariectomized rats. The possible mechanism is the dual activity of inhibiting bone resorption and improving bone formation.

目的 研究丹参酮ⅡA对去卵巢大鼠骨密度(bone mineral density,BMD)和骨形态计量学的影响,探讨丹参酮ⅡA对雌激素缺乏引起的骨质疏松症(osteoporosis,OP)的治疗作用.方法 3月龄SD雌性大鼠40只,采用数字表法随机分为假手术组(Sham)、单纯卵巢切除组(Ovx)、雌激素治疗组和丹参酮ⅡA治疗组,每组10只.手术后1周,雌激素组给予炔雌醇200 μg/kg;丹参酮ⅡA治疗组给予丹参酮ⅡA11mg/kg;Ovx组与Sham组每日给予与治疗组同等体积的0.9％氯化钠注射液,给药方式均为口服,1次/d.每2周称1次体质量,每个月检测全身BMD,3个月后处死所有实验动物并取材.测定椎骨和右侧股骨的离体BMD和生物力学性能;品红-苦味酸染色(Van-Gieson,VG)进行骨形态分析;大鼠处死前按时间依次皮下注射四环素、茜素红和钙黄绿素,镜下观察各组3种荧光之间的距离变化;计算子宫系数,并在光镜显微镜下观察子宫病理切片中各组子宫内膜厚度的变化.结果 Ovx组大鼠体质量(282.38±16.92)明显增加,给予丹参酮ⅡA治疗的大鼠体质量增长与Sham组差异无统计学意义(P＞0.05),雌激素组大鼠体质量(248.33±15.57)明显低于其他各组,差异有统计学意义(P＜0.01);丹参酮ⅡA组大鼠的子宫系数(0.925±0.553)与Ovx组(0.900±0.389)比较略高,差异无统计学意义(P＞0.05),但明显低于雌激素组(2.386±0.495),差异有统计学意义(P＜0.01);与Ovx组相比,丹参酮ⅡA组的全身BMD (0.149±0.008)、股骨三点弯曲试验和椎骨压缩试验的最大载荷(159.86±12.37、267.58±33.4)均有所提高,差异有统计学意义(P＜0.05),而丹参酮ⅡA组和雌激素组组间差异无统计学意义(P＞0.05);丹参酮ⅡA组的弹性模量与Ovx组之间差异无统计学意义(P＞0.05),但丹参酮ⅡA组均值高于Ovx组;骨形态计量分析结果显示丹参酮ⅡA组和雌激素组较Ovx组骨小梁明显变多变密,改善了松质骨的微结构;荧光标记观察表明口服丹参酮ⅡA和炔雌醇可加快骨的生长;子宫病理切片观察到Ovx组子宫内膜组织萎缩变薄,丹参酮ⅡA组与假手术组相差不大,雌激素组子宫内膜明显增厚,与子宫系数呈现相同的趋势.结论 丹参酮ⅡA可通过增加BMD,提高骨质量,改善骨微结构预防与治疗雌激素缺乏引起的OP,而刺激子宫组织增生的不良反应又远比雌激素小,对探索抗OP中药单体新药有非常重要的意义.

Objective To study the effect of the compound medicine of tanshinone 2A and resveratrol on peak bone mass in growing rats and to explore its possible mechanism,so as to explore anti-osteoporosis mechanisms of new traditional Chinese medicine (TCM) drugs. Methods Totally 40 1-month-old female Wistar rats were randomly divided into tanshinone 2A group,resveratrol group,compound group (tanshinone 2A and resveratrol),and normal control group,with 10 rats in each group. Body weight was measured once every two weeks,and the whole body bone mineral density was measured with dual-energy X-ray monthly. When the whole-body bone mineral density became statistically significant between medication groups and control group,all animals were sacrificed to determine the bone mineral density of vertebrae and right femoral bone. The biomechanical properties of femur and vertebrae were measured by AGS-X series universal test,then the bone morphology was analyzed with Fuchsin picric acid staining. Finally,the levels of tartrate-resistant acid phosphatase 5b and osteocalcin were measured with enzyme-linked immunosorbent assay.Results The body weights were not statistically significant among all groups (P>0.05). The whole-body bone mineral density showed no significant difference (P>0.05) after feeding for 1 month;however,two months later,it was significantly different between medication groups and control group;in particular,the whole-body (P=0.016),femoral (P=0.001),and vertebral bone mineral density (P=0.034),bone trabecular number (P=0.024),thickness (P=0.040),and area (P=0.038) were significantly increased in the compound group,along with the significantly decreased trabecular separation degree (P=0.032). Compared with the control group,the compound group had significantly increased osteocalcin (P=0.033) and tartrate-resistant acid phosphatase 5b (P=0.028) levels in serum.Conclusion The compound of tanshinone 2 A and resveratrol can improve the bone density and bone quality in rats,and such effect is higher than either tanshinone 2 A monomer or resveratrolmonomer.

Objective To explore whether sinusoidal electromagnetic field (SEMFs) can promote bone microstructure, bone strength and bone metabolism of hind-limb suspension (HLS) osteoporosis model.Methods A total of 30 female SD rats (180 ± 10 g body weight) were divided into control group, HLS group and HLS +SEMFs group.HLS+SEMFs group was treated daily for 1.5 hours at 50 Hz with 1.8 mT sinusoidal electromagnetic field interference and the rats were sacrificed after 4 weeks.Bone mineral density (BMD) and biomechanics of rat femur and vertebrae were measured.The levels of serum osteocalcin (OC) and tartrate-resistant acid phosphatase 5b (TRACP 5b) were determined by ELISA.The microstructure of cancellous bone was analyzed by micro-CT.The microstructure of left tibia was observed after picric acid staining.IPP 6.0 software was used to analyze trabecular bone parameters.The organ paraffin embedded sections were subjected to toxicological analysis by HE staining to evaluate the side effects of the electromagnetic field.Results There were no significant differences between the groups in body weight and organ coefficient during the experiment, and there were no abnormal pathological findings.The result showed that SEMFs could improve the bone mineral density of femur and vertebra after 4 weeks.Biomechanical experiments showed that SEMFs reduced the decrease of femoral and vertebral mechanical properties induced by HLS.In addition, SEMFs increased OC content and inhibited TRACP 5b.Micro-CT result showed that SEMFs partially inhibited the decrease of microstructure of cancellous bone caused by HLS.Compared with HLS group, trabecular number, trabecular thickness and trabecular bone area significantly increased, and the trabecular separation significantly decreased.Bone histomorphometric analysis also showed that SEMFs improved the microstructure of cancellous bone.Conclusion SEMFs alleviate bone loss caused by simulated microgravity in hind limbs by promoting bone formation and inhibiting bone resorption, thus SEMFs may be a potential biophysical treatment for osteoporosis.

Microgravity is one of the main threats to the health of astronauts. Pulsed electromagnetic fields (PEMFs) have been considered as one of the potential countermeasures for bone loss induced by space flight. However, the optimal therapeutic parameters of PEMFs have not been obtained and the action mechanism is still largely unknown. In this study, a set of optimal therapeutic parameters for PEMFs (50 Hz, 0.6 mT 50% duty cycle and 90 min/day) selected based on high‐throughput screening with cultured osteoblasts was used to prevent bone loss in rats induced by hindlimb suspension, a commonly accepted animal model to simulate the space environment. It was found that hindlimb suspension for 4 weeks led to significant decreases in femoral and vertebral bone mineral density (BMD) and their maximal loads, severe deterioration in bone micro‐structure, and decreases in levels of bone formation markers and increases in bone resorption markers. PEMF treatment prevented about 50% of the decreased BMD and maximal loads, preserved the microstructure of cancellous bone and thickness of cortical bone, and inhibited decreases in bone formation markers. Histological analyses revealed that PEMFs significantly alleviated the reduction in osteoblast number and inhibited the increase in adipocyte number in the bone marrow. PEMFs also blocked decreases in serum levels of parathyroid hormone and its downstream signal molecule cAMP, and maintained the phosphorylation levels of protein kinase A (PKA) and cAMP response element‐binding protein (CREB). The expression level of soluble adenylyl cyclases (sAC) was also maintained. It therefore can be concluded that PEMFs partially prevented the bone loss induced by weightless environment by maintaining bone formation through signaling of the sAC/cAMP/PKA/CREB pathway. Bioelectromagnetics. 39:569–584, 2018. © 2018 Wiley Periodicals, Inc.

OBJECTIVE:To study effects of 50 Hz 1.8 mT sinusoidal electromagnetic fields (SEMFs) on bone mineral density (BMD) in SD rats.METHODS:Thirty SD rats weighted(110±10) and aged 1 month were randomly divided into control group and electromagnetic field group, 15 in each group. Normal control group of 50 Hz 0 mT density and sinusoidal electromagnetic field group of 50 Hz 1.8 mT were performed respectively with 1.5 h/d and weighted weight once a week, and observed food-intake. Rats were anesthesia by intraperitoneal injection and dual energy X-ray absorptiometry were used to detect bone density of whole body, and detected bone density of femur and vertebral body. Osteocalcin and tartrate-resistant acid phosphatase 5b were detected by ELSA; weighted liver, kidney and uterus to calculate purtenance index, then detected pathologic results by HE.RESULTS:Compared with control group, there was no significant change in weight every week, food-intake every day; no obvious change of bone density of whole body at 2 and 4 weeks, however bone density of whole body, bone density of excised femur and vertebra were increased at 6 weeks. Expression of OC was increased, and TRACP 5b expression was decreased. No change of HE has been observed in liver, kidney and uterus and organic index.CONCLUSIONS:50 Hz 1.8 mT sinusoidal electromagnetic fields could improve bone formation to decrease relevant factors of bone absorbs, to improve peak bone density of young rats, in further provide a basis for clinical research electromagnetic fields preventing osteoporosis foundation.

Objective To investigate whether total flavonoid of epimedium capsules (TF) can prevent postmenopausal osteoporosis.Methods Forty-eight female Wistar female rats were randomly divided into four groups:sham group (Sham),ovariectomized group (OVX),ethinylestradiol group (EE) and total flavonoid of epimedium capsules (TF),each group of 12.Rats in each group underwent ovariectomy except sham group.The TF group received TF 265 mg/ (kg · d)and EE group received 200 μg/ (kg · d) EE.The OVX and Sham groups were only fed with equal volume of distilled water.All rats were sacrificed two months later.Bone mineral density,bone biomechanics,serum bone metabolism index test,Micro-CT scan and organ index of vital organs were calculated and pathologically observed.Results There were no significant differences in body weight,organ index of vital organs and pathological observation (P＞0.05),but the uterine index of OVX and TF groups were significantly lower than that of Sham group (P＜0.05).Femoral BMD (0.109 g/cm2) in OVX group was significantly lower than that in Sham group (0.128 g/cm2),EE group (0.125 g/cm2) and TF group (0.123 g/cm2),the differences were statistically significant (all P＜0.01).Vertebral changes among groups showed the same tendency as the femur.Biomechanical results showed that the maximum load of the femur and vertebrae in OVX group was 95.9 N and 201.9 N respectively,which were significantly lower than Sham group,while the maximum load of femur and vertebra in TF group was 122.1 N and 226.6 N respectively,which was significantly higher than that in OVX group,the changes of elastic modulus among all groups showed the same tendency with maximum load.Compared with Sham group,the number of bone trabecular in OVX group decreased,but trabecular separation increased.Compared with OVX group,the number of bone trabecular in EE and TF group increased,trabecular separation decreased.Serum bone metabolism index:The level of Osteocalcin in OVX group was 104.6 ng/mL,lower than that in Sham group (154.5 ng/mL),EE group (151.7 ng/mL) and TF group (136.9 ng/mL) (P＜0.05).The changes of the content of propeptide of type Ⅰ procollagen (P1NP) in type Ⅰ procollagen were consistent with those of OC.The levels of tartrate resistant acid phosphatase 5b (TRACP 5b) was significantly higher than that of Sham group (0.76 U/L),EE group (0.38 U/L) and TF group (0.95 U/L).The content of type Ⅰ collagen C-terminal peptide (CTX-Ⅰ) was consistent with that of TRACP 5b.Micro-CT scan results:The change trends of femur volume BMD,number of trabecular bone (Tb.N),trabecular bone thickness (Tb.Th),trabecular bone separation (Tb.Sp),bone volume/tissue volume (BV/TV) between the two groups were consistent with that of bone histomorphometry.There was no significant difference in femoral cortical bone between the two groups.Conclusion Epimedium flavonoids can prevent osteoporosis in ovariectomized rats.The possible mechanism is that they have the dual activity of inhibiting bone resorption and improving bone formation.

Objective: To compare the effects of 50 Hz 1.8 mT sinusoidal magnetic field (SEMF) and 50 Hz 0.6 mT pulsed electromagnetic field(PEMF) on the maturation and mineralization of rat calvaria osteoblasts. Methods: Primary cultured rat calvarial osteoblasts were divided into 3 groups:blank control group, SEMF group and PEMF group. The rats in SEMT and PEMT groups were treated with 50 Hz 1.8 mT SEMF or 50 Hz 0.6 mT PEMF for 90 min/d, respectively. Western blotting and Real-time RT-PCR were used to detect the protein and mRNA expressions of Collagen-1, bone morphogenetic protein 2 (BMP-2), osterix (OSX) and Runt-associated transcription factor 2(Runx-2). The alkaline phosphatase(ALP) activity was detected by ALP test kits at d6 and d9 after treatment, and by ALP staining using azo coupling at d10 after treatment. The formation of calcium nodules was observed by alizarin red staining. Results: Compared with blank control group, the protein and mRNA expressions of Collagen-1, BMP-2, OSX and Runx-2 in SEMT and PEMT groups were significantly increased (P <0.01 or P <0.05); while the mRNA expressions of Collagen-1 and BMP-2 in PEMF group were significantly higher than those in SEMF group. After 6 days treatment, the activity of ALP in PEMF group was significantly higher than that in blank control group (P<0.05), while such difference was not observed in SEMF group (P>0.05); after 9 days treatment, the activities of ALP in both PEMF and SEMP groups were significantly higher than that in blank control group (all P<0.05), but the difference between PEMF and SEMF groups was not significant (P>0.05). After 10 days treatment, ALP staining was increased in both PEMF and SEMF groups compared with that in blank control group (all P<0.01), and the stained area was bigger in PEMF group than that in SEMF group (P<0.05). After 12 days treatment, calcium nodules were increased in PEMF and SEMF groups compared with that in blank control group (all P<0.01), and more calcium nodules were observed in PEMF group than SEMF group (P<0.05). Conclusion: Both 50 Hz 1.8 mT that in SEMF and 50 Hz 0.6 mT PEMF can promote the maturation and mineralization of osteoblasts, and the effect of PEMF is more marked.

To study the effects of different frequency sinusoidal electromagnetic fields on the bone mineral density and bone histomorphometry of SD young rats, and to screen suitable electromagnetic field frequency. In total 32 female SD rats of 8 weeks old were randomly divided into 4 groups: control group, 15 Hz group, 30 Hz group and 45 Hz group. Except for the control group, rats in experimental groups were treated with corresponding frequency of 1.8 mT sinusoidal electromagnetic field 90 minutes every day. Rats were measured for bone mineral density after 8 weeks by dual energy X-ray absorptiometry. Rats' femur and vertebral bones were measured by analyzing the static and dynamic forms on the right tibia bone for morphometrics. Rat serum was measured to estimate the index of bone formation and bone resorption. Bone mineral density of rats from 15 Hz group and 45 Hz group was higher than that of the control group (P<0.05). Serum osteocalcin level of rats from 15 Hz group and 45 Hz group was higher than that of the control group (P<0.05). Double fluorescence spacing and static parameters of bone tissue in experimental group rat tibia were higher than that of the control group (P<0.05). Our findings imply that 15 Hz and 45 Hz sinusoidal electromagnetic fields can effectively increase bone mineral density in young rats for preventing osteoporosis.

Objective: To investigate the effect of resveratrol on peak bone mineral density and bone mass in growing rats. Methods: Thirty-six female healthy Wistar rats were randomly divided into control group, icariin group and resveratrol group with 12 rats in each group. Icariin (25 mg·kg-1·d-1), resveratrol (8.4 mg·kg-1·d-1) or equal volume of distilled water were given by gavage to icariin group, resveratrol group and control group, respectively. The rats were sacrificed after 12 weeks. The organ indexes were calculated and pathology sections were observed; the bone mineral density (BMD), bone biomechanics, serum bone metabolism index, and results of micro-CT scan were analyzed. Results: During the experiment, the body weight of rats showed an increasing trend and there was no significant difference among three groups (P>0.05). There were no significant differences in organ index of vital organs and pathological changes among the groups (all P>0.05). Compared with the control group, the whole body BMD, and the BMDs of femur and vertebrae in icariin and resveratrol groups were significantly increased after 12 weeks (all P<0.05). The maximum load values of femur and vertebrae, as well as elastic modulus of vertebrae in icariin and resveratrol groups were significantly higher than those in control group (P<0.05 or P<0.01). Micro-CT scan showed that the volumetric BMD, number of trabecular, trabecular thickness and bone volume/tissue volume of the cancellous bone in icariin and resveratrol groups were significantly higher and the trabecular separation was significantly lower than those in the control group (P<0.05 or P<0.01); while there was no significant difference in volumetric BMD of cortical bone for femur. The contents of osteocalcin in icariin and resveratrol groups were significantly higher than those in control group (all P<0.05), while the contents of tartarte-resistant acid phosphatase 5b (TRACP5b) were significantly lower than those in control group (all P<0.05).Conclusion: Resveratrol can inhibit bone resorption and enhance bone formation, so as to improve the peak bone mass and bone density, enhance bone strength and improve the microstructure of bone tissue in young rats.

目的 探讨蛇床子素对青年大鼠骨代谢的影响.方法 30只雌性wistar大鼠按体重随机分为A、B、C组,A组为正常对照组给予等体积蒸馏水灌胃,B组给予雌二醇灌胃,C组给予蛇床子素灌胃.3组分别在灌胃后4、8、12周分别测定全身骨密度,并在12周后检测左侧股骨和腰椎骨密度及右侧股骨和第4腰椎生物力学性能;检测血清抗酒石酸酸性磷酸酶-5b(TRAP-5b)和骨钙素(0C)含量,分析右侧胫骨骨形态计量学.结果 B、C组灌胃12周后全身骨密度及离体骨密度均高于A组,但C组低于B组(P＜0.01).B、C组生物力学性能指标均高于A组,C组股骨弹性模量和腰椎最大载荷低于B组(P＜0.05).B、C组TRAP-5b水平均低于A组,OC水平高于A组(P＜0.01);C组TRAP-5b水平高于B组,OC水平低于B组(P＜0.01).B、C组胫骨骨组织中骨小梁数量密集,骨小梁分离度低于A组,B组骨小梁数量高于C组,骨小梁分离度低于C组.结论 蛇床子素能够提高青年大鼠骨密度、生物力学性能及骨组织微结构,增加青年大鼠骨质量,但其作用效果略低于雌激素.

Objective: To investigate the effect of icariin total flavonoids capsules (ITFC) on bone mineral density (BMD) and bone histomorphometry in growing rats and its anti-osteoporosis mechanism. Methods: Thirty female SD rats were randomly divided into 3 groups:normal control group, ITFC-1 group and ITFC-2 group. Rats in ITFC-1 group and ITFC-2 group were fed with 50 mg·kg-1·d-1 or 100 mg·kg-1·d-1 ITFC, respectively, and those in normal control group were fed with equal volume of distilled water. The whole body BMD was measured after 4, 8 and 12 weeks, and BMDs of the right femur and lumbar vertebrae were measured after 12 weeks. The serum levels of tartaric acid phosphatase 5b (TRACP 5b) and bone alkaline phosphatase (BALP) were measured by ELISA. Bone morphometry was performed on the right tibia. Results: There were no significant differences in the body weight increase between normal control group and two ITFC groups (all P>0.05). There were also no significant differences in whole body BMDs after 4 and 8 weeks between normal control group and ITFC groups (all P>0.05). After 12 weeks, the whole body BMD, BMD of bone in vitro, serum BALP level and trabecular area in ITFC-1 group and ITFC-2 group were significantly higher, trabecular separation was significantly lower than that in normal control group (all P<0.05); and the trabecular width and the number in ITFC-2 group were also significantly higher, and serum TRACP 5b level was significantly lower than that in normal control group (all P<0.05). The BMD of bone in vitro, serum BALP level, trabecular number and area in ITFC-2 group were significantly higher, and serum TRACP 5b level was significantly lower than that in ITFC-1 group (all P<0.05). Conclusion: ITFC can prevent osteoporosis by increasing bone density and bone formation, decreasing bone resorption and improving microstructure of bone.