Pulsed electromagnetic fields promote bone formation by activating the sAC-cAMP-PKA-CREB signaling pathway.

The application of pulsed electromagnetic fields (PEMFs) in the prevention and treatment of osteoporosis has long been an area of interest. However, the clinical application of PEMFs remains limited because of the poor understanding of the PEMF action mechanism. Here, we report that PEMFs promote bone formation by activating soluble adenylyl cyclase (sAC), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and cAMP response element-binding protein (CREB) signaling pathways. First, it was found that 50 Hz 0.6 millitesla (mT) PEMFs promoted osteogenic differentiation of rat calvarial osteoblasts (ROBs), and that PEMFs activated cAMP-PKA-CREB signaling by increasing intracellular cAMP levels, facilitating phosphorylation of PKA and CREB, and inducing nuclear translocation of phosphorylated (p)-CREB. Blocking the signaling by adenylate cyclase (AC) and PKA inhibitors both abolished the osteogenic effect of PEMFs. Second, expression of sAC isoform was found to be increased significantly by PEMF treatment. Blocking sAC using sAC-specific inhibitor KH7 dramatically inhibited the osteogenic differentiation of ROBs. Finally, the peak bone mass of growing rats was significantly increased after 2 months of PEMF treatment with 90 min/day. The serum cAMP content, p-PKA, and p-CREB as well as the sAC protein expression levels were all increased significantly in femurs of treated rats. The current study indicated that PEMFs promote bone formation in vitro and in vivo by activating sAC-cAMP-PKA-CREB signaling pathway of osteoblasts directly or indirectly.

Pulsed electromagnetic fields prevented the decrease of bone formation in hindlimb-suspended rats by activating sAC/cAMP/PKA/CREB signaling pathway.

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.

[Effect of Compound Medicine of Tanshinone 2A and Resveratrol on Peak Bone Mass in Growing Rats].

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.

[Effect of Xianling Gubao capsule on bone metabolism in ovariectomized rats].

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.

[Resveratrol and puerarin on peak bone mass in young rats].

OBJECTIVE: To compare effects of resveratrol, puerarin and the compounds on peak bone mass in rats. METHODS: Forty SPF Wistar rats weighed 109.45 g to 119.44 g with an average of 115.87 g were selected. After 3 days' adaption, rats were divided into control group (the same volume of distilled water per day), puerarin group(15.4 mg/kg puerarin daily), resveratrol group (8.4 mg/kg resveratrol daily), compound drug group (daily dose of 8.4 resveratrol added 15.4 mg/kg of puerarin) and 10 in each group. The body weight of the rats was monitored at every 7 days and body bone density was measured at every month. All rats were sacrificed after 3 months. The bone mineral density of femur and vertebrae was detected by dual energy X-ray absorptiometry; bone biomechanics, VG staining was used to analyze bone histomorphometry;ELISA was used to detect serum bone metabolic index and microstructure of femur were scanned with Micro-CT scanner. RESULTS: There were no significant differences in body weight among groups during exoeriment. Bone mineral density results showed BMD of femur and vertebrae in the other three groups were significantly increased, and R+P group was significantly higher than PR group and RES group(P<0.05) by compared with CON group;three-point bending and compression test results showed compared with CON group, other three groups of femoral and vertebral maximum load values were significantly increased, and P+R group was higher than PR group and RES group, but elastic modulus was not statistically significant. Bone histomorphometry showed that number of trabecular bone in other three groups were significantly increased compared with CON group, separation of trabecular bone were significantly reduced, continuity was improved, and R+P group was significantly better than RES and PR group. The results of Micro-CT scan showed that separation of trabecular bone were significantly reduced, continuity were improved in other three groups, and R+P group was significantly better than RES and PR group. The numbers of trabecular bone (Tb.N), trabecular bone thickness (Tb.Th), volume of trabecular bone (BV/TV) in PR group, RES group and R+P group were significantly higher than CON group, but trabecular bone separation (Tb.Sp) was significantly reduced. Serum levels results showed, level of OC in the other three groups were higher than control group(P<0.05), content of TRACP 5b decreased, and level of OC in P+R group was significantly higher than PR group and RES group, content of TRACP 5b was no significant change. CONCLUSIONS: Compound of puerarin and resveratrol assigned in a 1:1 ratio could improve bone mineral density and bone mass in young rats, enhance biomechanical properties of bone, promote mineralization and maturation of osteoblasts, inhibit osteoblastic bone resorption, and is better than the role of their respective monomers. The paper showed that traditional Chinese medicine compound medicine will be used as a new way to prevent and treat osteoporosis.

Total flavonoid extract of Epimedium herb increases the peak bone mass of young rats involving enhanced activation of the AC10/cAMP/PKA/CREB pathway.

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.