Study on the effects and mechanisms of Wenzhong Bushen Formula in improving ovarian reserve decline in mice based on network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: The Wenzhong Bushen Formula (WZBSF) is a traditional Chinese medicine empirical formula known for its effects in tonifying qi, strengthening the spleen, warming the kidneys, promoting yang, regulating blood circulation, and balancing menstruation. Clinical evidence has demonstrated its significant efficacy in treating Diminished Ovarian Reserve (DOR) by improving ovarian reserves. However, the specific pharmacological mechanisms of WZBSF remain unclear. AIM OF THE STUDY: This study aims to investigate the mechanisms by which WZBSF improves ovarian reserve decline through network pharmacology and animal experiments. METHODS AND MATERIALS: WZBSF was analyzed using a dual UPLC-MS/MS and GC-MS platform. Effective components and targets of WZBSF were obtained from the TCMSP database and standardized using UniProt. Disease targets were collected from GeneCard, OMIM, PHARMGKB, and DisGeNET databases, with cross-referencing between the two sets of targets. A PPI protein interaction network was constructed using Cytoscape3.9.1 and STRING database, followed by KEGG and GO enrichment analysis using the Metascape database. Finally, an ovarian reserve decline model was established in mice, different doses of WZBSF were administered, and experimental validation was conducted through serum hormone detection, H&E staining, immunofluorescence (IF), immunohistochemistry (IHC), and Western blot analysis (WB). RESULTS: WZBSF shares 145 common targets with ovarian reserve decline. GO enrichment analysis revealed involvement in biological processes such as response to hormone stimulation and phosphatase binding, while KEGG analysis implicated pathways including the PI3K-AKT signaling pathway and FoxO signaling pathway. In mice with ovarian reserve decline, WZBSF restored weight gain rate, increased ovarian index, normalized estrous cycles, reversed serum hormone imbalances, restored various follicle counts, and improved ovarian morphology. Additionally, WZBSF reduced p-AKT and p-FOXO3a levels, preventing excessive activation of primordial follicles and maintaining ovarian reserve. CONCLUSION: WZBSF can ameliorate cyclophosphamide and busulfan-induced ovarian reserve decline, and its mechanism may be associated with the inhibition of the PI3K/AKT/FOXO3a signaling pathway.

Hydrocarbons removal and microbial community succession in petroleum-contaminated soil under hydrogen peroxide treatment.

Chemical oxidation as a pretreatment step coupled with bioremediation for petroleum-contaminated soil may pose serious impacts on indigenous microorganisms and the available nutrients. Petroleum-contaminated soil were treated by hydrogen peroxide (H2O2) at initial concentrations of 105 mM (HH), 21 mM (HL), and 105 mM in three equal amounts (HT) without adding any external catalyst. The contents of total petroleum hydrocarbons (TPH) and dissolved nutrients (total organic compounds, nitrogen, and phosphate), and the indigenous bacteria community succession (analyzed by high-throughput sequencing of 16S rDNA) were investigated over 50 days. Compared to the control treatment without H2O2 addition, H2O2 treatments for the petroleum-contaminated soil significantly promoted the TPH removal especially in the first 4 days and impacted the contents of dissolved nutrients. Both of chemical oxidation and nutrients contributed to microbial community structure changes in alpha diversity. Although the soil microbial community structure had undergone significant changes after different chemical oxidation pretreatments, Firmicutes, Proteobacteria, Gemmatimonadetes, and Actinobacteria were the main bacterial phyla. Compared with adding H2O2 at one time, H2O2 added in stepwise was beneficial to indigenous bacterial diversity recovery and TPH removal. H2O2 oxidation treatments showed a great influence on the microbial community structures in the start-up stage, while recovery time rather than the oxidation treatments presented greater effects on the composition of the microbial community structure with the incubation time extended. Therefore, adding H2O2 as pretreatment for petroleum-contaminated soil showed little effect on the structure of soil indigenous microbial community from a long-term scale, and was conducive to the continuous removal of TPH by indigenous microorganisms.

ZMYND8 expression combined with pN and pM classification as a novel prognostic prediction model for colorectal cancer: Based on TCGA and GEO database analysis.

BACKGROUND: Zinc finger MYND (Myeloid, Nervy and DEAF-1)-type containing 8 (ZMYND8) is closely correlated with tumor proliferation and invasiveness. However, its prognostic value has not been estimated in colorectal cancer (CRC). OBJECTIVE: We aimed to elucidate the prognostic significance of ZMYND8 expression and the pN and pM classification supplemented by its expression in CRCs. METHODS: The candidate gene ZMYND8 is identified by TCGA database and GEO database, and then we retrospectively evaluated the status and prognostic significance of ZMYND8 expression of 174 patients with CRC. RESULTS: Online data showed high expression of ZMYND8 is closely correlated with worse overall survival. Our study revealed high expression of ZMYND8 in CRC patients was significantly associated with worse overall and disease-free survival (P< 0.05), and was an independently adverse prognostic factor for overall survival (P< 0.001) and disease-free survival (P= 0.001) by univariate and multivariate analysis. C-index to combined prognostic model containing the pN, pM classification supplemented by the status of ZMYND8 expression showed improved predictive ability comparing with the pN and pM classification model (C-index of 0.597 vs. 0.545, respectively). CONCLUSION: The combined prognostic model could improve the ability to determine the clinical outcome of patients with CRC.

[Preventive effect and mechanism of puerarin on rat models of disuse osteoporosis].

To investigate the preventive effect and possible mechanism of puerarin(Pur) in rat model of disuse osteoporosis(DOP),thirty healthy Wistar female rats of 2 months old were randomly divided into control group(Control), hindlimb suspension group(HLS), and puerarin group(HLS+Pur) in hindlimb suspension, with 10 rats in each group. A disuse osteoporosis model was established by tail suspension method, and 15.4 mg·kg~(-1) puerarin suspension was administered to HLS+Pur group every day, and the same volume of distilled water was administered to Control group and HLS group respectively. After 28 days, the rats were sacrificed by abdominal aorta blood collection, the main organs of the rats were removed, and the bone tissues of the rats were dissected. The organ index of the rats was calculated and the histopathology of the organs was observed under microscope. Bone mineral density test and bone biomechanical experiment were performed. Bone histomorphometry results were observed after bone tissue sectioning, and serum biochemical markers of bone metabolism were determined. There was no significant difference in organ index between the groups. There was no obvious abnormality in the pathological examination of the organs. The results of bone mineral density showed that puerarin could significantly increase the bone density of the tibia and vertebrae caused by hindlimb suspension. The mechanical parameters experiments showed that puerarin could effectively increase the maximum load and elastic modulus of the tibia and vertebrae. Fluorescence labeling showed that the fluorosis interval increased and the bone formation increased during puerarin treatment. The VG staining results showed that compared with the HLS group, in the puerarin group, the number of trabecular bone increased, the thickness of the trabecular bone became thicker, and the bone separation became smaller, which greatly improved the bone microstructure after hindlinb suspension. In addition, serum biochemical indicators showed that puerarin could promote bone formation index bone calcium. The content of osteocalcin(OC) increased and inhibited the formation of tartrate-resistant acid phosphatase 5 b(TRACP 5 b). Puerarin has a preventive effect in the rat model of disuse osteoporosis and its effect is good, and its mechanism may be related to promoting bone formation and inhibiting bone resorption.

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.

The flavonol glycoside icariin promotes bone formation in growing rats by activating the cAMP signaling pathway in primary cilia of osteoblasts.

Icariin, a prenylated flavonol glycoside isolated from the herb Epimedium, has been considered as a potential alternative therapy for osteoporosis. Previous research has shown that, unlike other flavonoids, icariin is unlikely to act via the estrogen receptor, but its exact mechanism of action is unknown. In this study, using rat calvarial osteoblast culture and rat bone growth models, we demonstrated that icariin promotes bone formation by activating the cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) pathway requiring functional primary cilia of osteoblasts. We found that icariin increases the peak bone mass attained by young rats and promotes the maturation and mineralization of rat calvarial osteoblasts. Icariin activated cAMP/PKA/CREB signaling of the osteoblasts by increasing intracellular cAMP levels and facilitating phosphorylation of both PKA and CREB. Blocking cAMP/PKA/CREB signaling with inhibitors of the cAMP-synthesizing adenylyl cyclase (AC) and PKA inhibitors significantly inhibited the osteogenic effect of icariin in the osteoblasts. Icariin-activated cAMP/PKA/CREB signaling was localized to primary cilia, as indicated by localization of soluble AC and phosphorylated PKA. Furthermore, blocking ciliogenesis via siRNA knockdown of a cilium assembly protein, IFT88, inhibited icariin-induced PKA and CREB phosphorylation and also abolished icariin's osteogenic effect. Finally, several of these outcomes were validated in icariin-treated rats. Together, these results provide new insights into icariin function and its mechanisms of action and strengthen existing ties between cAMP-mediated signaling and osteogenesis.

Childhood cancer chemotherapy-induced bone damage: pathobiology and protective effects of resveratrol and other nutraceuticals.

Intensive cancer chemotherapy causes significant bone loss, for which the mechanisms remain unclear and effective treatments are lacking. This is a significant issue particularly for childhood cancers, as the most common ones have a >75% cure rate following chemotherapy; there is an increasing population of survivors who live with chronic bone defects. Studies suggest that these defects are the result of reduced bone from increased marrow fat formation and increased bone resorption following chemotherapy. These changes probably result from altered expression/activation of regulatory molecules or pathways regulating skeletal cell formation and activity. Treatment with methotrexate, an antimetabolite commonly used in childhood oncology, has been shown to increase levels of proinflammatory/pro-osteoclastogenic cytokines (e.g., enhanced NF-κB activation), leading to increased osteoclast formation and bone resorption, as well as to attenuate Wnt signaling, leading to both decreased bone and increased marrow fat formation. In recent years, understanding the mechanisms of action and potential health benefits of selected nutraceuticals, including resveratrol, genistein, icariin, and inflammatory fatty acids, has led to preclinical studies that, in some cases, indicate efficacy in reducing chemotherapy-induced bone defects. We summarize the supporting evidence.

Effects of Resveratrol Supplementation on Methotrexate Chemotherapy-Induced Bone Loss.

Intensive cancer chemotherapy is known to cause bone defects, which currently lack treatments. This study investigated the effects of polyphenol resveratrol (RES) in preventing bone defects in rats caused by methotrexate (MTX), a commonly used antimetabolite in childhood oncology. Young rats received five daily MTX injections at 0.75 mg/kg/day. RES was orally gavaged daily for seven days prior to, and during, five-day MTX administration. MTX reduced growth plate thickness, primary spongiosa height, trabecular bone volume, increased marrow adipocyte density, and increased mRNA expression of the osteogenic, adipogenic, and osteoclastogenic factors in the tibial bone. RES at 10 mg/kg was found not to affect bone health in normal rats, but to aggravate the bone damage in MTX-treated rats. However, RES supplementation at 1 mg/kg preserved the growth plate, primary spongiosa, bone volume, and lowered the adipocyte density. It maintained expression of genes involved in osteogenesis and decreased expression of adipogenic and osteoclastogenic factors. RES suppressed osteoclast formation ex vivo of bone marrow cells from the treated rats. These data suggest that MTX can enhance osteoclast and adipocyte formation and cause bone loss, and that RES supplementation at 1 mg/kg may potentially prevent these bone defects.

[Effects of 1.8 mT sinusoidal alternating electromagnetic fields of different frequencies on bone biomechanics of young rats].

Objective: To study the effects of 1.8 mT sinusoidal electromagnetic fields of different frequencies on bone mineral density (BMD) and biomechanical properties in young rats. Methods: A total of 32 female SD rats (6-week-old) were randomly divided into 4 groups (8 in each):control group, 10 Hz group, 25 Hz group and 40 Hz group. The experimental groups were given 1.8 mT sinusoidal electromagnetic field intervention 90 min per day. The whole body BMD of rats was detected with dual-energy X-ray absorptiometry after 4 and 8 weeks of intervention. After 8 weeks of intervention, all rats were sacrificed, and the BMD of femur and lumbar vertebra, the length and diameter of femur, the width between medial and lateral malleolus were measured. Electronic universal material testing machine was used to obtain biomechanical properties of femur and lumbar vertebra, and micro CT scan was performed to observe micro structures of tibial cancellous bone. Results: Compared with the control group, rats in 10 Hz and 40 Hz groups had higher whole body BMD, BMD of femur, maximum load and yield strength of femur, as well as maximum load and elastic modulus of lumbar vertebra (all P<0.05). But no significant differences in the length and diameter of femur, and the width between medial and lateral malleolus were observed between control group and experimental groups (all P>0.05). Micro CT scan showed that the trabecular number and separation degree, bone volume percentage were significantly increased in 10 Hz and 40 Hz groups (all P<0.01). Rats in 25 Hz group also had higher BMD and better in biomechanical properties than control group, but the differences were not statistically significant (all P>0.05). Conclusion: 10 and 40 Hz of 1.8 mT sinusoidal electromagnetic field can significantly improve the bone density, microstructure and biomechanical properties in young rats.

Sinusoidal electromagnetic fields promote bone formation and inhibit bone resorption in rat femoral tissues in vitro.

Effects of sinusoidal electromagnetic fields (SEMFs) on bone metabolism have not yet been well defined. The present study investigated SEMF effects on bone formation and resorption in rat femur bone tissues in vitro. Cultured femur diaphyseal (cortical bone) and metaphyseal (trabecular bone) tissues were treated with 50 Hz 1.8 mT SEMFs 1.5 h per day for up to 12 days and treatment effects on bone formation and resorption markers and associated gene expression were examined. Treatment with SEMFs caused a significant increase in alkaline phosphatase (ALP) activity and inhibited the tartrate-resistant acid phosphatase (TRACP) activity in the femoral diaphyseal or metaphyseal tissues. SEMFs also significantly increased levels of mRNA expression of osterix (OSX), insulin-like growth factor (IGF-1) and ALP in the bone tissues. SEMF treatment decreased glucose content and increased lactic acid contents in the culture conditioned medium. In addition, treatment with SEMFs decreased mRNA expression levels of bone resorption-related genes TRACP, macrophage colony stimulating factor (M-CSF) and cathepsin K (CTSK) in the cultured bone tissues. In conclusion, the current study demonstrated that treatment with 1.8 mT SEMFs at 1.5 h per day promoted bone formation, increased metabolism and inhibited resorption in both metaphyseal and diaphyseal bone tissues in vitro.

[Icariin enhances differentiation and maturation of rat calvarial osteoblasts in collagen hydrogel three-dimensional culture].

OBJECTIVE: To investigate the effects of icariin on the differentiation and maturation of rat calvarial osteoblasts(ROB) in collagen hydrogel three-dimensional culture. METHODS: ROB were obtained by enzyme digestion from the segregated neonatal SD rats skull and were embedded in 2 mg/mL rat tail collagen for three-dimensional culture. The growth state of ROB was observed by FDA/PI staining, HE staining and scanning electron microscopy. ROB were treated with icariin at the concentration of 1 × 10⁻4, 1 × 10⁻5, 1 × 10⁻6 and 1 × 10⁻7 mol/L respectively. The activity of alkaline phosphatase(ALP) was detected after 3, 6, 9 d of icariin treatment. Three-dimensional cultured ROB were treated with optimal concentration icariin for 12, 24, 36, 48 h and total RNA was extracted and the mRNA expressions of bone morphogenetic protein-2 (BMP-2), Runt-related transcription factor 2 (RUNX-2) and Osterix were detected by real time RT-PCR. The protein expression of BMP-2, RUNX-2 and Osterix were examined by Western-blotting. RESULTS: ROB were cultured in collagen hydrogel successfully. FDA/PI staining, HE staining, and scanning electron microscopy showed that ROB adhered with collagen tightly and distributed homogeneously. Icariin at final concentration of 1 × 10⁻5, 1 × 10⁻6 and 1×10⁻7 mol/L all enhanced the activity of ALP of collagen hydrogel three-dimensional cultured ROB, and 1 × 10⁻6 mol/L was the optimal concentration. Besides, icariin (1 × 10⁻6 mol/L) increased mRNA and protein expression of BMP-2、RUNX-2 and Osterix compared to control group. CONCLUSION: Icariin can enhance the expression of osteogenic markers of ROB in collagen hydrogel three-dimensional culture significantly.

Effects of pyrite bioleaching solution of Acidithiobacillus ferrooxidans on viability, differentiation and mineralization potentials of rat osteoblasts.

Iron pyrite, an important component of traditional Chinese medicine, has a poor solubility, bioavailability, and patient compliance due to a high dose required and associated side effects, all of which have limited its clinical applications and experimental studies on its action mechanisms in improving fracture healing. This study investigated Acidithiobacillus ferrooxidans (A.f)-bioleaching of two kinds of pyrites and examined bioactivities of the derived solutions in viability and osteogenic differentiation in rat calvarial osteoblasts. A.f bioleaching improved element contents (Fe, Mn, Zn, Cu, and Se) in the derived solutions and the solutions concentration-dependently affected osteoblast viability and differentiation. While the solutions had no effects at low concentrations and inhibited the osteoblast alkaline phosphatase (ALP) activity at high concentrations, they improved ALP activity at their optimal concentrations. The improved osteoblast differentiation and osteogenic function at optimal concentrations were also revealed by levels of ALP cytochemical staining, calcium deposition, numbers and areas of mineralized nodules formed, mRNA and protein expression levels of osteogenesis-related genes (osteocalcin, Bmp-2, Runx-2, and IGF-1), and Runx-2 nuclear translocation. Data from this study will be useful in offering new strategies for improving pyrite bioavailability and providing a mechanistic explanation for the beneficial effects of pyrite in improving bone healing.

Potential Effects of Phytoestrogen Genistein in Modulating Acute Methotrexate Chemotherapy-Induced Osteoclastogenesis and Bone Damage in Rats.

Chemotherapy-induced bone damage is a frequent side effect which causes diminished bone mineral density and fracture in childhood cancer sufferers and survivors. The intensified use of anti-metabolite methotrexate (MTX) and other cytotoxic drugs has led to the need for a mechanistic understanding of chemotherapy-induced bone loss and for the development of protective treatments. Using a young rat MTX-induced bone loss model, we investigated potential bone protective effects of phytoestrogen genistein. Oral gavages of genistein (20 mg/kg) were administered daily, for seven days before, five days during, and three days after five once-daily injections (sc) of MTX (0.75 mg/kg). MTX treatment reduced body weight gain and tibial metaphyseal trabecular bone volume (p < 0.001), increased osteoclast density on the trabecular bone surface (p < 0.05), and increased the bone marrow adipocyte number in lower metaphyseal bone (p < 0.001). Genistein supplementation preserved body weight gain (p < 0.05) and inhibited ex vivo osteoclast formation of bone marrow cells from MTX-treated rats (p < 0.001). However, MTX-induced changes in bone volume, trabecular architecture, metaphyseal mRNA expression of pro-osteoclastogenic cytokines, and marrow adiposity were not significantly affected by the co-administration of genistein. This study suggests that genistein may suppress MTX-induced osteoclastogenesis; however, further studies are required to examine its potential in protecting against MTX chemotherapy-induced bone damage.

Pulsed electromagnetic fields promote osteoblast mineralization and maturation needing the existence of primary cilia.

Although pulsed electromagnetic fields (PEMFs) have been approved as a therapy for osteoporosis, action mechanisms and optimal parameters are elusive. To determine the optimal intensity, exposure effects of 50 Hz PEMFs of 0.6-3.6 mT (0.6 interval at 90 min/day) were investigated on proliferation and osteogenic differentiation of cultured calvarial osteoblasts. All intensity groups stimulated proliferation significantly with the highest effect at 0.6 mT. The 0.6 mT group also obtained the optimal osteogenic effect as demonstrated by the highest ALP activity, ALP(+) CFU-f colony formation, nodule mineralization, and expression of COL-1 and BMP-2. To verify our hypothesis that the primary cilia are the cellular sensors for PEMFs, osteoblasts were also transfected with IFT88 siRNA or scrambled control, and osteogenesis-promoting effects of 0.6 mT PEMFs were found abrogated when primary cilia were inhibited by IFT88 siRNA. Thus primary cilia of osteoblasts play an indispensable role in mediating PEMF osteogenic effect in vitro.

Effects of resveratrol supplementation on bone growth in young rats and microarchitecture and remodeling in ageing rats.

Osteoporosis is a highly prevalent skeletal disorder in the elderly that causes serious bone fractures. Peak bone mass achieved at adolescence has been shown to predict bone mass and osteoporosis related risk fracture later in life. Resveratrol, a natural polyphenol compound, may have the potential to promote bone formation and reduce bone resorption. However, it is unclear whether it can aid bone growth and bone mass accumulation during rapid growth and modulate bone metabolism during ageing. Using rat models, the current study investigated the potential effects of resveratrol supplementation during the rapid postnatal growth period and in late adulthood (early ageing) on bone microarchitecture and metabolism. In the growth trial, 4-week-old male hooded Wistar rats on a normal chow diet were given resveratrol (2.5 mg/kg/day) or vehicle control for 5 weeks. In the ageing trial, 6-month-old male hooded Wistar rats were treated with resveratrol (20 mg/kg/day) or vehicle for 3 months. Treatment effects in the tibia were examined by µ-computer tomography (µ-CT) analysis, bone histomorphometric measurements and reverse transcription-polymerase chain reaction (RT-PCR) gene expression analysis. Resveratrol treatment did not affect trabecular bone volume and bone remodeling indices in the youth animal model. Resveratrol supplementation in the early ageing rats tended to decrease trabecular bone volume, Sirt1 gene expression and increased expression of adipogenesis-related genes in bone, all of which were statistically insignificant. However, it decreased osteocalcin expression (p = 0.03). Furthermore, serum levels of bone resorption marker C-terminal telopeptides type I collagen (CTX-1) were significantly elevated in the resveratrol supplementation group (p = 0.02) with no changes observed in serum levels of bone formation marker alkaline phosphatase (ALP). These results in rat models suggest that resveratrol supplementation does not significantly affect bone volume during the rapid growth phase but may potentially have negative effects on male skeleton during early ageing.