Effects of increased hypothalamic leptin gene expression on ovariectomy-induced bone loss in rats

Estrogen deficiency results in accelerated bone turnover with a net increase in bone resorption. Subcutaneous administration of leptin attenuates bone loss in ovariectomized (ovx) rats by reducing bone resorption. However, in addition to its direct beneficial effects, leptin has been reported to have indirect (central nervous system-mediated) antiosteogenic effects on bone, which may limit the efficacy of elevated serum leptin to prevent estrogen deficiency-associated bone loss. The present study evaluated the long-term effects of increased hypothalamic leptin transgene expression, using recombinant adeno-associated virus-leptin (rAAV-Lep) gene therapy, on bone mass, architecture, and cellular endpoints in sexually mature ovx Sprague-Dawley rats. Ovx rats were implanted with cannulae in the 3rd ventricle of the hypothalamus and injected with either rAAV-Lep or rAAV-GFP (control vector encoding green fluorescent protein) and maintained for 10 weeks. Additional controls consisted of ovary-intact rats and ovx rats pair-fed to rAAV-Lep rats. Lumbar vertebrae were analyzed by micro-computed tomography and tibiae by histomorphometry. Cancellous bone volume was lower and osteoclast perimeter, osteoblast perimeter, and bone marrow adipocyte density were greater in ovx rats compared to ovary-intact controls. In contrast, differences among ovx groups were not detected for any endpoint evaluated. In conclusion, whereas estrogen deficiency resulted in marked cancellous osteopenia, increased bone turnover and marrow adiposity, increasing hypothalamic leptin transgene expression in ovx rats had neither detrimental nor beneficial effects on bone mass, architecture, or cellular endpoints. These findings demonstrate that the antiresorptive effects of subcutaneous leptin administration in ovx rats are mediated through leptin targets in the periphery.     

Influence of hyperbaric oxygen therapy on bone metabolism in patients with neoplasm

BackgroundHyperbaric oxygen therapy (HBOT) is useful in the treatment of complications due to radiotherapy in patients with neoplasm. Its effects on bone metabolism are unclear. In our study, we analyzed the effects of HBOT on bone remodeling in oncological patients with radiotherapy.Materials and methodsProspective clinical study in 23 patients with neoplasms undergoing treatment with HBOT due to complications of radiotherapy (hemorrhagic cystitis, proctitis or radionecrosis) and 25 patients with chronic anal fissure. The average number of HBOT sessions was 20 ± 5 (100% oxygen, 2.3 atmospheres and 90 min per day). Serum levels of aminoterminal propeptide of type I collagen (P1NP), C terminal telopeptide of type I collagen (CTX), alkaline phosphatase (AP), 25hydroxyvitamin D (25-OHD), parathyroid hormone (PTH), were measured at 3 time points: T0 (before beginning HBOT), T1 (at the end of HBOT) and T2 (6 months after HBOT).ResultsAt baseline, the patients with neoplasm have higher bone turnover than those with anal fissure. These differences were 41% in CTX (0.238 ± 0.202 ng/mL in neoplasm and 0.141 ± 0.116 ng/mL in fissure; p = 0.04), 30% for PTH (46 ± 36 pg/mL in neoplasm and 32 ± 17 pg/mL in fissure; p = 0.04) and 15% for alkaline phosphatase (80 ± 24 U/L in neoplasm and 68 ± 16 U/L in fissure; p = 0.04). In the group with neoplasm, the values of P1NP decreased 6% after HBOT (T0: 49 ± 31 ng/mL, T2: 46 ± 12 ng/mL; p = 0.03). Also, there were non-significant decreases in PTH (−34%) and CTX (−30%).ConclusionsPatients with neoplasm and complications with radiotherapy have an increase in bone remodeling that may be diminished after HBOT.     

Bone turnover markers and novel biomarkers in lung cancer bone metastases

Context: Lung cancer still remains the leading cause of cancer-related mortality worldwide. Bone is one of preferred metastatic sites for lung cancer cells. So far, both accurate diagnosis and effective treatment of lung cancer bone metastases are difficult.

Objective: This review aimed to evaluate roles of bone turnover markers (BTMs), microRNAs (miRNAs), dickkopf1 (DKK1) and insulin like growth factor binding protein 3 (IGFBP-3) in lung cancer bone metastases.

Methods: We searched articles about these four biomarkers in lung cancer bone metastases mainly in PubMed.

Result: The levels of bone specific alkaline phosphatase (BALP), cross-linked carboxy-terminal telopeptide of type-I collagen (ICTP) and N-terminal telopeptides of type-I collagen (NTX) were reported to be significantly increased in lung cancer patients with bone metastases. ALP, NTX and bone sialoprotein were thought to be associated with prognosis of lung cancer patients with bone metastases. MiRNA-335, miRNA-33a, miRNA-21, DKK1 and IGFBP-3 were revealed to be novel biomarkers in lung cancer bone metastases.

Discussion and conclusion: Current researches have revealed that BTMs, miRNAs, DKK1 and IGFBP-3 may be useful in diagnosis, prognosis evaluation or treatment of lung cancer bone metastases. More studies about these biomarkers in lung cancer bone metastases are needed.     

Changes in protein turnover during gestation in the foetuses,placentas, liver,muscle and whole body of rats given a low-protein diet

(1)Protein synthesis and content have been studied in skeletal muscle, liver, foetuses and placentas of pregnant rats given a protein-deficient diet. Changes which occurred during the anabolic and subsequent catabolic phases of pregnancy are compared with those in well-fed pregnant and in protein-deficient non-pregnant rats. (2) The normal increase in liver protein did not occur during pregnancy in the protein-deficient group. (3) Protein deficiency affected protein content of the placenta earlier and more severely than that of the foetus. (4) Rates of protein synthesis in liver, placentas and foetuses were enhanced above control values by protein deficiency. (5)_Muscle protein increased normally during the anabolic phase of pregnancy but fell during the catabolic phase, unlike values for weel-fed animals. (6) Muscle protein synthesis rates rose by similar amounts in well-fed and protein-deficient animals during the anabolic phase of pregnancy. The fall to starting values during the catabolic phase was sharper and earlier in protein-deficient animals, which could reduce demands on the body amino acid pool by an amount equivalent to over 50% of the needs for protein deposition in foetuses and placentas. Thus, changes in muscle protein synthesis in both anabolic and catabolic phases of pregnancy may afford some protection to foetal protein synthesis.     

Candidate salivary biomarkers associated with alveolar bone loss: cross-sectional and in vitro studies

This cross-sectional study evaluated the association between radiographic evidence of alveolar bone loss and the concentration of host-derived bone resorptive factors (interleukin-1 beta, tumor necrosis factor-alpha, interleukin-6, prostaglandin-E2), and markers of bone turnover [pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP), osteocalcin, osteonectin] in stimulated human whole saliva collected from 110 untreated dental patients. Alveolar bone loss scores for each patient were derived from radiographic examination. Variables positively associated with increased bone loss score were: age, current smoking, use of bisphosphonate drugs, and salivary interleukin-1beta levels above the median. Salivary osteonectin levels above the median were associated with a decreased bone loss score. Additional in vitro studies were carried out to determine the fate of interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha added to whole and parotid saliva. All cytokines added to saliva were detected in significantly lower concentrations than when added to buffer alone. Protease inhibitors added to saliva did not prevent the reduction in detection of biomarkers. Variation in time of incubation, repeated cycles of freezing and thawing, or exposure to dimethylsulfoxide did not appreciably affect the measurement of cytokines in saliva. These results suggest that detection of biomarkers by conventional immunoassays may underestimate the actual quantity of molecules in saliva.     

Retracted: Icariin attenuates methotrexate chemotherapy-induced bone marrow microvascular damage and bone loss in rats

Methotrexate (MTX), a widely used antimetabolite in paediatric cancer to treatment, has been widely reported to cause bone loss and bone marrow (BM) microvascular (particularly sinusoids) damage. Investigations must now investigate how MTX-induced bone loss and microvasculature damage can be attenuated/prevented. In the present study, we examined the potency of icariin, an herbal flavonoid, in reducing bone loss and the dilation/damage of BM sinusoids in rats caused by MTX treatment. Groups of young rats were treated with five daily MTX injections (0.75 mg/kg) with and without icariin oral supplementation until Day 9 after the first MTX injection. Histological analyses showed a significant reduction in the bone volume/tissue volume (BV/TV) fraction (%) and trabecular number in the metaphysis trabecular bone of MTX-treated rats, but no significant changes in trabecular thickness and trabecular spacing. However, the BV/TV (%) and trabecular number were found to be significantly higher in MTX + icariin-treated rats than those of MTX alone-treated rats. Gene expression analyses showed that icariin treatment maintained expression of osteogenesis-related genes but suppressed the induction of adipogenesis-related genes in bones of MTX-treated rats. In addition, icariin treatment attenuated MTX-induced dilation of BM sinusoids and upregulated expression of endothelial cell marker CD31 in the metaphysis bone of icariin + MTX-treated rats. Furthermore, in vitro studies suggest that icariin treatment can potentially enhance the survival of cultured rat sinusoidal endothelial cells against cytotoxic effect of MTX and promote their migration and tube formation abilities, which is associated with enhanced production of nitric oxide.     

Effect of in ovo application of hydroxyapatite nanoparticles on chicken embryo development,oxidative status and bone characteristics

ABSTRACT

Intensive selection in modern lines of fast-growing chickens has caused several skeletal disorders. Therefore, current research is focused on methods to improve the bones of birds. A new potential solution is in ovo technology using nanoparticles with a high specificity for the bone tissue. Thus, the objective of the present study was to evaluate the effect of in ovo application of hydroxyapatite nanoparticles (HA-NP) in different concentrations (50, 100 and 500 μg/ml colloids) on chicken embryo development, with a particular focus on the oxidative status and bone characteristics of the embryo. The results showed that in ovo treatment with HA-NP did not negatively affect hatchability and body weight. However, bone weight was reduced in 500 μg/ml group. The concentrations of calcium, phosphorus and crude ash were not affected. The modulatory effect of HA-NP was observed on the basis of antioxidative markers – superoxide dismutase, total antioxidant status, malondialdehyde in serum and selected tissues. Glutathione concentration in serum suggested higher metabolic stress. Among bone turnover markers, the concentration of osteocalcin was found to be significantly affected by HA-NP injection. Thus, the in ovo application of HA-NP could modify the molecular responses at the stage of embryogenesis but these changes were not reflected in embryo growth and even slowed down bone development. Nevertheless, the question for the follow-up research is whether in ovo administration of HA-NP would affect the antioxidative status and bone turnover resulting in improved bone conditions and body gain in post hatch chickens.     

The 32-kDa glycoprotein of Chlamydia trachomatis is an acidic protein that may be involved in the attachment process

Abstract The 32-kDa glycoprotein of Chlamydia trachomatis was shown to have a p I of 6.2 to 6.4 which distinguished this protein from the chlamydial histone-like protein of similar molecular mass that has a p I of > 10. The initial interaction of the glycan of 32 kDa glycoprotein and HeLa cells was also investigated. Glycan was cleaved from the protein backbone by N -glycanase and radiolabeled with tritium by sodium borohydride reduction. Competition assays showed the binding of glycan to HeLa cells was inhibited by galactose, mannose, and N -acetylglucosamine but not by sedoheptulose and fructose. Untreated and UV-treated organisms inhibited the binding, while heat-inactivated organisms did not. Binding was blocked by rabbit antiserum against whole organisms but not by rabbit anti-155-kDa antiserum or monoclonal antibodies against the lipopolysaccharide and major outer membrane protein.     

Attainment of peak bone mass and bone turnover rate in relation to estrous cycle, pregnancy and lactation in colony-bred Sprague-Dawley rats: suitability for studies on pathophysiology of bone and therapeutic measures for its management

Alteration in biochemical markers of bone turnover and bone mineral density (BMD) of whole body and isolated femur and tibia in relation to age, estrous cycle, pregnancy and lactation and suitability of use of rat as model for studies on pathophysiology of bone and therapeutic measures for its management were investigated. Immature rats (1, 1.5 and 2 month of age; weighing, respectively, 39.3 ± 1.0, 67.8 ± 2.4 and 87.2 ± 5.2 g) exhibited high rate of bone turnover, as evidenced by high serum osteocalcin and alkaline phosphatase and urine calcium/creatinine ratio. However, their BMD (whole body or of isolated long bones) was below measurable levels. Marked increase in body weight at 3 months (185.5 ± 5.2 g) was associated with low serum osteocalcin and alkaline phosphatase and urine calcium/creatinine ratio. Biochemical markers and BMD attained at puberty at 3 months were maintained until 36 month of age. No significant change in serum calcium was observed with increasing age or on any of the biomarkers during estrous cycle, and BMD of femur and tibia isolated during proestrus and diestrus stages was almost similar. Onset of pregnancy was associated with significant increase in serum total alkaline phosphatase and osteocalcin levels, but serum calcium, urine calcium/creatinine ratio or BMD of whole body or isolated long bones were not significantly different from that at proestrus stage. No marked change, except increase in body weight (P < 0.05), was also evident in these parameters between days 5 and 19 of pregnancy, irrespective of number of implantations in the uterus. A significant decrease in BMD of isolated femur (neck and mid-shaft regions) was observed on days 5 and 21 of lactation as compared to that during pregnancy or diestrus/proestrus stages of estrous cycle; the decrease being almost similar in females lactating two or six young ones. BMD of isolated tibia (global and region proximal to tibio-fibular separation point), though generally lower than that during cycle and pregnancy, was statistically non-significant. However, clear evidence of occurrence of osteoporosis during lactation, with decrease in BMD of >2.5 × S.D. in isolated femur (global, neck and mid-shaft) as well as tibia (global) was observed only when BMD data was analysed on T-/Z-score basis. Serum biochemical markers of bone turnover, too, were significantly increased in comparison to cyclic rats. Findings demonstrate marked increase in body weight and bone turnover during first 3 months of age, direct correlation between peak bone mass and onset of puberty at 3 months of age and increase in bone resorption rate during lactation. Finding of the study while might suggests possible use of rat as useful model for studies on bone turnover rate during lactation and post-weaning periods and extrapolation of the result to the human situation, but not in relation to ageing.     

Variable effects of tyrosine kinase inhibitors on avian osteoclastic activity and reduction of bone loss in ovariectomized rats

We compared the effects of the tyrosine kinase inhibitor genistein, a naturally occurring isoflavone, to those of tyrphostin A25, tyrphostin A47, and herbimycin on avian osteoclasts in vitro. Inactive analogs daidzein and tyrphostin A1 were used to control for nonspecific effects. None of the tyrosine kinase inhibitors inhibited bone attachment. However, bone resorption was inhibited by genistein and herbimycin with ID₅₀s of 3 μM and 0.1 μM, respectively; tyrphostins and daidzein were inactive at concentrations below 30 μM, where nonspecific effects were noted. Genistein and herbimycin thus inhibit osteoclastic activity via a mechanism independent of cellular attachment, and at doses approximating those inhibiting tyrosine kinase autophosphorylation in vitro; the tyrphostins were inactive at meaningful doses. Because tyrosine kinase inhibitors vary widely in activity spectrum, effects of genistein on cellular metabolic processes were compared to herbimycin. Unlike previously reported osteoclast metabolic inhibitors which achieve a measure of selectivity by concentrating on bone, neither genistein nor herbimycin bound significantly to bone. Osteoclastic protein synthesis, measured as incorporation of ³H-leucine, was significantly inhibited at 10 μM genistein, a concentration greater than that inhibiting bone degradation, while herbimycin reduced protein synthesis at 10 nM. These data suggested that genistein may reduce osteoclastic activity at pharmacologically attainable levels, and that toxic potential was lower than that of herbimycin. To test this hypothesis in a mammalian system, bone mass was measured in 200 g ovariectomized rats treated with 44 μmol/day genistein, relative to untreated controls. During 30 d of treatment, weights of treated and control group animals were indistinguishable, indicating no toxicity, but femoral weight in the treated group was 12% greater than controls (P < 0.05). Our data indicate that the isoflavone inhibitor genistein suppresses osteoclastic activity in vitro and in vivo at concentrations consistent with its ID₅₀s on tyrosine kinases, with a low potential for toxicity. © 1996 Wiley-Liss, Inc.     

The effects of different exercise modes for preventing endothelial dysfunction of arteries and bone loss in ovariectomized rats

[Purpose]

Several epidemiological studies have demonstrated that there are positive correlations between vascular disorders and bone loss in postmenopausal women. The aim of the present study was to examine the effect of different types of exercise (e.g., climbing and swimming) for preventing endothelial dysfunction of arteries and bone loss in ovariectomized rats.

[Methods]

Twenty Sprague-Dawley female rats were randomly divided into three groups: ovariectomy (OVX) plus treatment with vitamin D₃ and nicotine (VDN) (control rats [Con], n = 7), which is an animal model for endothelial dysfunction and bone loss; voluntary climbing resistance exercise with OVX plus VDN (climbing rats [Clim], n = 6), and swimming exercise with OVX plus VDN (swimming rats [Swim], n = 7). The period of exercise training was 8 weeks.

[Results]

The endothelin-1 (ET-1) protein levels were significantly lower in the Clim and Swim groups than in the Con. The endothelial nitric oxide synthase protein levels were significantly higher in the Swim group than in the Con, but they did not differ between the Clim and Con groups. The cortical bone mineral density in the tibia and breaking energy of the femur were significantly higher in the Clim group than in the Con, but this positive effect was not seen in the Swim group.

[Conclusion]

Voluntary climbing exercise decreased arterial ET-1 protein levels and prevented bone loss in a postmenopause-model rat combining OVX and VDN. Conversely, swimming suppressed endothelial dysfunction of the arteries but did not prevent bone loss. Thus, the type of exercise should be cautiously chosen for enhancing vascular function and bone status, especially in females after menopause.     

p62/SQSTM1 indirectly mediates remote multipotent mesenchymal cells and rescues bone loss and bone marrow integrity in ovariectomized rats

Intramuscular administration of p62/SQSTM1 (sequestosome1)-encoding plasmid demonstrated an anticancer effect in rodent models and dogs as well as a high safety profile and the first evidence of clinical benefits in humans. Also, an anti-inflammatory effect of the plasmid was reported in several rodent disease models. Yet, the mechanisms of action for the p62 plasmid remain unknown. Here, we tested a hypothesis that the p62-plasmid can act through the modulation of bone marrow multipotent mesenchymal cells (MSCs). We demonstrated that a p62 plasmid can affect MSCs indirectly by stimulating p62-transfected cells to secrete an active ingredient(s) sensed by untransfected MSCs. When we transfected MSCs with the p62-plasmid, collected their supernatant, and added it to an untransfected MSCs culture, it switched the differentiation state and prompt osteogenic responses of the untransfected MSCs. According to an accepted viewpoint, ovariectomy leads to bone pathology via dysregulation of MSCs, and restoring the MSC homeostasis would restore ovariectomy-induced bone damage. To validate our in vitro observations in a clinically relevant in vivo model, we administered the p62 plasmid to ovariectomized rats. It partially reversed bone loss and notably reduced adipogenesis with concurrent reestablishing of the MSC subpopulation pool within the bone marrow. Overall, our study suggests that remote modulation of progenitor MSCs via administering a p62-encoding plasmid may constitute a mechanism for its previously reported effects and presents a feasible disease-preventing and/or therapeutic strategy.     

The effects of royal jelly protein on bone mineral density and strength in ovariectomized female rats

[Purpose]Sex hormones deficiency leads to dramatically bone loss in particular postmenopausal women. Royal jelly has anti-osteoporosis effect due to maintain bone volume in that condition. We hypothesized that royal jelly protein (RJP, a latent residue after extracting royal jelly) also prevents bone deficient in ovariectomized (OVX) female rats, the animal model of postmenopausal women. [Methods]Female Sprague-Dawley rats (n = 30, 6 weeks age old) were sham operated (Sham; sham operated group, n = 7), OVX control group (OC, n = 7), OVX with low RJP intake group (ORL, n = 8), and OVX with high RJP intake group (ORH, n = 8) during 8 weeks experimental periods. In the end point of this experiment, the bone samples (lumbar spine, tibia, and femur) were surgically removed under anesthesia. These bone samples were evaluated bone mineral density (BMD) and bone strength.[Results]BMD of lumbar spine in RJP intake groups (ORL, ORH) were higher than that in OC group (p < 0.05 and p < 0.01) in RJP intake volume dependent manner. BMD of tibial proximal metaphysis and diaphysis in RJP intake groups were also higher than these in OC group (p < 0.01 and p < 0.01 / p < 0.05 and p < 0.001). In addition, breaking force of femur in RJP intake groups were significantly increase compared with that in OC group (p < 0.001 respectively). [Conclusion]These findings indicate that RJP contribute to prevent sex hormone related bone abnormality