Extracellular ATP-induced calcium oscillations regulating the differentiation of osteoblasts through aerobic oxidation metabolism pathways.

INTRODUCTION: The increase of ATP concentration in the extracellular space represents one of the effective signals that stimulate the physiological activities of cells when the bone is exposed to external mechanical stimulation such as stretching and shear stress force throughout life. However, the effects of ATP on osteoblast differentiation and related mechanisms are not well understood. MATERIALS AND METHODS: In this study, the roles of extracellular ATP on osteoblast differentiation, intracellular calcium ([Ca2+]i) levels, metabolomics, and the expression of proteins related to energy metabolism were investigated. RESULTS: Our results showed that 100 µM extracellular ATP initiated intracellular calcium ([Ca2+]i) oscillations via the calcium-sensing receptor (P2R) and promoted the differentiation of MC3T3-E1 cells. Metabolomics analysis showed that the differentiation of MC3T3-E1 cells depended on aerobic oxidation, but little glycolysis. Moreover, the differentiation of MC3T3-E1 cells and aerobic oxidation were suppressed with the inhibition of AMP-activated protein kinase (AMPK). CONCLUSION: These results indicate that calcium oscillations triggered by extracellular ATP can activate aerobic oxidation through AMPK-related signaling pathways and thus promote osteoblast differentiation.

Extracellular Calcium-Induced Calcium Transient Regulating the Proliferation of Osteoblasts through Glycolysis Metabolism Pathways.

During bone remodeling, high extracellular calcium levels accumulated around the resorbing bone tissue as soon as the activation of osteoclasts. However, if and how calcium is involved in the regulation of bone remodeling remains unclear. In this study, the effect of high extracellular calcium concentrations on osteoblast proliferation and differentiation, intracellular calcium ([Ca2+]i) levels, metabolomics, and the expression of proteins related to energy metabolism were investigated. Our results showed that high extracellular calcium levels initiated a [Ca2+]i transient via the calcium-sensing receptor (CaSR) and promoted the proliferation of MC3T3-E1 cells. Metabolomics analysis showed that the proliferation of MC3T3-E1 cells was dependent on aerobic glycolysis, but not the tricarboxylic acid cycle. Moreover, the proliferation and glycolysis of MC3T3-E1 cells were suppressed following the inhibition of AKT. These results indicate that calcium transient triggered by high extracellular calcium levels activated glycolysis via AKT-related signaling pathways and ultimately promoted the proliferation of osteoblasts.

KIF2 mediates the neuroprotection in cerebral ischaemia injury by affecting NF-κB pathway.

Stroke is the most common cerebrovascular disease with high morbidity and mortality around the world. However, the underlying mechanisms involved in nerve injury and cerebral ischaemia/reperfusion (I/R) during cerebrovascular disease are still not completely clear. In the present study, we investigate the role of kinesin family member 2 (KIF2) in the neuroprotection after cerebral I/R injury. KIF2 was aberrantly expressed in the cerebral tissues from middle cerebral artery occlusion (MCAO) rat model in a time dependent manner. A similar changing pattern was found in the cultured hypoxic neurons as well as SK-N-SH cells in vitro. Compared to the control, KIF2 inhibition significantly increased the level of malonic dialdehyde (MDA), and reduced the level of superoxide dismutase (SOD) as well as glutathione peroxidase (GSH-px) activity in cerebral tissues of MCAO rat model. The reactive oxygen species (ROS) level was also up-regulated after KIF2 siRNA knockdown in cultured hypoxic SK-N-SH cells. The apoptosis rates of hypoxic neurons and SK-N-SH cells as well as activated-caspase-3 level were obviously increased after KIF2 silencing. Furthermore, we found that the nuclear factor-kappa B (NF-κB) pathway was involved in KIF2-mediated neuroprotection after cerebral I/R injury, and induced apoptosis of hypoxic SK-N-SH cells by KIF2 silencing could be attenuated by the specific inhibitor BAY11-7082 of NF-κB. In conclusion, we demonstrate that KIF2 could mediate the neuroprotection in cerebral I/R injury by inhibiting activation of NF-κB pathway. This might provide a novel therapeutic target for cerebral I/R injury.

Daphnetin inhibits RANKL-induced osteoclastogenesis in vitro.

Osteoporosis is a common orthopedic disease which is associated with hyper-activated osteoclastogenesis. Daphnetin is a natural coumarin derivative isolated from Genus Daphne, which possesses antiarthritis effect. However, the role of daphnetin in osteoclastogenesis has not been illustrated. This study aimed to investigate the effects of daphnetin on receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis in vitro. Our results showed that the osteoclast formation was significantly suppressed by daphnetin treatment in bone marrow-derived macrophages (BMMs), which was illustrated by reduced number of tartrate-resistant acid phosphatase positive multinucleated osteoclasts and decreased expression levels of tumor necrosis factor receptor-associated factors (TRAF6), c-Fos, nuclear factor of activated T cells c1, and cathepsin K. RANKL caused significant induction effects in reactive oxygen species (ROS) generation and nicotinamide adenine dinucleotide phosphate oxidase activity, whereas the induction was dramatically reduced after pretreatment with daphnetin. In addition, daphnetin prevented the RANKL-induced activation of NF-κB and Akt/GSK-3ß pathways in BMMs. These findings indicated that daphnetin exhibited an inhibitory effect on RANKL-induced osteoclastogenesis in vitro. The effect of daphnetin might be mediated by inhibiting ROS signal transduction, as well as preventing the activation of NF-κB and Akt/GSK-3ß signaling pathways. These findings indicated that daphnetin might be considered as a new therapeutic approach for the osteoporosis treatment.

The calcium transient characteristics induced by fluid shear stress affect the osteoblast proliferation.

Ca2+ signaling is essential for bone metabolism. Fluid shear stress (FSS), which can induce a rapid release of calcium from endoplasmic reticulum (ER) to produce calcium transients, plays a significant role in osteoblast proliferation and differentiation. However, it is still unclear of how calcium transients induced by FSS activating a number of downstream signals which subsequently regulate cell functions. In this study, we performed a group of Ca2+ transients models, which were induced by FSS to investigate the effects of different magnitudes of Ca2+ transients in osteoblast proliferation. Further, we performed a global proteomic profile of MC3T3-E1 cells in different Ca2+ transients models stimulated by FSS. GO enrichment and KEGG pathway analysis revealed that the TCA cycle was activated in the proliferating process. The activation of TCA needed mitochondrial Ca2+ uptake which were influenced by the amplitude of Ca2+ transients induced by FSS. Our work elucidate that osteoblast proliferation induced by FSS was related to the magnitude of calcium transients, which further activated energetic metabolism signaling pathway. This work revealed further understanding the mechanism of osteoblast proliferation induced by mechanic loading and help us to design new methods for osteoporosis therapy.

Ladder-Climbing Training Prevents Bone Loss and Microarchitecture Deterioration in Diet-Induced Obese Rats.

Resistance exercise has been proved to be effective in improving bone quality in both animal and human studies. However, the issue about whether resistance exercise can inhibit obesity-induced bone loss has not been previously investigated. In the present study, we have evaluated the effects of ladder-climbing training, one of the resistance exercises, on bone mechanical properties and microarchitecture in high-fat (HF) diet-induced obese rats. Twenty-four rats were randomly assigned to the Control, HF + sedentary (HF-S) and HF + ladder-climbing training (HF-LCT) groups. Rats in the HF-LCT group performed ladder-climbing training for 8 weeks. The results showed that ladder-climbing training significantly reduced body and fat weight, and increased muscle mass along with a trend toward enhanced muscle strength in diet-induced obese rats. MicroCT analysis demonstrated that obesity-induced bone loss and architecture deterioration were significantly mitigated by ladder-climbing training, as evidenced by increased trabecular bone mineral density, bone volume over total volume, trabecular number and thickness, and decreased trabecular separation and structure model index. However, neither HF diet nor ladder-climbing training had an impact on femoral biomechanical properties. Moreover, ladder-climbing training significantly increased serum adiponectin, decreased serum leptin, TNF-α, IL-6 levels, and downregulated myostatin (MSTN) expression in diet-induced obese rats. Taken together, ladder-climbing training prevents bone loss and microarchitecture deterioration in diet-induced obese rats through multiple mechanisms including increasing mechanical loading on bone due to improved skeletal muscle mass and strength, regulating the levels of myokines and adipokines, and suppressing the release of pro-inflammatory cytokines. It indicates that resistance exercise may be a promising therapy for treating obesity-induced bone loss.

Combination of Weight-Bearing Training and Anti-MSTN Polyclonal Antibody Improve Bone Quality In Rats.

Weight-bearing exercise is beneficial to bone health. Myostatin (MSTN) deficiency has a positive effect on bone formation. We wondered if a combination of weight-bearing training and polyclonal antibody for MSTN (MsAb) would augment bone formation to a greater degree than single treatment. In this study, rats were randomly assigned to four groups: Control, weight-bearing training (WT), MsAb, and WT+MsAb. The trained rats ran at 15 m/min bearing with 35% of their body weight, 40 min/day (2 min of running followed by 2 min of rest), 6 days/week, for 8 weeks. The rats with MsAb were injected once a week with MsAb for 8 weeks. MicroCT analysis showed that compared with the MsAb group, WT+MsAb significantly enhanced cortical bone mineral density (BMD) (p < .01), bone volume over total volume (BV/TV) (p < .01), trabecular thickness (p < .05), and reduced trabecular separation (Tb.Sp) (p < .01). Compared with the WT group, WT+MsAb significantly increased trabecular BMD (p < .05), BV/TV (p < .05), and decreased Tb.Sp (p < .05). Three-point bending test demonstrated that MsAb failed to improve bone biomechanical properties (p > .05), weight-bearing training significantly increased energy absorption (p < .05) and elastic modulus (p < .05). However, when they combined, biomechanical properties including maximum load (p < .05), stiffness (p < .05), elastic modulus (p < .01) and energy absorption (p < .01) were all significantly enhanced. In conclusion, the combination of weight-bearing training and MsAb have a greater positive effect on bone than treatment with either MsAb or weight-bearing training alone, suggesting that resistance training in combination with MSTN antagonists could be an effective approach for improving bone health and reducing osteoporosis risk.

Exercise ameliorates anxious behavior and promotes neuroprotection through osteocalcin in VCD-induced menopausal mice.

AIMS: As the ovaries age and women transition to menopause and postmenopause, reduced estradiol levels are associated with anxiety and depression. Exercise contributes to alleviate anxiety and depression and the bone-derived hormone osteocalcin has been reported to be necessary to prevent anxiety-like behaviors. The aim of this study was to investigate the effects of exercise on anxiety behaviors in climacteric mice and whether it was related to osteocalcin. METHODS: Menopausal mouse model was induced by intraperitoneal injection of 4-vinylcyclohexene diepoxide (VCD). Open field, elevated plus maze, and light-dark tests were used to detect anxious behavior in mice. The content of serum osteocalcin was measured and its correlation with anxiety behavior was analyzed. BRDU and NEUN co-localization cells were detected with immunofluorescence. Western blot was applied to obtain apoptosis-related proteins. RESULTS: The VCD mice showed obvious anxiety-like behaviors and 10 weeks of treadmill exercise significantly ameliorated the anxiety and increased circulating osteocalcin in VCD mice. Exercise increased the number of BRDU and NEUN co-localization cells in hippocampal dentate gyrus, reduced the number of impaired hippocampal neurons, inhibited the expression of BAX, cleaved Caspase3, and cleaved PARP, promoted the expression of BCL-2. Importantly, circulating osteocalcin levels were positively associated with the improvements of anxiety, the number of BRDU and NEUN co-localization cells in hippocampal dentate gyrus and negatively related to impaired hippocampal neurons. CONCLUSION: Exercise ameliorates anxiety behavior, promotes hippocampal dentate gyrus neurogenesis, and inhibits hippocampal cell apoptosis in VCD-induced menopausal mice. They are related to circulating osteocalcin, which are increased by exercise.

Improvement of Osteoporosis in Rats With Hind-Limb Unloading Treated With Pulsed Electromagnetic Field and Whole-Body Vibration.

OBJECTIVE: Physical factors have been used to address disuse osteoporosis, but their effects and mechanism remain unclear. The purpose of this study was to determine the effects of pulsed electromagnetic field (PEMF) and whole-body vibration (WBV) on disuse osteoporosis to increase knowledge about treating osteoporosis. METHODS: A disuse osteoporosis rat model was developed by hind-limb unloading (HU) for 6 weeks. Forty 4-month-old female Sprague-Dawley rats were divided into 5 groups and given the following interventions: HU, HU treated with PEMF (HUP), HU treated with WBV (HUW), HU treated with both PEMF and WBV (HUPW), and no intervention (controls). After 8 weeks of intervention, measurements were taken. RESULTS: HU induced a decrease in bone mineral density (BMD), whereas HUP, HUW, and HUPW increased it. Moreover, the bone resorption markers tartrate-resistant acid phosphatase (TRAP) and C-terminal peptide of type 1 collagen in the HU group significantly increased, whereas the osteogenesis markers osteocalcin and N-terminal propeptide of type 1 procollagen significantly decreased. The markers osteocalcin and N-terminal propeptide of type 1 procollagen significantly increased, but TRAP and C-terminal peptide of type 1 collagen significantly decreased in the HUPW, HUP, and HUW groups compared with the HU group. In particular, HUPW effectively increased osteocalcin and decreased TRAP compared with HUP and WBV. Microcomputed tomography analysis of the femur indicated that HUPW improved trabecular number, bone volume over total volume, bone surface over bone volume, trabecular separation, and the structure model index compared with HUP and that it improved bone surface over bone volume, trabecular separation, and structure model index compared with HUW. The HUPW group showed a significant increase in maximum load compared with the HUW group and a significant increase in elastic modulus compared with the HUP group. CONCLUSION: PEMF, WBV, and their combination all attenuated bone resorption and enhanced osteogenesis. WBV and the combination of treatments have great potential to improve osteogenesis compared with PEMF. In addition, HUPW significantly attenuated bone resorption compared with HUW and HUP. IMPACT: The results of this study indicated that HUPW could effectively improve disuse osteoporosis compared with HUP, given that trabecular number and bone volume over total volume are associated with disuse osteoporosis. Moreover, BMD recovered well with HUP, HUW, and HUPW but the bone structure-especially mechanical performance-did not, indicating that osteoporosis should be evaluated with BMD and mechanical performance, not with BMD in isolation.

Stretch-induced sarcoplasmic reticulum calcium leak is causatively associated with atrial fibrillation in pressure-overloaded hearts.

AIMS: Despite numerous reports documenting an important role of hypertension in the development of atrial fibrillation (AF), the detailed mechanism underlying the pathological process remains incompletely understood. Here, we aim to test the hypothesis that diastolic sarcoplasmic reticulum (SR) Ca2+ leak in atrial myocytes, induced by mechanical stretch due to elevated pressure in the left atrium (LA), plays an essential role in the AF development in pressure-overloaded hearts. METHODS AND RESULTS: Isolated mouse atrial myocytes subjected to acute axial stretch displayed an immediate elevation of SR Ca2+ leak. Using a mouse model of transverse aortic constriction (TAC), the relation between stretch, SR Ca2+ leak, and AF susceptibility was further tested. At 36 h post-TAC, SR Ca2+ leak in cardiomyocytes from the LA (with haemodynamic stress), but not right atrium (without haemodynamic stress), significantly increased, which was further elevated at 4 weeks post-TAC. Accordingly, AF susceptibility to atrial burst pacing in the 4-week TAC mice were also significantly increased, which was unaffected by inhibition of atrial fibrosis or inflammation via deletion of galectin-3. Western blotting revealed that type 2 ryanodine receptor (RyR2) in left atrial myocytes of TAC mice was oxidized due to activation and up-regulation of Nox2 and Nox4. Direct rescue of dysfunctional RyR2 with dantrolene or rycal S107 reduced diastolic SR Ca2+ leak in left atrial myocytes and prevented atrial burst pacing stimulated AF. CONCLUSION: Our study demonstrated for the first time the increased SR Ca2+ leak mediated by enhanced oxidative stress in left atrial myocytes that is causatively associated with higher AF susceptibility in pressure-overloaded hearts.

Dilute acid catalyzed fractionation and sugar production from bamboo shoot shell in γ-valerolactone/water medium.

Overcoming the recalcitrance barrier of cellulosic biomass for efficient production of fermentable sugars at low cost is the current limitation for the industrialization of lignocellulosic biorefineries. In the present work, a two-step non-enzymatic strategy was developed for the fractionation of the main components in bamboo shoot shell (BSS) and conversion of polysaccharides into fermentable sugars by dilute acid in a γ-valerolactone (GVL)/H2O solvent system. About 86.0% of lignin and 87.4% of hemicelluloses were removed in the first step by 0.6% H2SO4 under 140 °C for 1 h with the addition of 60% GVL. The residue solids enriched with cellulose were then subjected to acid hydrolysis employing 0.05% H2SO4 as the catalyst in 80% GVL at 180 °C for 20 min. The maximum total soluble sugar yield achieved in the acid hydrolysate was 70.7%. This research could provide valuable insights into the valorization of lignocellulosic biomass and become a promising alternative to the biomass-derived carbohydrate production scheme.

Kinetics study of levulinic acid production from corncobs by tin tetrachloride as catalyst.

Levulinic acid (LA) is an ideal platform chemical that can be produced through acid-catalyzed dehydration and hydrolysis of hexose sugars obtained from lignocellulosic materials. In this study, SnCl4 was identified as an efficient catalyst for LA production and the reaction kinetics was investigated in a single water phase under different reaction conditions. The Box-Behnken design response surface methodology (RSM) was applied to determine the optimized reaction conditions and three individual variables including reaction temperature, duration, and catalyst concentration were evaluated. An appealing LA yield of 76.0% was achieved at 193 °C and 17 min with 82 mM SnCl4 catalyst. A kinetics model was developed to predict the yields of glucose, HMF, and LA, which are tally with the experimental results. The analysis of the related kinetic parameters and the results of the RSM experiment helped to provide insights into the interplay between various reaction steps with SnCl4 as catalysts.

Mild alkaline presoaking and organosolv pretreatment of corn stover and their impacts on corn stover composition, structure, and digestibility.

An efficient strategy was developed in current work for biochemical conversion of carbohydrates of corn stover into monosaccharides. Corn stover was first presoaked in mild alkaline solution (1% Na2S) under 40°C for 4h, after which about 35.3% of the lignin was successfully removed while the specific surface area was notably enlarged. Then the presoaked solids were subjected to organosolv pretreatment that employed 20% methanol with an addition of 0.2% HCl as catalyst at 160°C for 20min, and the maximum total sugar yield of the pretreated corn stover achieved was 98.6%. The intact structure of corn stover was disrupted by this two-step process, which resulted in a porous but crystalline structure of the regenerated solids that were mainly composed of cellulose. The enlarged specific surface area and increased accessibility made the regenerated solids highly digestible by a moderate enzyme loading.

Catalytic conversion of corncob and corncob pretreatment hydrolysate to furfural in a biphasic system with addition of sodium chloride.

Catalytic conversion of corncob pretreatment hydrolysate and raw corncob into furfural in a modified biphasic system by SO42-/SnO2- MMT solid catalyst has been developed. The influence of the organic solvent type, organic to water phase ratio, sodium chloride concentration, reaction temperature and time on the furfural production were comparatively evaluated. The results showed that furfural yields of 81.7% and 66.1% were achieved at 190°C for 15mins and 190°C for 20mins, respectively, for corncob pretreatment hydrolysate and raw corncob by this solid catalyst. The solid catalyst used in this study exhibited good stability and high efficiency applied in the modified biphasic system in addition to excellent recyclability. The proposed catalytic system displayed high performance for catalytic conversion of lignocellulosic biomass into important platform chemicals and has great potential in industrial application.

Inhibiting myostatin signaling prevents femoral trabecular bone loss and microarchitecture deterioration in diet-induced obese rats.

Besides resulting in a dramatic increase in skeletal muscle mass, myostatin (MSTN) deficiency has a positive effect on bone formation. However, the issue about whether blocking MSTN can inhibit obesity-induced bone loss has not been previously investigated. In the present study, we have evaluated the effects of MSTN blocking on bone quality in high-fat (HF), diet-induced obese rats using a prepared polyclonal antibody for MSTN (MsAb). Twenty-four rats were randomly assigned to the Control, HF and HF + MsAb groups. Rats in the HF + MsAb group were injected once a week with purified MsAb for eight weeks. The results showed that MsAb significantly reduced body and fat weight, and increased muscle mass and strength in the HF group. MicroCT analysis demonstrated that obesity-induced bone loss and architecture deterioration were significantly mitigated by MsAb treatment, as evidenced by increased bone mineral density, bone volume over total volume, trabecular number and thickness, and decreased trabecular separation and structure model index. However, neither HF diet nor MsAb treatment had an impact on femoral biomechanical properties including maximum load, stiffness, energy absorption and elastic modulus. Moreover, MsAb significantly increased adiponectin concentrations, and decreased TNF-α and IL-6 levels in diet-induced obese rats. Taken together, blocking MSTN by MsAb improves bone quality in diet-induced obese rats through a mechanotransduction pathway from skeletal muscle, and the accompanying changes occurring in the levels of circulating adipokines and pro-inflammatory cytokines may also be involved in this process. It indicates that the administration of MSTN antagonists may be a promising therapy for treating obesity and obesity-induced bone loss.