Gut microbial alterations in arginine metabolism determine bone mechanical adaptation.

Although mechanical loading is essential for maintaining bone health and combating osteoporosis, its practical application is limited to a large extent by the high variability in bone mechanoresponsiveness. Here, we found that gut microbial depletion promoted a significant reduction in skeletal adaptation to mechanical loading. Among experimental mice, we observed differences between those with high and low responses to exercise with respect to the gut microbial composition, in which the differential abundance of Lachnospiraceae contributed to the differences in bone mechanoresponsiveness. Microbial production of L-citrulline and its conversion into L-arginine were identified as key regulators of bone mechanoadaptation, and administration of these metabolites enhanced bone mechanoresponsiveness in normal, aged, and ovariectomized mice. Mechanistically, L-arginine-mediated enhancement of bone mechanoadaptation was primarily attributable to the activation of a nitric-oxide-calcium positive feedback loop in osteocytes. This study identifies a promising anti-osteoporotic strategy for maximizing mechanical loading-induced skeletal benefits via the microbiota-metabolite axis.

Rescuing SERCA2 pump deficiency improves bone mechano-responsiveness in type 2 diabetes by shaping osteocyte calcium dynamics.

Type 2 diabetes (T2D)-related fragility fractures represent an increasingly tough medical challenge, and the current treatment options are limited. Mechanical loading is essential for maintaining bone integrity, although bone mechano-responsiveness in T2D remains poorly characterized. Herein, we report that exogenous cyclic loading-induced improvements in bone architecture and strength are compromised in both genetically spontaneous and experimentally-induced T2D mice. T2D-induced reduction in bone mechano-responsiveness is directly associated with the weakened Ca2+ oscillatory dynamics of osteocytes, although not those of osteoblasts, which is dependent on PPARα-mediated specific reduction in osteocytic SERCA2 pump expression. Treatment with the SERCA2 agonist istaroxime was demonstrated to improve T2D bone mechano-responsiveness by rescuing osteocyte Ca2+ dynamics and the associated regulation of osteoblasts and osteoclasts. Moreover, T2D-induced deterioration of bone mechano-responsiveness is blunted in mice with osteocytic SERCA2 overexpression. Collectively, our study provides mechanistic insights into T2D-mediated deterioration of bone mechano-responsiveness and identifies a promising countermeasure against T2D-associated fragility fractures.

Early protection against bone stress injuries by mobilization of endogenous targeted bone remodeling.

Bone stress injuries are common overuse injuries, especially in soldiers, athletes, and performers. In contrast to various post-injury treatments, early protection against bone stress injuries can provide greater benefit. This study explored the early protection strategies against bone stress injuries by mobilization of endogenous targeted bone remodeling. The effects of various pharmaceutical/biophysical approaches, individual or combinational, were investigated by giving intervention before fatigue loading. We optimized the dosage and administration parameters and found that early intervention with pulsed electromagnetic field and parathyroid hormone (i.e., PEMF+PTH) resulted in the most pronounced protective effects among all the approaches against the bone stress injuries. In addition, the mechanisms by which the strategy mobilizes targeted bone remodeling and enhances the self-repair capacity of bone were systematically investigated. This study proposes strategies to reduce the incidence of bone stress injuries in high-risk populations (e.g., soldiers and athletes), particularly for those before sudden increased physical training.

Bone Deterioration in Response to Chronic High-Altitude Hypoxia Is Attenuated by a Pulsed Electromagnetic Field Via the Primary Cilium/HIF-1α Axis.

Chronic high-altitude hypoxia induces irreversible abnormalities in various organisms. Emerging evidence indicates that hypobaric hypoxia markedly suppresses bone mass and bone strength. However, few effective means have been identified to prevent such bone deficits. Here, we assessed the potential of pulsed electromagnetic fields (PEMFs) to noninvasively resist bone deterioration induced by hypobaric hypoxia. We observed that exogenous PEMF treatment at 15 Hz and 20 Gauss (Gs) improved the cancellous and cortical bone mass, bone microstructure, and skeletal mechano-properties in rats subjected to chronic exposure of hypobaric hypoxia simulating an altitude of 4500 m for 6 weeks by primarily modulating osteoblasts and osteoblast-mediated bone-forming activity. Moreover, our results showed that whereas PEMF stimulated the functional activity of primary osteoblasts in hypoxic culture in vitro, it had negligible effects on osteoclasts and osteocytes exposed to hypoxia. Mechanistically, the primary cilium was found to function as the major electromagnetic sensor in osteoblasts exposed to hypoxia. The polycystins PC-1/PC-2 complex was identified as the primary calcium channel in the primary cilium of hypoxia-exposed osteoblastic cells responsible for the detection of external PEMF signals, and thereby translated these biophysical signals into intracellular biochemical events involving significant increase in the intracellular soluble adenylyl cyclase (sAC) expression and subsequent elevation of cyclic adenosine monophosphate (cAMP) concentration. The second messenger cAMP inhibited the transcription of oxygen homeostasis-related hypoxia-inducible factor 1-alpha (HIF-1α), and thus enhanced osteoblast differentiation and improved bone phenotype. Overall, the present study not only advances our understanding of bone physiology at high altitudes, but more importantly, proposes effective means to ameliorate high altitude-induced bone loss in a noninvasive and cost-effective manner. © 2023 American Society for Bone and Mineral Research (ASBMR).

Glucose- and glutamine-dependent bioenergetics sensitize bone mechanoresponse after unloading by modulating osteocyte calcium dynamics.

Disuse osteoporosis is a metabolic bone disease resulting from skeletal unloading (e.g., during extended bed rest, limb immobilization, and spaceflight), and the slow and insufficient bone recovery during reambulation remains an unresolved medical challenge. Here, we demonstrated that loading-induced increase in bone architecture/strength was suppressed in skeletons previously exposed to unloading. This reduction in bone mechanosensitivity was directly associated with attenuated osteocytic Ca2+ oscillatory dynamics. The unloading-induced compromised osteocytic Ca2+ response to reloading resulted from the HIF-1α/PDK1 axis-mediated increase in glycolysis, and a subsequent reduction in ATP synthesis. HIF-1α also transcriptionally induced substantial glutaminase 2 expression and thereby glutamine addiction in osteocytes. Inhibition of glycolysis by blockade of PDK1 or glutamine supplementation restored the mechanosensitivity in those skeletons with previous unloading by fueling the tricarboxylic acid cycle and rescuing subsequent Ca2+ oscillations in osteocytes. Thus, we provide mechanistic insight into disuse-induced deterioration of bone mechanosensitivity and a promising therapeutic approach to accelerate bone recovery after long-duration disuse.

Pulsed Electromagnetic Field Protects Against Brain Injury After Intracerebral Hemorrhage: Involvement of Anti-Inflammatory Processes and Hematoma Clearance via CD36.

Intracerebral hemorrhage causes high mortality and morbidity, but its therapy methods are limited. In the present study, pulsed electromagnetic field (PEMF) was demonstrated to have beneficial effects on an intracerebral hemorrhage (ICH) model. This study explored the effects and underlying mechanisms of PEMF in a mouse model of ICH and cultured BV2 cells. PEMF was applied 4 hours after collagenase-induced ICH at day 0 and 4 hours per day for seven consecutive days. The expression levels of proinflammatory factors were assessed by ELISA kits and western blotting. Hematoma volume was measured by histological analysis. The effects of PEMF on phagocytosis of the erythrocytes were observed in cultured BV2 cells and ICH mouse models. Seven days after ICH, the hematoma volume was significantly reduced in PEMF-treated animals compared to nontreated mice. We found that PEMF decreased the hematoma volume and the expression levels of proinflammatory factors after ICH. Moreover, PEMF enhanced the erythrophagocytosis of microglia via CD36. Furthermore, we found that downregulation CD36 with Genistein blocked the effects of PEMF-induced hematoma clearance and anti-inflammations effects. Thus, the PEMF-mediated promotion of neurological functions may at least partly involve anti-inflammatory processes and hematoma clearance. These results suggest that PEMF treatment promoted the hematoma clearance and alleviated the inflammation after ICH.

High-specificity protection against radiation-induced bone loss by a pulsed electromagnetic field.

Radiotherapy increases tumor cure and survival rates; however, radiotherapy-induced bone damage remains a common issue for which effective countermeasures are lacking, especially considering tumor recurrence risks. We report a high-specificity protection technique based on noninvasive electromagnetic field (EMF). A unique pulsed-burst EMF (PEMF) at 15 Hz and 2 mT induces notable Ca2+ oscillations with robust Ca2+ spikes in osteoblasts in contrast to other waveforms. This waveform parameter substantially inhibits radiotherapy-induced bone loss by specifically modulating osteoblasts without affecting other bone cell types or tumor cells. Mechanistically, primary cilia are identified as major PEMF sensors in osteoblasts, and the differentiated ciliary expression dominates distinct PEMF sensitivity between osteoblasts and tumor cells. PEMF-induced unique Ca2+ oscillations depend on interactions between ciliary polycystins-1/2 and endoplasmic reticulum, which activates the Ras/MAPK/AP-1 axis and subsequent DNA repair Ku70 transcription. Our study introduces a previously unidentified method against radiation-induced bone damage in a noninvasive, cost-effective, and highly specific manner.

Semaphorins as Potential Immune Therapeutic Targets for Cancer.

Semaphorins are a large class of secreted or membrane-bound molecules. It has been reported that semaphorins play important roles in regulating several hallmarks of cancer, including angiogenesis, metastasis, and immune evasion. Semaphorins and their receptors are widely expressed on tumor cells and immune cells. However, the biological role of semaphorins in tumor immune microenvironment is intricate. The dysregulation of semaphorins influences the recruitment and infiltration of immune cells, leading to abnormal anti-tumor effect. Although the underlying mechanisms of semaphorins on regulating tumor-infiltrating immune cell activation and functions are not fully understood, semaphorins can notably be promising immunotherapy targets for cancer.

Radiotherapy-induced bone deterioration is exacerbated in diabetic rats treated with streptozotocin.

Following radiotherapy, patients have decreased bone mass and increased risk of fragility fractures. Diabetes mellitus (DM) is also reported to have detrimental effects on bone architecture and quality. However, no clinical or experimental study has systematically characterized the bone phenotype of the diabetic patients following radiotherapy. After one month of streptozotocin injection, three-month-old male rats were subjected to focal radiotherapy (8 Gy, twice, at days 1 and 3), and then bone mass, microarchitecture, and turnover as well as bone cell activities were evaluated at 2 months post-irradiation. Micro-computed tomography results demonstrated that DM rats exhibited greater deterioration in trabecular bone mass and microarchitecture following irradiation compared with the damage to bone structure induced by DM or radiotherapy. The serum biochemical, bone histomorphometric, and gene expression assays revealed that DM combined with radiotherapy showed lower bone formation rate, osteoblast number on bone surface, and expression of osteoblast-related markers (ALP, Runx2, Osx, and Col-1) compared with DM or irradiation alone. DM plus irradiation also caused higher bone resorption rate, osteoclast number on bone surface, and expression of osteoclast-specific markers (TRAP, cathepsin K, and calcitonin receptor) than DM or irradiation treatment alone. Moreover, lower osteocyte survival and higher expression of Sost and DKK1 genes (two negative modulators of Wnt signaling) were observed in rats with combined DM and radiotherapy. Together, these findings revealed a higher deterioration of the diabetic skeleton following radiotherapy, and emphasized the clinical importance of health maintenance.

Oxygen Enrichment Mitigates High-Altitude Hypoxia-Induced Hippocampal Neurodegeneration and Memory Dysfunction Associated with Attenuated Tau Phosphorylation.

Cai, Jing, Junyong Ruan, Xi Shao, Yuanjun Ding, Kangning Xie, Chi Tang, Zedong Yan, Erping Luo, and Da Jing. Oxygen enrichment mitigates high-altitude hypoxia-induced hippocampal neurodegeneration and memory dysfunction associated with attenuated tau phosphorylation. High Alt Med Biol. 22:274-284, 2021. Background: Brain is predominantly vulnerable to high-altitude hypoxia (HAH), resulting in neurodegeneration and cognitive impairment. The technology of oxygen enrichment has proven effective to decrease the heart rate and improve the arterial oxygen saturation by reducing the equivalent altitude. However, the efficacy of oxygen enrichment on HAH-induced cognitive impairments remains controversial based on the results of neuropsychological tests, and its role in HAH-induced hippocampal morphological and molecular changes remains unknown. Therefore, this study aims to systematically investigate the effects of oxygen enrichment on the memory dysfunction and hippocampal neurodegeneration caused by HAH. Materials and Methods: Fifty-one male Sprague-Dawley rats were equally assigned to three groups: normal control, HAH, and HAH with oxygen enrichment (HAHO). Rats in the HAH and HAHO groups were exposed to hypoxia for 3 days in a hypobaric hypoxia chamber at a simulated altitude of 6,000 m. Rats in the HAHO group were supplemented with oxygen-enriched air, with 12 hours/day in the hypobaric hypoxia chamber. Results: Our results showed that oxygen enrichment improved the locomotor activity of HAH-exposed rats. The Morris water maze test revealed that oxygen enrichment significantly ameliorated HAH-induced spatial memory deficits. Oxygen enrichment also improved morphological alterations of pyramidal cells and the ultrastructure of neurons in the hippocampal CA1 region in rats exposed to acute HAH. Tau hyperphosphorylation at Ser396, Ser262, Thr231, and Thr181 was also significantly attenuated by oxygen enrichment in HAH-exposed rats. Conclusions: Together, our study reveals that oxygen enrichment can ameliorate HAH-induced cognitive impairments associated with improved hippocampal morphology and molecular expression, and highlights that oxygen enrichment may become a promising alternative treatment against neurodegeneration for humans ascending to the plateau.

Pulsed Electromagnetic Fields Ameliorate Skeletal Deterioration in Bone Mass, Microarchitecture, and Strength by Enhancing Canonical Wnt Signaling-Mediated Bone Formation in Rats with Spinal Cord Injury.

Spinal cord injury (SCI) leads to extensive bone loss and high incidence of low-energy fractures. Pulsed electromagnetic fields (PEMF) treatment, as a non-invasive biophysical technique, has proven to be efficient in promoting osteogenesis. The potential osteoprotective effect and mechanism of PEMF on SCI-related bone deterioration, however, remain unknown. The spinal cord of rats was transected at vertebral level T12 to induce SCI. Thirty rats were assigned to the control, SCI, and SCI+PEMF groups (n = 10). One week after surgery, the SCI+PEMF rats were subjected to PEMF (2.0 mT, 15 Hz, 2 h/day) for eight weeks. Micro-computed tomography results showed that PEMF significantly ameliorated trabecular and cortical bone microarchitecture deterioration induced by SCI. Three-point bending and nanoindentation assays revealed that PEMF significantly improved bone mechanical properties in SCI rats. Serum biomarker and bone histomorphometric analyses demonstrated that PEMF enhanced bone formation, as evidenced by significant increase in serum osteocalcin and P1NP, mineral apposition rate, and osteoblast number on bone surface. The PEMF had no impact, however, on serum bone-resorbing cytokines (TRACP 5b and CTX-1) or osteoclast number on bone surface. The PEMF also attenuated SCI-induced negative changes in osteocyte morphology and osteocyte survival. Moreover, PEMF significantly increased skeletal expression of canonical Wnt ligands (Wnt1 and Wnt10b) and stimulated their downstream p-GSK3ß and ß-catenin expression in SCI rats. This study demonstrates that PEMF can mitigate the detrimental consequence of SCI on bone quantity/quality, which might be associated with canonical Wnt signaling-mediated bone formation, and reveals that PEMF may be a promising biophysical approach for resisting osteopenia/osteoporosis after SCI in clinics.

Radiotherapy-induced bone deterioration is exacerbated in diabetic rats treated with streptozotocin

Following radiotherapy, patients have decreased bone mass and increased risk of fragility fractures. Diabetes mellitus (DM) is also reported to have detrimental effects on bone architecture and quality. However, no clinical or experimental study has systematically characterized the bone phenotype of the diabetic patients following radiotherapy. After one month of streptozotocin injection, three-month-old male rats were subjected to focal radiotherapy (8 Gy, twice, at days 1 and 3), and then bone mass, microarchitecture, and turnover as well as bone cell activities were evaluated at 2 months post-irradiation. Micro-computed tomography results demonstrated that DM rats exhibited greater deterioration in trabecular bone mass and microarchitecture following irradiation compared with the damage to bone structure induced by DM or radiotherapy. The serum biochemical, bone histomorphometric, and gene expression assays revealed that DM combined with radiotherapy showed lower bone formation rate, osteoblast number on bone surface, and expression of osteoblast-related markers (ALP, Runx2, Osx, and Col-1) compared with DM or irradiation alone. DM plus irradiation also caused higher bone resorption rate, osteoclast number on bone surface, and expression of osteoclast-specific markers (TRAP, cathepsin K, and calcitonin receptor) than DM or irradiation treatment alone. Moreover, lower osteocyte survival and higher expression of Sost and DKK1 genes (two negative modulators of Wnt signaling) were observed in rats with combined DM and radiotherapy. Together, these findings revealed a higher deterioration of the diabetic skeleton following radiotherapy, and emphasized the clinical importance of health maintenance.

Melatonin suppresses ER stress-dependent proapoptotic effects via AMPK in bone mesenchymal stem cells during mitochondrial oxidative damage.

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) have been used as important cell-based tools for clinical applications. Oxidative stress-induced apoptosis causes a low survival rate after transplantation, and the underlying mechanisms remain unknown. The endoplasmic reticulum (ER) and mitochondria are vital organelles regulated by adenosine monophosphate (AMP)-activated protein kinase (AMPK), especially during oxidative stress injury. Melatonin exerts an antioxidant effect by scavenging free radicals. Here, we aimed to explore whether cytoprotective melatonin relieves ER stress-mediated mitochondrial dysfunction through AMPK in BMSCs after oxidative stress injury. METHODS: Mouse BMSCs were isolated and exposed to H2O2 in the absence or presence of melatonin. Thereafter, cell damage, oxidative stress levels, mitochondrial function, AMPK activity, ER stress-related proteins, and apoptotic markers were measured. Additionally, the involvement of AMPK and ER stress in the melatonin-mediated protection of BMSCs against H2O2-induced injury was investigated using pharmacologic agonists and inhibitors. RESULTS: Melatonin improved cell survival and restored mitochondrial function. Moreover, melatonin intimately regulated the phosphorylation of AMPK and molecules associated with ER stress pathways. AMPK activation and ER stress inhibition following melatonin administration improved the mitochondrial membrane potential (MMP), reduced mitochondria-initiated oxidative damage, and ultimately suppressed apoptotic signaling pathways in BMSCs. Cotreatment with N-acetyl-L-cysteine (NAC) significantly enhanced the antioxidant effect of melatonin. Importantly, pharmacological AMPK activation/ER stress inhibition promoted melatonin-induced cytoprotection, while pharmacological AMPK inactivation/ER stress induction conferred resistance to the effect of melatonin against H2O2 insult. CONCLUSIONS: Our data also reveal a new, potentially therapeutic mechanism by which melatonin protects BMSCs from oxidative stress-mediated mitochondrial apoptosis, possibly by regulating the AMPK-ER stress pathway.

Pulsed electromagnetic fields modify the adverse effects of glucocorticoids on bone architecture, bone strength and porous implant osseointegration by rescuing bone-anabolic actions.

Long-term glucocorticoid therapy is known to induce increased bone fragility and impaired skeletal regeneration potential. Growing evidence suggests that pulsed electromagnetic fields (PEMF) can accelerate fracture healing and increase bone mass both experimentally and clinically. However, how glucocorticoid-treated bone and bone cells respond to PEMF stimulation remains poorly understood. Here we tested the effects of PEMF on bone quantity/quality, bone metabolism, and porous implant osseointegration in rabbits treated with dexamethasone (0.5 mg/kg/day, 6 weeks). The micro-CT, histologic and nanoindentation results showed that PEMF ameliorated the glucocorticoid-mediated deterioration of cancellous and cortical bone architecture and intrinsic material properties. Utilizing the new porous titanium implant (Ti2448) with low toxicity and low elastic modulus, we found that PEMF stimulated bone ingrowth into the pores of implants and enhanced peri-implant bone material quality during osseous defect repair in glucocorticoid-treated rabbits. Dynamic histomorphometric results revealed that PEMF reversed the adverse effects of glucocorticoids on bone formation, which was confirmed by increased circulating osteocalcin and P1NP. PEMF also significantly attenuated osteocyte apoptosis, promoted osteoblast-related osteocalcin, Runx2 and Osx expression, and inhibited osteocyte-specific DKK1 and Sost expression (negative regulators of osteoblasts) in glucocorticoid-treated skeletons, revealing improved functional activities of osteoblasts and osteocytes. Nevertheless, PEMF exerted no effect on circulating bone-resorbing cytokines (serum TRAcP5b and CTX-1) or skeletal gene expression of osteoclast-specific markers (TRAP and cathepsin K). PEMF also significantly upregulated skeletal gene expression of canonical Wnt ligands (Wnt1, Wnt3a and Wnt10b), whereas PEMF did not alter non-canonical Wnt5a expression. This study demonstrates that PEMF treatment improves bone mass, strength and porous implant osseointegration in glucocorticoid-treated rabbits by promoting potent bone-anabolic action, which is associated with canonical Wnt-mediated improvement in osteoblast and osteocyte functions. This study provides a new treatment alternative for glucocorticoid-related bone disorders in a convenient and non-invasive manner.

Spatiotemporal characterization of microdamage accumulation and its targeted remodeling mechanisms in diabetic fatigued bone.

The skeleton of type 1 diabetes mellitus (T1DM) has deteriorated mechanical integrity and increased fragility, whereas the mechanisms are not fully understood. Load-induced microdamage naturally occurs in bone matrix and can be removed by initiating endogenous targeted bone remodeling. However, the microdamage accumulation in diabetic skeleton and the corresponding bone remodeling mechanisms remain poorly understood. Herein, streptozotocin-induced T1DM rats and age-matched non-diabetic rats were subjected to daily uniaxial ulnar loading for 1, 4, 7, and 10 days, respectively. The SPECT/CT and basic fuchsin staining revealed significant higher-density spatial accumulation of linear and diffuse microdamage in diabetic ulnae than non-diabetic ulnae. Linear microcracks increased within 10-day loading in diabetic bone, whereas peaked at Day 7 in non-diabetic bone. Moreover, diabetic fatigued ulnae had more severe disruptions of osteocyte canaliculi around linear microcracks. Immunostaining results revealed that diabetes impaired targeted remodeling in fatigued bone at every key stage, including increased apoptosis of bystander osteocytes, decreased RANKL secretion, reduced osteoclast recruitment and bone resorption, and impaired osteoblast-mediated bone formation. This study characterizes microdamage accumulation and abnormal remodeling mechanisms in the diabetic skeleton, which advances our etiologic understanding of diabetic bone deterioration and increased fragility from the aspect of microdamage accumulation and bone remodeling.

Differential skeletal response in adult and aged rats to independent and combinatorial stimulation with pulsed electromagnetic fields and mechanical vibration.

Pulsed electromagnetic fields (PEMFs) and whole-body vibration (WBV) are proved to partially preserve bone mass/strength in hindlimb-unloaded and ovariectomized animals. However, the potential age-dependent skeletal response to either PEMF or WBV has not been fully investigated. Moreover, whether the coupled "mechano-electro-magnetic" signals can induce greater osteogenic potential than single stimulation remains unknown. Herein, 5-month-old or 20-month-old rats were assigned to the Control, PEMF, WBV, and PEMF + WBV groups. After 8-week treatment, single PEMF/WBV enhanced bone mass, strength, and anabolism in 5-month-old rats, but not in 20-month-old rats. PEMF + WBV induced greater increase of bone quantity, quality, and anabolism than single PEMF/WBV in young adult rats. PEMF + WBV also inhibited bone loss in elderly rats by primarily improving osteoblast and osteocyte activity, but had no effects on bone resorption. PEMF + WBV upregulated the expression of various canonical Wnt ligands and downstream molecules (p-GSK-3ß and ß-catenin), but had no impacts on noncanonical Wnt5a expression in aged skeleton, revealing the potential involvement of canonical Wnt signaling in bone anabolism of PEMF + WBV. This study not only reveals much weaker responsiveness of aged skeleton to single PEMF/WBV relative to young adult skeleton, but also presents a novel noninvasive approach based on combinatorial treatment with PEMF + WBV for improving bone health and preserving bone quantity/quality (especially for age-related osteoporosis) with stronger anabolic effects.

Oxygen Enrichment Ameliorates Cardiorespiratory Alterations Induced by Chronic High-Altitude Hypoxia in Rats.

Chronic high-altitude hypoxia (HAH) results in compensatory pathological adaptations, especially in the cardiorespiratory system. The oxygen enrichment technology can provide long-lasting oxygen supply and minimize oxygen toxicity, which has proven to be effective to increase oxygen saturation, decrease heart rate, and improve human exercise performance after ascending to high altitudes. Nevertheless, it remains unknown whether oxygen enrichment can resist chronic HAH-induced cardiorespiratory alterations. Thirty-six male rats were equally assigned to the normal control (NC), HAH, and HAH with oxygen enrichment (HAHO) groups. The HAH and HAHO rats were housed in a hypobaric hypoxia chamber equivalent to 5,000 m for 4 weeks. The HAHO rats were exposed to oxygen-enriched air for 8 h/day. We found that oxygen enrichment mitigated the augmented skin blood flow and improved the locomotor activity of HAH-exposed rats. Oxygen enrichment inhibited HAH-induced increase in the production of red blood cells (RBCs). The hemodynamic results showed that oxygen enrichment decreased right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) in HAH-exposed rats. HAH-associated right ventricular hypertrophy and cardiomyocyte enlargement were ameliorated by oxygen enrichment. Oxygen enrichment inhibited HAH-induced excessive expression of cytokines associated with cardiac hypertrophy and myocardial fibrosis [angiotensin-converting enzyme (ACE)/angiotensin-converting enzyme 2 (ACE2), angiotensin II (Ang II), collagen type I alpha 1 (Col1α1), collagen type III alpha 1 (Col3α1), and hydroxyproline] in the right ventricle (RV). Oxygen enrichment inhibited medial thickening, stenosis and fibrosis of pulmonary arterioles, and cytokine expression related with fibrosis (Col1α1, Col3α1, and hydroxyproline) and pulmonary vasoconstriction [endothelin-1(ET-1)] in HAH-exposed rats. This study represents the first effort testing the efficacy of the oxygen enrichment technique on cardiopulmonary structure and function in chronic HAH animals, and we found oxygen enrichment has the capability of ameliorating chronic HAH-induced cardiopulmonary alterations.

Effects of mechanical vibration on cell morphology, proliferation, apoptosis, and cytokine expression/secretion in osteocyte-like MLO-Y4 cells exposed to high glucose.

Diabetic patients exhibit significant bone deterioration. Our recent findings demonstrate that mechanical vibration is capable of resisting diabetic bone loss, whereas the relevant mechanism remains unclear. We herein examined the effects of mechanical vibration on the activities and functions of osteocytes (the most abundant and well-recognized mechanosensitive cells in the bone) exposed to high glucose (HG). The osteocytic MLO-Y4 cells were incubated with 50 mM HG for 24 h, and then stimulated with 1 h/day mechanical vibration (0.5 g, 45 Hz) for 3 days. We found that mechanical vibration significantly increased the proliferation and viability of MLO-Y4 cells under the HG environment via the MTT, BrdU, and Cell Viability Analyzer assays. The apoptosis detection showed that HG-induced apoptosis in MLO-Y4 cells was inhibited by mechanical vibration. Moreover, increased cellular area, microfilament density, and anisotropy in HG-incubated MLO-Y4 cells were observed after mechanical vibration via the F-actin fluorescence staining. The real-time polymerase chain reaction and western blotting results demonstrated that mechanical vibration significantly upregulated the gene and protein expression of Wnt3a, ß-catenin, and osteoprotegerin (OPG) and decreased the sclerostin, DKK1, and receptor activator for nuclear factor-κB ligand (RANKL) expression in osteocytes exposed to HG. The enzyme-linked immunosorbent assay assays showed that mechanical vibration promoted the secretion of prostaglandin E2 and OPG, and inhibited the secretion of tumor necrosis factor-α and RANKL in the supernatant of HG-treated MLO-Y4 cells. Together, this study demonstrates that mechanical vibration improves osteocytic architecture and viability, and regulates cytokine expression and secretion in the HG environment, and implies the potential great contribution of the modulation of osteocytic activities in resisting diabetic osteopenia/osteoporosis by mechanical vibration.

Spatiotemporal Distribution of Linear Microcracks and Diffuse Microdamage Following Daily Bouts of Fatigue Loading of Rat Ulnae.

Microdamage accumulation contributes to impaired skeletal mechanical integrity. The bone can remove microdamage by initiating targeted bone remodeling. However, the spatiotemporal characteristics of microdamage initiation and propagation and their relationship with bone remodeling in response to fatigue loading, especially for more physiologically relevant daily bouts of compressive loading, remain poorly understood. The right forelimbs of 24 rats were cyclically loaded with a ramp waveform for 1,500 cycles/day, and contralateral ulnae were not loaded as the controls. The rats were divided into four equal groups and loaded for 1, 4, 7, and 10 days, respectively. We demonstrated that linear microcracking accumulation exhibited a non-linear time-varying process within 10 days of loading with peaked microcrack density at Day 7. Disrupted canaliculi surrounding linear microcracks showed high similarity with the temporal changes of linear microcracking accumulation. Observable intracortical resorption regions were found on Day 10. We found more linear microcracks accumulated in the tensile cortex, but longer cracks were observed in the compressive sides. Increased accumulation of diffuse microdamage was observed from Day 4, but no obvious peak was observed within the 10-day loading period. Diffuse damage first initiated in the compressive cortices but extended to tension from Day 7. The diffuse damage exhibited no impacts on the surrounding osteocyte integrity. Together, our findings revealed a time-dependent, bone remodeling-mediated varying process of linear microcracking accumulation following daily bouts of fatigue loading (with observable peak at Day 7 under our loading regime). Our study also identified distinct spatial accumulation of linear and diffuse microdamage in rat ulnae with tensile and compressive strains. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2112-2121, 2019.

Pulsed electromagnetic fields regulate osteocyte apoptosis, RANKL/OPG expression, and its control of osteoclastogenesis depending on the presence of primary cilia.

Growing evidence has shown that pulsed electromagnetic fields (PEMF) can modulate bone metabolism in vivo and regulate the activities of osteoblasts and osteoclasts in vitro. Osteocytes, accounting for 95% of bone cells, act as the major mechanosensors in bone for transducing external mechanical signals and producing cytokines to regulate osteoblastic and osteoclastic activities. Targeting osteocytic signaling pathways is becoming an emerging therapeutic strategy for bone diseases. We herein systematically investigated the changes of osteocyte behaviors, functions, and its regulation on osteoclastogenesis in response to PEMF. The osteocyte-like MLO-Y4 cells were exposed to 15 Hz PEMF stimulation with different intensities (0, 5, and 30 Gauss [G]) for 2 hr. We found that the cell apoptosis and cytoskeleton organization of osteocytes were regulated by PEMF with an intensity-dependent manner. Moreover, PEMF exposure with 5 G significantly inhibited apoptosis-related gene expression and also suppressed the gene and protein expression of the receptor activator of nuclear factor κB ligand/osteoprotegerin (RANKL/OPG) ratio in MLO-Y4 cells. The formation, maturation, and osteoclastic bone-resorption capability of in vitro osteoclasts were significantly suppressed after treated with the conditioned medium from PEMF-exposed (5 G) osteocytes. Our results also revealed that the inhibition of osteoclastic formation, maturation, and bone-resorption capability induced by the conditioned medium from 5 G PEMF-exposed osteocytes was significantly attenuated after abrogating primary cilia in osteocytes using the polaris siRNA transfection. Together, our findings highlight that PEMF with 5 G can inhibit cellular apoptosis, modulate cytoskeletal distribution, and decrease RANKL/OPG expression in osteocytes, and also inhibit osteocyte-mediated osteoclastogenesis, which requires the existence of primary cilia in osteocytes. This study enriches our basic knowledge for further understanding the biological behaviors of osteocytes and is also helpful for providing a more comprehensive mechanistic understanding of the effect of electromagnetic stimulation on bone and relevant skeletal diseases (e.g., bone fracture and osteoporosis).