Hindlimb-unloaded Mice Research Articles

RNAseq and RNA molecular barcoding reveal differential gene expression in cortical bone following hindlimb unloading in female mice.

Disuse-induced bone loss is seen following spinal cord injury, prolonged bed rest, and exposure to microgravity. We performed whole transcriptomic profiling of cortical bone using RNA sequencing (RNAseq) and RNA molecular barcoding (NanoString) on a hindlimb unloading (HLU) mouse model to identify genes whose mRNA transcript abundances change in response to disuse. Eleven-week old female C57BL/6 mice were exposed to ambulatory loading or HLU for 7 days (n = 8/group). Total RNA from marrow-flushed femoral cortical bone was analyzed on HiSeq and NanoString platforms. The expression of several previously reported genes associated with Wnt signaling and metabolism was altered by HLU. Furthermore, the increased abundance of transcripts, such as Pfkfb3 and Mss51, after HLU imply these genes also have roles in the cortical bone’s response to altered mechanical loading. Our study demonstrates that an unbiased approach to assess the whole transcriptomic profile of cortical bone can reveal previously unidentified mechanosensitive genes and may eventually lead to novel targets to prevent disuse-induced osteoporosis.

Knockdown of MACF1 inhibits the migration and cytoskeletal arrangement of pre-osteoclasts induced by simulated microgravity

Bone loss remains a major health concern for astronauts during space flight. Increased osteoclast activity is one of the main causes of bone loss in astronauts subjected to microgravity conditions. However, the underlying molecular mechanisms remain unclear. Microtubule actin crosslinking factor 1 (MACF1) has been implicated in the regulation of cytoskeletal distribution, cell migration, and cell differentiation. Our previous studies have shown that MACF1 promotes the differentiation of pre-osteoclasts. However, whether MACF1 regulates the migration and cytoskeletal arrangement of pre-osteoclasts under microgravity conditions has not yet been elucidated. In this study, we used a hind-limb unloading (HLU) mouse model and a random positioning machine (RPM) to simulate the effects of microgravity on pre-osteoclasts. In the HLU mouse model, the expression of MACF1 was upregulated in primary pre-osteoclasts of mice, accompanied by enhanced migration in vivo. Moreover, simulated microgravity using the RPM also promoted MACF1 expression and migration of pre-osteoclasts in vitro. Additionally, knockdown of MACF1 disrupted cytoskeletal arrangement (F-actin and microtubules) and further inhibited the migration of pre-osteoclasts via the RhoA/ROCK1 signaling pathway. We further demonstrated that knockdown of MACF1 disrupted the enhanced migration and cytoskeleton arrangement of pre-osteoclasts induced by simulated microgravity. These data demonstrate that MACF1 positively regulates the migration and cytoskeletal organization of pre-osteoclasts under simulated microgravity, suggesting that MACF1 may be a therapeutic target for the treatment of bone loss induced by microgravity.

Mammalian and Invertebrate Models as Complementary Tools for Gaining Mechanistic Insight on Muscle Responses to Spaceflight.

Bioinformatics approaches have proven useful in understanding biological responses to spaceflight. Spaceflight experiments remain resource intensive and rare. One outstanding issue is how to maximize scientific output from a limited number of omics datasets from traditional animal models including nematodes, fruitfly, and rodents. The utility of omics data from invertebrate models in anticipating mammalian responses to spaceflight has not been fully explored. Hence, we performed comparative analyses of transcriptomes of soleus and extensor digitorum longus (EDL) in mice that underwent 37 days of spaceflight. Results indicate shared stress responses and altered circadian rhythm. EDL showed more robust growth signals and Pde2a downregulation, possibly underlying its resistance to atrophy versus soleus. Spaceflight and hindlimb unloading mice shared differential regulation of proliferation, circadian, and neuronal signaling. Shared gene regulation in muscles of humans on bedrest and space flown rodents suggest targets for mitigating muscle atrophy in space and on Earth. Spaceflight responses of C. elegans were more similar to EDL. Discrete life stages of D. melanogaster have distinct utility in anticipating EDL and soleus responses. In summary, spaceflight leads to shared and discrete molecular responses between muscle types and invertebrate models may augment mechanistic knowledge gained from rodent spaceflight and ground-based studies.

BCAAs and Di-Alanine supplementation in the prevention of skeletal muscle atrophy: preclinical evaluation in a murine model of hind limb unloading

Skeletal muscle atrophy occurs in response to various pathophysiological stimuli, including disuse, aging, and neuromuscular disorders, mainly due to an imbalance of anabolic/catabolic signaling. Branched Chain Amino Acids (BCAAs: leucine, isoleucine, valine) supplements can be beneficial for counteracting muscle atrophy, in virtue of their reported anabolic properties. Here, we carried out a proof-of-concept study to assess the in vivo/ex vivo effects of a 4-week treatment with BCAAs on disuse-induced atrophy, in a murine model of hind limb unloading (HU). BCAAs were formulated in drinking water, alone, or plus two equivalents of L-Alanine (2 ALA) or the dipeptide L-Alanyl-L-Alanine (Di-ALA), to boost BCAAs bioavailability. HU mice were characterized by reduction of body mass, decrease of soleus – SOL – muscle mass and total protein, alteration of postural muscles architecture and fiber size, dysregulation of atrophy-related genes (Atrogin-1, MuRF-1, mTOR, Mstn). In parallel, we provided new robust readouts in the HU murine model, such as impaired in vivo isometric torque and ex vivo SOL muscle contractility and elasticity, as well as altered immune response. An acute pharmacokinetic study confirmed that L-ALA, also as dipeptide, enhanced plasma exposure of BCAAs. Globally, the most sensitive parameters to BCAAs action were muscle atrophy and myofiber cross-sectional area, muscle force and compliance to stress, protein synthesis via mTOR and innate immunity, with the new BCAAs + Di-ALA formulation being the most effective treatment. Our results support the working hypothesis and highlight the importance of developing innovative formulations to optimize BCAAs biodistribution.

Green Tea Catechin, Epigallocatechin Gallate, Prevents Bone Loss in Hindlimb-Unloading Mice

Abstract Objectives Osteoporosis is a major health problem in the elderly characterized by bone loss and micro-architectural deterioration of bone tissue and associated with an increased risk of fracture. Prolonged bed rest, or physical inactivity during space flight causes rapid and marked bone loss. The effects of catechin, the main ingredient of Japanese green tea, on the bone are currently under study. It has been shown that green tea catechin modulates bone resorption in osteoclasts. However, there is no evidence supporting its inhibitory effect on bone loss during physical inactivity. In the present study, we investigated whether green tea catechin prevented bone loss through skeletal hindlimb-unloading in mice. Methods Female 8-week-old ddY mice were divided into five groups (n = 6–8 each) and subjected to: (1) normal housing fed a control diet, (2) sham unloading fed a control diet, (3) hind limb-unloading fed a control diet, (4) hind limb-unloading fed a 0.05% epigallocatechin gallate (EGCG)-containing diet, and (5) hind limb-unloading fed a 0.25% EGCG-containing diet for three weeks. Purified EGCG (97%) was used for green tea catechin. Results Bone mineral density of the tibia significantly decreased in hind limb-unloading mice. Treatment with 0.25% EGCG prevented bone loss and maintained trabecular bone mineral density more significantly than in cortical bones. The 0.25% EGCG diet inhibited decrease in the gene expression of alkaline phosphatase, a marker of bone formation, in the bone marrow in hind limb-unloading mice. Conclusions These results suggest that EGCG has ability to prevent bone loss induced by hindlimb-unloading in mice. These osteoprotective effects of EGCG may result from the inhibition of unloading-induced decrease in bone formation. Funding Sources This work was supported by the Honjo International Scholarship Foundation of Japan.

Exosomes derived from cyclic mechanical stretch-exposed bone marrow mesenchymal stem cells inhibit RANKL-induced osteoclastogenesis through the NF-κB signaling pathway.

BackgroundSkeletal unloading usually induces severe disuse osteoporosis (DOP), which often occurs in patients subjected to prolonged immobility or in spaceflight astronauts. Increasing evidence suggests that exosomes are important mediators in maintaining the balance between bone formation and resorption. We hypothesized that exosomes play an important role in the maintenance of bone homeostasis through intercellular communication between bone marrow mesenchymal stem cells (BMSCs) and osteoclasts under mechanical loading.MethodsCells were divided into cyclic mechanical stretch (CMS)-treated BMSCs and normal static-cultured BMSCs, and exosomes were extracted by ultracentrifugation. After incubation with CMS-treated BMSC-derived exosomes (CMS_Exos) or static-cultured BMSC-derived exosomes (static_Exos), the apoptosis rates of bone marrow macrophages (BMMs) were determined by flow cytometry, and cell viability was detected with a Cell Counting Kit-8 (CCK-8) assay. Osteoclast differentiation was determined with an in vitro osteoclastogenesis assay. Signaling pathway activation was evaluated by western blotting and immunofluorescence staining. Hindlimb unloading (HU)-induced DOP mouse models were prepared to evaluate the function of exosomes in DOP.ResultsBoth CMS_Exos and static_Exos could be internalized by BMMs, and CMS_Exos did not affect BMM viability or increase apoptosis. The CMS_Exos effectively suppressed receptor activator of nuclear factor kappa-B ligand (RANKL)-mediated osteoclastogenesis and F-actin ring formation. Further molecular investigation demonstrated that CMS_Exos impaired osteoclast differentiation via inhibition of the RANKL-induced nuclear factor kappa-B (NF-κB) signaling pathway. Both CMS_Exos and static_Exos partly rescued the osteoporosis caused by mechanical unloading; however, the CMS_Exo group showed more obvious rescue. Treatment with CMS_Exos significantly decreased the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts. Exosomes derived from CMS-treated BMSCs strongly inhibited osteoclast differentiation by attenuating the NF-κB signaling pathway in vitro and rescued osteoporosis caused by mechanical unloading in an HU mouse model in vivo.ConclusionsIn this research, we demonstrated that Exosomes derived from CMS-treated BMSCs inhibited osteoclastogenesis by attenuating NF-κB signaling pathway activity in vitro and ameliorated bone loss caused by mechanical unloading in an HU mouse model, providing new insights into intercellular communication between osteoblasts and osteoclasts under mechanical loading.

MiR-138–5p negatively regulates osteoblast differentiation through inhibiting β-catenin under simulated microgravity in MC3T3-E1 cells

With the increasing interest in exploring the deep-space environment, the problems of health and safety of astronauts' bone induced by microgravity warrants an investigation. Recent studies have discovered that several microRNAs (miRNAs) have played a vital role in osteoblast differentiation and bone formation under microgravity, whereas the in-depth signaling pathway mechanisms are not yet completely understood. Here, we performed the hind limb unloading (HLU) mice model and 3D clinostat-random position machine (RPM) to simulate the effects of microgravity on bone at animal and cellular level, respectively. Firstly, we screened the different expressed miRNAs under simulated microgravity by miRNA sequencing, we then identified the highest different expressed miRNA (miR-138–5p) that was up-regulated under simulated microgravity and inhibited osteoblast differentiation in MC3T3-E1 cells. Moreover, we analyzed miR-138's targets during osteogenic differentiation and we found that these targets regulated osteogenic differentiation through Wnt/β-catenin signaling, a key signaling pathway in controlling osteoblast differentiation. The association between miR-138–5p and β-catenin activity was determined by a luciferase reporter assay. Further experiments confirmed that miR-138–5p suppressed β-catenin expression and β-catenin activity. Moreover, β-catenin overexpression attenuated osteoblast differentiation reduction induced by increased miR-138–5p level in miR-138–5p overexpression osteoblastic cells. In addition, inhibition of miR-138–5p counteracted the negative effects of simulated microgravity on osteoblast differentiation and β-catenin expression in MC3T3-E1 cells. Our findings demonstrated that a signaling pathway mechanism of miR-138–5p in regulating osteoblast differentiation under simulated microgravity, which may reveal a novel mechanism for astronauts' bone loss induced by microgravity and provide a potential therapeutic target for disused osteoprosis.

Signature molecular changes in the skeletal muscle of hindlimb unloaded mice

Hind-limb unloaded (HU) mouse is a well-recognized model of muscle atrophy; however, the molecular changes in the skeletal muscle during unloading are poorly characterized. We have used Raman spectroscopy to evaluate the structure and behavior of signature molecules involved in regulating muscle structural and functional health. The Raman spectroscopic analysis of gastrocnemius muscles was compared between 16-18 weeks old HU c57Bl/6J mice and ground-based controls. The spectra showed that the signals for asparagine and glutamine were reduced in HU mice, possibly indicating increased catabolism. The peaks for hydroxyproline and proline were split, pointing towards molecular breakdown and reduced tendon repair. We also report a consistently increased intensity in> 1300 cm-1 range in the Raman spectra along with a shift towards higher frequencies in the HU mice, indicating activation of sarcoplasmic reticulum (SR) stress during HU.

Long noncoding RNA HCG18 inhibits the differentiation of human bone marrow-derived mesenchymal stem cells in osteoporosis by targeting miR-30a-5p/NOTCH1 axis

BackgroundRecent studies have demonstrated that long non-coding RNAs (LncRNAs) can influence bone cell differentiation and formation. However, it is unclear whether lncRNA HCG18 is involved in osteoporosis (OP). This study was conducted to investigate the regulation of HCG18 in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).MethodsBMSCs were isolated and cultured from mouse pathological models and osteoporosis patients. RT-qPCR was performed to detect the expression of HCG18 and miR-30a-5p in BMSCs. The interaction between HCG18 and miR-30a-5p was analyzed by dual luciferase assay and RNA pulldown assay. The interaction between miR-30a-5p and NOTCH1 3′-UTR was analyzed by dual luciferase assay. RT-qPCR and Western blotting were used to detect the expression of osteogenic genes Runx2, OCN and OPN. Hindlimb-unloaded (HU) mice model was established, and HCG18 was knocked down on bone-formation surfaces by using lentivirus mediated shRNA transfection.ResultsThe expression of HCG18 was increased in BMSCs of OP patients, while the expression of miR-30a-5p was decreased. The expression of HCG18 and miR-30a-5p was negatively correlated in BMSCs. During the differentiation from BMSCs to osteoblasts, the expression of HCG18 was significantly downregulated, and the expression of miR-30a-5p was significantly upregulated. Overexpression of HCG18 was able to reverse the osteogenic-induced upregulation of miR-30a-5p expression, and knockdown of HCG18 further promoted the expression of miR-30a-5p. In addition, miR-30a-5p partially abolished the effect of HCG18 on osteogenic differentiation of BMSCs. NOTCH1 was a target protein of miR-30a-5p, and upregulation of NOTCH1 reversed the effect of miR-30a-5p on osteogenic differentiation of BMSCs. Furthermore, this study found that lentivirus mediated HCG18 knockdown on the bone-formation surfaces of hindlimb-unloaded (HU) mice partially alleviated unloading-induced bone lossConclusionsHCG18 inhibited osteogenic differentiation of BMSCs induced by OP via the miR-30a-5p/NOTCH1 axis. HCG18 can be identified as a regulator of osteogenic differentiation of BMSCs.

Neutrophil-to-Lymphocyte Ratio: A Biomarker to Monitor the Immune Status of Astronauts.

A comprehensive understanding of spaceflight factors involved in immune dysfunction and the evaluation of biomarkers to assess in-flight astronaut health are essential goals for NASA. An elevated neutrophil-to-lymphocyte ratio (NLR) is a potential biomarker candidate, as leukocyte differentials are altered during spaceflight. In the reduced gravity environment of space, rodents and astronauts displayed elevated NLR and granulocyte-to-lymphocyte ratios (GLR), respectively. To simulate microgravity using two well-established ground-based models, we cultured human whole blood-leukocytes in high-aspect rotating wall vessels (HARV-RWV) and used hindlimb unloaded (HU) mice. Both HARV-RWV simulation of leukocytes and HU-exposed mice showed elevated NLR profiles comparable to spaceflight exposed samples. To assess mechanisms involved, we found the simulated microgravity HARV-RWV model resulted in an imbalance of redox processes and activation of myeloperoxidase-producing inflammatory neutrophils, while antioxidant treatment reversed these effects. In the simulated microgravity HU model, mitochondrial catalase-transgenic mice that have reduced oxidative stress responses showed reduced neutrophil counts, NLR, and a dampened release of selective inflammatory cytokines compared to wildtype HU mice, suggesting simulated microgravity induced oxidative stress responses that triggered inflammation. In brief, both spaceflight and simulated microgravity models caused elevated NLR, indicating this as a potential biomarker for future in-flight immune health monitoring.

The coupling of reduced type H vessels with unloading-induced bone loss and the protection role of Panax quinquefolium saponin in the male mice.

Unloading-induced bone loss is a critical complication characterized by the imbalance of bone formation and resorption induced by long-term confinement in bed or spaceflight. CD31hiEmcnhi (type H) vessel is a specific subtype of capillary, which was coupled with osteogenesis. However, the change of type H vessel and its contributions to the unloading-induced bone loss remains undisclosed. Herein, we found that bone formation and the number of type H vessels were synchronously reduced in the hindlimb-unloading (HU) mice. Panax quinquefolium saponin (PQS) could increase bone mass, osteoblast function and the number of type H vessels in the HU mice. In vitro, PQS treatment accelerated HMECs migration, augmented the total tube loops and increased the secretion of VEGF and Noggin. Primary osteoblasts function was obviously increased when treated with supernatant from PQS-treated HMECs. These effects of PQS were substantially counteracted when VEGF and Noggin in HMECs were knocked down by siRNA. These results demonstrated that unloading-induced bone loss is coupled with reduction of type H vessels and PQS performs preventive function via promoting type H vessel angiogenesis, which is closely associated with endothelial cell-derived VEGF and Noggin.

A Comprehensive Analysis of MicroRNAs in Human Osteoporosis.

MicroRNAs (miRNAs) are single-stranded RNA molecules that control gene expression in various processes, such as cancers, Alzheimer’s disease, and bone metabolic diseases. However, the regulatory roles of miRNAs in osteoporosis have not been systematically analyzed. Here, we performed a comprehensive analysis to identify the differentially expressed miRNAs involved in osteoporosis. MiRNAs associated with osteoporosis were collected through literature retrieval and further screened based on specific inclusion and exclusion criteria. The osteoporosis therapeutic targets of miRNAs were obtained by the integration of miRWalk 3.0 database and five human disease therapeutic target databases. Then, the network analysis and functional enrichment analysis of miRNAs and their targets were performed. As a result, 11 eligible miRNAs were identified highly associated with osteoporosis. MiRNA-mRNA network demonstrated there were the complex mutual interactions between miRNAs and their targets. Besides, ADRB2, AR, ESR1, FGFR1, TRAF6, etc., were identified as the top hub genes in protein-protein interaction (PPI) network. Functional enrichment analysis revealed that miRNAs and their targets were mainly mapped on processes associated with bone and immune system, such as bone remolding, bone mineralization, PI3K/AKt, TNF signaling pathways and Th17 cell differentiation. RT-PCR results showed that the expression of miR-335-3p was significantly down-regulated in hind limb unloading (HLU) mice tibia samples compared with controls, the remaining 10 miRNAs were significantly up-regulated after HLU (P < 0.01). In summary, we identified 11 differentially expressed miRNAs and their hub target genes in osteoporosis, which may be novel diagnostic biomarkers for osteoporosis.

MUSCLE FUNCTION & HOMEOSTASIS / MOLECULAR THERAPEUTIC APPROACHES: P.76 Validation of in vivo and ex vivo readouts in a murine model of hind limb unloading to optimize translational research on skeletal muscle atrophy

Skeletal muscle atrophy is a major consequence of several pathophysiological conditions, including neuromuscular diseases, disuse (limb immobilization, space flight), aging (sarcopenia) and other wasting conditions (i.e. cancer cachexia). Basically, a disequilibrium between anabolic/catabolic pathways leads to progressive loss of muscle mass and functional impairment. The aim of translational research in this field is to identify anabolic compounds able to support protein synthesis and muscle mass, restoring muscle function and metabolism. Therefore, the validation of adequate readout parameters to assess atrophy and efficacy of therapeutics in reliable animal models is pivotal. Here, we focused on a detailed preclinical investigation of the hind limb unloading (HU) murine model, in which disuse leads to severe atrophy, dysfunction and damage in postural soleus (SOL) muscle. 10-week-old, male C57BL/6J mice underwent a 2-week HU protocol (T0–T2), in comparison to age-, sex-matched non-HU mice for a wide array of in vivo/ex vivo outcomes. At T0 (pre-HU), all mice had comparable body weight (BW). At T2, HU mice BW was significantly lower with respect to T0 (-9.7%) and to non-HU mice (-17.3%). We disclosed for the first time that in vivo torque of hind limb plantar flexor muscles (i.e. SOL, gastrocnemius) was severely impaired in HU mice, as well as SOL muscle contractile and elastic properties, measured ex vivo. This was paralleled by a significant decrease in SOL muscle mass (-39%) and total protein content (-47%). SOL muscle histology was also seriously compromised by HU protocol, with clear signs of myofiber damage and atrophy (significant decrease in fiber cross-sectional area vs non-HU condition). Finally, we found a remarkable -61% reduction of salivary IgA levels, which suggests a HU-induced negative immune response modulation, in line with previous observations from other models of hind limb suspension. Overall, our findings support the usefulness of the HU mouse model for preclinical translational research on skeletal muscle atrophy.

Simulated microgravity suppresses MAPK pathway-mediated innate immune response to bacterial infection and induces gut microbiota dysbiosis.

During spaceflight, astronauts are subjected to various physical stressors including microgravity, which could cause immune dysfunction and thus potentially predispose astronauts to infections and illness. However, the mechanisms by which microgravity affects innate immunity remain largely unclear. In this study, we conducted RNA-sequencing analysis to show that simulated microgravity (SMG) suppresses the production of inflammatory cytokines including tumor necrosis factor (TNF) and interleukin-6 (IL-6) as well as the activation of the innate immune signaling pathways including the p38 mitogen-activated protein kinase (MAPK) and the Erk1/2 MAPK pathways in the Enteropathogenic escherichia coli (EPEC)-infected macrophage cells. We then adopted hindlimb-unloading (HU) mice, a model mimicking the microgravity of a spaceflight environment, to demonstrate that microgravity suppresses proinflammatory cytokine-mediated intestinal immunity to Citrobacter rodentium infection and induces the disturbance of gut microbiota, both of which phenotypes could be largely corrected by the introduction of VSL#3, a high-concentration probiotic preparation of eight live freeze-dried bacterial species. Taken together, our study provides new insights into microgravity-mediated innate immune suppression and intestinal microbiota disturbance, and suggests that probiotic VSL#3 has great potential as a dietary supplement in protecting individuals from spaceflight mission-associated infections and gut microbiota dysbiosis.

Prevention of bone loss and improvement of pain-related behavior in hind limb-unloaded mice by administration of teriparatide and bisphosphonate

Objectives There are few reports on the comparison between teriparatide (PTH) and bisphosphonate (BP) in terms of osteoporosis pain-related behavior and immunohistochemical findings. The aims of this study were to evaluate skeletal pain associated with osteoporosis and to examine the inhibitory effect of PTH and BP on pain and bone loss in hind limb-unloaded (HU) mice. The mechanism of osteoporotic pain in HU mice was evaluated by examining pain-related behavior and immunohistochemical findings. The effects of PTH and alendronate (ALN), a potent osteoclast inhibitor, on these parameters were also assessed. Methods Eight-week-old male ddY mice were tail-suspended for 2 weeks and assigned to four groups: hind limb-loaded (HL) mice with only tail suspension treated with vehicle; HU mice with tail suspension treated with vehicle; HU mice treated with PTH; and HU mice treated with ALN. Starting immediately after reloading, vehicle, PTH, or ALN was injected subcutaneously. After a 2-week treatment, mechanical sensitivity was examined using von Frey filaments. Bilateral hind limbs were removed for micro-computed tomography, immunohistochemical analysis, and messenger RNA (mRNA) expression analysis. Results HU mice with tail suspension developed bone loss and mechanical hyperalgesia in the hind limbs. The HU mice showed an increased osteoclasts and sclerostin-positive cells in the hind limb bone. Furthermore, PTH and ALN both prevented HU-induced bone loss and mechanical hyperalgesia in the osteoporotic animal models. Histological examination of the hind limb bone revealed that, similar to ALN, PTH inhibited the osteoclasts and sclerostin-positive cells. The mRNA levels of TNFα and IL-6 tended to decrease with ALN or PTH treatment compared with those without any treatment. Conclusions Treatment with PTH as well as BP prevented bone loss, mechanical hyperalgesia, osteoclast increase, and osteocyte increase. Similar to BP, the inhibitory effect of PTH on osteoclasts might contribute to the improvement of skeletal pain.

The Combination of Soy Isoflavones and Resveratrol Preserve Bone Mineral Density in Hindlimb-Unloaded Mice

It is well known that physical inactivity during space flight or prolonged bed rest causes rapid bone loss. Soy isoflavones (ISOs) and resveratrol (RES) have been reported to be useful to maintain a positive balance for bone turnover. Therefore, we examined the combined effects of ISO and RES on bone loss that was induced by hindlimb-unloading in mice. Female eight-week-old ddY mice were divided into the following six groups (n = 6–8 each): normally housed mice, loading mice, hindlimb-unloading (UL) mice fed a control diet, UL mice fed a 0.16% ISO conjugates, UL mice fed a 0.15% RES diet, and UL mice fed a 0.16% ISO and 0.15% RES diet. After three weeks, femoral bone mineral density was markedly decreased in unloading mice. The combination of ISO and RES prevented bone loss and especially maintained the trabecular bone mineral density more effectively compared with cortical bones. ISO and/or RES inhibited the increase in the RANKL/OPG expression ratio in bone marrow cells in UL mice. These results suggest that the combination of ISO and RES had a preventive effect against bone loss induced by hindlimb-unloading in mice. These osteoprotective effects of ISO and RES may result from the inhibition of bone resorption.

Functional Block of Interleukin-6 Reduces a Bone Pain Marker but Not Bone Loss in Hindlimb-Unloaded Mice.

Interleukin-6 (IL-6) is widely accepted to stimulate osteoclasts. Our aim in this study was to examine whether the inhibitory effect of IL-6 on bone loss and skeletal pain associated with osteoporosis in hindlimb-unloaded (HU) mice in comparison with bisphosphonate. Eight-week-old male ddY mice were tail suspended for 2 weeks. Starting immediately after reload, vehicle (HU group), alendronate (HU-ALN group), or anti-IL-6 receptor antibody (HU-IL-6i group) was injected subcutaneously. After a 2-week treatment, pain-related behavior was examined using von Frey filaments. The bilateral distal femoral and proximal tibial metaphyses were analyzed three-dimensionally with micro-computed tomography. Calcitonin gene-related peptide (CGRP) expressions in dorsal root ganglion (DRG) neurons innervating the hindlimbs were examined using immunohistochemistry. HU mice with tail suspension developed bone loss. The HU mice showed mechanical hyperalgesia in the hindlimbs and increased CGRP immunoreactive neurons in the L3-5 DRG. Treatment with IL-6i and ALN prevented HU-induced mechanical hyperalgesia and upregulation of CGRP expressions in DRG neurons. Furthermore, ALN but not IL-6i prevented HU-induced bone loss. In summary, treatment with IL-6i prevented mechanical hyperalgesia in hindlimbs and suppressed CGRP expressions in DRG neurons of osteoporotic models. The novelty of this research suggests that IL-6 is one of the causes of immobility-induced osteoporotic pain regardless improvement of bone loss.

Bone-targeted lncRNA OGRU alleviates unloading-induced bone loss via miR-320-3p/Hoxa10 axis

Unloading-induced bone loss is a threat to human health and can eventually result in osteoporotic fractures. Although the underlying molecular mechanism of unloading-induced bone loss has been broadly elucidated, the pathophysiological role of long noncoding RNAs (lncRNAs) in this process is unknown. Here, we identified a novel lncRNA, OGRU, a 1816-nucleotide transcript with significantly decreased levels in bone specimens from hindlimb-unloaded mice and in MC3T3-E1 cells under clinorotation-unloading conditions. OGRU overexpression promoted osteoblast activity and matrix mineralization under normal loading conditions, and attenuated the suppression of MC3T3-E1 cell differentiation induced by clinorotation unloading. Furthermore, this study found that supplementation of pcDNA3.1(+)–OGRU via (DSS)6–liposome delivery to the bone-formation surfaces of hindlimb-unloaded (HLU) mice partially alleviated unloading-induced bone loss. Mechanistic investigations demonstrated that OGRU functions as a competing endogenous RNA (ceRNA) to facilitate the protein expression of Hoxa10 by competitively binding miR-320-3p and subsequently promote osteoblast differentiation and bone formation. Taken together, the results of our study provide the first clarification of the role of lncRNA OGRU in unloading-induced bone loss through the miR-320-3p/Hoxa10 axis, suggesting an efficient anabolic strategy for osteoporosis treatment.

The Immunomodulatory Effect of Radiofrequency Electromagnetic Field on Serum Cytokine Levels in A Mouse Model of Hindlimb Unloading.

Objective Astronauts are exposed to a wide range of environmental stresses during spaceflights that reduce their immune responses and make them more susceptible to infections and malignancies. Exposure to a low dose of a certain stress induces an adaptive response, which leads to resistance to higher doses of the same or other types of stress. We designed this study to investigate the effect of radiofrequency electromagnetic field (RF-EMF)-induced adaptive response on immune system modulation in a mouse model of hindlimb unloading (HU) as a ground-based animal model of spaceflight conditions. Materials and Methods In this experimental study, serum levels of T helper (Th)-mediated cytokines were determined by the multiplex cytometric bead assay in four groups of mice (n=10 per group): HU mice, RF-EMF-treated mice, HU mice pre-exposed to RF-EMF; and untreated controls. Mice were exposed to 2450 MHz RF-EMF with SAR 0.478 W/ kg for 12 hours/day for three successive days. Results Tumor necrosis factor-alpha (TNF-α), interleukin-9 (IL-9) and IL-22 were significantly decreased in HU mice. Comparison between HU mice and RF-EMF-treated mice showed an opposite change in IL-6, while IL-9, IL-22, IFN-γ and TNF-α decreased in both groups. However, just interferon gamma (IFN-γ) was significantly decreased in HU mice that were pre-exposed to RF-EMF compared to the control group. ConclusionThe effect of RF-EMF in elevating IL-6 and reducing IL-9 in opposite directions in HU mice suggest a modulating effect of RF-EMF on HU-induced changes in these cytokines, as Th2 and Th9 eventually returned to normal levels and balances in cytokine ratios were also restored in HU mice pre-exposed to RF-EMF.

Recombinant Irisin Prevents the Reduction of Osteoblast Differentiation Induced by Stimulated Microgravity through Increasing β-Catenin Expression.

Background: Irisin, a novel exercise-induced myokine, was shown to mediate beneficial effects of exercise in osteoporosis. Microgravity is a major threat to bone homeostasis of astronauts during long-term spaceflight, which results in decreased bone formation. Methods: The hind-limb unloading mice model and a random position machine are respectively used to simulate microgravity in vivo and in vitro. Results: We demonstrate that not only are bone formation and osteoblast differentiation decreased, but the expression of fibronectin type III domain-containing 5 (Fdnc5; irisin precursor) is also downregulated under simulated microgravity. Moreover, a lower dose of recombinant irisin (r-irisin) (1 nM) promotes osteogenic marker gene (alkaline phosphatase (Alp), collagen type 1 alpha-1(ColIα1)) expressions, ALP activity, and calcium deposition in primary osteoblasts, with no significant effect on osteoblast proliferation. Furthermore, r-irisin could recover the decrease in osteoblast differentiation induced by simulated microgravity. We also find that r-irisin increases β-catenin expression and partly neutralizes the decrease in β-catenin expression induced by simulated microgravity. In addition, β-catenin overexpression could also in part attenuate osteoblast differentiation reduction induced by simulated microgravity. Conclusions: The present study is the first to show that r-irisin positively regulates osteoblast differentiation under simulated microgravity through increasing β-catenin expression, which may reveal a novel mechanism, and it provides a prevention strategy for bone loss and muscle atrophy induced by microgravity.