Nano-curcumin mitigates muscle impairment in hypoxic hindlimb-unloaded mice.

Background: Hindlimb-unloaded (HU) mice are animal models of simulated microgravity that exhibit pathological changes in several organs; however, relevant findings regarding the kidneys remain elusive. We investigated the possible contribution of protein dysregulation through endoplasmic reticulum (ER) stress to renal pathology in HU mice. Methods: We categorized male C57BL/6j mice into ground-based control and HU groups and treated them daily with a placebo or 4-phenylbutyrate (4PBA, an ER stress inhibitor), respectively, for 3 weeks. HU mice showed reduced body weight, whereas kidney weight remained unchanged. Results: Treatment with 4PBA increased the kidney weight of HU mice. The histopathological changes in HU mouse kidneys, including the widening of the Bowman’s capsule and increased glomerular area, were reversed through 4PBA treatment, accompanied by a 4PBA-induced reduction in the expression of several ER stress markers. Particularly, we found a reduction in the splicing of the X-box binding protein, indicating reduced ER stress in 4PBA-treated HU mice. Thus, 4PBA may use additional mechanisms to suppress ER stress and prevent renal pathology in HU mice. Conclusion: Our findings are the first to suggest that HU causes disruption of renal microarchitecture and that 4PBA may be a potent drug for kidney restoration under HU. Our preliminary findings have translational potential for conditions that mimic HU, such as prolonged bed rest.

The altered gut microbes of astronauts during space travel may contribute to health issues after their return to Earth. Previously, an association between the elevated endoplasmic reticulum (ER) stress and gut microbial dysbiosis has been described. Herein, we induced gut microbial changes in mice under a simulated microgravity environment in an established model of hindlimb unloaded (HU) mice. The intestinal metabolomic profiles under microgravity conditions using the HU model were examined, along with the potential role of 4-phenylbutyric acid (4-PBA), a potent ER stress inhibitor. For a microgravity environment, the mice were suspended in special cages individually for three weeks. Mice were sacrificed, and gut dissections were performed, followed by amplicon sequencing analysis of bacterial species via DNA extraction and 16S rRNA analysis. The results indicate that the gut bacterial communities of mice differed under gravity and microgravity conditions. Principal component analyses revealed differences in the bacterial community structure in all groups. Around 434 operational taxonomic units (OTUs) were specific to mice seen in controls, while 620 OTUs were specific to HU mice. Additionally, 321 bacterial OTUs were specific to HU mice treated with 4-PBA. When the relative abundance of taxa was analyzed, Bacteroidetes dominated the gut of control and HU mice treated with 4-PBA.. In contrast, the untreated HU mice were dominated by Firmicutes. At the genus level, a reduction in beneficial species of Akkermansia and Lactobacillus was observed in HU but not the unloaded–treated and control mice. Furthermore, an increase in the relative abundance of Lachnospiraceae and Enterorhabdus, associated with inflammation, was observed in HUmice but not in controls and unloaded-treated mice. Following treatment with 4-PBA, the ratio of Firmicutes to Bacteroidetes was restored in unloaded–treated mice, comparable to controls. Of note, beneficial microbes such as Akkermansia and Lactobacillus were observed in unloaded–treated mice but not or in lesser relative abundance in HU mice. Nonetheless, microbial diversity was reduced in unloaded–treated mice compared to controls, and future studies are needed to mitigate this finding. These may comprise the addition of pre-/pro- and postbiotic species in the diet to increase microbial diversity. Overall, the findings suggest that 4-PBA, a potent ER stress inhibitor, may have therapeutic value in treating patients on prolonged bed rest or astronauts during spaceflight.

Rapamycin relieves anxious emotion and synaptic plasticity deficits induced by hindlimb unloading in mice

Space travel and prolonged bed rest cause bone loss due to musculoskeletal disuse. In space, radiation fields may also have detrimental consequences because charged particles traversing the tissues of the body can elicit a wide range of cytotoxic and genotoxic lesions. The effects of heavy-ion radiation exposure in combination with musculoskeletal disuse on bone cells and tissue are not known. To explore this, normally loaded 16-week-old male C57BL/6 mice were exposed to (56)Fe ions (1 GeV/nucleon) at doses of 0 cGy (sham), 10 cGy, 50 cGy or 2 Gy 3 days before tissue harvest. Additional mice were hindlimb unloaded by tail traction continuously for 1 week to simulate weightlessness and exposed to (56)Fe-ion radiation (0 cGy, 50 cGy, 2 Gy) 3 days before tissue harvest. Despite the short duration of this study, low-dose (10, 50 cGy) irradiation of normally loaded mice reduced trabecular volume fraction (BV/TV) in the proximal tibiae by 18% relative to sham-irradiated controls. Hindlimb unloading together with 50 cGy radiation caused a 126% increase in the number of TRAP(+) osteoclasts on cancellous bone surfaces relative to normally loaded, sham-irradiated controls. Together, radiation and hindlimb unloading had a greater effect on suppressing osteoblastogenesis ex vivo than either treatment alone. In sum, low-dose exposure to heavy ions (50 cGy) caused rapid cancellous bone loss in normally loaded mice and increased osteoclast numbers in hindlimb unloaded mice. In vitro irradiation also was more detrimental to osteoblastogenesis in bone marrow cells that were recovered from hindlimb unloaded mice compared to cells from normally loaded mice. Furthermore, irradiation in vitro stimulated osteoclast formation in a macrophage cell line (RAW264.7) in the presence of RANKL (25 ng/ml), showing that heavy-ion radiation can stimulate osteoclast differentiation even in the absence of osteoblasts. Thus heavy-ion radiation can acutely increase osteoclast numbers in cancellous tissue and, under conditions of musculoskeletal disuse, can enhance the sensitivity of bone cells, in particular osteoprogenitors, to the effects of radiation.

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

Prolonged bed rest or microgravity exposure, as experienced during spaceflight, profoundly impacts cardiac health. Yet, the molecular mechanisms driving these detrimental changes remain largely elusive. Hindlimb unloading (HLU), a model of simulated microgravity, induces endoplasmic-reticulum (ER) stress, and its role in maladaptive cardiac remodeling remains unknown. To investigate the impact of HLU, its underlying molecular mechanisms and the therapeutic potential of ER stress suppression, C57BL6 mice were assigned to grounded control (GC) or HLU group. HLU mice received daily vehicle or 4-phenylbutyrate (4-PBA), an ER stress inhibitor, for 21 days. Additionally, HL-1 cardiomyocytes were treated with the ER stress inducer thapsigargin, with or without 4-PBA, to explore the cross-communication between ER stress and mitochondrial and metabolic dysfunction in vitro. Cardiac transcriptomic analysis revealed significant gene dysregulation in HLU compared to GC hearts. In HLU hearts, downregulated genes were mainly enriched for mitochondrial function and metabolic pathways, while upregulated genes were linked to extracellular matrix (ECM) pathways. Conversely, HLU mice treated with 4-PBA showed upregulation of mitochondrial function-related genes and downregulation of ECM-related genes. The oxidative phosphorylation (OXPHOS), which was downregulated in HLU hearts, became one of the most upregulated pathways following 4-PBA treatment. Consistent with the in vivo findings, thapsigargin-induced ER stress significantly compromised mitochondrial function, whereas co-treatment with 4PBA significantly preserved mitochondrial function. Together, our findings strongly suggest that prolonged bed rest or microgravity exposure triggers ER stress-induced mitochondrial and metabolic dysfunction in the heart, and pharmacological suppression of ER stress limits these detrimental cellular effects.

Enhancing microbial diversity as well as multi-organ health in hind-limb unloaded mice

Muscle disuse produces severe atrophy and a slow-to-fast phenotype transition in the postural Soleus (Sol) muscle of rodents. Antioxidants, amino-acids and growth factors were ineffective to ameliorate muscle atrophy. Here we evaluate the effects of nandrolone (ND), an anabolic steroid, on mouse skeletal muscle atrophy induced by hindlimb unloading (HU). Mice were pre-treated for 2-weeks before HU and during the 2-weeks of HU. Muscle weight and total protein content were reduced in HU mice and a restoration of these parameters was found in ND-treated HU mice. The analysis of gene expression by real-time PCR demonstrates an increase of MuRF-1 during HU but minor involvement of other catabolic pathways. However, ND did not affect MuRF-1 expression. The evaluation of anabolic pathways showed no change in mTOR and eIF2-kinase mRNA expression, but the protein expression of the eukaryotic initiation factor eIF2 was reduced during HU and restored by ND. Moreover we found an involvement of regenerative pathways, since the increase of MyoD observed after HU suggests the promotion of myogenic stem cell differentiation in response to atrophy. At the same time, Notch-1 expression was down-regulated. Interestingly, the ND treatment prevented changes in MyoD and Notch-1 expression. On the contrary, there was no evidence for an effect of ND on the change of muscle phenotype induced by HU, since no effect of treatment was observed on the resting gCl, restCa and contractile properties in Sol muscle. Accordingly, PGC1α and myosin heavy chain expression, indexes of the phenotype transition, were not restored in ND-treated HU mice. We hypothesize that ND is unable to directly affect the phenotype transition when the specialized motor unit firing pattern of stimulation is lacking. Nevertheless, through stimulation of protein synthesis, ND preserves protein content and muscle weight, which may result advantageous to the affected skeletal muscle for functional recovery.

Muscle disuse in the hindlimb unloaded (HU) mice causes significant atrophy and weakness. However, the cellular and molecular mechanisms driving disuse-muscle atrophy remain elusive. We investigated the potential contribution of proteins dysregulation by sarcoplasmic reticulum (SR), a condition called SR stress, to muscle loss during HU. Male, c57BL/6j mice were assigned to ground-based controls or HU groups treated with vehicle or 4-phenylbutyrate (4-PBA), a potent inhibitor of SR stress, once a day for three weeks. We report that the 4-PBA reduced the SR stress and partly reversed the muscle atrophy and weakness in the HU mice. Transcriptome analysis revealed that several genes were switched on (n = 3688) or differentially expressed (n = 1184) due to HU. GO, and KEGG term analysis revealed alterations in pathways associated with the assembly of cilia and microtubules, extracellular matrix proteins regulation, calcium homeostasis, and immune modulation during HU. The muscle restoration with 4-PBA partly reversed these changes along with differential and unique expression of several genes. The analysis of genes among the two comparisons (HU-v vs. control and HU-t vs. HU-v.) shows 841 genes were overlapped between the two comparisons and they may be regulated by 4-PBA. Altogether, our findings suggest that the pharmacological suppression of SR stress may be an effective strategy to prevent disuse-induced muscle weakness and atrophy.

The human body is adapted to Earth’s gravity, but space microgravity significantly alters kidney function, with the hindlimb unloaded (HU) mouse model serving as a valuable tool for studying these effects. During space travel, changes in gut microbiota can lead to health-related issues. In this study, we explored the protective role of crocodile gut microbiome media on kidney health in a HU mouse model, given the known impact of space travel on gut microbiota and related health issues. Male C57BL/6 mice (four months old) were divided into a ground-based control group (GC), HU mice fed with distilled water (HU), and HU mice fed with crocodile bacterial conditioned media (HU-CP). All groups were maintained in a controlled environment for three weeks. At the end of the experiment, mice were euthanized, kidney tissues were dissected for histopathological examination and transcriptomic analysis. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test, with p < 0.05 considered significant. Transcriptomic analysis revealed distinct gene expression profiles across GC, HU, and HU-CP groups, with HU-CP inducing both unique (4325 genes) and differential (975 genes) expression compared to HU group. The treatment partially restored glomerular morphology, reduced inflammation, and reversed gene expression alterations associated with oxidative stress, apoptosis, fibrosis, and inflammation. The crocodile bacterial conditioned media demonstrated potential therapeutic benefits in mitigating renal injury induced by simulated microgravity in HU mice. Further research is needed to elucidate the specific mechanisms involved and explore the potential clinical applications of this approach.Supplementary InformationThe online version contains supplementary material available at 10.1186/s42523-025-00503-y.

Objectives Hindlimb unloading (HU), widely used to simulate microgravity effects, is known to induce a stress response. However, as single-housed animals are usually used in such experiments, social isolation (SI) stress can affect experimental results. In the present study, we aimed to delineate stressful effects of 3-day HU and SI in mice. Methods Three animal groups, HU, SI, and group-housed (GH) control mice, were recruited. A comprehensive analysis of stress-related markers was performed using ELISA, western blotting, and immunohistochemistry. Results Our results showed that blood corticosterone and activity of glucocorticoid receptors and cAMP response element-binding protein (CREB) in the hippocampus of SI and HU animals did not differ from GH control. However, SI mice demonstrated upregulation of the hippocampal corticotropin-releasing hormone (CRH), inducible NO synthase (iNOS), vesicular glutamate transporter 1 (VGLUT1), and glutamate decarboxylases 65/67 (GAD65/67) along with activation of Fos-related antigen 1 (Fra-1) in the amygdala confirming the expression of stress. In HU mice, the same increase in GAD65/67 and Fra-1 indicated the contribution of SI. The special HU effect was expressed only in neurogenesis attenuation. Discussion Thus, our data indicated that 3-day HU could not be characterized as physiological stress, but SI stress contributed to the negative effects of HU.

Post-traumatic osteoarthritis progression is diminished by early mechanical unloading and anti-inflammatory treatment in mice

Simulated microgravity accurately models long-duration spaceflight effects on bone and skeletal muscle in skeletally immature mice☆

Disuse skeletal muscle atrophy in humans. Proteomic and molecular adaptations

Background:Disuse osteoporosis is a prevalent complication among patients with rheumatoid arthritis (RA). The treatment of disuse osteoporosis has been reported to diminish the efficacy of conventional osteoporosis drugs. Therefore, alternative...