Hindlimb Unloading Rats Research Articles

Structural Modification and Pharmacological Evaluation of (Thiadiazol-2-yl)pyrazines as Novel Piezo1 Agonists for the Intervention of Disuse Osteoporosis.

Piezo1 plays a pivotal role in regulating bone remodeling and homeostasis and has emerged as a promising target for chemical intervention in disuse osteoporosis. Nevertheless, the development of small-molecule Piezo1 agonists is still in its infancy, and highly efficacious Piezo1 agonists are urgently required. In this study, by shedding light on the structural novelty of the canonical Piezo1 agonist Yoda1, we initiated a structural optimization campaign based on the (thiadiazol-2-yl)pyrazine scaffold. A deuterated compound 12a was identified to be the most potent candidate against Piezo1 with an EC50 value of 2.21 μM, which was over 20-fold more potent than the reference Yoda1. This compound effectively activated Piezo1 and initiated Ca2+ influx in MSCs and promoted MSC osteogenesis via activating the Ca2+-related Erk signaling pathway. Furthermore, compound 12a was found to alleviate disuse osteoporosis with a desirable safety profile in a HU (hindlimb-unloading) rat model, thus warranting it as a potential probe for further investigation.

Salidroside alleviates simulated microgravity-induced bone loss by activating the Nrf2/HO-1 pathway

BackgroundBone loss caused by microgravity exposure presents a serious threat to the health of astronauts, but existing treatment strategies have specific restrictions. This research aimed to investigate whether salidroside (SAL) can mitigate microgravity-induced bone loss and its underlying mechanism.MethodsIn this research, we used hindlimb unloading (HLU) and the Rotary Cell Culture System (RCCS) to imitate microgravity in vivo and in vitro.ResultsThe results showed that salidroside primarily enhances bone density, microstructure, and biomechanical properties by stimulating bone formation and suppressing bone resorption, thereby preserving bone mass in HLU rats. In MC3T3-E1 cells cultured under simulated microgravity in rotary wall vessel bioreactors, the expression of osteogenic genes significantly increased after salidroside administration, indicating that salidroside can promote osteoblast differentiation under microgravity conditions. Furthermore, the Nrf2 inhibitor ML385 diminished the therapeutic impact of salidroside on microgravity-induced bone loss. Overall, this research provides the first evidence that salidroside can mitigate bone loss induced by microgravity exposure through stimulating the Nrf2/HO-1 pathway.ConclusionThese findings indicate that salidroside has great potential for treating space-related bone loss in astronauts and suggest that Nrf2/HO-1 is a viable target for counteracting microgravity-induced bone damage.

Simulated Microgravity Alters Gene Regulation Linked to Immunity and Cardiovascular Disease.

Microgravity exposure induces a cephalad fluid shift and an overall reduction in physical activity levels which can lead to cardiovascular deconditioning in the absence of countermeasures. Future spaceflight missions will expose crew to extended periods of microgravity among other stressors, the effects of which on cardiovascular health are not fully known. In this study, we determined cardiac responses to extended microgravity exposure using the rat hindlimb unloading (HU) model. We hypothesized that exposure to prolonged simulated microgravity and subsequent recovery would lead to increased oxidative damage and altered expression of genes involved in the oxidative response. To test this hypothesis, we examined hearts of male (three and nine months of age) and female (3 months of age) Long-Evans rats that underwent HU for various durations up to 90 days and reambulated up to 90 days post-HU. Results indicate sex-dependent changes in oxidative damage marker 8-hydroxydeoxyguanosine (8-OHdG) and antioxidant gene expression in left ventricular tissue. Three-month-old females displayed elevated 8-OHdG levels after 14 days of HU while age-matched males did not. In nine-month-old males, there were no differences in 8-OHdG levels between HU and normally loaded control males at any of the timepoints tested following HU. RNAseq analysis of left ventricular tissue from nine-month-old males after 14 days of HU revealed upregulation of pathways involved in pro-inflammatory signaling, immune cell activation and differential expression of genes associated with cardiovascular disease progression. Taken together, these findings provide a rationale for targeting antioxidant and immune pathways and that sex differences should be taken into account in the development of countermeasures to maintain cardiovascular health in space.

Sex similarities and divergences in systemic and muscle iron metabolism adaptations to extreme physical inactivity in rats.

Previous data in humans suggest that extreme physical inactivity (EPI) affects iron metabolism differently between sexes. Our objective was to deepen the underlying mechanisms by studying rats of both sexes exposed to hindlimb unloading (HU), the reference experimental model mimicking EPI. Eight-week-old male and female Wistar rats were assigned to control (CTL) or hindlimb unloading (HU) conditions (n=12/group). After 7days of HU, serum, liver, spleen, and soleus muscle were removed. Iron parameters were measured in serum samples, and ICP-MS was used to quantify iron in tissues. Iron metabolism genes and proteins were analysed by RT-qPCR and Western blot. Compared with control males, control females exhibited higher iron concentrations in serum (+43.3%, p<0.001), liver (LIC; +198%, P<0.001), spleen (SIC; +76.1%, P<0.001), and transferrin saturation (TS) in serum (+53.3%, P<0.001), contrasting with previous observations in humans. HU rat males, but not females, exhibited an increase of LIC (+54% P<0.001) and SIC (+30.1%, P=0.023), along with a rise of H-ferritin protein levels (+60.9% and +134%, respectively, in liver and spleen; P<0.05) and a decrease of TFRC protein levels (-36%; -50%, respectively, P<0.05). HU males also exhibited an increase of splenic HO-1 and NRF2 mRNA levels, (p<0.001), as well as HU females (P<0.001). Concomitantly to muscle atrophy observed in HU animals, the iron concentration increased in soleus in females (+26.7, P=0.004) while only a trend is observed in males (+17.5%, P=0.088). In addition, the H-ferritin and myoglobin protein levels in soleus were increased in males (+748%, P<0.001, +22%, P=0.011, respectively) and in females (+369%, P<0.001, +21.9%, P=0.007, respectively), whereas TFRC and ferroportin (FPN) protein levels were reduced in males (-68.9%, P<0.001, -76.8%, P<0.001, respectively) and females (-75.9%, P<0.001, -62.9%, P<0.001, respectively). Interestingly, in both sexes, heme exporter FLVCR1 mRNA increased in soleus, while protein levels decreased (-39.9% for males P=0.010 and -49.1% for females P<0.001). Taken together, these data support that, in rats (1) extreme physical inactivity differently impacts the distribution of iron in both sexes, (2) splenic erythrophagocytosis could play a role in this iron misdistribution. The higher iron concentrations in atrophied soleus from both sexes are associated with a decoupling between the increase in iron storage proteins (i.e., ferritin and myoglobin) and the decrease in levels of iron export proteins (i.e., FPN and FLVCR1), thus supporting an iron sequestration in skeletal muscle under extreme physical inactivity.

Lactoferrin Application for the Prevention and Restoration of Bone Tissue Alterations during Hindlimb Unloading in Rats

Lactoferrin Application for the Prevention and Restoration of Bone Tissue Alterations during Hindlimb Unloading in Rats

Prazosin improves insulin-induced anabolic signaling by protecting capillary regression in the soleus muscle of hindlimb-unloaded rats

Prazosin improves insulin-induced anabolic signaling by protecting capillary regression in the soleus muscle of hindlimb-unloaded rats

Celecoxib attenuates hindlimb unloading-induced muscle atrophy via suppressing inflammation, oxidative stress and ER stress by inhibiting STAT3.

Improving inflammation may serve as useful therapeutic interventions for the hindlimb unloading-induced disuse muscle atrophy. Celecoxib is a selective non-steroidal anti-inflammatory drug. We aimed to determine the role and mechanism of celecoxib in hindlimb unloading-induced disuse muscle atrophy. Celecoxib significantly attenuated the decrease in soleus muscle mass, hindlimb muscle function and the shift from slow- to fast-twitch muscle fibers caused by hindlimb unloading in rats. Importantly, celecoxib inhibited the increased expression of inflammatory factors, macrophage infiltration in damaged soleus muscle. Mechanistically, Celecoxib could significantly reduce oxidative stress and endoplasmic reticulum stress in soleus muscle of unloaded rats. Furthermore, celecoxib inhibited muscle proteolysis by reducing the levels of MAFbx, MuRF1, and autophagy related proteins maybe by inhibiting the activation of pro-inflammatory STAT3 pathway in vivo and in vitro. This study is the first to demonstrate that celecoxib can attenuate disuse muscle atrophy caused by hindlimb unloading via suppressing inflammation, oxidative stress and endoplasmic reticulum stress probably, improving target muscle function and reversing the shift of muscle fiber types by inhibiting STAT3 pathways-mediated inflammatory cascade. This study not only enriches the potential molecular regulatory mechanisms, but also provides new potential therapeutic targets for disuse muscle atrophy.

Mechanical interaction of myosin and native thin filament in the disused rat soleus muscle

The disuse of skeletal limb muscles occurs in a variety of conditions, yet our comprehension of the molecular mechanisms involved in adaptation to disuse remains incomplete. We studied the mechanical characteristics of actin-myosin interaction using an in vitro motility assay and isoform composition of myosin heavy and light chains by dint of SDS-PAGE in soleus muscle of both control and hindlimb-unloaded rats. 14 days of hindlimb unloading led to the increased maximum sliding velocity of actin, reconstituted, and native thin filaments over rat soleus muscle myosin by 24 %, 19 %, and 20 %, respectively. The calcium sensitivity of the “pCa-velocity” relationship decreased. There was a 26 % increase in fast myosin heavy chain IIa (MHC IIa), a 22 % increase in fast myosin light chain 2 (MLC 2f), and a 13 % increase in fast MLC 1f content. The content of MLC 1s/v, typical for slow skeletal muscles and cardiac ventricles did not change. At the same time, MLC 1s, typical only for slow skeletal muscles, disappeared. The maximum velocity of soleus muscle native thin filaments was 24 % higher compared to control ones sliding over the same rabbit myosin. Therefore, both myosin and native thin filament kinetics could influence the mechanical characteristics of the soleus muscle. Additionally, the MLC 1s and MLC 1s/v ratio may contribute to the mechanical characteristics of slow skeletal muscle, along with MHC, MLC 2, and MLC 1 slow/fast isoforms ratio.

Effects of short-term simulated microgravity on changes in extracellular space structure and substance diffusion and clearance

Substance transportation and clearance in the brain extracellular space (ECS) is crucial to brain function, but simulated microgravity disrupts substance diffusion and clearance in the brain ECS and there is limited knowledge regarding these disruptions. This study employed a tracer-based magnetic resonance imaging (MRI) technique to examine the impact of simulated microgravity on hippocampal ECS structure and the drainage of brain interstitial fluid (ISF) in a tail-suspended hindlimb-unloading rat model. The results demonstrated that under simulated microgravity conditions for 3, 7, and 14 days, the volume fraction of the hippocampal ECS increased in hindlimb unloading rats, and short-term (3 days) simulated microgravity expedited the drainage of the hippocampal ISF, but the drainage slowed under longer (7 or 14 days) simulated microgravity. These findings suggested that simulated microgravity alters the structure of the brain ECS and the speed of brain ISF drainage, which may have implications for space-flight-related brain injuries and relevant neuroprotective approaches via the brain ECS.

Cordymin alleviates osteoporosis induced by hindlimb unloading via regulating the gut - microelements -bone axis --for non-clinical studies

IntroductionThe purpose of this study was to evaluate the protective effects of cordymin on osteoporosis induced by hindlimb unloading(HLU) in rats and whether cordymin can prevent bone loss from HLU.Materials and methodsWe employed the hindlimb suspension rats model to mimic physiological changes concomitant with space travel.The mechanical strength in the femoral neck,cancellous bone volume, gut microbiota structure,serum calcium and phosphorus contents, bone mineral content and bone mineral content can be changed after hindlimb unloading. Oral cordymin was administered for 4 weeks,cordymin treatment significantly increased the mechanical strength through elevated bone volume/tissue volume (BV/TV), trabecular number (Tb. N), trabecular thickness (Tb. Th) and decreased trabecular separation (Tb. Sp).ResultsImportantly, 16 S rRNA sequencing showed cordymin treatment regulated the various genera that were imbalanced in hindlimb unloading rats. At the same time,The plasma total calcium and inorganic phosphate concentrations in hindlimb unloading rats decreased and bone mineral content in the lumbar vertebrae and femur increased after treatment with cordymin.ConclusionThese data indicate that the cordymin might exert bone protective effects indirectly via modulating the complex relationship between gut microbiota, microelements and bone loss.

Effects of exercise prehabilitation on muscle atrophy and contractile properties in hindlimb-unloaded rats.

Effective strategies for rapid recovery after surgery are needed. Therefore, we investigated the effects of exercise prehabilitation (EP) and hindlimb unloading (HU) on muscle loss and contractility. Twenty-two Sprague-Dawley rats (12 wk old) were divided into normal control (NCON, n = 5), hindlimb unloading control (HCON, n = 10), and exercise prehabilitation followed by hindlimb unloading (Ex-preH, n = 7) groups. Ex-PreH performed exercise training for 14 days before hindlimb unloading for 14 days. Body composition was evaluated, along with muscle strength and function. The soleus (SOL) and extensor digitorum longus (EDL) muscle contractile properties were analyzed at the whole-muscle level. The titin concentration and myosin heavy chain (MHC) type composition were analyzed. There were no effects of Ex-preH on total mass, lean mass, or muscle weight. Physical function was significantly higher in the Ex-preH group than in the HCON group (39.5° vs. 35.7°). The SOL twitch force (19.6 vs. 7.1 mN/m2 ) and specific force (107.3 vs. 61.2 mN/m2 ) were greater in Ex-preH group than in HCON group. EDL shortening velocity was higher in Ex-preH group than in HCON group (13.2 vs. 5.0 FL/s). The SOL full-length titin level was higher in Ex-preH group than in HCON group. Exercise prehabilitation did not prevent muscle mass loss followed by muscle wasting, although it minimized the reduction of physical function. Therefore, exercise prehabilitation should be considered for rapid functional recovery after disuse due to surgery and injuries.

Isorhamnetin Alleviates the Depression Induced by Hindlimb Unloading in Rats

Isorhamnetin Alleviates the Depression Induced by Hindlimb Unloading in Rats

The Impact of SRT2104 on Skeletal Muscle Mitochondrial Function, Redox Biology, and Loss of Muscle Mass in Hindlimb Unloaded Rats.

Mechanical unloading during microgravity causes skeletal muscle atrophy and impairs mitochondrial energetics. The elevated production of reactive oxygen species (ROS) by mitochondria and Nox2, coupled with impairment of stress protection (e.g., SIRT1, antioxidant enzymes), contribute to atrophy. We tested the hypothesis that the SIRT1 activator, SRT2104 would rescue unloading-induced mitochondrial dysfunction. Mitochondrial function in rat gastrocnemius and soleus muscles were evaluated under three conditions (10 days): ambulatory control (CON), hindlimb unloaded (HU), and hindlimb-unloaded-treated with SRT2104 (SIRT). Oxidative phosphorylation, electron transfer capacities, H2O2 production, and oxidative and antioxidant enzymes were quantified using high-resolution respirometry and colorimetry. In the gastrocnemius, (1) integrative (per mg tissue) proton LEAK was lesser in SIRT than in HU or CON; (2) intrinsic (relative to citrate synthase) maximal noncoupled electron transfer capacity (ECI+II) was lesser, while complex I-supported oxidative phosphorylation to ECI+II was greater in HU than CON; (3) the contribution of LEAK to ECI+II was greatest, but cytochrome c oxidase activity was lowest in HU. In both muscles, H2O2 production and concentration was greatest in SIRT, as was gastrocnemius superoxide dismutase activity. In the soleus, H2O2 concentration was greater in HU compared to CON. These results indicate that SRT2104 preserves mitochondrial function in unloaded skeletal muscle, suggesting its potential to support healthy muscle cells in microgravity by promoting necessary energy production in mitochondria.

Nicotinamide mononucleotide supplementation mitigates osteopenia induced by modeled microgravity in rats.

Exposure to weightlessness causes severe osteopenia, resulting in raised fracture risk. The current study aimed to investigate whether nicotinamide mononucleotide (NMN) supplementation protected against the osteopenia in hindlimb unloading (HLU) rats in vivo and modeled microgravity-induced osteoblastic dysfunction in vitro. The 3-mo-old rats were exposed to HLU and intragastrically administered NMN every 3days (500mg/kg body weight) for 4weeks. NMN supplementation mitigated HLU-induced bone loss, evidenced by greater bone mass and biomechanical properties and better trabecular bone structure. NMN supplementation mitigated HLU-induced oxidative stress, evidenced by greater levels of nicotinamide adenine dinucleotide and activities of superoxide dismutase 2 and lesser malondialdehyde levels. Modeled microgravity stimulation using rotary wall vessel bioreactor in MC3T3-E1 cells inhibited osteoblast differentiation, which was reversed by NMN treatment. Furthermore, NMN treatment mitigated microgravity-induced mitochondrial impairments, evidenced by lesser reactive oxygen species generation and greater adenosine triphosphate production, mtDNA copy number, and activities of superoxide dismutase 2 and Complex I and II. Additionally, NMN promoted activation of AMP-activated protein kinase (AMPK), evidenced by greater AMPKα phosphorylation. Our research suggested that NMN supplementation attenuated osteoblastic mitochondrial impairment and mitigated osteopenia induced by modeled microgravity.

Hawthorn leaves flavonoids attenuate cardiac remodeling induced by simulated microgravity

Context Hawthorn leaves are a kind of widely used medicinal plant in China. The major ingredient, hawthorn leaves flavonoids (HLF), have cardiotonic, cardioprotective, and vascular protective effects. Objective The study evaluated the protective role of HLF in cardiac remodelling and the underlying mechanisms under simulated microgravity by hindlimb unloading rats. Materials and methods Adult male Sprague-Dawley rats were divided into control, HLF, HU (hindlimb unloading) and HU + HLF groups (n = 8). After HU and daily intragastric administration at the dose of 100 mg/kg/d for 8 weeks, cardiac function and structure were evaluated by biochemical indices and histopathology. We identified the main active compounds and mechanisms involved in the cardioprotective effects of HLF via bioinformatics and molecular docking analysis, and relative signalling pathway activity was verified by Western blot. Results HLF treatment could reverse the HU-induced decline in LV-EF (HU, 55.13% ± 0.98% vs. HU + HLF, 71.16% ± 5.08%), LV-FS (HU, 29.44% ± 0.67% vs. HU + HLF, 41.62% ± 4.34%) and LV mass (HU, 667.99 ± 65.69 mg vs. HU + HLF, 840.02 ± 73.00 mg). Furthermore, HLF treatment significantly increased NPRA expression by 135.39%, PKG by 51.27%, decreased PDE5A by 20.03%, NFATc1 by 41.68% and Rcan1.4 by 54.22%. Conclusions HLF plays a protective effect on HU-induced cardiac remodelling by enhancing NPRA-cGMP-PKG pathway and suppressing the calcineurin-NFAT pathway, which provides a theoretical basis for use in clinical therapies.

Hydrogen Inhalation Ameliorates Oxidative Stress and Glucose Metabolism Disorder in the Brain of Hindlimb Unloading Rats

Many studies have shown that spaceflight causes oxidative stress and induces brain disorder in astronauts, but the counter measurements are lacking. Increasing evidence demonstrated that hydrogen can act as a therapeutic antioxidant. In this study, rats were treated with or without about 5% hydrogen under hindlimb unloading or normal conditions for 28 d. We assessed rat’s brain function by open-field test, step-down passive avoidance test, the neurotransmitter’s level detected by liquid chromatography with tandem mass spectrometry, and Nissl and hematoxylin–eosin staining analysis. We also assessed the oxidative damage by changes of malondialdehyde level, the ratio of reduced glutathione to oxidized glutathione, and superoxide dismutase and catalase activity. Glucose metabolism disorder was disclosed through glucose metabolomic analysis. The underlying mechanism of the effects of hydrogen was analyzed by mRNA sequencing and detecting mRNA and protein levels. Our data showed that hindlimb unloading caused oxidative damage and glucose metabolism disorder in brain tissues and decreased brain function in rats. Hydrogen inhalation reduced oxidative damage, ameliorated glucose metabolism disorder, and alleviated the dysfunction of rat brain function. Peroxisome-proliferator-activated receptor gamma coactivator 1α and brain-derived neurotrophic factor, the key regulators of glucose metabolism and brain function, were obviously affected. This research confirms the protective effect of hydrogen inhalation on declining brain function under hindlimb unloading conditions and discloses the underlying mechanism, which provides a potential strategy for astronauts’ health.

Metformin Attenuates Slow-to-Fast Fiber Shift and Proteolysis Markers Increase in Rat Soleus after 7 Days of Rat Hindlimb Unloading

Muscle unloading leads to signaling alterations that cause muscle atrophy and weakness. The cellular energy sensor AMPK can regulate myofiber-type shift, calcium-dependent signaling and ubiquitin-proteasome system markers. We hypothesized that the prevention of p-AMPK downregulation during the first week of muscle unloading would impede atrophy development and the slow-to-fast shift of soleus muscle fibers, and the aim of the study was to test this hypothesis. Thirty-two male Wistar rats were randomly assigned to four groups: placebo control (C), control rats treated with metformin (C + M), 7 days of hindlimb suspension (HS) + placebo (7HS), and 7 days of HS + metformin administration (7HS + M). In the soleus of the 7HS rats, we detected a slow-to-fast fiber-type shift as well as a significant downregulation of MEF-2D and p300 in the nuclei. In the 7HS group, we also found decreases in p-ACC (AMPK target) protein level and in the expression of E3 ubiquitin ligases and p-CaMK II protein level vs. the C group. The 7-day metformin treatment for soleus muscle unloading (1) prevented slow-to-fast fiber-type shift; (2) counteracted changes in the p-ACC protein level; (3) hindered changes in the nuclear protein level of the slow myosin expression activators MEF-2D and p300, but did not affect NFATc1 signaling; and (4) attenuated the unloading-induced upregulation of MuRF-1, atrogin-1, ubiquitin and myostatin mRNA expression, but did not prevent soleus muscle atrophy. Thus, metformin treatment during muscle disuse could be useful to prevent the decrease in the percentage of slow-type fatigue-resistant muscle fibers.

Muscle Atrophy Evaluation via Radiomics Analysis Using Ultrasound Images: A Cohort Data Study.

Existing methods for muscle atrophy evaluation based on muscle size measures from ultrasound images are inadequate in precision. Radiomics has been widely used in various medical studies, but its validity for the evaluation of muscle atrophy has not been fully explored. This study presents a radiomics analysis for muscle atrophy evaluation using ultrasound images. The hindlimb unloading rat model was developed to simulate weightlessness muscle atrophy and ultrasound images of the hind limbs were acquired for both the hindlimb unloaded (HU) and control groups during a 21-day HU period. A total of 368 radiomics features were extracted and the stable and informative features were selected through a two-stage feature selection procedure. The feature change trajectory of the stable features was analyzed using the hierarchical clustering method. Finally, an adaptive longitudinal feature selection and grading network, ALNet, was developed to evaluate muscle atrophy. The clustering trajectories of ultrasound image features showed similar trends to the changes in muscle atrophy at the molecular level. The best grading accuracy achieved by the ALNet was 79.5% for the Soleus (Sol) muscle and 82.6% for the Gastrocnemius (Gas) muscle. The test-retest is essential in performing radiomics analysis on ultrasound images. The longitudinal feature selection is important for muscle atrophy grading. The ultrasound image features of the Gas muscle have better discrimination ability than that of the Sol muscle. This study proves for the first time the capability of ultrasound image features for muscle atrophy evaluation.

Formononetin ameliorates simulated microgravity-induced bone loss by suppressing bone turnover in rats

Rapid bone loss exposed to spaceflight is one of the highest risk factors for astronauts which can increase the occurrence of fracture risk and lead to serious health problems, but the therapeutic effects of the current countermeasures still have certain limitations and deficiencies. This study aims to find novel potential drugs for the prevention of the bone loss caused by microgravity. Therefore, the systems pharmacology was utilized to discover the active natural compound formononetin in Radix Astragali as the potential drug for the prevention of bone loss. Further, we investigated the therapeutic effects of formononetin in the prevention of bone loss induced by microgravity. Here, the hind limb unloading (HLU) rats’ model was established to simulate the weightlessness, and the HLU rats were treated with formononetin (30 mg· kg−1·day−1) for 4 weeks. The results showed that formononetin treatment clearly ameliorated the microstructure of the trabecular bone, accelerated bone mineral apposition rate and increased bone mineral density, additionally improving biomechanical properties of the bone. Furthermore, the results of the bone turnover biomarkers in serum indicated that formononetin administration effectively decreased the levels of both the bone formation biomarkers and bone resorption biomarkers. In addition, we observed the levels of bone remodeling cytokines including IFN-γ, IL-6, IL-8, IL-12, IL-4, IL-10 and TNF-α were partially recovered by formononetin administration. All of the above indicated that formononetin administration could prevent bone loss induced by HLU treatment due to its ability to inhibit bone turnover and regulate bone remodeling cytokines. These results may contribute to elucidate the novel effects of formononetin against bone loss and further illustrate its potential application value as a leading compound in drug development.

Calycosin prevents bone loss induced by hindlimb unloading

Bone loss induced by microgravity exposure seriously endangers the astronauts’ health, but its countermeasures still have certain limitations. The study aims to find potential protective drugs for the prevention of the microgravity-induced bone loss. Here, we utilized the network pharmacology approach to discover a natural compound calycosin by constructing the compound-target interaction network and analyzing the topological characteristics of the network. Furthermore, the hind limb unloading (HLU) rats’ model was conducted to investigate the potential effects of calycosin in the prevention of bone loss induced by microgravity. The results indicated that calycosin treatment group significantly increased the bone mineral density (BMD), ameliorated the microstructure of femoral trabecular bone, the thickness of cortical bone and the biomechanical properties of the bone in rats, compared that in the HLU group. The analysis of bone turnover markers in serum showed that both the bone formation markers and bone resorption markers decreased after calycosin treatment. Moreover, we found that bone remodeling-related cytokines in serum including IFN-γ, IL-6, IL-8, IL-12, IL-4, IL-10 and TNF-α were partly recovered after calycosin treatment compared with HLU group. In conclusion, calycosin partly recovered hind limb unloading-induced bone loss through the regulation of bone remodeling. These results provided the evidence that calycosin might play an important role in maintaining bone mass in HLU rats, indicating its promising application in the treatment of bone loss induced by microgravity.