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Calycosin prevents bone loss induced by hindlimb unloading

Calycosin prevents bone loss induced by hindlimb unloading www.nature.com/npjmgrav ARTICLE OPEN 1,2,3,6 1,2,3,4,6 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 Xiang Jin , Hong Wang , Xuechao Liang , Kang Ru , Xiaoni Deng , Shuo Gao , Wuxia Qiu , Ying Huai , 1,2,3 1,2,3 5 1,2,3 1,2,3✉ 1,2,3✉ Jiaqi Zhang , Linbin Lai , Fan Li , Zhiping Miao , Wenjuan Zhang and Airong Qian 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. npj Microgravity (2022) 8:23 ; https://doi.org/10.1038/s41526-022-00210-x INTRODUCTION disorder after spinal cord injury . However, due to the complexity of its ingredients and the limitations of traditional drug research Bone loss induced by spaceflight has become one of the most methods, it remains difficult to find the effective compounds for important risk factors for astronauts, which threatens astronauts’ 1,2 the treatment of bone loss. At present, the network pharmacology health and limits space exploration . Among the 60 American has been applied to comprehensively determine the potential and Russian astronauts who participated in the Mir Space Station 14,15 active compounds and targets of complex drugs . Network (Mir) and the International Space Station (ISS) long-duration space pharmacology integrates network biology analysis, gene con- missions (average 176 ± 45 days), about 92% of the astronauts nectivity and redundancy, and gene pleiotropy on the basis of suffered from more than 5% bone loss in at least one skeletal part, systems biology and multi-directional pharmacology , which 40% of the astronauts suffered from more than 10% bone loss in 3 provides a new idea for drug discovery and mechanism at least one skeletal part . When the Soviet astronauts flew in 17,18 research . space for 75 to 184 days, the bone mineral density (BMD) of the In this study, we initially used the network pharmacology to calcaneus decreased 0.9–19.8%, and the spine BMD decreased identify the effective natural compounds of Radix Astragali for the about 0.3–10.8% for the astronauts who stayed at the Salute 4 prevention of bone loss. Furthermore, the HLU rat model was used Space Station for 5 to 7 months . At present, several preventative to evaluate the function of the compounds screened by network and therapeutic strategies have shown good efficacy for bone loss pharmacology analysis in bone loss. As a result, we found that induced by spaceflight, such as physical exercise, mechanical 5,6 calycosin partly recovered HLU-induced bone loss through the stimulation and drug therapy , but still have certain limitations regulation of bone remodeling. This research will provide a and significant deficiencies. Among them, drug treatments such as promising candidate for the treatment of bone loss induced by bisphosphonates have significant protective effects on bone loss spaceflight. induced by microgravity, but there were the inevitable side 7–9 effects, such as osteonecrosis of the jaw, headache and nausea . Therefore, new safer compounds could be found to prevent and METHODS treat bone loss induced by spaceflight. Active compounds and targets of Radix Astragali Recent studies demonstrated that natural small molecule drugs derived from Traditional Chinese Medicine (TCM) for homology of The chemical components of the herb in Radix Astragali were medicine and food, have good prospects in the treatment of determined through the Traditional Chinese Medicine Systems osteoporosis because of their safety, low side effects and low cost, Pharmacology Database and Analysis Platform (TCMSP, 10–12 such as curcumin, lycopene, and resveratrol, etc . Radix https://tcmspw.com/tcmsp.php): a common database for the Astragali, as a kind of TCM for homology of medicine and food, study of TCM and the components of Chinese herbal medicine. has been reported to have a positive therapeutic effect on bone To further obtain the effective compounds from Radix Astragali, Lab for Bone Metabolism, Xi’an Key Laboratory of Special Medicine and Health Engineering; Key Lab for Space Biosciences and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China. Research Center for Special Medicine and Health Systems Engineering, School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China. NPU-UAB Joint Laboratory for Bone Metabolism, School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, 4 5 China. Research Center for toxicological and biological effects, Institute for Hygiene of Ordnance Industry, Xi’an 710065, China. Hospital of Northwestern Polytechnical University, Xi’an, Shaanxi 710072, China. These authors contributed equally: Xiang Jin, Hong Wang. email: wenjuan_zhang2017@nwpu.edu.cn; qianair@nwpu.edu.cn Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; X. Jin et al. we performed an in silico ADME approach, which integrated approved by the Institutional Ethics Committee of Northwestern predict drug-likeness (PreDL), predict oral bioavailability (PreOB) Polytechnical University. and predict Caco-2 permeability (PreCaco-2). Oral bioavailability (OB), as one of the most important pharmacokinetic parameters, BMD analysis can determine whether a small molecule compound has drug After the rats were injected intraperitoneally with anesthesia and activity. Herein, we employed an in-house system OBioavail1.1 to deeply anesthetized, they were neatly arranged on test bench of perform the OB screening . Additionally, Caco-2 permeability the bone densitometer. We scanned the whole body of the rats by prediction model PreCaco-2 was developed to predict absorption the small animal scan mode using dual-energy X-ray absorptio- of the drug through a robust in silico. And the PreDL was metry (DEXA) assay (Lunar Prodigy Advance DXA, GE healthcare, developed to classify drug-like and nondrug-like chemicals by the Madison, WI, USA) . After all scans were completed, then we molecular descriptors and Tanimoto coefficient (as shown in Eq. 20 selected the femurs as the region of interest (ROI) to analyze the (1)) . BMD value using GE’s purpose-designed software (enCORE 2006, A  B GE Healthcare, Madison, WI, USA). TðA; BÞ¼ (1) 2 2 jj A þjj B A  B Micro-CT scanning where A denotes the molecular properties of ingredients in herb, The right femur fixed with 4% paraformaldehyde (PFA) was fixed and B represents the average drug-likeness index of all on the template and scanned along the long axis with a micro CT compounds in DrugBank database (http://www.drugbank.ca/). In scanner (Skyscan 1276, Bruker microCT, Kontich, Belgium) . The this work, the compounds matching OB ≥ 30%, DL ≥ 0.18 and specific parameters during the scanning: voltage was 70 kVp, Caco-2 ≥ -0.4 were screened as bioactive compounds. Further, we conduct the prediction and discovery of the targets current was 114 μA, the image pixel was 10 μm, and layer spacing of these active compounds based on a new computational model was 10 μm. Later, the scanning files were analyzed to reconstruct termed SysDT through two powerful algorithms of Random Forest and analyze the three-dimensional image. And the trabecular (RF) and Support Vector Machine (SVM), which integrates large- bone with a 2 mm thickness and 1 mm in the growth plate was scale information of chemistry, genomics and pharmacology . selected as the area of interest for further data analysis. The 3D Subsequently, we individually convert these predicted target parameters for qualitative analysis of the trabecular bone were as names into the standard gene symbols through the Uniprot follows: the bone volume per tissue volume (BV/TV; %), trabecular database (https://www.uniprot.org). number (Tb.N; 1/mm), trabecular spacing (Tb.Sp; mm), trabecular thickness (Tb.Th; mm), and trabecular pattern factor (Tb.Pf; 1/mm). The cortical bone with a 0.5 mm thickness and 4 mm below the Disease target femoral growth plate was selected as the region of interest for The targets associated to bone loss, were identified from the further data analysis. The 3D parameters for qualitative analysis of GeneCards database (https://www.genecards.org/), using “osteo- the cortical bone were the cortical thickness (Cr.Th; mm). Among porosis” as the search term, and all the genes found in the them, the 3D parameters of the trabecular bone and cortical bone database were considered as the target set of osteoporosis. were qualitatively analyzed by CTan software, and the 3D images of the trabecular bone and cortical bone were constructed by Network construction and topological analysis CTvol software and CTan software (Skyscan 1276, Bruker microCT, To analyze and find potential drugs in the treatment of bone loss, we Kontich, Belgium). constructed and analyzed the visualized compound-target (C-T) network in this study. Using all bioactive compounds of Radix Three-point bending mechanical test Astragali and the common targets of these active compounds with The mechanical properties of the left femurs were tested using a bone loss disease, a network diagram of compound-target interac- universal material testing machine (Intron company, USA). Before tions was generated, in which a compound and a target were linked mechanical testing, the left femurs stored at −80 °C were soaked with each other. By analyzing the topological properties of the C-T in saline solution to thaw for about 4 h, and then measured them network, we identified the key compounds according to the degree on the material testing machine. Each sample was placed on two values and neighborhood connectivity of nodes. The visualization of supports separated by a distance of 20 mm. The load was applied the network was achieved by the Cytoscape software. to the middle of the diaphysis and pressed down at a speed of 2 mm/min until fracture occurred . The load and displacement Animal experiments data were then recorded by acquisition computer. According to Twenty-four healthy Sprague-Dawley male rats weighing the load-displacement curve and the internal and external 200 ± 20 g (SPF standard) were kept at a suitable temperature of diameters of the fractured bone, the mechanical properties were 25 ± 3 °C with a 12 h light-dark cycle. After 7 days of adaptation, all calculated by Matlab software, including: the maximum load (N), rats were randomly divided into four groups with six rats in each 2 toughness (J/mm ), stiffness (N/mm), maximum stress (MPa) and group, including baseline group, ground control group, HLU Young’s modulus (GPa). −1 −1 group, HLU treatment group with calycosin, 30 mg· kg ·day . Among them, the rats in the baseline group were sacrificed before Dynamic bone histomorphometry hind limb unloading and preserved their tissues for further The femurs were fixed in 4% PFA and dehydrated in examination. The rats in the control group were free to move 60–70–80–90–95% alcohol, with each step taking for 24 to 72 h. without HLU, but the rats tails in the HLU and HLU+Calycosin It was then dehydrated twice with for 12 h to 24 h each time, as groups were suspended to unload their hindlimbs as suggested was xylene. Next, the femurs were put into the embedding bottle, by MoreyHolton and Globus . Calycosin was intragastrically administered once a day in the HLU+Calycosin group for 4 weeks; and adding an appropriate amount of embedding agent. Routine and an equal volume of 0.5% CMC-Na was received in the control sectioning about 10 µm by the hard tissue slicer (Leica Microtome, and HLU groups, daily, for 4 weeks. Besides, the rats of each group HistoCore AUTOCUT) and place sections in an oven at 60 °C were injected with 5 mg/kg calcein subcutaneously on days 13 overnight. The dynamic parameters of mineral deposition rate and 3 before necropsy. All procedures strictly followed the (MAR) (micrometers per day) were quantified using CaseViewer Guidelines for the Care and Use of Laboratory Animals, and were software. npj Microgravity (2022) 23 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; X. Jin et al. Fig. 1 The analysis of Network pharmacology. a Venn diagram of targets for active compounds of Radix Astragali and osteoporosis-related targets. b The visualization network of compound-target was built by Cytoscape 3.8.0. It is composed of 148 target nodes (diamond, purple), 16 compound nodes (quadrilateral, green) and 1 Chinese herbal medicine node (round, red). Serum analysis accounting for 18.4% of all 87 ingredients of Radix Astragali. Detailed information of the compounds is presented in Supple- The rats’ blood was collected from the abdominal aorta. Serum mentary Table 2. was obtained by centrifuging at 1200 rpm for 10 min at 4 °C. The concentrations of bone turnover markers and cytokines of bone remodeling in serum were measured by using rat ELISA kits, Intersection of drug-disease targets including NTX, TRACP5b, PINP, RANKL, OPG, BGP, ALP, IL-4, IL-6, IL- To obtain the potential targets of each compound in Radix 8, IL-10, IL-12, TNF-α and IFN-γ. All testing procedures were carried Astragali for the treatment of bone loss, we took the intersection out according to the manufacturer’s instructions. of compound targets and bone disease targets. Totally, we obtained 203 candidate targets of 16 compounds targets and Statistical analysis 4613 targets for the disease based on the SysDT model and Genecards database, respectively. The construction of Venn All the results are presented as the mean ± SD. Statistical analyses diagram revealed a total of 148 targets as the common target were carried out using GraphPad Prism 8.0 (GraphPad Software genes between compounds and osteoporosis disease, as shown in Inc., San Diego, CA, USA). Statistical significant differences were performed by two-tailed Student’s t test analysis between two Fig. 1a and Supplementary Table 3. In addition, a majority of groups. P < 0.05 was considered statistically significant. natural products were shown to be hit by numerous targets, indicating that the multiple pharmacological effects of these compounds. For instance, calycosin (MOL000417) can interact Reporting summary with 23 targets, and Jaranol (MOL000239) targeted on 8 different Further information on research design is available in the Nature proteins related to osteoporrosis. Among them, the information of Research Reporting Summary linked to this article. 23 interacting targets of calycosin related to osteoporosis are shown in Table 1. RESULTS Building of compound-target network to screen key The screening of active compounds in Radix Astragali compound In order to screen the potential compounds in Radix Astragali,we To further determine the key compounds in the treatment of bone initially collected all the compounds through the TCMSP database, loss, a compound-target (C-T) interaction network was constructed and then a systematic pharmacology model, combining the andanalyzedbyCytoscape software.Asshown in Fig. 1b, the absorption, distribution, metabolism, and excretion (ADME) compound-target interaction network was built, which including 149 screening model, were further employed . In total, 87 com- targets and 16 compounds. The C-T interaction network visually pounds were collected from the Radix Astragali (as displayed in Supplementary Table 1). The ADME qualities of each component, displayed the regulatory relationship between the compounds and including oral bioavailability (OB), drug-likeness (DL), and Caco-2 targets. As shown in Supplementary Table 2, the topology analysis of permeability (Caco-2), were to be examined in order to further the network displayed that quercetin (MOL000098, degree= 118), obtain potential natural products with desirable pharmacological kaempferol (MOL000422, degree= 44), 7-O-methylisomucronulatol properties. As a result, 16 potential bioactive compounds with (MOL000378, degree= 29), formononetin (MOL000392, degree= OB ≥ 30%, DL ≥ 0.18 and Caco-2 ≥ −0.40 were screened out, 28), isorhamnetin (MOL000354, degree= 26) and calycosin Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 23 X. Jin et al. Table 1. The information of 23 interacting targets of calycosin related to osteoporosis. Target name Name Uniprot ID Organism 1 ADRA1D Alpha-1D adrenergic receptor P25100 Homo sapiens 2 ADRA2C Alpha-2C adrenergic receptor P18825 Homo sapiens 3 ADRB1 Beta-1 adrenergic receptor P08588 Homo sapiens 4 HTR3A 5-hydroxytryptamine receptor 3A P46098 Homo sapiens 5ACHE Acetylcholinesterase P22303 Homo sapiens 6 ADRA1B Alpha-1B adrenergic receptor P35368 Homo sapiens 7 ADRB2 Beta-2 adrenergic receptor P07550 Homo sapiens 8 CAMKK2 Calmodulin Q96RR4 Homo sapiens 9 PDE3A CGMP-inhibited 3’,5’-cyclic phosphodiesterase A Q14432 Homo sapiens 10 ESR1 Estrogen receptor P03372 Homo sapiens 11 GABRA1 Gamma-aminobutyric acid receptor subunit alpha-1 P14867 Homo sapiens 12 CHRM1 Muscarinic acetylcholine receptor M1 P11229 Homo sapiens 13 CHRM3 Muscarinic acetylcholine receptor M3 P20309 Homo sapiens 14 NOS2 Nitric oxide synthase, inducible P35228 Homo sapiens 15 NOS3 Nitric oxide synthase, endothelial P29474 Homo sapiens 16 NCOA2 Nuclear receptor coactivator 2 Q15596 Homo sapiens 17 PTGS1 Prostaglandin G/H synthase 1 P23219 Homo sapiens 18 PTGS2 Prostaglandin G/H synthase 2 P35354 Homo sapiens 19 RXRA Retinoic acid receptor RXR-alpha P19793 Homo sapiens 20 SCN5A Sodium channel protein type 5 subunit alpha Q14524 Homo sapiens 21 F2 Prothrombin P00734 Homo sapiens 22 PRSS1 Trypsin-1 P07477 Homo sapiens 23 OPRM1 Mu-type opioid receptor P35372 Homo sapiens (MOL000417, degree= 24) exhibited the higher degree number of cortical bone thickness significantly (P < 0.01), whereas the treatment of HLU rats with calycosin significantly increased the interactions with various protein targets. Among these 6 compounds cortical bone thickness (P < 0.01) (Fig. 2i). The changes of BMD with higher degree values, calycosin showed the highest neighbor- after various treatments are shown in Fig. 2j. After hind limb hood connectivity value. Therefore, we speculated that calycosin unloading, the BMD of the HLU group were decreased signifi- might be the key compound in the treatment of bone loss. cantly compared with that in the control group (P < 0.01), indicating HLU gave rise to the reduction of bone mass in rats. Effects of calycosin on bone microstructure and BMD Rats treated with calycosin obviously increased the BMD values Based on the above results of network pharmacology, a natural compared with HLU rats (P < 0.01), indicating that calycosin might bioactive compound calycosin for the protection against bone loss play an important role in maintaining bone mass of tail suspended was screened out. To directly investigate the effects of calycosin rats. against bone loss in vivo, the HLU rat model was employed to mimic the bone loss induced by spaceflight microgravity. The Effect of calycosin on bone biomechanics microarchitectural changes in the femoral diaphysis were assessed To further evaluate the effect of calycosin on the biomechanical using Micro-CT, as a measure of bone quality. The representative properties, we performed a three-point bending test on the mid- three-dimensional (3D) images of the cancellous bone and cortical diaphysis femurs of rats. The results of three-point bending test bone of the femoral metaphysis in the right femur are shown in are shown in Fig. 3. Compared with the control group, the Fig. 2a, b, respectively. The trabecular Tb. Th, BV/TV and femurs biomechanical parameters of maximum stress, maximum load, bone mineral density (BMD) of the control group were increased stiffness, Young’s modulus and toughness in the HLU group were compared to baseline and the trabecular Tb. Pf in the control obviously decreased (P < 0.05 or P < 0.01) (Fig. 3a–e). Furthermore, group were decreased compared with baseline group, indicating treatment with calycosin significantly prevented the HLU-induced that the skeleton grows in the normal environment during decrease of Young’s modulus, toughness and stiffness (P < 0.05), 4 weeks (Fig. 2e–g, j). The trabecular bones of the control group while the maximum stress and maximum load did not change are tightly connected and the mesh structure is complete. notably (P > 0.05). Compared with the control group, the trabecular bone parameters BV/TV, Tb. Th, Tb. N were significantly decreased and Tb. Sp, Tb. Pf were significantly increased in the HLU group (P < 0.01), indicating Effect of calycosin on bone histomorphometry that hind limb unloading caused severe damage to the cancellous Bone histomorphology can dynamically reflect the histophysiolo- bone (Fig. 2d–h). However, the trabecular bone parameters of BV/ gical characteristics and changes of bone, and accurately depict TV, Tb. Th and Tb. N were significantly increased by calycosin the pathological dynamics process of bone repair and reconstruc- treatment (P < 0.01) and the Tb. Sp, Tb. Pf were significantly tion. The distance between two labeled calcein fluorescence lines reduced compared with HLU rats (P < 0.01), indicating that the in bone tissue was monitored dynamically in this work to microarchitectural properties were improved after treatment with determine bone formation. Figure 4a shows the representative calycosin. In addition, the analysis of cortical bone characteristics images of bone deposition line in each group. As shown in Fig. 4b, in the diaphyseal region showed that HLU caused a reduction of further quantitative analysis of calcein double labeling npj Microgravity (2022) 23 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA X. Jin et al. Fig. 2 Effects of calycosin on bone microstructure and BMD. Representative Micro-CT 3D images of trabecular bone of distal femurs (a) and cortical bone of diaphyseal femurs (b) within various groups. c Representative section images of trabecular bones of distal femurs. d Quantitative analysis of the trabecular number (Tb.N) (d), trabecular thickness (Tb.Th) (e), bone volume per tissue volume (BV/TV) (f), trabecular bone pattern factor (Tb.Pf) (g), trabecular spacing (Tb.Sp) (h), cortical thickness (Cr.Th) (i) in cortical bone and BMD (j) in trabecular bone. Here and in succeeding bar charts with dot, the centre line of the whiskers, which is also the upper bound of the box, depicts the group mean, and the whiskers correspond to the standard deviation (SD). The results are represented as the mean ± SD; n = 6/group. P-values # ## calculated using a two-tailed Student’s t test (^P < 0.05, ^^P < 0.01 v.s. baseline; P < 0.05, P < 0.01 v.s. control; *P < 0.05, **P < 0.01 v.s. HLU). demonstrated that the rats in the HLU group displayed an turnover biomarkers in the rats’ serum were detected. As shown in obviously lower mineral apposition rate (MAR) compared with that Fig. 5, in comparison, the serum levels of PINP, BGP, ALP, RANKL, in the control group (P < 0.01). Yet, calycosin administration NTX, TRACP5b and the ratio of RANKL/OPG in the HLU group increased the rate of calcification in the HLU + Calycosin group significantly increased compared that in the control group compared with that in the HLU group (P < 0.01). (P < 0.01 or P < 0.05), indicating that the lack of load caused the bone metabolism of rats to be in a state of high bone turnover (Fig. 5a–g). However, compared with HLU rats, treatment with Effect of calycosin on bone turnover markers calycosin did not significantly alter the levels of ALP (P > 0.05), but The results above showed that calycosin might play an important obviously suppressed the HLU-induced content elevation of role in maintaining bone mass of tail suspended rats. To further RANKL, PINP, BGP, NTX, TRACP5b and ratio elevation of RANKL/ elucidate the underlying mechanism, the changes of the bone Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 23 X. Jin et al. Fig. 3 Effect of calycosin on bone biomechanics. Evaluation parameters in the four groups of rats included the following: maximum stress (a), Young’s modulus (b), maximum load (c), stiffness (d), and toughness (e). The results are represented as the mean ± SD; n = 6/group. P- # ## values calculated using a two-tailed Student’s t test (^P < 0.05, ^^P < 0.01 v.s. baseline; P < 0.05, P < 0.01 v.s. control; *P < 0.05, **P < 0.01 v.s. HLU). Fig. 4 Effect of calycosin on bone histomorphology. a Representative fluorescence micrographs of trabecular bone sections showing green calcein labels within various groups (scale bar: 200 μm). b Quantitative analysis of the mineral deposition rate (MAR). The results are represented as the mean ± SD; n = 3/group. P-values calculated using a two-tailed Student’s t test (^P < 0.05, ^^P < 0.01 v.s. baseline; P < 0.05, ## P < 0.01 v.s. control; *P < 0.05, **P < 0.01 v.s. HLU). OPG in the serum (P < 0.01 or P < 0.05), indicating that calycosin calycosin significantly decreased the levels of TNF-α,IFN-γ,IL-6, could reduce the high bone turnover caused by tail suspension to IL-8 and IL-12, and increased the content of IL-4 and IL-10 a certain extent (Fig. 5a–g). notably compared with that in the HLU group (P <0.01). The above results showed that calycosin administration could up- regulate the content of IL-4 and IL-10 and down-regulate the Effect of calycosin on bone remodeling-related cytokines content of TNF-α,IFN-γ, IL-6, IL-8 and IL-12 in HLU rats. To evaluate the effects of calycosin on cytokines related to According to the above results, it could be seen that calycosin bone remodeling, the levels of IL-4, IL-6, IL-8, IL-10, IL-12, TNF-α reduced the ratio of RANKL/OPG in serum. While IL-10, IL-12 and IFN-γ in serum were detected using ELISA (Fig. 6). An and IFN-γ likely affect the secretion of RANKL/RANK/OPG, increasing trend in the content of TNF-α,IFN-γ, IL-6, IL-8 and IL- further regulating the process of bone remodeling. These 12 wasrecordedinthe HLU groupascomparedtothose in the results suggested that calycosin might regulate the process of control group (P < 0.01), while the levels of IL-4 and IL-10 were bone metabolism through bone immunity. declined (P <0.01) (Fig. 6a–g). Nevertheless, treatment with npj Microgravity (2022) 23 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA X. Jin et al. Fig. 5 Effect of calycosin on bone turnover markers. The bone turnover biomarkers levels in rats serum of ALP (a), BGP (b), PINP (c), TRACP 5b (d), RANKL (e), NTX (f) and RANKL/OPG (g) in the four groups. The results are represented as the mean ± SD; n = 6/group. P-values # ## calculated using a two-tailed Student’s t test (^P < 0.05, ^^P < 0.01 v.s. baseline; P < 0.05, P < 0.01 v.s. control; *P < 0.05, **P < 0.01 v.s. HLU). DISCUSSION that it is effective and feasible to discover new drugs through the method of network pharmacology. In this research, we utilized the Bone loss induced by microgravity has become one of the network pharmacology approach to uncover a potential small important threats to the health of astronauts and the major molecule compound-calycosin for treating bone loss. obstacle to long-term space flight. Therefore, the study aims to Previous studies have shown that calycosin have a dose- find potential protective drugs with low side effects for the dependent therapeutic effect on postmenopausal osteoporosis treatment of bone loss induced by microgravity. In the present rats and regulates the expression of OPG/RANKL via MAPK work, we preliminarily screened calycosin as a potential com- signaling . However, there has been no direct evidence of the pound for the treatment of bone loss through the network protective effects of calycosin on bone loss induced by micro- pharmacology, and further found calycosin might play an gravity in hind limb unloading rats. In this study, the Micro CT important role in maintaining bone mass in HLU rats due to the analysis showed that calycosin administration increased BMD, the improvement of bone mineral density, microstructure, ameliorated the microstructure of femoral trabecular bone and biomechanical properties and mineral apposition rate of bone; increased the thickness of cortical bone in HLU rats. The MAR of the effects were mainly owing to inhibit bone turnover and reduce bone is reflected by the distance between two injections of enhanced bone remodeling by HLU. The findings may provide a calcein fluorescent labeling in bone tissue. The results demon- potential and promising candidate in the treatment of weightless- strated that the mineral apposition rate of the bone was increased induced bone loss. by calycosin treatment, which illustrated its role in promoting the The discovery of new small molecule drugs for weightless bone bone formation to a certain extent. Meanwhile, the analysis of loss not only provides effective medical protection for astronauts, three-point bending test showed that calycosin treatment could but also has positive significance for the development of increase the bone biomechanical parameters toughness, stiffness aerospace medicine and aerospace industry. The network pharmacology provides a new idea and strategy for drug and Young’s modulus, but there were no significant changes in discovery. For example, Zhiguo Zhong identified quercetin as Max-load and Max-stress, indicating that calycosin administration the potential ingredient of Erzhi Pill (EZP) for the treatment of could improve biomechanical properties to a certain degree. bone loss through the method of system pharmacology, and Taken together, these findings suggested that calycosin might further in vivo research verified that quercetin owned positive play an important role in maintaining bone mass of tail effects on osteoblast differentiation of skull . This demonstrate suspended rats. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 23 X. Jin et al. Fig. 6 Effect of calycosin on bone remodeling-related cytokines. The levels of bone remodeling-related cytokines in rats serum IFN-γ (a), IL-6 (b), IL-8 (c), IL-12 (d), TNF-α (e), IL-4 (f) and IL-10 (g) in the four groups. The results are represented as the mean ± SD; n = 6/group. P-values # ## calculated using a two-tailed Student’s t test (^P < 0.05, ^^P < 0.01 v.s. baseline; P < 0.05, P < 0.01 v.s. control; *P < 0.05, **P < 0.01 v.s. HLU). Aside from the well-documented development of bone loss ,it metabolism induced by HLU. Based on the results, it could be is also well known that there is deleterious changes in immune observed that calycosin administration reduced the enhanced 29,30 function and metabolic disruptions caused by spacelight . bone remodeling caused by HLU. Recent studies have shown that Thence, to further investigate the underlying mechanism of the bone system can be regulated by the immune system, since calycosin in the prevention of bone loss, we detected the both of them shared the same bone marrow microenviron- 32–35 expressions of bone metabolism markers and bone remodeling ment . Specifically, the immune T cells and B cells regulate cytokines. Serum bone turnover biomarkers can not only reflect osteocytes through the secreted positive and negative cytokines, the therapeutic efficacy of drugs that prevent bone loss, but also which in turn directly or indirectly regulates the coupling balance reveal the pathogenesis of metabolic bone. Bone turnover of bone formation and bone resorption, affecting bone metabo- 34–36 biomarkers include bone formation markers and bone resorption lism . For example, T helper 17 (Th-17) cells can not only markers . Among them, RANKL, NTX and TRACP5b are markers of induce osteoblasts to express RANKL to promote bone resorption bone resorption, and BGP, ALP and PINP are markers of bone by secreting IL-17, but also stimulate immune cells to produce IL- formation. In this study, both the levels of bone resorption and 1, IL-6 and other inflammatory cytokines to enhance bone bone formation markers were increased in the HLU group absorption through activating RANK signaling, resulting in bone 37,38 compared with that in the control group, which revealed that loss . In this study, the levels of IL-4 and IL-10 in the HLU group the lack of load led to an increase in bone turnover, indicating that were decreased compared with the control group, whereas the bone homeostasis and the bone metabolism were altered in a concentration of IL-6, IL-8, IL-12, TNF-α and IFN-γ in the HLU group state of high bone turnover. And the elevated levels of bone were increased compared with that in the control group. The formation biomarkers in the HLU group might be due to that changes of cytokines indicated that the lack of load might initiate osteoblasts attempted to make a compensatory for bone loss the inflammatory changes of bone microenvironment state, induced by tail suspension. Calycosin administration obviously inducing a rapid phase of bone loss. Treatment with calycosin inhibited the increase in the serum levels of RANKL, NTX, significantly increased the level of IL-4 in serum, indicating that TRACP5b, BGP, and PINP caused by HLU, whereas did not calycosin might induce Treg cells to produce IL-4, further significantly change the expression of ALP. The main reason regulating the balance of the bone remodeling system . might be that there was a compensatory response in the rats Calycosin significantly increased the content of IL-10, which could body. These results above suggested that calycosin might play an up-regulate the expression of OPG and down-regulate the important role in suppressing the high bone turnover and bone expression of RANKL, and inhibit the differentiation and npj Microgravity (2022) 23 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA X. Jin et al. maturation of osteoclasts, further regulated bone remodeling 11. Gezici, S. & Sekeroglu, N. Current perspectives in the application of medicinal plants against cancer: novel therapeutic agents. Anticancer. Agents Med. Chem. through the pathway RANKL/RANK/OPG . In addition, IL-10 also 19, 101–111 (2019). could inhibit the secretion of cytokines IL-6 and TNF-α that 12. Gullett, N. P. et al. Cancer prevention with natural compounds. Semin. Oncol. 37, promoted bone resorption . In general, unlike the aforemen- 258–281 (2010). tioned IL-6 and TNF-α, IL-10 is a cytokine that protects bone 13. Zhang, Y. N. et al. 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ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China under Open Access This article is licensed under a Creative Commons No. 81901917 and No. 82072106; the Key Research and Development Project of Attribution 4.0 International License, which permits use, sharing, Shaanxi Province under No. 2022SF-295 and No. 2018SF-363; the Fundamental adaptation, distribution and reproduction in any medium or format, as long as you give Research Funds for the Central Universities under No. D5000210746; the National appropriate credit to the original author(s) and the source, provide a link to the Creative Program of Innovation and Entrepreneurship for Undergraduates under No. Commons license, and indicate if changes were made. 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Calycosin prevents bone loss induced by hindlimb unloading

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www.nature.com/npjmgrav ARTICLE OPEN 1,2,3,6 1,2,3,4,6 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 1,2,3 Xiang Jin , Hong Wang , Xuechao Liang , Kang Ru , Xiaoni Deng , Shuo Gao , Wuxia Qiu , Ying Huai , 1,2,3 1,2,3 5 1,2,3 1,2,3✉ 1,2,3✉ Jiaqi Zhang , Linbin Lai , Fan Li , Zhiping Miao , Wenjuan Zhang and Airong Qian 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. npj Microgravity (2022) 8:23 ; https://doi.org/10.1038/s41526-022-00210-x INTRODUCTION disorder after spinal cord injury . However, due to the complexity of its ingredients and the limitations of traditional drug research Bone loss induced by spaceflight has become one of the most methods, it remains difficult to find the effective compounds for important risk factors for astronauts, which threatens astronauts’ 1,2 the treatment of bone loss. At present, the network pharmacology health and limits space exploration . Among the 60 American has been applied to comprehensively determine the potential and Russian astronauts who participated in the Mir Space Station 14,15 active compounds and targets of complex drugs . Network (Mir) and the International Space Station (ISS) long-duration space pharmacology integrates network biology analysis, gene con- missions (average 176 ± 45 days), about 92% of the astronauts nectivity and redundancy, and gene pleiotropy on the basis of suffered from more than 5% bone loss in at least one skeletal part, systems biology and multi-directional pharmacology , which 40% of the astronauts suffered from more than 10% bone loss in 3 provides a new idea for drug discovery and mechanism at least one skeletal part . When the Soviet astronauts flew in 17,18 research . space for 75 to 184 days, the bone mineral density (BMD) of the In this study, we initially used the network pharmacology to calcaneus decreased 0.9–19.8%, and the spine BMD decreased identify the effective natural compounds of Radix Astragali for the about 0.3–10.8% for the astronauts who stayed at the Salute 4 prevention of bone loss. Furthermore, the HLU rat model was used Space Station for 5 to 7 months . At present, several preventative to evaluate the function of the compounds screened by network and therapeutic strategies have shown good efficacy for bone loss pharmacology analysis in bone loss. As a result, we found that induced by spaceflight, such as physical exercise, mechanical 5,6 calycosin partly recovered HLU-induced bone loss through the stimulation and drug therapy , but still have certain limitations regulation of bone remodeling. This research will provide a and significant deficiencies. Among them, drug treatments such as promising candidate for the treatment of bone loss induced by bisphosphonates have significant protective effects on bone loss spaceflight. induced by microgravity, but there were the inevitable side 7–9 effects, such as osteonecrosis of the jaw, headache and nausea . Therefore, new safer compounds could be found to prevent and METHODS treat bone loss induced by spaceflight. Active compounds and targets of Radix Astragali Recent studies demonstrated that natural small molecule drugs derived from Traditional Chinese Medicine (TCM) for homology of The chemical components of the herb in Radix Astragali were medicine and food, have good prospects in the treatment of determined through the Traditional Chinese Medicine Systems osteoporosis because of their safety, low side effects and low cost, Pharmacology Database and Analysis Platform (TCMSP, 10–12 such as curcumin, lycopene, and resveratrol, etc . Radix https://tcmspw.com/tcmsp.php): a common database for the Astragali, as a kind of TCM for homology of medicine and food, study of TCM and the components of Chinese herbal medicine. has been reported to have a positive therapeutic effect on bone To further obtain the effective compounds from Radix Astragali, Lab for Bone Metabolism, Xi’an Key Laboratory of Special Medicine and Health Engineering; Key Lab for Space Biosciences and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China. Research Center for Special Medicine and Health Systems Engineering, School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China. NPU-UAB Joint Laboratory for Bone Metabolism, School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, 4 5 China. Research Center for toxicological and biological effects, Institute for Hygiene of Ordnance Industry, Xi’an 710065, China. Hospital of Northwestern Polytechnical University, Xi’an, Shaanxi 710072, China. These authors contributed equally: Xiang Jin, Hong Wang. email: wenjuan_zhang2017@nwpu.edu.cn; qianair@nwpu.edu.cn Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; X. Jin et al. we performed an in silico ADME approach, which integrated approved by the Institutional Ethics Committee of Northwestern predict drug-likeness (PreDL), predict oral bioavailability (PreOB) Polytechnical University. and predict Caco-2 permeability (PreCaco-2). Oral bioavailability (OB), as one of the most important pharmacokinetic parameters, BMD analysis can determine whether a small molecule compound has drug After the rats were injected intraperitoneally with anesthesia and activity. Herein, we employed an in-house system OBioavail1.1 to deeply anesthetized, they were neatly arranged on test bench of perform the OB screening . Additionally, Caco-2 permeability the bone densitometer. We scanned the whole body of the rats by prediction model PreCaco-2 was developed to predict absorption the small animal scan mode using dual-energy X-ray absorptio- of the drug through a robust in silico. And the PreDL was metry (DEXA) assay (Lunar Prodigy Advance DXA, GE healthcare, developed to classify drug-like and nondrug-like chemicals by the Madison, WI, USA) . After all scans were completed, then we molecular descriptors and Tanimoto coefficient (as shown in Eq. 20 selected the femurs as the region of interest (ROI) to analyze the (1)) . BMD value using GE’s purpose-designed software (enCORE 2006, A  B GE Healthcare, Madison, WI, USA). TðA; BÞ¼ (1) 2 2 jj A þjj B A  B Micro-CT scanning where A denotes the molecular properties of ingredients in herb, The right femur fixed with 4% paraformaldehyde (PFA) was fixed and B represents the average drug-likeness index of all on the template and scanned along the long axis with a micro CT compounds in DrugBank database (http://www.drugbank.ca/). In scanner (Skyscan 1276, Bruker microCT, Kontich, Belgium) . The this work, the compounds matching OB ≥ 30%, DL ≥ 0.18 and specific parameters during the scanning: voltage was 70 kVp, Caco-2 ≥ -0.4 were screened as bioactive compounds. Further, we conduct the prediction and discovery of the targets current was 114 μA, the image pixel was 10 μm, and layer spacing of these active compounds based on a new computational model was 10 μm. Later, the scanning files were analyzed to reconstruct termed SysDT through two powerful algorithms of Random Forest and analyze the three-dimensional image. And the trabecular (RF) and Support Vector Machine (SVM), which integrates large- bone with a 2 mm thickness and 1 mm in the growth plate was scale information of chemistry, genomics and pharmacology . selected as the area of interest for further data analysis. The 3D Subsequently, we individually convert these predicted target parameters for qualitative analysis of the trabecular bone were as names into the standard gene symbols through the Uniprot follows: the bone volume per tissue volume (BV/TV; %), trabecular database (https://www.uniprot.org). number (Tb.N; 1/mm), trabecular spacing (Tb.Sp; mm), trabecular thickness (Tb.Th; mm), and trabecular pattern factor (Tb.Pf; 1/mm). The cortical bone with a 0.5 mm thickness and 4 mm below the Disease target femoral growth plate was selected as the region of interest for The targets associated to bone loss, were identified from the further data analysis. The 3D parameters for qualitative analysis of GeneCards database (https://www.genecards.org/), using “osteo- the cortical bone were the cortical thickness (Cr.Th; mm). Among porosis” as the search term, and all the genes found in the them, the 3D parameters of the trabecular bone and cortical bone database were considered as the target set of osteoporosis. were qualitatively analyzed by CTan software, and the 3D images of the trabecular bone and cortical bone were constructed by Network construction and topological analysis CTvol software and CTan software (Skyscan 1276, Bruker microCT, To analyze and find potential drugs in the treatment of bone loss, we Kontich, Belgium). constructed and analyzed the visualized compound-target (C-T) network in this study. Using all bioactive compounds of Radix Three-point bending mechanical test Astragali and the common targets of these active compounds with The mechanical properties of the left femurs were tested using a bone loss disease, a network diagram of compound-target interac- universal material testing machine (Intron company, USA). Before tions was generated, in which a compound and a target were linked mechanical testing, the left femurs stored at −80 °C were soaked with each other. By analyzing the topological properties of the C-T in saline solution to thaw for about 4 h, and then measured them network, we identified the key compounds according to the degree on the material testing machine. Each sample was placed on two values and neighborhood connectivity of nodes. The visualization of supports separated by a distance of 20 mm. The load was applied the network was achieved by the Cytoscape software. to the middle of the diaphysis and pressed down at a speed of 2 mm/min until fracture occurred . The load and displacement Animal experiments data were then recorded by acquisition computer. According to Twenty-four healthy Sprague-Dawley male rats weighing the load-displacement curve and the internal and external 200 ± 20 g (SPF standard) were kept at a suitable temperature of diameters of the fractured bone, the mechanical properties were 25 ± 3 °C with a 12 h light-dark cycle. After 7 days of adaptation, all calculated by Matlab software, including: the maximum load (N), rats were randomly divided into four groups with six rats in each 2 toughness (J/mm ), stiffness (N/mm), maximum stress (MPa) and group, including baseline group, ground control group, HLU Young’s modulus (GPa). −1 −1 group, HLU treatment group with calycosin, 30 mg· kg ·day . Among them, the rats in the baseline group were sacrificed before Dynamic bone histomorphometry hind limb unloading and preserved their tissues for further The femurs were fixed in 4% PFA and dehydrated in examination. The rats in the control group were free to move 60–70–80–90–95% alcohol, with each step taking for 24 to 72 h. without HLU, but the rats tails in the HLU and HLU+Calycosin It was then dehydrated twice with for 12 h to 24 h each time, as groups were suspended to unload their hindlimbs as suggested was xylene. Next, the femurs were put into the embedding bottle, by MoreyHolton and Globus . Calycosin was intragastrically administered once a day in the HLU+Calycosin group for 4 weeks; and adding an appropriate amount of embedding agent. Routine and an equal volume of 0.5% CMC-Na was received in the control sectioning about 10 µm by the hard tissue slicer (Leica Microtome, and HLU groups, daily, for 4 weeks. Besides, the rats of each group HistoCore AUTOCUT) and place sections in an oven at 60 °C were injected with 5 mg/kg calcein subcutaneously on days 13 overnight. The dynamic parameters of mineral deposition rate and 3 before necropsy. All procedures strictly followed the (MAR) (micrometers per day) were quantified using CaseViewer Guidelines for the Care and Use of Laboratory Animals, and were software. npj Microgravity (2022) 23 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; X. Jin et al. Fig. 1 The analysis of Network pharmacology. a Venn diagram of targets for active compounds of Radix Astragali and osteoporosis-related targets. b The visualization network of compound-target was built by Cytoscape 3.8.0. It is composed of 148 target nodes (diamond, purple), 16 compound nodes (quadrilateral, green) and 1 Chinese herbal medicine node (round, red). Serum analysis accounting for 18.4% of all 87 ingredients of Radix Astragali. Detailed information of the compounds is presented in Supple- The rats’ blood was collected from the abdominal aorta. Serum mentary Table 2. was obtained by centrifuging at 1200 rpm for 10 min at 4 °C. The concentrations of bone turnover markers and cytokines of bone remodeling in serum were measured by using rat ELISA kits, Intersection of drug-disease targets including NTX, TRACP5b, PINP, RANKL, OPG, BGP, ALP, IL-4, IL-6, IL- To obtain the potential targets of each compound in Radix 8, IL-10, IL-12, TNF-α and IFN-γ. All testing procedures were carried Astragali for the treatment of bone loss, we took the intersection out according to the manufacturer’s instructions. of compound targets and bone disease targets. Totally, we obtained 203 candidate targets of 16 compounds targets and Statistical analysis 4613 targets for the disease based on the SysDT model and Genecards database, respectively. The construction of Venn All the results are presented as the mean ± SD. Statistical analyses diagram revealed a total of 148 targets as the common target were carried out using GraphPad Prism 8.0 (GraphPad Software genes between compounds and osteoporosis disease, as shown in Inc., San Diego, CA, USA). Statistical significant differences were performed by two-tailed Student’s t test analysis between two Fig. 1a and Supplementary Table 3. In addition, a majority of groups. P < 0.05 was considered statistically significant. natural products were shown to be hit by numerous targets, indicating that the multiple pharmacological effects of these compounds. For instance, calycosin (MOL000417) can interact Reporting summary with 23 targets, and Jaranol (MOL000239) targeted on 8 different Further information on research design is available in the Nature proteins related to osteoporrosis. Among them, the information of Research Reporting Summary linked to this article. 23 interacting targets of calycosin related to osteoporosis are shown in Table 1. RESULTS Building of compound-target network to screen key The screening of active compounds in Radix Astragali compound In order to screen the potential compounds in Radix Astragali,we To further determine the key compounds in the treatment of bone initially collected all the compounds through the TCMSP database, loss, a compound-target (C-T) interaction network was constructed and then a systematic pharmacology model, combining the andanalyzedbyCytoscape software.Asshown in Fig. 1b, the absorption, distribution, metabolism, and excretion (ADME) compound-target interaction network was built, which including 149 screening model, were further employed . In total, 87 com- targets and 16 compounds. The C-T interaction network visually pounds were collected from the Radix Astragali (as displayed in Supplementary Table 1). The ADME qualities of each component, displayed the regulatory relationship between the compounds and including oral bioavailability (OB), drug-likeness (DL), and Caco-2 targets. As shown in Supplementary Table 2, the topology analysis of permeability (Caco-2), were to be examined in order to further the network displayed that quercetin (MOL000098, degree= 118), obtain potential natural products with desirable pharmacological kaempferol (MOL000422, degree= 44), 7-O-methylisomucronulatol properties. As a result, 16 potential bioactive compounds with (MOL000378, degree= 29), formononetin (MOL000392, degree= OB ≥ 30%, DL ≥ 0.18 and Caco-2 ≥ −0.40 were screened out, 28), isorhamnetin (MOL000354, degree= 26) and calycosin Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 23 X. Jin et al. Table 1. The information of 23 interacting targets of calycosin related to osteoporosis. Target name Name Uniprot ID Organism 1 ADRA1D Alpha-1D adrenergic receptor P25100 Homo sapiens 2 ADRA2C Alpha-2C adrenergic receptor P18825 Homo sapiens 3 ADRB1 Beta-1 adrenergic receptor P08588 Homo sapiens 4 HTR3A 5-hydroxytryptamine receptor 3A P46098 Homo sapiens 5ACHE Acetylcholinesterase P22303 Homo sapiens 6 ADRA1B Alpha-1B adrenergic receptor P35368 Homo sapiens 7 ADRB2 Beta-2 adrenergic receptor P07550 Homo sapiens 8 CAMKK2 Calmodulin Q96RR4 Homo sapiens 9 PDE3A CGMP-inhibited 3’,5’-cyclic phosphodiesterase A Q14432 Homo sapiens 10 ESR1 Estrogen receptor P03372 Homo sapiens 11 GABRA1 Gamma-aminobutyric acid receptor subunit alpha-1 P14867 Homo sapiens 12 CHRM1 Muscarinic acetylcholine receptor M1 P11229 Homo sapiens 13 CHRM3 Muscarinic acetylcholine receptor M3 P20309 Homo sapiens 14 NOS2 Nitric oxide synthase, inducible P35228 Homo sapiens 15 NOS3 Nitric oxide synthase, endothelial P29474 Homo sapiens 16 NCOA2 Nuclear receptor coactivator 2 Q15596 Homo sapiens 17 PTGS1 Prostaglandin G/H synthase 1 P23219 Homo sapiens 18 PTGS2 Prostaglandin G/H synthase 2 P35354 Homo sapiens 19 RXRA Retinoic acid receptor RXR-alpha P19793 Homo sapiens 20 SCN5A Sodium channel protein type 5 subunit alpha Q14524 Homo sapiens 21 F2 Prothrombin P00734 Homo sapiens 22 PRSS1 Trypsin-1 P07477 Homo sapiens 23 OPRM1 Mu-type opioid receptor P35372 Homo sapiens (MOL000417, degree= 24) exhibited the higher degree number of cortical bone thickness significantly (P < 0.01), whereas the treatment of HLU rats with calycosin significantly increased the interactions with various protein targets. Among these 6 compounds cortical bone thickness (P < 0.01) (Fig. 2i). The changes of BMD with higher degree values, calycosin showed the highest neighbor- after various treatments are shown in Fig. 2j. After hind limb hood connectivity value. Therefore, we speculated that calycosin unloading, the BMD of the HLU group were decreased signifi- might be the key compound in the treatment of bone loss. cantly compared with that in the control group (P < 0.01), indicating HLU gave rise to the reduction of bone mass in rats. Effects of calycosin on bone microstructure and BMD Rats treated with calycosin obviously increased the BMD values Based on the above results of network pharmacology, a natural compared with HLU rats (P < 0.01), indicating that calycosin might bioactive compound calycosin for the protection against bone loss play an important role in maintaining bone mass of tail suspended was screened out. To directly investigate the effects of calycosin rats. against bone loss in vivo, the HLU rat model was employed to mimic the bone loss induced by spaceflight microgravity. The Effect of calycosin on bone biomechanics microarchitectural changes in the femoral diaphysis were assessed To further evaluate the effect of calycosin on the biomechanical using Micro-CT, as a measure of bone quality. The representative properties, we performed a three-point bending test on the mid- three-dimensional (3D) images of the cancellous bone and cortical diaphysis femurs of rats. The results of three-point bending test bone of the femoral metaphysis in the right femur are shown in are shown in Fig. 3. Compared with the control group, the Fig. 2a, b, respectively. The trabecular Tb. Th, BV/TV and femurs biomechanical parameters of maximum stress, maximum load, bone mineral density (BMD) of the control group were increased stiffness, Young’s modulus and toughness in the HLU group were compared to baseline and the trabecular Tb. Pf in the control obviously decreased (P < 0.05 or P < 0.01) (Fig. 3a–e). Furthermore, group were decreased compared with baseline group, indicating treatment with calycosin significantly prevented the HLU-induced that the skeleton grows in the normal environment during decrease of Young’s modulus, toughness and stiffness (P < 0.05), 4 weeks (Fig. 2e–g, j). The trabecular bones of the control group while the maximum stress and maximum load did not change are tightly connected and the mesh structure is complete. notably (P > 0.05). Compared with the control group, the trabecular bone parameters BV/TV, Tb. Th, Tb. N were significantly decreased and Tb. Sp, Tb. Pf were significantly increased in the HLU group (P < 0.01), indicating Effect of calycosin on bone histomorphometry that hind limb unloading caused severe damage to the cancellous Bone histomorphology can dynamically reflect the histophysiolo- bone (Fig. 2d–h). However, the trabecular bone parameters of BV/ gical characteristics and changes of bone, and accurately depict TV, Tb. Th and Tb. N were significantly increased by calycosin the pathological dynamics process of bone repair and reconstruc- treatment (P < 0.01) and the Tb. Sp, Tb. Pf were significantly tion. The distance between two labeled calcein fluorescence lines reduced compared with HLU rats (P < 0.01), indicating that the in bone tissue was monitored dynamically in this work to microarchitectural properties were improved after treatment with determine bone formation. Figure 4a shows the representative calycosin. In addition, the analysis of cortical bone characteristics images of bone deposition line in each group. As shown in Fig. 4b, in the diaphyseal region showed that HLU caused a reduction of further quantitative analysis of calcein double labeling npj Microgravity (2022) 23 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA X. Jin et al. Fig. 2 Effects of calycosin on bone microstructure and BMD. Representative Micro-CT 3D images of trabecular bone of distal femurs (a) and cortical bone of diaphyseal femurs (b) within various groups. c Representative section images of trabecular bones of distal femurs. d Quantitative analysis of the trabecular number (Tb.N) (d), trabecular thickness (Tb.Th) (e), bone volume per tissue volume (BV/TV) (f), trabecular bone pattern factor (Tb.Pf) (g), trabecular spacing (Tb.Sp) (h), cortical thickness (Cr.Th) (i) in cortical bone and BMD (j) in trabecular bone. Here and in succeeding bar charts with dot, the centre line of the whiskers, which is also the upper bound of the box, depicts the group mean, and the whiskers correspond to the standard deviation (SD). The results are represented as the mean ± SD; n = 6/group. P-values # ## calculated using a two-tailed Student’s t test (^P < 0.05, ^^P < 0.01 v.s. baseline; P < 0.05, P < 0.01 v.s. control; *P < 0.05, **P < 0.01 v.s. HLU). demonstrated that the rats in the HLU group displayed an turnover biomarkers in the rats’ serum were detected. As shown in obviously lower mineral apposition rate (MAR) compared with that Fig. 5, in comparison, the serum levels of PINP, BGP, ALP, RANKL, in the control group (P < 0.01). Yet, calycosin administration NTX, TRACP5b and the ratio of RANKL/OPG in the HLU group increased the rate of calcification in the HLU + Calycosin group significantly increased compared that in the control group compared with that in the HLU group (P < 0.01). (P < 0.01 or P < 0.05), indicating that the lack of load caused the bone metabolism of rats to be in a state of high bone turnover (Fig. 5a–g). However, compared with HLU rats, treatment with Effect of calycosin on bone turnover markers calycosin did not significantly alter the levels of ALP (P > 0.05), but The results above showed that calycosin might play an important obviously suppressed the HLU-induced content elevation of role in maintaining bone mass of tail suspended rats. To further RANKL, PINP, BGP, NTX, TRACP5b and ratio elevation of RANKL/ elucidate the underlying mechanism, the changes of the bone Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 23 X. Jin et al. Fig. 3 Effect of calycosin on bone biomechanics. Evaluation parameters in the four groups of rats included the following: maximum stress (a), Young’s modulus (b), maximum load (c), stiffness (d), and toughness (e). The results are represented as the mean ± SD; n = 6/group. P- # ## values calculated using a two-tailed Student’s t test (^P < 0.05, ^^P < 0.01 v.s. baseline; P < 0.05, P < 0.01 v.s. control; *P < 0.05, **P < 0.01 v.s. HLU). Fig. 4 Effect of calycosin on bone histomorphology. a Representative fluorescence micrographs of trabecular bone sections showing green calcein labels within various groups (scale bar: 200 μm). b Quantitative analysis of the mineral deposition rate (MAR). The results are represented as the mean ± SD; n = 3/group. P-values calculated using a two-tailed Student’s t test (^P < 0.05, ^^P < 0.01 v.s. baseline; P < 0.05, ## P < 0.01 v.s. control; *P < 0.05, **P < 0.01 v.s. HLU). OPG in the serum (P < 0.01 or P < 0.05), indicating that calycosin calycosin significantly decreased the levels of TNF-α,IFN-γ,IL-6, could reduce the high bone turnover caused by tail suspension to IL-8 and IL-12, and increased the content of IL-4 and IL-10 a certain extent (Fig. 5a–g). notably compared with that in the HLU group (P <0.01). The above results showed that calycosin administration could up- regulate the content of IL-4 and IL-10 and down-regulate the Effect of calycosin on bone remodeling-related cytokines content of TNF-α,IFN-γ, IL-6, IL-8 and IL-12 in HLU rats. To evaluate the effects of calycosin on cytokines related to According to the above results, it could be seen that calycosin bone remodeling, the levels of IL-4, IL-6, IL-8, IL-10, IL-12, TNF-α reduced the ratio of RANKL/OPG in serum. While IL-10, IL-12 and IFN-γ in serum were detected using ELISA (Fig. 6). An and IFN-γ likely affect the secretion of RANKL/RANK/OPG, increasing trend in the content of TNF-α,IFN-γ, IL-6, IL-8 and IL- further regulating the process of bone remodeling. These 12 wasrecordedinthe HLU groupascomparedtothose in the results suggested that calycosin might regulate the process of control group (P < 0.01), while the levels of IL-4 and IL-10 were bone metabolism through bone immunity. declined (P <0.01) (Fig. 6a–g). Nevertheless, treatment with npj Microgravity (2022) 23 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA X. Jin et al. Fig. 5 Effect of calycosin on bone turnover markers. The bone turnover biomarkers levels in rats serum of ALP (a), BGP (b), PINP (c), TRACP 5b (d), RANKL (e), NTX (f) and RANKL/OPG (g) in the four groups. The results are represented as the mean ± SD; n = 6/group. P-values # ## calculated using a two-tailed Student’s t test (^P < 0.05, ^^P < 0.01 v.s. baseline; P < 0.05, P < 0.01 v.s. control; *P < 0.05, **P < 0.01 v.s. HLU). DISCUSSION that it is effective and feasible to discover new drugs through the method of network pharmacology. In this research, we utilized the Bone loss induced by microgravity has become one of the network pharmacology approach to uncover a potential small important threats to the health of astronauts and the major molecule compound-calycosin for treating bone loss. obstacle to long-term space flight. Therefore, the study aims to Previous studies have shown that calycosin have a dose- find potential protective drugs with low side effects for the dependent therapeutic effect on postmenopausal osteoporosis treatment of bone loss induced by microgravity. In the present rats and regulates the expression of OPG/RANKL via MAPK work, we preliminarily screened calycosin as a potential com- signaling . However, there has been no direct evidence of the pound for the treatment of bone loss through the network protective effects of calycosin on bone loss induced by micro- pharmacology, and further found calycosin might play an gravity in hind limb unloading rats. In this study, the Micro CT important role in maintaining bone mass in HLU rats due to the analysis showed that calycosin administration increased BMD, the improvement of bone mineral density, microstructure, ameliorated the microstructure of femoral trabecular bone and biomechanical properties and mineral apposition rate of bone; increased the thickness of cortical bone in HLU rats. The MAR of the effects were mainly owing to inhibit bone turnover and reduce bone is reflected by the distance between two injections of enhanced bone remodeling by HLU. The findings may provide a calcein fluorescent labeling in bone tissue. The results demon- potential and promising candidate in the treatment of weightless- strated that the mineral apposition rate of the bone was increased induced bone loss. by calycosin treatment, which illustrated its role in promoting the The discovery of new small molecule drugs for weightless bone bone formation to a certain extent. Meanwhile, the analysis of loss not only provides effective medical protection for astronauts, three-point bending test showed that calycosin treatment could but also has positive significance for the development of increase the bone biomechanical parameters toughness, stiffness aerospace medicine and aerospace industry. The network pharmacology provides a new idea and strategy for drug and Young’s modulus, but there were no significant changes in discovery. For example, Zhiguo Zhong identified quercetin as Max-load and Max-stress, indicating that calycosin administration the potential ingredient of Erzhi Pill (EZP) for the treatment of could improve biomechanical properties to a certain degree. bone loss through the method of system pharmacology, and Taken together, these findings suggested that calycosin might further in vivo research verified that quercetin owned positive play an important role in maintaining bone mass of tail effects on osteoblast differentiation of skull . This demonstrate suspended rats. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 23 X. Jin et al. Fig. 6 Effect of calycosin on bone remodeling-related cytokines. The levels of bone remodeling-related cytokines in rats serum IFN-γ (a), IL-6 (b), IL-8 (c), IL-12 (d), TNF-α (e), IL-4 (f) and IL-10 (g) in the four groups. The results are represented as the mean ± SD; n = 6/group. P-values # ## calculated using a two-tailed Student’s t test (^P < 0.05, ^^P < 0.01 v.s. baseline; P < 0.05, P < 0.01 v.s. control; *P < 0.05, **P < 0.01 v.s. HLU). Aside from the well-documented development of bone loss ,it metabolism induced by HLU. Based on the results, it could be is also well known that there is deleterious changes in immune observed that calycosin administration reduced the enhanced 29,30 function and metabolic disruptions caused by spacelight . bone remodeling caused by HLU. Recent studies have shown that Thence, to further investigate the underlying mechanism of the bone system can be regulated by the immune system, since calycosin in the prevention of bone loss, we detected the both of them shared the same bone marrow microenviron- 32–35 expressions of bone metabolism markers and bone remodeling ment . Specifically, the immune T cells and B cells regulate cytokines. Serum bone turnover biomarkers can not only reflect osteocytes through the secreted positive and negative cytokines, the therapeutic efficacy of drugs that prevent bone loss, but also which in turn directly or indirectly regulates the coupling balance reveal the pathogenesis of metabolic bone. Bone turnover of bone formation and bone resorption, affecting bone metabo- 34–36 biomarkers include bone formation markers and bone resorption lism . For example, T helper 17 (Th-17) cells can not only markers . Among them, RANKL, NTX and TRACP5b are markers of induce osteoblasts to express RANKL to promote bone resorption bone resorption, and BGP, ALP and PINP are markers of bone by secreting IL-17, but also stimulate immune cells to produce IL- formation. In this study, both the levels of bone resorption and 1, IL-6 and other inflammatory cytokines to enhance bone bone formation markers were increased in the HLU group absorption through activating RANK signaling, resulting in bone 37,38 compared with that in the control group, which revealed that loss . In this study, the levels of IL-4 and IL-10 in the HLU group the lack of load led to an increase in bone turnover, indicating that were decreased compared with the control group, whereas the bone homeostasis and the bone metabolism were altered in a concentration of IL-6, IL-8, IL-12, TNF-α and IFN-γ in the HLU group state of high bone turnover. And the elevated levels of bone were increased compared with that in the control group. The formation biomarkers in the HLU group might be due to that changes of cytokines indicated that the lack of load might initiate osteoblasts attempted to make a compensatory for bone loss the inflammatory changes of bone microenvironment state, induced by tail suspension. Calycosin administration obviously inducing a rapid phase of bone loss. Treatment with calycosin inhibited the increase in the serum levels of RANKL, NTX, significantly increased the level of IL-4 in serum, indicating that TRACP5b, BGP, and PINP caused by HLU, whereas did not calycosin might induce Treg cells to produce IL-4, further significantly change the expression of ALP. The main reason regulating the balance of the bone remodeling system . might be that there was a compensatory response in the rats Calycosin significantly increased the content of IL-10, which could body. 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Fluorosis increases the risk of postmenopausal Reprints and permission information is available at http://www.nature.com/ osteoporosis by stimulating interferon gamma. Biochem. Bioph. Res. Co. 479, reprints 372–379 (2016). 47. Schacht, E. Osteoporosis in rheumatoid arthritis - rationale for alfacalcidol in Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims prevention and therapy. Z. Rheumatol. 59,10–20 (2000). in published maps and institutional affiliations. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China under Open Access This article is licensed under a Creative Commons No. 81901917 and No. 82072106; the Key Research and Development Project of Attribution 4.0 International License, which permits use, sharing, Shaanxi Province under No. 2022SF-295 and No. 2018SF-363; the Fundamental adaptation, distribution and reproduction in any medium or format, as long as you give Research Funds for the Central Universities under No. D5000210746; the National appropriate credit to the original author(s) and the source, provide a link to the Creative Program of Innovation and Entrepreneurship for Undergraduates under No. Commons license, and indicate if changes were made. The images or other third party S202110699538, No. XN2021031 and No. S202010699126; and the grant under material in this article are included in the article’s Creative Commons license, unless BKJ17J004. indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly AUTHOR CONTRIBUTIONS from the copyright holder. To view a copy of this license, visit http:// Data curation: X.J.; Formal analysis: X.J., W.Z., and A.Q.; Experiment validation: H.W., creativecommons.org/licenses/by/4.0/. X.L., K.R., X.D., W.Q., Y.H., J.Z., L.L., S.G., F.L., Z.M., and X.J.; Funding acquisition: A.Q., W.Z., and Z.M.; Methodology: X.J., H.W., and W.Z.; Writing-original draft: X.J.; Writing- © The Author(s) 2022 review & editing: W.Z., A.Q., and H.W. X.J. and H.W. contributed equally to this work. npj Microgravity (2022) 23 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA

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