Temporal effects of three-dimensional clinostat treatment on bone and serum metabolome in C57BL/J mice: a new method of simulating microgravity effect

doi:10.21203/rs.3.rs-1861083/v1

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Temporal effects of three-dimensional clinostat treatment on bone and serum metabolome in C57BL/J mice: a new method of simulating microgravity effect

https://doi.org/10.21203/rs.3.rs-1861083/v1

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Microgravity is an unavoidable aspect of space flight. A three-dimensional (3D) clinostat is a great method to simulate the effects of microgravity on Earth. The impact of microgravity on plants, cells, and Caenorhabditis elegans has been extensively studied. However, no study has used a 3D clinostat apparatus to simulate the effects of microgravity on mouse models. Therefore, we conducted a study to produce a space microgravity mouse models using 3D clinostat treatment and explored the time effect of 3D clinostat treatment on the skeleton and metabolome of C57BL/J mice. Thirty 8-week-old male C57BL/J mice were randomly assigned to three groups: mice in individually ventilated cages (MC group, n = 6), mice in survival boxes (SB group, n = 12), and mice in survival boxes receiving 3D clinostat treatment (CS group, n = 12). Bone loss was assessed using microcomputed tomography of the left femur, whereas the changes in serum metabolites were monitored using untargeted metabolomics. A marked reduction in the trabecular number (p < 0.05) and an increased trabecular spacing (p < 0.1) were observed to occur in a time-dependent manner in the CS group compared with the SB group. Compared with the metabolome of the SB group, the metabolome of the CS group showed significant differences at Ⅰ and Ⅳ stages. Furthermore, the common pathways involved in differential metabolites in the Ⅰ and Ⅳ stages were valine, leucine, isoleucine degradation, and 2-oxocarboxylic acid metabolism. The KEGG pathways in the Ⅳ stages were mainly related to the nervous system, which was a result of microgravity.

microgravity

three-dimensional clinostat (3D clinostat)

bone loss

trabecular number

untargeted metabolomics

animal model

C57BL/J mice

The complex environment of space, including microgravity, radiation, changes in circadian rhythm, and extreme temperatures, can impact several biological processes ^1–6. Microgravity is one of the critical problems that astronauts face in space. Many studies ^7–12 have shown that long-term weightlessness can lead to various physiological and pathological changes, including in the immune, nervous, reproductive, and cardiovascular systems and in skeletal muscle tissues. Focusing on these physiological and pathological changes, many researchers previously studied a series of behavioral and dietary interventions ^13–18, such as training, increasing muscle and bone strength, and increasing the intake of dietary protein, amino acids, and minerals in weightlessness. Astronauts live in a closed system consuming foods that as in accordance with the in-flight macronutrient guidelines ¹⁹ of the National Aeronautics and Space Agency (55% carbohydrate, 30% fat, and 15% protein). However, the bone loss occurred when the intake of omega-3 fatty acids, beta-alanine, and carnosine were reduced. Therefore, researchers focused on dietary interventions, such as ensuring the quality and freshness of food during flight ¹⁷.

Previous studies ^20–22 have shown that long-term space operations can lead to bone loss and fracture, muscle atrophy, and metabolic changes. The bone parameters most frequently studied under weightless conditions are trabecular number and spacing ^21,23. Studies have shown ^21,24,25 that the number of bone trabeculae decreases and bone spacing increases during long-term weightlessness or bed rest among humans ²⁶. Currently, weightlessness studies are mainly selected by relying on animal models ^27,28, and the rodent hindlimb unloading model is the most widely used ^29,30 to study spatial effects, of which, bone loss and metabolic changes are the most significant. However, this model has certain limitations in that it can only be applied locally and not to the whole experimental animal ³¹.

Clinostats have invited the attention of researchers because experiments involving microgravity in space are expensive and conducted infrequently, and the time frame for producing a microgravity environment on ground is generally too short to meet the requirements of most biological experiments. A previous study has shown that the three-dimensional (3D) clinostat is the best method to simulate the effect of microgravity on ground, and the tested object experinces zero total force in the entire experimental process through random clinostat ³². The clinostat is not like a drop tower that only provides a microgravity environment but it can also be used as an effect of microgravity simulation equipment ³². Previous studies ^33–40 have used the 3D clinostat to simulate the effect of microgravity on plants, cells, and Caenorhabditis elegans but not on mice. Therefore, we conducted a study to establish a space microgravity model of C57BL/J mice using a 3D clinostat to achieve the effect of simulated microgravity and explored the representative bone loss parameters of the mice and metabolic changes.

2.1 Structure of the 3D Clinostat

The Chinese Academy of Sciences, Beijing, China, manufactured a 3D clinostat apparatus in accordance with the original design by Hoson ⁴¹, and its operation was controlled via a rotation control system (Fig. 1a,b).

The clinostat comprised a base frame, a supporting frame, an outer fram, and an inter frame (Fig. 1a). This is similar to the 3D clinostat developed by Hoson ⁴¹. The sample stage and illumination are placed on both sides of the inter-frame in parallel, and they are 170 mm away from the shaft. The inner frame was fixed on the outer frame, which rotated about the horizontal axis with its rotating axes perpendicular to each other. Additionally, the rotational motion of the two rotating frames was driven by stepper motors. A controller with a single-chip microcomputer regulated the motor rotational speed, direction, and running duration. A slip ring was used to perform all incoming and outgoing electrical wiring. Moreover, the rotating frames were the outer frame and middle frame. It can rotate independently and can be used to simulate the microgravity effect of larger plants, seeds, and microorganisms. Specific parameters: Loading platform: area, 400 × 300 mm; distance from the center, 170 mm; bearing capacity, 0–3 kg; and illumination, 400 mm × 300 mm. The sample stage can hold up to 12 sample containers with a diameter of 90 mm. Opposite the sample stage distance from the center point, 170 mm, removable. The lighting power is adjustable in the range of 0–50 w, and the light source is a white LED divided into two groups. The light is adjustable (5 cm under the light plate, 80–800 µmol/m²/s). The rotation modes are as follows: (1) random rotation: speed, 0–10 rpm; speed resolution, 0.1 rpm; (2) constant speed rotation: 0–10 rpm; speed resolution, 0.1 rpm; 10–30 rpm; resolution, 1 rpm. Laboratory environment: long term (≥ 30 d). The overall size of the 3D clinostat is 1000 mm × 912 mm × 1250 mm, and the total weight is 35 kg.

2.2 Mice and Treatments

The Ethics Committee of the Laboratory of Animal Science of Peking Union Medical College approved all experimental procedures. The Animal Ethics is IACUC-20220415. Animal care was provided in accordance with institutional guidelines and all animal studies were performed in compliance with the ARRIVE guidelines. Thirty 8-week-old male C57BL/J mice were housed under a 12-h light/dark cycle with unlimited access to water and food. The mice were randomly divided into three groups: mice in individually ventilated cages (MC group, n = 6), mice in survival boxes (SB group, n = 12), and mice in survival boxes receiving 3D clinostat treatment (CS group, n = 12). As the 3D clinostat rotates 360°, we took advantage of the burrowing habits of the mouse and designed a survival box to keep the mice fixed on the 3D clinostat (Fig. 1c). From Fig. 1c, 1 represents the free movement area of the mouse, 2 represents the rest area of the mouse, 3 represents the food and water supply area. In the third area, we put feed and solid AGAR to provide water. Meanwhile, only one mouse can be placed in one survival box; therefore, fighting and biting among mice are avoided.

The activity of the mice makes it impossible for it to be uniform, and the total force cannot be zero. However, binding or other ways to limit movement will result in stress injury to the mouse. When using a clinostat to simulate microgravity, the mice could drill into the survival box and stay in the rest area. It can be used as a mass point to ensure that the total force applied to the experimental animal is near zero and to perform simulated microgravity experiments in 3D clinostat safely and stably (Fig. 1d, e).

Before the study, we conducted adaptive training for the mice in the CS group to relieve stress. The adaptive training process involved the following: the mice in the CS group were placed on the 3D clinostat, and we provided food and solid AGAR in the survival boxes. The CS group underwent adaptive training for 5 days and rotated for 1 h, 2 h, 4 h, 8 h, and 12 h each day and rested for 2 days before starting the study. Meanwhile, the MC group was always placed in IVCs and the SB group was always placed in survival boxes without any treatment. Water and food were freely available, and all mice were observed daily.

2.3 Simulated Microgravity

The three groups were studied at the same time. The CS group experienced simulated microgravity on clinostat for 1 d and rested for 1 d; the SB group was always placed in the survival boxes; the MC group was always placed in the individually ventilated cages. The experiment lasted for 12 weeks. Left femur from the CS and/or SB groups were collected weekly. Serum and left femur from the MC group were collected every 2 weeks. The left femur was wrapped in saline-soaked gauze and stored at − 20℃ for ex vivo microcomputed tomography analysis, and the serum was stored at − 20℃ for metabolomics. The samples were anesthetized by intraperitoneal injection of pentobarbital sodium (50mg/kg) before collection, and then collected. Finally, the mice were killed by cervical vertebra twisting.

2.4 Micro-CT Imaging of Bone

CT imaging was conducted using a Siemens INVEON scanner with the following settings: tube voltage, 60 kV; tube current, 400 µA; and exposure time, 800 ms over 360^o rotation. The Feldkamp filtered back-projection algorithm was used to reconstruct the images. Images acquired from the scanner were viewed and analyzed using INVEON Workplace software.

2.5 Extraction of Metabolites

To extract metabolites from serum samples, 400 µL of cold extraction solvent methanol/acetonitrile/H₂O (2:2:1, v/v/v) was added to 100 mg of sample and adequately vortexed. After vortexing, the samples were incubated on ice for 20 min and then centrifuged at 14,000 g for 20 min at 4℃. Finally, the samples were redissolved in 100 µL of acetonitrile/water (1:1, v/v) solvent for liquid chromatography–mass spectrometry (LC–MS) analysis and transferred to LC vials.

2.6 LC–MS Analysis

For the untargeted metabolomics of polar metabolites, extracts were analyzed using a quadrupole time-of-flight mass spectrometer (Sciex Triple TOF 6600) coupled to hydrophilic interaction chromatography via electrospray ionization by the Shanghai Applied Protein Technology Co. Ltd. The LC separation was on an ACQUIY UPLC BEH Amide column (2.1 × 100 mm, 1.7-µm particle size) (Waters, Ireland) using a gradient of solvent A (25-mM ammonium acetate and 25 mM ammonium hydroxide in water) and solvent B (acetonitrile). The gradient was 85% B for 1 min and was linearly reduced to 65% in 11 min, and then was reduced to 40% in 0.1 min and maintained for 4 min, and then increased to 85% in 0.1 min, with a 5-min re-equilibration period. The flow rate was 0.4 mL/min, the column temperature was 25℃, the autosampler temperature was 5℃, and the injection volume was 2 µL. The mass spectrometer was operated in both negative and positive ionization modes. The ESI source conditions were set as follows: ion source gas 1 (gas 1) as 60, ion source gas 2 (gas 2) as 60, curtain gas (CUR) as 30, source temperature: 600℃, IonSpray Voltage Floating (ISVF) ± 5500 V. In auto MS/MS acquisition, the instrument was set to acquire over the m/z range 25 to 1,000 Da, and the accumulation time for production scan was set at 0.005 s/spectra. The production scan was acquired using information-dependent acquisition with selected high sensitivity mode. The parameters were set as follows: the collision energy was fixed at 35 V with ± 15 eV; declustering potential 60 V (+) and − 60 V (–); exclude isotopes within 4 Da; candidate ions to monitor per cycle: 10.

2.7 Data Analysis

The raw MS data (wiff. scan files) were converted to MzXML files using ProteoWizard MSConvert before importing into freely available XCMS software. For peak picking, the following parameters were used: centWave m/z = 25 ppm, peak width = c (10, 60), prefilter = c (10,100). For peak grouping, bw = 5, mzwid = 0.025, and minfrac = 0.5 were used. Only the variables with more than 50% of the nonzero measurement values in the extracted ion features were kept in at least one group. MS/MS spectra identified metabolite compounds with an in-house database established with available authentic standards. After normalization to total peak intensity, the processed data were uploaded before importing into SIMCA-P (version 14.1, Umetrics, Umea, Sweden). They were subjected to multivariate data analysis, including Pareto-scaled Principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA). Sevenfold cross-validation and response permutation testing were used to evaluate the robustness of the model.

2.8 Analysis of Bioinformatics

For Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation of pathways, the metabolites were blasted against the online KEGGdatabase to retrieve their COs and were subsequently mapped to pathways in KEGG.

2.9 Statistical Analysis

The data are shown as means ± SEM. T-tests evaluated the significance of differences in the mouse body weight between groups. One sample t-test analyzed the trend of time change. GraphPad Prism 9.0 software was used for statistical analysis. A difference was considered significant if the P-value was < 0.05. Nonmetric multidimensional scaling (NMDS) was conducted based on the Bray–Curtis distance. The corresponding KEGG pathway enrichment analyses were applied based on Fisher's exact test, considering the total metabolites of each pathway as a background dataset. Only pathways with P-values < 0.05 were considered significantly changed pathways.

3.1 Effects on Femur Parameters of SB-Group Mice

The mice in the SB or MC groups were observed for 12 weeks without treatment. None of the mice escaped from the survival boxes or individually ventilated cages. No apparent physical damage to the mice was observed throughout the study. Figure 2 showed the results of the analysis of femur parameters, including bone surface area/bone volume, bone volume/total volume, trabecular number, trabecular spacing, and trabecular thickness. We examined the effect of time on femur parameters. The results showed that no significant difference in time-dependent femur parameters between the two groups (Fig. 2a-e). It suggested when mice were stimulated with the survival boxes, time-dependent bone volume/total volume, trabecular thickness, trabecular spacing, trabecular number, and bone surface area/bone volume were unaffected.

3.2 Metabolic Patterns in the SB Group and the MC Group

Studies have shown ^42–44 that microgravity changes metabolites in the body, and therefore we examined the metabolomics of the SB and MC groups. Serum specimens were subjected to untargeted metabolomics analysis to identify the hematic metabolic profiles among the two groups tested (Fig. 3). NMDS analysis showed that there was no significant differences between SB and MC groups (p > 0.5). It suggested that survival boxes did not effect the metabolic pattern of C57BL/J mice.

3.3 Effects of 3D clinostat on body weight and bone parameters in mice.

Throughout the study, we have a professional veterinarian who checks the mice daily. At the end of the study, no physical damage was observed in the mice. Conversely, the coat is smooth and shiny, without vertical hair phenomenon, and in a good mental state. The limbs of mice had no trauma, bending, dislocation, or swelling. No oral bleeding, salivation, and no signs of diarrhea, bloody stools, or anal prolapse. In Fig. 4a, the weight of mice in the CS group showed an upward trend. It suggested that 3D clinostat did not cause stress injury in mice. We placed mice in survival boxes, using the 3D clinostat to simulate the effect of microgravity and to explore the effect of microgravity on femur parameters. There was a declining trend in time-dependent trabecular numbers (p < 0.05) and trabecular spacing was an increasing trend (p = 0.078) in the CS group (Fig. 4b, c). Our results indicated that simulating microgravity with a 3D clinostat led to bone loss. The longer the simulation period of microgravity with a 3D clinostat, the more severe the bone loss.

3.4 Differences Characteristics of Metabolic Patterns in Mice in Different Time Regions

We performed analysis for metabolic patterns in the SB group and CS group in different time regions (Fig. 5a). NMDS analysis showed that compared with the SB group, Ⅰ and Ⅳ stages have a separation in the CS group (p < 0.5). Ⅱ and Ⅲ stages did not separate between the SB and CS groups. Differences between groups were shown in Fig. 5b, our results showed that p value of the Ⅰ and Ⅳ stage was 0.1. Next, we examined the total differential metabolites numbers (rate) and known differential metabolites numbers (rate) (Table 1). Table 1 showed that the differential metabolites changed most significantly in the Ⅰ stage, and in the Ⅱ stage, the differential metabolites changed decreased. Meanwhile, compared with the Ⅱ stage, the differential metabolites of the Ⅲ and Ⅳ stages were increasing with time-dependent. It suggested that after 3D clinostat treatment, mice have a stress response in the Ⅰ stage and need to adapt and the number of differential metabolites in mice increased with the time of microgravity simulation. Meanwhile, we observed that common differential metabolites were most in the Ⅰ and Ⅳ stage in the Venn (Fig. 6).

Table 1

Differential metabolite statistics
		Ⅰ	Ⅱ	Ⅲ	Ⅳ	Total metabolites
All	Changed	3036(0.199)	246(0.016)	365(0.023)	867(0.056)	15226
	Up	1766(0.115)	148(0.009)	195(0.012)	377(0.024)	2486
	Down	1270(0.103)	98(0.006)	170(0.011)	490(0.032)	2028
Matched	Changed	233 (0.156)	21(0.014)	30(0.020)	56(0.037)	1492
	Up	150(0.100)	16(0.010)	12(0.008)	35(0.023)	213
	Down	83(0.055)	5(0.003)	18(0.012)	21(0.014)	127
Note: The numbers outside the brackets represented the number of differential metabolites; the numbers in parentheses represented the ratio of differential metabolites to total metabolites.

3.5 The differential metabolites were involved in the KEGG pathway in the Ⅰ stage

Given the significant expression of differential metabolites in the Ⅰ stage in Table 1 and Fig. 6. We then analyzed the KEGG pathway, in which the differential metabolites known in the Ⅰ stage were enriched (Fig. 7). Here, we found metabolic pathways associated with stress responses, including Retrograde endocannabinoid signaling. It suggested that the 3D clinostat treatment produced a stress response in the Ⅰ stage.

3.6 Differential metabolites shared by stage I and stage IV

We observed that the common differential metabolites were the most in the I and IV stages (Fig. 6). Therefore, we measured these common differential metabolites and these metabolites were enriched in the KEGG pathway (Table 1, Fig. 8). Table 2 showed that the differential metabolites mainly focus on the tumor, neurotoxicity and inflammation. Figure 8 showed that most of the I stage metabolic pathways are activated. The KEGG metabolic pathways in the Ⅱ and Ⅲ stages were inhibited and the metabolic pathways with significant differences gradually decreased.

With the prolongation of the 3D clinostat processing time, 8 signaling pathways were observed to be activated in the Ⅳ stage, including 2-Oxocarboxylic acid metabolism, Valine, leucine and isoleucine degradation, Alcoholism, Amphetamine addiction, Cocaine addiction, Dopaminergic synapse, Melanogenesis, Prolactin signaling pathway. And a pathway was inhibited, including Fatty acid biosynthesis. Here, we found that 2-Oxocarboxylic acid metabolism and Valine, leucine and isoleucine degradation overlapped in the I and IV stages. It suggested that in the I stage, the mice developed a stress response in the 3D clinostat, and gradually adapted to the Ⅱ and Ⅲ stages. Meanwhile, with the extension of simulated microgravity time, the metabolic pathways of mice changed significantly.

Table 2

The common differential metabolites in the stage I and stage IV
Name	I fold change	IV fold change	Function	Related disease	Reference
Ginsenoside re	2.96	1.64	nerve protection, anti-cancer	cancer, neurodegeneration disease	⁴⁵
Hexadecanedioic acid, 3,3,14,14-tetramethyl-	2.49	0.70	-	-	-
Methylmalonic acid	2.43	1.30	drive tumor aggressiveness	tumor	⁴⁶
6-phospho-d-gluconate	2.01	1.26	glycolysis intermediate	systemic lupus erythematosus	⁴⁷
Kynurenic acid	1.94	4.45	nerve protection	Alzheimer's disease	⁴⁸
L-Tyrosine	1.78	2.08	melanin formation substrate	melanin	⁴⁹
Delsoline	1.57	1.30	gangliolysis	muscle tension and overexertion	⁵⁰
Coproporphyrin i	1.47	0.81	biomarker of OATP1B Activity	Rheumatoid Arthritis	⁵¹
Cis,cis-muconic acid	1.02	0.84	C6 dicarboxylic acid platform chemical	the production of drugs	⁵²
Propylene glycol propyl ether	-0.32	-0.23	toxicity	tumors	⁵³
Chlorhexidine	-0.50	-0.74	antimicrobial	gingivitis	⁵⁴
Pentadecanoic acid	-0.61	-0.48	promoted glucose uptake	type 2 diabetes	⁵⁵
Norharmane	-0.67	-0.70	protective against neurodegenerative diseases	Alzheimer’s disease	⁵⁶
Oxindole	-0.78	-0.79	kinase inhibitors	cancer	⁵⁷
Gln-val	-0.84	-0.89	melanoma cells to metastasize to the liver	melanoma	⁵⁸
2,5-dimethoxy-4-propylphenethylamine	-0.86	-0.79	induce neurotoxicity	neuroinflammation	⁵⁹
Trimethylamine n-oxide	-0.94	-0.77	inducing M1 macrophage polarization	graft-versus-host disease	⁶⁰
Phosphatidylcholine lyso alkyl 16:0	-0.98	-0.98	-	-	-

Weightlessness is inevitable on a space mission and contributes to a series of damaging effects. Economics and technology have limited our ability to study the effects of weightlessness in space. Therefore, models that simulate the effects of microgravity (such as hindlimb unloading models) have become the preferred tools for studying weightlessness. However, these models also have some limitations ³¹. For example, weightlessness occurs when the entire object floats completely in space, whereas hindlimb unloading usually simulates partial weightlessness ³¹. This method may not make the subject accept the overall weightlessness.

In our study, we used a 3D clinostat to simulate the effects of microgravity. On a clinostat, an organism is in a gravitational field and is subjected to a constant gravity vector, but because of the rotation of the clinostat, the direction of the gravity vector acting on the organism continuously changes, and the vector sum generated by one rotation (360°) is equal to zero, i.e., zero gravity ^32,61. The direction of the gravity vector changes rapidly so that the organism does not experience gravity, and the result is an effect similar to that of a microgravity environment ^32,62.

To explore the effects of microgravity simulated by the 3D clinostat, we selected the bones and metabolites most affected by weightlessness. Theoretically, it is possible to simulate microgravity on the ground by processing samples with 3D clinostat. It has been used on plants, cells, and Caenorhabditis Elegans ^33–40. However, it was difficult for mice to keep immobile, considered as a mass point. Thus, we took advantage of the burrowing habits of the mouse and designed a survival box to keep the mice fixed on the 3D clinostat which we thought is a great idea for mice. To investigate whether the survival box affected bone parameters in mice, we compared the MC and SB group mice. Excluding the effect of the survival box on bone loss, our results indicated that bone volume/total bone trabecular thickness, trabecular number, trabecular spacing, and bone surface area/bone volume did not change with time. Subsequently, we analyzed the metabolomics of mice in the SB and MC groups, and the results showed no significant differences between the two groups. It suggested that the survival box would not interfere with our experimental results.

Bone is unique among tissues in that most of its volume consists of a mineralized extracellular matrix ⁶³. Structurally, there are two major types of bone ⁶⁴: cortical bone and cancellous bone (also called trabecular or spongy bone). Cancellous bone is often affected first and most dramatically by endocrine status alterations or mechanical loading reductions because of its massive surface area relative to its volume ⁶³. Some studies showed that weightlessness causes bone loss in humans and mice. Bone parameters (e.g. bone trabeculae) were reduced ^21,65,66. Coulombe et al. ⁶⁷ selected bone parameters to study microgravity using the hindlimb unloading model. J R Gardner et al. ⁶⁸described the use of magnetic resonance (MR) microscopy to examine changes in tibial trabecular bone structure in mice following 28 days of hindlimb suspension. In this first MR study involving mice, analysis of 3D images showed that apparent bone volume fraction, trabecular number, and trabecular thickness were decreased, and apparent trabecular spacing increased, significantly (P < 0.05) in hindlimb-suspended mice compared to controls. These changes agreed well with light microscopy measurements from an independent study and also with actual spaceflight experiments with rats ⁶⁸. Kevin A Maupin et al. ⁶⁹showed that 9-week-old male C57BL/6 mice resided in spaceflight for ~ 4 week, and spaceflight had major negative effects on trabecular bone mass of the following weight-bearing bones. Therefore, after ruling out the influence of the survival boxes, we also selected bones for detection. We conducted a follow-up inquiry to compare the SB and the CS groups results. And the results showed that the CS group using microgravity simulated by the 3D clinostat reduced trabecular number and increased trabecular spacing with time-dependent. It suggested that 3D clinostat could cause bone loss by simulated microgravity.

An increasing body of studies has shown that bone trabecular number and connectivity are key contributors to bone strength and mechanical competency ^70–72. Some studies have found that the trabecular bone is one of the main structures that support mechanical loads ^73–75. The trabecular bone can enhance the mechanical strength of skeletal bones ^76–78. Trabecular bone undergoes time-dependent mechanical changes when subjected to a sustained load ^79–81, consistent with our results. In both humans and animal models following space flight, bone structure (i.e., trabecular bone) is diminished, leading to an increased fracture risk upon returning to gravitational loading ²¹. Bone formation is observed to decrease in most space flight studies, and bone resorption increases in humans in space and rodent models ^82–89. For bone-growing mice, increased sensitivity to changes in bone formation during space flight was more attractive. Therefore, in our study, the decrease in trabecular number and increase in trabecular spacing may have been due to the combined effect of decreased bone formation and increased bone resorption and was time-dependent. We also observed no damage in the mice throughout our study. This result suggested that the method using the 3D clinostat plus survival boxes completely simulated weightlessness in mice without causing damage to the mice.

Changes in human metabolites are also inevitable during space flight missions. Our results showed that mice treated with a 3D clinostat developed a stress response during the Ⅰ stage, leading to a significant increase in the Ⅰ stage differential metabolites compared to other stages. Retrograde Endocannabinoid signaling is primarily affected. Studies showed ⁹⁰ that CB1 receptor-endocannabinoid signaling was activated by stress and played a role in buffing or inhibiting the behavioral and endocrine effects of acute stress. Sachin Patel et. al found ⁹¹ that activation of CB1 cannabinoid receptors reduced anxiety-like behaviors in mice and further supported an anxiolytic role for endogenous cannabinoid signaling.

Subsequently, our results showed that KEGG signaling pathway was inhibited in the Ⅱ and Ⅲ stages, and the number of affected signaling pathways decreased. It suggested that the mice adapted to the 3D clinostat intervention in the Ⅱ and Ⅲ stages. Weight results and the number of differential metabolites also supported this point. During long-term space operations ⁶¹, microgravity may affect intracranial physiological functions, such as intracranial pressure, spinal and neurocognitive performance, and brain edema induced by microgravity ⁹², resulting in neuro-ocular syndrome ⁶². Irina Mikheeva et.al observed ⁹³ that 30-day spaceflight had a significant effect on the structure of motoneurons of the trochlear nerve nucleus in mice. Xiao Wen Mao et.al found that the mice exposure to the spaceflight environment (Space Shuttle Atlantis, STS-135, 13 day) could induce significant changes in protein expression related to neuronal structure and metabolic function. Furthermore, the results also found that the nervous system was the most to be affected by long-term microgravity in the Ⅳ stage. Therefore, the mouse models built with 3D clinostat is an excellent model to explore the effect of microgravity.

Our results also showed two overlapping metabolic pathways in the Ⅰ and Ⅳ stages: valine, leucine, and isoleucine degradation and 2-oxocarboxylic acid metabolism. Valine, leucine, and isoleucine ⁹⁴, also known as branched-chain amino acids, usually act as nitrogen carriers to assist in synthesizing other amino acids required for muscle formation. Branched-chain amino acids have both synthetic and antide composition effects, which can help prevent protein decomposition and muscle loss ^94,95. Frederico Gerlinger-Romero et al found ⁹⁶ that Beta-hydroxy-beta-methylbutyrate (a leucine metabolite) can improve skeletal muscle function and protect bone from the harmful effects of fasting in Wister rats after fasting. 2-oxocarboxylic acid metabolism^97–99 was closely related to osteoarthritis, myocardial infarction and bronchial asthma. Naiqiang Zhu et al demonstrated ⁹⁸ that 2-oxocarboxylic acid metabolism was highly correlated with osteoarthritis and can be used as a biomarker for early diagnosis of osteoarthritis. It suggested that throughout the process of simulating microgravity, valine, leucine, and isoleucine degradation and 2-oxocarboxylic acid metabolism always play their roles. Therefore, Valine, leucine, and isoleucine degradation and 2-oxocarboxylic acid metabolism were potential targets for using 3D clinostat to simulate microgravity effects.

It was feasible to simulate the effect of microgravity on C57BL/J mice by 3D clinostat treatment, and we established a mouse model experiencing microgravity. Bone loss (a reduction in the trabeculae number) agreed with previous studies. Additionally, serum metabolomes were changed with increasing 3D clinostat treatment time. The effects of microgravity simulated by a long time 3D clinostat are most affected in the nervous system.

Conflict of Interest

The authors declare that they have no conflicts of interest.

Authors' contributions

J.G. designed the study; C.S., T.K., and M.Z. performed the experiments; C.S., K.G., and X.S. analyzed the data; J.G. and C. S. wrote the manuscript.

Funding

This research was supported by the National Natural Science Foundation of China (General Program，82070103), the National Key R&D Program of China (2021YFF0703400), CAMS Innovation Fund for Medical Science (CIFMS, 2021-I2M-1-036).

Acknowledgments

We are grateful for the serum metabolome analysis provided by the Shanghai Applied Protein Technology Company.

Ethical approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Data Availability

The data that supports the findings of this study are available from the corresponding author upon request.

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Temporal effects of three-dimensional clinostat treatment on bone and serum metabolome in C57BL/J mice: a new method of simulating microgravity effect

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Abstract

Figures

1 Introduction

2 Methods

2.1 Structure of the 3D Clinostat

2.2 Mice and Treatments

2.3 Simulated Microgravity

2.4 Micro-CT Imaging of Bone

2.5 Extraction of Metabolites

2.6 LC–MS Analysis

2.7 Data Analysis

2.8 Analysis of Bioinformatics

2.9 Statistical Analysis

3 Results

3.1 Effects on Femur Parameters of SB-Group Mice

3.2 Metabolic Patterns in the SB Group and the MC Group

3.3 Effects of 3D clinostat on body weight and bone parameters in mice.

3.4 Differences Characteristics of Metabolic Patterns in Mice in Different Time Regions

3.5 The differential metabolites were involved in the KEGG pathway in the Ⅰ stage

3.6 Differential metabolites shared by stage I and stage IV

4 Discussion

5 Conclusions

Declarations

References

Additional Declarations

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