Simulated Space Environmental Factors of Weightlessness, Noise and Low Air Pressure Differentially Affect the Circadian Rhythm and Gut Microbiome

Abstract


Background
Astronauts encounter environmental conditions that are dramatically different from those on the Earth's surface, including natural factors (gravity, radiation, magnetic eld, lighting conditions, etc.) and social factors (shift work, con nement, emergency tasks, etc) [1][2][3].In space, many astronauts' physiological and behavioral aspects are prone to be in uenced by the environment, including uid shift, body weight and nutrition, digesta propulsion and digestive function, cardiovascular functions, in ammation, transcriptional and metabolic changes, lipid level changes, microbiome response, and cognitive function [3][4][5].
It has also been demonstrated that the circadian rhythms of diverse organisms display signi cant changes in space [6][7][8][9][10].Among different space environmental factors, the effects of microgravity and lighting conditions on circadian rhythms have been extensively investigated [9,[11][12][13][14][15].However, the potential effects of other factors, such as noise and low atmospheric pressure, on circadian rhythms remain elusive.
In the manned space capsule, although the goal is to maintain a 1.0 atmospheric pressure (atm), the atmospheric pressure is usually lower, e.g., ~ 0.9 atm, which is not considered to be unharmful to human health.During the Apollo missions, the environment inside the lunar module was 0.34 atm (5.0 psi) and 100% oxygen.It has been proposed that a lunar habitat will maintain an atmospheric pressure of 8-psi, which is approximately 0.54 atm [2,16].Brown et al proposed that diurnal and seasonal changes in atmospheric pressure play roles in modulating the metabolic rhythms of potato, and the rhythms of oxygen consumption in atworm Dugesia [17].In 2019, Kitahara et al revealed that higher hydrostatic pressure led to shortened circadian periods of the cyanobacteria circadian clock [18].Low atmospheric pressure may cause disturbances in circadian rhythms due to decreased levels of dissolved oxygen in blood [19][20][21].Hypoxia regulates the circadian clock by slowing the circadian cycle and dampening the amplitude through HIF1A, a key hypoxia-inducible factor.Low atmospheric pressure has been known to modulate circadian rhythms by slowing the circadian period and attenuating the amplitude due to hypoxia, which results in increased levels of HIF1A [19][20].These ndings suggest that hydrostatic or atmospheric pressure may affect circadian parameters.
The operation of various instruments and equipment on the space station results in environmental noise.According to a 2003 report, the average level of noise in the International Space Station was approximately 70 dB with a maximum of over 90 dB during sleep episodes [2].Environmental noise, including noise in industrial settings may cause a broad range of physical consequences, including cardiac problems, sickness-related absenteeism, self-reported fatigue and disturbed sleep [22][23][24].It has been reported that the night noise of road tra c noise affects blood glucose homeostasis due to disruption of circadian rhythms in diabetic patients but not nondiabetic patients [24].However, the effects of noise on circadian rhythm and health remain to be further addressed.
The latest decade has seen rapid advances in the study of the gut microbiomes in different species.Gastrointestinal microorganisms play key regulatory roles in host physiological processes including metabolic immunity and neuroendocrine pathways [25][26][27].In addition, accumulating evidence has demonstrated that host circadian rhythms modulate microbial oscillations, and vice versa [28,29].
In space, the transcriptional and metabolic changes and microbiome responses between the twin astronauts are widely affected in the long-term space environment [4].The changes in the gut microbiome of the mice were observed on a space shuttle mission (STS-135) for 13 days and after a 37-d space ight onboard the International Space Station [5,30].However, the effects of space ight on the rhythms of the gut microbiome are unclear.
The present study aimed to investigate the impacts of several simulated space environmental conditions, noise, low temperature and weight loss, on mouse circadian rhythms and the gut microbiome.The results demonstrate that these factors may impact circadian rhythms and the gut microbiome through modi cation of the circadian clock.

Results
Combinational conditions of low air pressure and noise impact on locomotor rhythms In this study, 6 free-moving mice in the capsule and 6 free-moving mice in the animal room were raised for recording of locomotion rhytmicity.For hypothalamus and feces sampling, 3 mice were raised for each time point either in the capsule or animal room (Fig. 1a).Uniform eld noise of 85 dbA and ~ 0.9 atmospheric pressure were loaded in the simulated space capsule.We recorded the locomotion rhythms of control mice but not those of mice undergoing hind limb unloading (HU) to mimic microgravity, in both the simulated space capsule and animal room.Compared to the mice in the animal room, the mice in the simulated space capsule showed a decrease in amplitude of locomotion during the days after the initiation of exposure to noise and low atmospheric pressure (Fig. 1b-d).Furthermore, it took the control mice in the capsule took 5.8 ± 0.5 d to adjust to the 6-h advance of the LD cycles, which was signi cantly faster than that of the control mice in the animal room (7.7 ± 0.8 d) (Fig. 1b,c,e,f).These data suggest that noise and low atmospheric pressure may affect the function of the circadian system.

Simulated space environmental factors differentially affect global gene expression
To address the effects of different simulated space environmental factors (noise, low air pressure and microgravity) on global gene expression, the hypothalamus tissues containing suprachiasmatic nuclei (SCN) from HU and control mice, in the capsule and animal room in the control (day 6) and experiment (day 36) periods, were collected for RNA sequencing (RNA-seq), respectively.Global changes in gene expression were observed (Fig. 2a,b).Notably, the quantities of differentially expressed genes (DEGs) in the capsule were much lower than those in the animal room (Fig. 2b,c).For instance, of the detected genes (Table S1), 22 were upregulated at the early stage and 151 were upregulated at the late stage of the control mice in the capsule while 884 were upregulated at the early stage and 4350 were upregulated at the late stage of the control mice in the animal room.Similarly, the number of DEGs was much higher in the HU mice (Fig. 2b-d; Figure S1b-g).It is worth noting that the number of DEGs in animals in SSC was much lower than those in AR, for both HU or control mice.
Gene enrichment and functional annotation by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that a number of important biological processes associated with diseases were altered in the capsule mice, such as viral protein infection in HU mice and prion diseases in the control mice in the capsule.Pathways implicated in metabolism, neural systems, cell physiology, and diseases and cancers were also found.The pathway of circadian entrainment was enriched in the HU and control mice in the animal room (late vs. early) but not in the mice in the simulated space capsule.(Fig. 2e-f; Figure S1h-k; Table S3).

Simulated space environmental factors differentially modify the rhythmicity of global gene expression
JTK_Cycle analysis revealed differential reprogramming patterns of global circadian expression of hypothalamus transcripts between early and late stages under different conditions.For instance, 828 and 1558 genes of HU mice in the simulated space capsule showed rhythmicity in the early and late stages, respectively, and 78 genes showed rhymicity at both stages (Fig. 3a,b; Figure S2a-f; Table S2).Compared to HU mice in the animal room, the phase of global gene expression of HU mice in the capsule was limited in much narrower ranges at either early or late stages (Fig. 3b).
We next predicted the enriched pathways of those genes with signi cant changes in their rhythmicities under different conditions by KEGG pathway analysis.The pathways implicated in hypertrophic cardiomyopathy (HCM) and dilate cardiomyopathy (DCM) were identi ed between HU and control mice at the early stage in the animal room and between the early and late stages of HU mice in the animal room.Alterations in the rhythmicity of genes implicated in the pathway of circadian rhythm were identi ed between HU and control mice in the animal room at the late stage.Furthermore, the pathway associated with human papillomavirus infection was found in HU and noise and low atmospheric conditions.Additionally, pathways associated with cancers, cell physiology, immune defense, digestion, and neurodegenerative diseases were also found under the respective conditions (Fig. 3g-j; Figure S3; Table S4).

Simulated space environmental factors differentially affect the expression of core circadian clock genes
Together, the changes in locomotion rhythms (Fig. 1b-e) and pathways associated with circadian entrainment in the animal room (Fig. 3a-d) suggest that circadian rhythms may be affected at the molecular level.RNA-seq analysis con rmed that the average levels of circadian clock genes showed differentially altered gene expression patterns (Fig. 4a,b).
The average levels of many core circadian clock genes showed signi cant changes in mice in the animal room but not simulated space capsule (Fig. 4a-c).In the animal room, the levels of Clock, Per1, Per2, Per3, Cry1, Cry2, Nr1d1, Arntl and Npas2 were upregulated at the late stage in control mice, and Clock, Per2, Per3, Cry1, Cry2, Nr1d1 and Npas2 were upregulated in the HU mice.In contrast, none of these genes showed signi cant changes in the mice in the capsule.Arntl2 showed no signi cant change in either HU or control mice (Fig. 4a,b).Relative levels of some circadian clock genes (late levels normalized to early levels), including Clock, Per1, Per2, Cry1, Cry2 and Nr1d1, were decreased in HU compared to control mice in the animal room.In contrast, the levels of these genes were comparable between HU and control mice in the simulated space capsule (Fig. 4c).Together, these data suggest that microgravity and noise and low atmospheric pressure may repress the expression and function of circadian clock genes.Changes in circadian rhythmicity (amplitude or phase) of these clock genes were also observed in most of these clock genes (Fig. 4d-k; Figure S4a-l).
The pressure was 0.9 atm in the capsule from day 7 to the end in the present study.The RNA-seq data showed no signi cant difference in the hypothalamus Hif1a level in control mice between the early and late stages in the capsule (Figure S4m,n).We further measured the HIF1A protein levels in liver samples, and revealed no signi cant change between these two stages; together, these data suggest that the low atmospheric pressure in the capsule is not su cient to elicit hypoxia stress.However, a signi cant increase in HIF1A levels was observed in control mice at the late stage in the animal room and a signi cant decrease was observed in HU mice at the late stage in the capsule (Figure S4o).

Simulated space environmental factors differentially affect the abundance of the gut microbiome
Stool samples from the transverse colon were subjected to 16S rRNA sequencing.In total, the sequenced microbiome consisted of 25 phyla, 58 classes, 82 orders, 129 families, and 172 genera of microorganisms, of which Bacteroidetes and Firmicutes were the most dominant.Together with Proteobacteria, Verrucomicrobia, Cyanobacteria, Actinobacteria, Deferribacteres, and Tenericutes, these phyla constituted over 99% of the known phylogenetic categories (Fig. 5a,b; Table S5).
The α-diversity between the data sets showed no signi cant difference (Fig. 5a-c).The PCoA results based on the OTU abundance revealed different separation under the indicated conditions (Figure S5a-h).Interestingly, a clear separation was visualized between the early and late stages of the HU mice gut microbiome in the animal room but not the HU mice in the simulated space capsule (Fig. 5d-g; Figure S5f,h), suggesting that microgravity imposes long-term impacts on the structure of the gut microbial community, which could be repressed by superimposition with noise and low atmospheric pressure.
Among the detected phyla, changes in the abundance of many bacteria were observed (Fig. 6a-d; Figure S6).For instance, interestingly, Adlercreutzia was increased at the late stage of HU mice in the animal room but not in the capsule(Figure S6a,c).Verrucomicrobia abundance was increased at the late stage of control mice in the capsule and the animal room but was decreased at the late stage of HU mice in the capsule and the animal room (Fig. 6a-d; Figure S6a-g).
PICRUSt analysis revealed that microbial pathways were signi cantly correlated with host cardiovascular functions.For instance, pathways of cardiac muscle contraction and HCM were identi ed between the early and late stages of control mice in both the animal room and the simulated space capsule, such as the renin-angiotensin system and HCM between the HU and control mice at the early stage in the simulated space capsule and cardiac muscle contraction, viral myocarditis, renin-angiotensin system and HCM pathways between the HU and control mice at the late stage in the simulated space capsule, and between HU and control mice at the late stage in the simulated space capsule.In addition, the pathway of amyotrophic lateral sclerosis was identi ed between the HU and control mice at the late stage in the simulated space capsule (Fig. 6e,f; Figure S7; Table S7).Other important pathways implicated in immune defense, metabolism, cancers, cell physiology, and other processes were also found under different conditions (Fig. 6e,f; Figure S7; Table S7).

Simulated space environmental factors differentially affect the diel oscillation of gut microbiome
Most of prokaryotic organisms possess no endogenous circadian systems; however, they exhibit diurnal rhythms owing to daily cycling environmental factors or the host internal milieu [31].It has been revealed that mammalian oral and gut microbiomes undergo diurnal changes in the abundance of many microorganisms [32,33].
The composition of some taxa displayed overt diurnal patterns over a day, including Akkermansia, Allobaculum, Bacteroides, Prevotella, Sutterella and so on (Fig. 7a).We next looked at the compositional changes of some bacteria at the genus level and found that the rhythmicities were changed under some conditions, such as Cetobacterium, Kaistobacter, Rhodoplanes, Acinetobacter, Corynebulcterium, Paraprevotella, Enterobacter, Devosia, etc (Fig. 7b-o; Figure S8; Table S6).Changes in phase or amplitude were found in some of these bacteria.For instance, Prevotella showed a higher amplitude in the HU mice in the capsule at the late stage than at the early, and a higher amplitude of Corynebacterium was seen in the control mice in the capsule.Rhodoplanes showed a higher amplitude in the HU mice in the capsule at the late stage.And Paraprevotella showed a dramatic increase in the oscillation in the HU mice at the late stage while it showed induction in abundance only at night in the control mice at the late stage in the animal room (Fig. 7b-o; Figure S8; Table S6).

Discussion
It is known that the space environment causes alterations in the circadian parameters of heart rate, body temperature, urinal variables, sleep-wake cycles and alertness [1,13,14,[34][35][36][37]. Head-down bed rest in humans and hind limb unloading experiments in rodents are comparable ground-based approaches to mimic microgravity which leads to cephalad redistribution of body uid which is similar to what occurs in space [38].Head-down bed rest leads to changes in circadian/diel rhythms of physiological, metabolic and locomotion [39][40][41][42][43].At the molecular level, upregulation of genes involved in circadian rhythm was found during and after 84 days of head-down tilt bed rest [12].
In space, the astronauts also encounter noise and low atmospheric pressure in the simulated space capsule in addition to microgravity; however, whether these two factors in uence circadian rhythms remains elusive.In this work, we demonstrate that HU and the combination of noise and low atmospheric pressure differentially impact the expression of circadian clock genes and the rhythmicity of global gene expression.
In mammals, circadian clock gene expression, architecture and consequent physiological and behavioral rhythms change along with development and aging [44].The changes in clock gene expression between the early and late stages of mice in the animal room represent the development of molecular expression and the function of the circadian clock system in hypothalamus (Fig. 4).However, exposure to noise and low atmospheric pressure repressed the changes in global gene expression including those genes playing a role in the regulation of circadian rhythms.Moreover, we observed more concentrated phases of the oscillating genes of mice in the simulated space capsule.These data suggest that noise and low atmospheric pressure may hamper adaptation to microgravity at the transcriptomic level.The changed rhythms in locomotor and gene expression might be modulated through the master oscillator because the SCN resides in the hypothalamus [44].
Honda et al found that weight loss simulated by clinostate showed no effects on the expression of HIF1A in Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells [45].Consistently, there was no signi cant change in Hif1a in mouse embryonic stem cells (mESCs) between space ight and ground control [46].In contrast, the expression of HIF1A and HIF1-dependent transcripts in T lymphocytes and cells of the monocyte-macrophage system were changed in altered gravity simulated by parabolic ights [47].Wang et al found that in a 28-d tail-suspension (30°), there was a signi cant increase in HIF1A in mouse hippocampus [48].In this study we showed that 0.9 atm is not su cient to cause hypoxia effects on circadian rhythms as the HIF1A level remained unchanged.We found signi cant increases in the control mice in the animal room for both Hif1a mRNA in hypothalamus and HIF1A protein in the liver and a signi cant increase in HIF1A protein in the liver in the control mice in the animal room, while Hif1a mRNA was not changed in hypothalamus.Together, these data suggest that HIF1A expression differs among tissues/cells, and more importantly, it is modulated by hypoxia-independent pathways.Microgravity and its analogs cause comprehensive physiological changes including autonomic imbalances, cardiac atrophy, vascular impairment and microcirculatory dysfunction, which account for cardiovascular deconditioning [49][50][51].In this work, we revealed that several pathways involving genes showing altered rhythmicities are associated with cardiovascular deconditioning (Fig. 3; Figure S3; Table S4), suggesting that rhythmicity is important for the genes associated with cardiovascular functions.
Noise is one of the globally prevalent environmental and occupational hazards, such as in cement plants and textile factories [52][53][54].Accumulating evidence has demonstrated that dysbiosis of the gut microbiome is associated with the pathology of some cardiovascular disorders including atherosclerosis, hypertension and vascular dysfunction [55,56].It has been reported that the diversity of the gut microbiome of White-Crowned Sparrow (Zonotrichia leucophrys) living in San Francisco Bay was altered upon exposure to environmental noise [57].In this study, we showed that pathways associated with cardiovascular functions were enriched in many conditions including either HU or control and in either the simulated space capsule or the animal room (Fig. 6e,f; Figure S7; Table S7), suggesting that it was not speci cally caused by HU or noise and low atmospheric pressure.However, it is worth noting that amyotrophic lateral sclerosis was speci cally present between the early and late stages of HU mice in the simulated space capsule.
Different gravitational conditions from 1 g on the surface of Earth and simulated microgravity have been shown to affect the gut microbiome in humans and rodents [5,48,[58][59][60][61].In this study we detected no signi cant change in α-diversity between all detected conditions (Fig. 5a).In contrast, signi cant changes in α-diversity have been reported in other studies under altered gravity or simulated microgravity conditions [58,60].This inconsistency could be attributed to differences in sampling and calculation as we harvested the stool samples at six time points across a day, and the averaged values were used for αdiversity calculation.
Changes in the abundance or rhythmicity of a number of bacteria were found in this work (Fig. 6; Fig. 7; Figure S8; Table S6).Among these microorganisms, Adlercreutzia can metabolize diadzein from soybean food to equol in the gut, which is considered to prevent from bone loss [62].Therefore, the increase in gut Adlercreutzia at the late stage of HU mice in animal room suggests that it might be an adaptation to the HU condition, which might be repressed in by other factors like noise and low atmospheric pressure.A decrease in Prevotellaceae occurred in rats with uremia [63], Prevotella strains are associated with chronic in ammatory conditions including arthritis and HIV-1[64], and some Corynebacteria species are linked with a number of syndromes, such as granulomatous lymphadenitis, pneumonitis, pharyngitis, cutaneous infections and endocarditis [65].Therefore, changes in rhythmicity may impose critical physiological effects under simulated microgravity, noise and low atmospheric pressure conditions.The changes in microbiome rhymicity are likely due to the modi cation of the mouse clock by environmental factors.

Conclusions
In this work we demonstrate that noise and low atmospheric pressure may also contribute to circadian disturbance and dysbiosis of the gut microbiome in space.In the future it will be critical to systematically study the effects of superimposition of these factors on the physiology, cognition and performance of astronauts.These ndings will help further our understanding of complex space conditions on circadian rhythms, microbial alterations and their correlation with the physiology and health of the astronauts, and will improve the welfare of other professionals working in similar settings, e.g., environments with high levels of noise.

Animals and bioethics
Two-month-old C57BL/6N male mice were provided by Vital River Laboratories, Beijing.Half of these mice were raised in a simulated space capsule and the other half were raised in a normal animal room.Drinking water and food were provided by SPF Biotechnology Co., Ltd.(Beijing, China) and were provided ad libitum.
There were six mice in the capsule and six in the animal room for locomotion monitoring.In both the capsule and the animal room, there were 36 mice undergoing HU and 36 free-moving mice as controls for sampling tissues along with some spare mice.

Experimental conditions
The simulated space capsule and the animal room are located in the Chinese Astronaut Research and Training Center.The simulated space capsule is a ground simulated platform of a simulated space capsule, in which temperature, humidity, air pressure and lighting conditions can be accurately controlled.
The light regime in the simulated capsule and animal room was a cycle alternating with 12 h of light and 12 h of dark (LD12:12).The illumination intensity of white light (full spectrum light) was approximately 200 lux.The conditions in the animal room were normal atmospheric pressure and weak noise 30-45 dBA throughout the experiment.In the simulated space capsule, the normal atmospheric pressure condition and weak noise (30-45 dBA) were set for days 1-6; a 0.9 atmospheric pressure environment (atm) and noise (85 ± 2 dBA) were set for days 7-49 (Fig. 1a).On day 7 in the simulated space capsule, the 85 dbA uniform eld noise and 0.9 atm were added from 9:00 AM to the end of the experiment.The partial pressure of oxygen in the capsule after loading noise and low atmospheric pressure was 22 ± 2 kPa, the partial pressure of carbon dioxide was ≯ 1 kPa and the ammonia concentration was ≯ 20 ppm.

Protocols and arrangements
The hind limb unloading (HU) approach was employed to mimic the effects of weightlessness, which is a widely accepted ground-based weightlessness simulation [Ge et al 2019].The device for hind limb unloading allows free movement throughout the cage with the body and the ground at 30° [48].
The hypothalamus, liver and transverse colon containing stool of mice in both the capsule and the animal room were sampled at early and late stages, the early stage was from day 6 to day 7 (prior to the loading of noise and low atmospheric pressure in the capsule) and the late stage was from day 36 to day 37.The tissues were sampled every 4 h beginning at 9:00 AM and ending at 5:00 AM the next day.At each time point, three HU mice and three control mice were sacri ced and sampled.

Monitoring and analysis of locomotor activity
Six mice housed in the simulated space capsule and six mice in the animal room were living in mouse cages equipped with running wheels.Clocklab Analysis Software for Circadian Biology (Actimetrics Co. Ltd., USA) acquisition and analysis system collected the running wheel data of mice.The animal room data for days 38-39 were missing.

RNA sequencing of the hypothalamus tissues
The hypothalamic tissues were isolated and total RNA was extracted by using Trizol Reagent and subjected to RNA sequencing which was performed on a NovaSeq 6000 sequencer (Illumina, Co., Ltd., USA) by Forevergen Co. Ltd (Guangzhou, China).The reads length was PE150bp, and the quali ed reads were obtained after removing raw reads with adapters or low quality bases, and then aligned to the mouse genome (NCBI37/MM9) by SOAP with default parameters.RSeQC was used to calculate the gene expression level represented as reads per kilobase per million mapped reads (RPKM) [66,67].The JTK_CYCLE software was used to analyze and screen rhythm genes based on the RPKM values, and threshold for ADJ.P ≤ 0.05 and PER ≥ 20 h and ≤ 24 h [68], and an analysis model with circadian repetition was used.To identify pathways with signi cant enrichment, pathway enrichment analysis was performed based on the KEGG database and the result of the analysis model with circadian repetition.KEGG pathways ful lling the criterion of a hypergeometric p < 0.05 & log 2 (FoldChange) > 1 or < -1 were de ned as signi cantly enriched in DEGs and were evaluated using DAVID (https://david.ncifcrf.gov/tools.jsp) to convert the ID to Entrez Gene ID for the species Mus musculus and the pathways were visualized using KOBAS (http://kobas.cbi.pku.edu.cn/kobas3/genelist/)[69].

16S rRNA sequencing and analysis
The feces from the transverse colon lumen were harvested at xed time points and genomic DNA was extracted per the manufacturer's protocol (QiaAmp DNA Stool Mini Kit).The DNA was then used as a template for polymerase chain reaction ampli cation of bacterial 16S rRNA genes.The primers 338F: 5'-ACTCCTACGGGAGGCAGCA-3' and 806R: 5'-GGACTACHVGGGTWTCTAAT-3' were used to amplify the high-variation region (V3 ~ V4) of 16S rRNA.High-throughput sequencing was performed on an Illumina HiSeq 2500 in paired-ended mode by Forevergen Co. Ltd (Guangzhou, China).
The bioinformatics analysis of these sequences was used for OTU sequence clustering, clustering with operational taxonomic units (OUTs).OTU sequence clustering was performed with a consistency of 97%.
Clustering methods for upper bioinformatics analysis included OTU community and species analysis, beta diversity analysis, and signi cance analysis of species differences.Beta diversity on unweighted UniFrac was calculated by Quantitative Insights Into Microbial Ecology (QIIME) software (version 1.7.0;Novogene).Principal coordinate analysis (PCoA) was conducted to obtain principal coordinates and visualize from complex, multidimensional data.Unweighted Pair-Group Method with Arithmetic Euclidean distance was used to interpret the distance matrix using average linkage and was conducted by QIIME software (version 1.7.0;Novogene).LEfSe uses linear discriminant analysis (LDA) to estimate the effect of species abundance on the indicated conditions.KEGG functional pro les were predicted according to the taxonomic information with the phylogenetic information by reconstructing the whole microbial community, and Statistical Analysis of Metagenomic Pro les (STAMP) (v2.1.3,Welch's t-test, two-sided, post hoc comparisons (Benjamini-Hochberg FDR) p < 0.05) [70,71].The JTK_CYCLE software was used to analyze and screen rhythmicity of the microbiome, and threshold for ADJ.P ≤ 0.05 and PER ≥ 20 h and ≤ 24 h [68].The experiments in the simulated space capsule and the animal room were carried out simultaneously.The hind limb unloaded (HU) mice or control mice were placed in both simulated space capsule and animal room.The gray squares represent the dark episodes, the white squares represent the light episodes, the green dotted lines indicate that the capsule was being operated and was loaded with conditions of low atmospheric pressure and noise, the red circles represent the time points of sampling of tissues and feces every 4 h.Speakers symbol and speakers symbol with crosses denote the start and end of noise, respectively, simultaneous with exposure to the low atmospheric pressure.(b,c) Representative double-plotted actograms of the wheel-running activity of mice in the (b) simulated space capsule (c) and animal room.The mice were subjected to a 6-h phase advance in LD cycles from day 38 to the end (n=6).

Western blot analysis of HIF1A protein in mouse liver
Crosses in blue denote the missing data on days 38-39.(d) The number of activities of mice per minute in the simulated space capsule and the animal room.(e) Daily onset of locomotion in the 6-h phase advance after change in lighting regime (n=6).(f) The days to adjust to the 6-h phase advance.(d) were calculated and analyzed within days 7-16 of the experiment.One-way ANOVA was used to compare the differences between groups: (e-f) are the mean (SE),* p < 0.05, ** p < 0.05, # p < 0.001.SSC: simulated space capsule; AR: animal room; HU: hind limb unloading; Ctrl: control.

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Figure 3 Changes
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Figure 4 Changes
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