Abstract

摘要

A study was conducted to evaluate the three-dimensional clinostat simulated microgravity effect on mouse models, focusing on the central nervous system. Eighteen mice were divided into three groups: control, survival box, and clinostat + survival box. Behavioral tests, femur micro-CT, brain transcriptomics, serum metabolomics, and fecal microbiomics were performed.

进行了一项研究，以评估三维回转器模拟微重力对小鼠模型的影响，重点在中枢神经系统。十八只小鼠被分为三组：对照组、生存盒组和回转器+生存盒组。进行了行为测试、股骨微CT、脑转录组学、血清代谢组学和粪便微生物组学分析。

Results showed decreased activity, altered gait, enhanced fear memory, bone loss, immune/endocrine changes in brain transcriptome, and altered metabolic pathways in serum and gut microbiota in clinostat-treated mice. The model closely mimics spaceflight-induced transcriptome changes, suggesting its value in studying microgravity-related neurological alterations and highlighting the need for attention to emotional changes in space..

结果显示，在旋转器处理的小鼠中，活动减少、步态改变、恐惧记忆增强、骨质流失、脑转录组的免疫/内分泌变化，以及血清和肠道微生物群中代谢通路的改变。该模型紧密模拟了太空飞行引起的转录组变化，表明其在研究微重力相关的神经学变化中的价值，并强调了对太空中情绪变化关注的必要性。

Introduction

介绍

Space exploration is a long-term dream and pursuit of humanity, and it is also one of the important fields of technological development. In long-term space flight, astronauts face many problems. One of them is the reduction of gravitational force. Terrestrial life has evolved under the influence of gravity for millions of years, and long-term microgravity represents unusual stress experiences that can cause physiological and morphological changes.

空间探索是人类长期的梦想与追求，也是技术发展的重要领域之一。在长期的太空飞行中，宇航员面临许多问题，其中之一就是引力的减少。地球生命在重力影响下已经进化了数百万年，长期微重力代表着非同寻常的压力体验，可能引起生理和形态上的变化。

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. Numerous studies have shown that long-term weightlessness can lead to various physiological and pathological changes, such as the skeletal and muscular system, cardiovascular system, immune system, and nervous system

众多研究表明，长期失重会引起骨骼肌肉系统、心血管系统、免疫系统、神经系统等出现各种生理及病理性改变。

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. Prolonged exposure to microgravity during extended space missions poses various physiological challenges to astronauts, necessitating thorough research into the effects of microgravity on the human body. One of the crucial issues is the impact of long-term spaceflight on brain function. Studies have shown that astronauts experience changes such as ataxia, posture disorders, perceptual illusions, fatigue, and cognitive impairment.

长期太空任务期间，长时间暴露在微重力环境下会给宇航员带来各种生理挑战，因此有必要深入研究微重力对人体的影响。其中一个关键问题是长期太空飞行对大脑功能的影响。研究表明，宇航员会出现共济失调、姿势障碍、感知错觉、疲劳和认知障碍等变化。

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. The high cost and technical issues related to spaceflight have stimulated the development of ground models that reasonably simulate the effects of space flight. Currently, tail-suspension hindlimb unloading is the most classic and widely used model, particularly those affecting the musculoskeletal system.

。与太空飞行相关的高成本和技术问题促使了地面模型的发展，这些模型能够合理模拟太空飞行的影响。目前，尾部悬吊后肢卸载是最经典和广泛使用的模型，特别是那些影响肌肉骨骼系统的模型。

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. However, this model has certain limitations, that is, it can only be applied locally, and cannot be applied to the whole experimental animal. Besides, there are physiological differences between hindlimb unloading models and spaceflight, especially in terms of somatosensory input, spatial orientation, and vestibular disruption.

然而，该模型有一定的局限性，即只能局部应用，不能应用于整个实验动物。此外，后肢卸载模型与太空飞行之间存在生理差异，尤其是在体感输入、空间定向和前庭紊乱方面。

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Three-dimensional (3D) clinostat is a device that generates multi-directional G forces. By controlling the rotation on both axes, it can offset the cumulative gravity vector at the center of the device

三维（3D）回转器是一种产生多方向G力的装置。通过控制两个轴上的旋转，它可以抵消设备中心的累积重力矢量。

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. Previous studies have shown that 3D clinostat have been widely used on plants, cells and Caenorhabditis elegans to simulate microgravity effects

以往的研究表明，3D回转器已广泛用于植物、细胞和秀丽隐杆线虫，以模拟微重力效应。

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. Our previous studies suggest that the central nervous system probably changed in mice trained with 3D clinostat

我们之前的研究表明，在使用3D旋转器训练的小鼠中，中枢神经系统可能发生了变化。

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. Therefore, the aim of our work is to systematically assess 3D clinostat simulated microgravity effect in mice by behavioral and multi group analysis, focusing on the changes in central nervous system and cognitive function.

因此，我们工作的目的是通过行为和多组分析，系统地评估3D旋转器模拟微重力对小鼠的影响，重点关注中枢神经系统和认知功能的变化。

Methods

方法

Mice and treatment

小鼠与治疗

10-week-old male C57BL/6J mice were purchased from Beijing HFK Bioscience Co., Ltd and kept in the experimental room at temperature 22 ± 2 °C, humidity 40 ± 5%, under a 12-h light/dark cycle. The mice were randomly divided into three groups: (1) MC group with mice housed in ordinary cage (MC, n = 6); (2) SB group with mice housed in survival box (SB, n = 6); (3) CS group with mice housed in survival box receiving 3D clinostat treatment (CS, n = 6).

10周龄雄性C57BL/6J小鼠购自北京华阜康生物科技股份有限公司，饲养在实验室内，温度22 ± 2°C，湿度40 ± 5%，12小时光照/黑暗循环。小鼠被随机分为三组：(1) 普通笼养组（MC，n = 6）；(2) 生存盒组（SB，n = 6）；(3) 3D回转器处理生存盒组（CS，n = 6）。

The structure of the 3D clinostat and the survival box (shown in Supplementary Figure S1) has been described in detail in our previous study.

3D旋转器和生存箱的结构（见补充图S1）已在我们之前的研究中详细描述。

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. The rotation modes are as follows: random rotation speed, 0–10 rpm; speed resolution, 0.1 rpm. Before the study, all mice were acclimated to the facility environment for 2 days and then adaptive training was carried out for 5 days to alleviate stress and adapt to the jelly like diet containing 59% water, 1% agar, and 40% purified feed.

旋转模式如下：随机旋转速度，0–10转/分钟；速度分辨率，0.1转/分钟。在研究之前，所有小鼠都适应设施环境2天，然后进行5天的适应性训练，以减轻压力并适应含59%水、1%琼脂和40%纯化饲料的果冻状饮食。

During training, the CS group underwent adaptive rotation for 1 h, 2 h, 4 h, 8 h, and 12 h each day, while the MC group was always placed in ordinary cage and the SB group was always placed in survival boxes without any treatment. The formal experiments began at 11 weeks old. The CS group experienced continuous simulated microgravity effect on clinostat for 8 weeks with the exception of cleaning cages and changing food for approximately 20 min per day; the other two groups of mice always lived in corresponding living facilities in the same room.

在训练期间，CS组每天进行1小时、2小时、4小时、8小时和12小时的自适应旋转，而MC组始终放置在普通笼子中，SB组始终放置在生存箱中且不进行任何处理。正式实验从11周龄开始。CS组在旋转器上连续经历8周的模拟微重力效应，除了每天清洁笼子和更换食物约20分钟外；其他两组小鼠始终生活在同一房间内的相应生活设施中。

To avoid the interference of acute stress, weight monitoring showed that the body weight of mice in the CS group gradually returned to the control group level after 28 days, suggesting that the mice had fully adapted to the 3D clinostat. Starting from the fifth week, mice underwent a series of behavioral experiments.

为了避免急性应激的干扰，体重监测显示，CS组小鼠的体重在28天后逐渐恢复到对照组水平，这表明小鼠已完全适应3D回转器。从第5周开始，小鼠进行了一系列行为学实验。

Following each behavioral experiment, the mice were promptly placed into a 3D clinostat to ensure continuous microgravity effect stimulation. After 8 weeks, the mice were euthanized dissected for sampling at the end of the experiment (Fig. .

每次行为实验后，立即将小鼠置于3D回转器中，以确保持续的微重力效应刺激。8周后，实验结束时对小鼠实施安乐死并进行解剖取样（图）。

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a). Animal care was provided in accordance with institutional guidelines and all animal studies were performed in compliance with the ARRIVE guidelines and were approved by the Ethics Committee of the Laboratory of Animal Science of Peking Union Medical College (IACUC-20220415).

a). 根据机构指南提供了动物护理，所有动物研究均符合ARRIVE指南，并经北京协和医学院动物科学实验室伦理委员会批准（IACUC-20220415）。

Fig. 1

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(

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) Flowchart of the experimental procedures and the behavioral experiments are conducted in the order of arrangement; (

实验程序的流程图和行为实验按照安排的顺序进行；

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) Changes in mice body weight; (

) 小鼠体重的变化； (

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) Total moving distance of mice in open field test; (

) 小鼠在旷场试验中的总移动距离；(

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) Exercise time of mice in open field test; (

) 小鼠在旷场试验中的运动时间；(

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) Recognition index of mice in novel object recognition test; (

新物体识别测试中老鼠的识别指数；

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) Spontaneous alternations index in Y maze; (

) Y迷宫中的自发交替指数；(

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) Latency to the platform time in Morris water maze. MC: ordinary cage control group; SB: survival box control group; CS: 3D clinostat model group. The data are presented as the mean ± SD, *

在Morris水迷宫中到达平台的时间。MC：普通笼子对照组；SB：生存箱对照组；CS：3D旋转器模型组。数据以平均值±标准差表示，*

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< 0.05, **

< 0.05, **

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< 0.01, ***

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< 0.001, n = 6 for each group.

< 0.001，每组n = 6。

Full size image

全尺寸图像

Mouse behavioral tests

小鼠行为测试

The sequence of the behavioral experiments was arranged as follows: Grip strength experiment (day 29), Open field test (day 30), Novel object recognition test (day 30–32), Y-Maze (day 33), Elevated plus maze (day 34), Tail suspension test (day 35), Gait analysis (day 36–40), Rotarod test (day 42–44), Morris water maze (day 45–51), Forced swim test (day 52) and Fear conditioning test (day 53–56).

行为实验的顺序安排如下：握力实验（第29天）、旷场试验（第30天）、新物体识别试验（第30-32天）、Y迷宫（第33天）、高架十字迷宫（第34天）、尾悬挂试验（第35天）、步态分析（第36-40天）、转棒试验（第42-44天）、莫里斯水迷宫（第45-51天）、强迫游泳试验（第52天）和恐惧条件试验（第53-56天）。

Each mouse was tested individually and then put back into the 3D clinostat as soon as possible after the test to minimize the time spent disengaging from the rotation..

每只小鼠单独进行测试，然后在测试结束后尽快放回3D旋转器中，以尽量减少脱离旋转的时间。

Detailed methods of behavioral testing are as follows:

行为测试的详细方法如下：

Grip strength experiment

握力实验

The evaluation of grip strength was conducted using a grip strength meter. Each animal was individually placed on the grip strength board and pulled by the tail, prompting them to grasp the bar. The rodents instinctively grasp anything in order to prevent this involuntary backward movement until the pulling force exceeds their grip strength.

使用握力计进行握力评估。每只动物被单独放置在握力板上，通过拉尾巴促使它们抓住横杆。啮齿动物会本能地抓住任何东西，以防止这种不自主的向后移动，直到拉力超过它们的握力。

The maximum value of grip strength was recorded using the Bioseb system. Each animal was tested three times repeatedly, and the mean value was calculated. During the behavioral testing time, each mouse was removed from the 3D clinostat device for about 15 min for testing, and then quickly returned to the 3D clinostat device after testing..

使用Bioseb系统记录握力的最大值。每只动物重复测试三次，并计算平均值。在行为测试期间，每只小鼠从3D回转器装置中取出约15分钟进行测试，测试后迅速返回到3D回转器装置中。

Open field test

开放场地测试

The open field was an empty square environment, with a size of 50 cm × 50 cm × 30 cm. A central area was marked in the open field, centered in the middle of the empty space, with a square length of 16 cm zone. The fringe area was 10 cm wide. The animals were placed in the middle of the central area and observed for five minutes.

旷场是一个50厘米×50厘米×30厘米的空旷方形环境。在旷场中央标记了一个中心区域，位于空旷空间的正中间，为一个边长16厘米的正方形区域。外围区域宽度为10厘米。将动物放置在中心区域的中央，并观察五分钟。

Behavioral parameters including velocity, distance traveled, and time spent in the central and fringe areas were recorded by a computerized video tracking system (Ethovision XT; Noldus, Information Technology, The Netherlands). The open field apparatus was cleaned after each session using 70% ethanol and allowed to dry between tests.

包括速度、行进距离以及在中心和边缘区域所花费的时间等行为参数，通过计算机化的视频跟踪系统（Ethovision XT；Noldus，信息技术，荷兰）进行记录。每次实验后，使用70%的乙醇清洁旷场装置，并在测试之间让其自然干燥。

During the behavioral testing time, each mouse was removed from the 3D clinostat device for about 20 min for testing, and then quickly returned to the 3D clinostat device after testing..

在行为测试期间，每只小鼠被从3D回转器装置中取出约20分钟进行测试，测试后迅速返回到3D回转器装置中。

Novel object recognition test

新颖物体识别测试

The novel object recognition test was used to assess short- and long-term recognition memory. The experimental procedure consisted of three stages. On the first day, there was an adaptation period without any toy bricks. On the second day, familiarization took place with two toy bricks (2 cm × 2 cm) in opposite corners, both with the same shape, size, and color.

新物体识别测试用于评估短期和长期识别记忆。实验程序包括三个阶段。第一天，有一个适应期，没有任何玩具积木。第二天，熟悉过程在对角放置的两块玩具积木（2厘米×2厘米）进行，这两块积木形状、大小和颜色相同。

On the third day, the test period occurred, with one of the toy bricks changed to a different shape and color. The camera on top of the box was used to record how long it took for a mouse to examine the new object (TN) and the familiar object (TF), allowing a total of five minutes for exploring the two distinct objects.

在第三天，测试期到来，其中一块玩具积木被换成了不同的形状和颜色。盒子顶部的摄像机用于记录小鼠检查新物体（TN）和熟悉物体（TF）所花费的时间，总共给予五分钟来探索这两个不同的物体。

The recognition index was calculated as follows: Recognition index = (TN/(TN + TF)) × 100%. A higher recognition index reflected better short- and long-term recognition memory abilities in animals within this group. The behavioral test time was about 20 min per day for each mouse to be removed from the 3D clinostat for testing, and then quickly returned to the 3D clinostat after testing..

识别指数的计算方法如下：识别指数 = (TN / (TN + TF)) × 100%。较高的识别指数反映了该组动物在短期和长期识别记忆能力上表现更好。行为测试时间约为每天20分钟，每只小鼠从3D旋转器中取出进行测试，测试后迅速返回到3D旋转器中。

Y-maze

Y迷宫

In the Y-maze test, each mouse was placed at the end of one arm of the 'Y' and allowed to explore the maze freely for five minutes. The sequence of entry of the mice in the arms was recorded to calculate the percentage of change. The mouse entered three different arms in turn, which was counted as one alternation.

在Y迷宫测试中，每只小鼠被放置在“Y”型迷宫的一个臂的末端，并允许其自由探索迷宫五分钟。记录小鼠进入各臂的顺序以计算变化百分比。小鼠依次进入三个不同的臂，这被计为一次交替。

Spontaneous alternations (%) = Number of spontaneous alternations/(Total number of arm entries − 2) *100%. Higher spontaneous alternations indicated better learning and memory ability. During the behavioral testing time, each mouse was removed from the 3D clinostat device for about 20 min for testing, and then quickly returned to the 3D clinostat device after testing..

自发交替率 (%) = 自发交替次数 / (总臂数进入次数 - 2) * 100%。较高的自发交替率表明学习和记忆能力更好。在行为测试期间，每只小鼠从3D回转器设备中取出约20分钟进行测试，测试后迅速返回到3D回转器设备中。

Elevated plus maze

高架十字迷宫

During the Elevated Plus Maze (EPM) test, the maze was composed of four arms (30 cm × 5 cm). Two opposite arms were closed, enclosed by lateral walls (depth 5 cm), and the remaining two arms were open and lacked walls. The four arms were connected by a central area (5 cm × 5 cm), and the maze was elevated 50 cm from the floor.

在高架十字迷宫（EPM）测试中，迷宫由四条臂（30厘米 × 5厘米）组成。两条相对的臂是封闭的，由侧壁包围（深度5厘米），其余两条臂是开放的且没有墙壁。四条臂通过一个中央区域（5厘米 × 5厘米）相连，迷宫离地面50厘米高。

During the test, mice were placed on the central square platform and allowed to explore the maze for 5 min. The following parameters were recorded and analyzed: the number of entries into open/closed arms, the time spent in the open/closed arms, and the total number of arm entries. These data were recorded by a camera and analyzed using a computerized video tracking system..

在测试期间，将小鼠放置在中央方形平台上，让其在迷宫中探索5分钟。记录并分析以下参数：进入开放/封闭臂的次数、在开放/封闭臂中停留的时间，以及进入臂的总次数。这些数据通过摄像机记录，并使用计算机化的视频跟踪系统进行分析。

The anxiety index was calculated as the ratio of open arm time and entries to total time and total entries. The open arm entries percent was calculated as entries into the open arm divided by the total number of arm entries (entries into the open arm + entries into the closed arm). During the behavioral testing time, each mouse was removed from the 3D clinostat device for about 20 min for testing, and then quickly returned to the 3D clinostat device after testing..

焦虑指数计算为开放臂时间和进入次数与总时间和总进入次数的比值。开放臂进入百分比计算为进入开放臂的次数除以总臂进入次数（进入开放臂次数＋进入封闭臂次数）。在行为测试期间，每只小鼠从3D回转器装置中取出约20分钟进行测试，测试结束后迅速返回到3D回转器装置中。

Tail suspension test

尾悬挂试验

The Tail Suspension Test was performed as previously described

尾悬挂试验按之前描述的方法进行。

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. Adhesive tape was applied to the tail, approximately 1 cm from the tip, and the mouse was suspended for 6 min. After a 2 min habituation period, the immobility time was recorded during the final 4 min using Tail Suspension Real-Time Analysis (System-2.0). During the behavioral testing time, each mouse was removed from the 3D clinostat device for about 20 min for testing, and then quickly returned to the 3D clinostat device after testing..

. 将胶带贴在尾部，距离尾尖约1厘米处，然后将小鼠悬挂6分钟。在2分钟的适应期后，使用尾部悬挂实时分析系统（System-2.0）记录最后4分钟内的静止时间。在行为测试期间，每只小鼠从3D回转器装置中取出约20分钟进行测试，测试结束后迅速放回3D回转器装置。

Gait analysis

步态分析

The gait of naturally moving mice was analyzed using CatWalk XT (Noldus Information Technology, Netherlands), as previously described

使用CatWalk XT（荷兰Noldus信息技术公司）分析自然移动小鼠的步态，如前所述。

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. This gait analysis system consists of a glass walkway floor illuminated with a green light that is completely internally reflected in the glass, a standard charge-coupled device (CCD) camera underneath, and software for recording and analyzing the data obtained from mouse paws. A darkroom box was used at the end of the walkway to create an incentive for the mice to cross the field, where the entrance to their home cage is placed.

该步态分析系统由一块玻璃走道地板组成，地板被绿光照射并在玻璃中完全内反射，下方有一台标准电荷耦合器件（CCD）相机，以及用于记录和分析从小鼠爪子获取数据的软件。走道尽头使用了一个暗箱来激励小鼠穿过场地，其中放置了它们的家庭笼子入口。

The mice were placed in the front of the start zone of the walkway and were trained to cross the walkway with five accomplished runs per day on five consecutive days. The test was started on the fifth day, and each mouse was tested three times. The successful run was defined as a mouse walking across the runway without any hesitation.

小鼠被放置在步道起点区域的前端，并接受训练，连续五天每天完成五次穿越步道的任务。测试在第五天开始，每只小鼠测试三次。成功穿越定义为小鼠毫无犹豫地走过跑道。

The walkway was cleaned thoroughly with 70% ethanol between each animal. Gait parameters of footprints were automatically generated (left front, LF; left hind, LH; right front, RF; and right hind, RH). The behavioral test time was about 20 min per day for each mouse to be removed from the 3D clinostat for testing, and then quickly returned to the 3D clinostat after testing..

每次动物实验之间，通道都用70%的乙醇彻底清洁。足迹的步态参数自动生成（左前，LF；左后，LH；右前，RF；右后，RH）。行为测试时间约为每天20分钟，每只小鼠从3D旋转器中取出进行测试，然后在测试结束后迅速返回到3D旋转器。

Rotarod test

转棒测试

The rotarod apparatus (Model: 47600, Italy) was used to assess the locomotor abilities of rodents, particularly their coordination and endurance. Mice were subjected to a training session of four trials per day for 3 days, the rotarod gradually accelerated from 5 to 40 rpm within 120 s until the mouse fell off.

使用旋转棒装置（型号：47600，意大利）评估啮齿动物的运动能力，特别是它们的协调性和耐力。小鼠连续3天每天进行4次训练，旋转棒在120秒内从5转/分钟逐渐加速到40转/分钟，直到小鼠掉落。

Starting from the 4th day after training, formal testing experiments were conducted. During test trial, the rotarod gradually accelerated from 5 to 40 rpm within 300 s. The latency at which the mouse fell from the rod (or a maximum of 5 min) was recorded. The average latency from three trials was determined.

从训练后的第4天开始，进行正式的测试实验。在测试过程中，转棒在300秒内从5 rpm逐渐加速到40 rpm。记录小鼠从转棒上掉落的潜伏期（或最长5分钟）。计算三次试验的平均潜伏期。

During the behavioral testing time, each mouse was removed from the 3D clinostat device for about 30 min for testing, and then quickly returned to the 3D clinostat device after testing..

在行为测试期间，每只小鼠从3D旋转器装置中取出约30分钟进行测试，测试后迅速返回到3D旋转器装置中。

Morris water maze

莫里斯水迷宫

The water maze was divided into four quadrants and contained a platform that remained submerged under 1–1.5 cm of water. During the training trials, mice were placed in the water from the opposite quadrant of the platform. Successful navigation to the platform was considered when the mouse found it within 1 min.

水迷宫被划分为四个象限，并包含一个平台，该平台始终淹没在1-1.5厘米深的水下。在训练试验中，将小鼠从平台对面的象限放入水中。如果小鼠在1分钟内找到平台，则认为其成功导航到平台。

If the mouse failed to find the platform within this time limit, a guidance rod was used to guide the mouse to stay on the platform for 10 s and the latency was recorded as 60 s. Training trials were conducted for three trial sessions each day for six consecutive days. During probe trials, the platform was removed from the maze.

如果老鼠在此时间限制内未能找到平台，则使用引导杆引导老鼠停留在平台上10秒，并将潜伏期记录为60秒。训练试验每天进行三次，连续六天。在探测试验中，平台被移出迷宫。

Frequency of visits to the platform, time spent in the platform quadrant, cumulative duration in the target zone, and latency to the platform were used as indicators of memory performance. The behavioral test time was about 30 min per day for each mouse to be removed from the 3D clinostat for testing, and then quickly returned to the 3D clinostat after testing..

平台访问频率、平台象限停留时间、目标区域累计持续时间以及到达平台的延迟被用作记忆表现的指标。行为测试时间约为每天每只小鼠从3D回转器中取出进行测试30分钟，测试后迅速返回3D回转器。

Forced swim test

强迫游泳试验

The forced swim test was performed as previous studies

强迫游泳测试按照以前的研究进行。

13

13

. Each mouse was individually placed into a non-transparent cylindrical plastic container (height of 25 cm; diameter of 18 cm) containing 15 cm of water at 23–25 °C. The test duration was 6 min, and immobility was analyzed during the last 4 min. Immobility behavior was considered by inactive time when the animal remained floating passively without any movements, with the exception of minimal movements to keep the head above water.

每只小鼠被单独放置在一个不透明的圆柱形塑料容器中（高度为25厘米，直径为18厘米），容器内装有15厘米深、温度在23-25°C的水。测试持续时间为6分钟，并在最后4分钟内分析静止不动的行为。当动物被动漂浮、无任何动作时（除保持头部在水面上所需的最小动作外），该行为被视为静止不动。

During the behavioral testing time, each mouse was removed from the 3D clinostat device for about 20 min for testing, and then quickly returned to the 3D clinostat device after testing..

在行为测试期间，每只小鼠被从3D回转器中取出约20分钟进行测试，测试结束后迅速放回3D回转器。

Fear conditioning test

恐惧条件反射测试

Both contextual and cued fear conditioning protocols were implemented as previous studies

结合了之前研究的背景性和提示性恐惧条件协议都被实施了。

14

14

. On the first day, mice were introduced to a Plexiglas training cage under constant illumination for 5 min. On the second day, animals were allowed to adapt for 180 s in the box before commencing three to five rounds of circulation training. Auditory stimulation (30 s, 5 kHz, 70 dB) was then administered, followed by the delivery of electric current stimulation (0.65 mA, 1 s).

第一天，将小鼠置于持续光照的有机玻璃训练笼中5分钟。第二天，让动物在箱中适应180秒后，开始进行三到五轮循环训练。随后施加听觉刺激（30秒，5千赫，70分贝），接着进行电流刺激（0.65毫安，1秒）。

The intervals between each training session were 30–60 s. Animals were allowed to remain in the cage for 30 s before being returned to their home cage. Twenty-four hours later, a contextual fear conditioning test was conducted by placing animals back into the same training cage without any sound or electric current stimulation, and their freezing behavior was recorded over a 330 s period.

每次训练间隔时间为30-60秒。动物在被送回其饲养笼之前，允许在笼中停留30秒。24小时后，进行情境恐惧条件测试，将动物重新放回相同的训练笼中，不伴有任何声音或电流刺激，并记录它们在330秒内的冻结行为。

On the fourth day, cued fear conditioning began. The background and smell of the boxes were changed, and after 180 s of adaptation in the box, only auditory stimulation (5 kHz, 70 dB) was presented. Freezing behavior was then recorded over a 420 s period. Prior to each animal’s placement, the fear-conditioning box was thoroughly cleaned with 70% ethanol.

在第四天，开始进行提示恐惧条件反射。盒子的背景和气味被改变，在盒子中适应180秒后，仅呈现听觉刺激（5 kHz，70 dB）。随后记录420秒内的冻结行为。在每次放置动物之前，用70%的乙醇彻底清洁恐惧条件反射盒。

The behavioral test time was about 20 min per day for each mouse to be removed from the 3D clinostat for testing, and then quickly returned to the 3D clinostat after testing..

每只小鼠每天进行行为测试的时间约为20分钟，测试时从小型3D回转器中取出，测试后迅速放回3D回转器。

Brain transcriptomics

大脑转录组学

Brain samples were bisected along the plane separating the hemispheres. For each brain, one hemisphere was selected for the generation of mRNA sequencing data, following a methodology similar to that described in the previously published paper

大脑样本沿分离半球的平面被切成两半。对于每个大脑，选择一个半球用于生成mRNA测序数据，所采用的方法类似于先前发表的论文中描述的方法。

15

15

. Total RNA extraction was carried out using the TRIzol reagent (Invitrogen, CA, USA), following the manufacturer’s protocol. To assess the quality and quantity of the extracted RNA, the NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) was employed. The integrity of the RNA was then evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

总RNA提取使用TRIzol试剂（Invitrogen，美国加利福尼亚）按照制造商的方案进行。为了评估提取RNA的质量和数量，使用了NanoDrop 2000分光光度计（Thermo Scientific，美国）。随后使用Agilent 2100生物分析仪（Agilent Technologies，美国加利福尼亚圣克拉拉）评估RNA的完整性。

Subsequently, RNA libraries were constructed with the VAHTS Universal V6 RNA-seq Library Prep Kit, strictly adhering to the manufacturer’s instructions..

随后，使用VAHTS Universal V6 RNA-seq Library Prep Kit构建RNA文库，并严格遵循制造商的说明。

The transcriptome sequencing and subsequent analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). The libraries were sequenced on an Illumina Novaseq 6000 platform, generating 150 bp paired-end reads. The raw reads in fastq format underwent an initial processing step using fastp. During this process, low-quality reads were filtered out to obtain clean reads suitable for further analysis.

转录组测序及后续分析由上海欧易生物医学科技有限公司完成。文库在Illumina Novaseq 6000平台上进行测序，生成150 bp的双端读段。原始fastq格式的读段使用fastp进行了初步处理，在此过程中，低质量的读段被过滤掉，以获得适合进一步分析的高质量读段。

The clean reads were then mapped to the reference genome using HISAT2. To quantify gene expression levels, the FPKM of each gene was calculated and the read counts for each gene were obtained via HTSeq-count. To evaluate the biological duplication of the samples, Principal Component Analysis (PCA) was performed using the R programming language (version 3.2.0).

然后使用HISAT2将清洁读段映射到参考基因组。为了量化基因表达水平，计算了每个基因的FPKM，并通过HTSeq-count获得了每个基因的读取计数。为了评估样本的生物学重复性，使用R编程语言（版本3.2.0）进行了主成分分析（PCA）。

For the differential expression analysis, the DESeq2 package was utilized. Genes were considered significantly differentially expressed if they met the criteria of a .

对于差异表达分析，使用了DESeq2包。如果基因符合标准，则被认为显著差异表达。

P

P

value < 0.05 and a fold change > 1.5 or fold change < 0.667. Bioinformatic analysis and graphics was performed using the OECloud tools at

值 < 0.05 且倍数变化 > 1.5 或倍数变化 < 0.667。生物信息学分析和图形使用 OECloud 工具完成。

https://cloud.oebiotech.com/task/

https://cloud.oebiotech.com/task/

.

。

The hindlimb unloading mice and real spaceflight microgravity mice brain transcriptome data have been sourced from published data. Specifically, the brain RNA-seq data of hindlimb unloading mice, which underwent hindlimb unloading for 2 weeks and had their total RNA extracted from each brain using Trizol for transcriptome analysis (GeneLab ID: GLDS-32, .

后肢卸载小鼠和真实太空飞行微重力小鼠的脑转录组数据来源于已发表的数据。具体来说，后肢卸载小鼠的脑RNA-seq数据是通过使用Trizol从每只小鼠的大脑中提取总RNA进行转录组分析获得的，这些小鼠经历了2周的后肢卸载（GeneLab ID：GLDS-32）。

https://doi.org/10.26030/jpyz-fn46

https://doi.org/10.26030/jpyz-fn46

), and the data from spaceflight mice, that were housed in the Rodent Habitat for 39–42 days, euthanized in space (mice were anesthetized by ketamine/xylazine/acepromazine and subjected to cardiac puncture followed by cervical dislocation), and had their whole carcasses stored at − 80 °C during the Rodent Research-3 mission (GeneLab ID: GLDS-352, .

），这些数据来自于在太空飞行的小鼠，它们被安置在啮齿动物栖息地39至42天，在太空中实施安乐死（小鼠通过氯胺酮/甲苯噻嗪/乙酰丙嗪麻醉后进行心脏穿刺和颈椎脱臼），并在啮齿动物研究-3任务期间将其整个尸体保存在-80°C（GeneLab ID：GLDS-352， 。

https://doi.org/10.26030/jm59-zy54

https://doi.org/10.26030/jm59-zy54

), are both accessible within the NASA Open Science Data Repository (

），都可以在NASA开放科学数据存储库（

https://osdr.nasa.gov/bio

https://osdr.nasa.gov/bio

). Differential expression analysis was performed using the DESeq2.

）。使用DESeq2进行差异表达分析。

P

P

value < 0.05 and foldchange > 1.5 or foldchange < 0.667 was set as the threshold for significantly differential expression gene. Bioinformatic analysis and graphics was performed using the OECloud tools at

将值 < 0.05 且倍数变化 > 1.5 或倍数变化 < 0.667 设为显著差异表达基因的阈值。生物信息学分析和图表使用 OECloud 工具完成。

https://cloud.oebiotech.com/task/

https://cloud.oebiotech.com/task/

.

。

To compare the differences between three models, namely the 3D clinostat model (CS), hindlimb unloading model (HU), and space flight mice (FL), we conducted a distance comparison of transcriptome changes. Select shared genes from transcriptome data for analysis, and obtain log

为了比较三种模型之间的差异，即3D回转器模型（CS）、后肢卸载模型（HU）和太空飞行小鼠（FL），我们进行了转录组变化的距离比较。从转录组数据中选择共享基因进行分析，并获取log值。

2

2

FoldChang values for each gene, namely FC

每个基因的FoldChang值，即FC

CS

计算机科学

(1,2,3…n), FC

(1,2,3…n), FC

HU

匈牙利语

(1,2,3…n), FC

(1,2,3…n), 全连接层

FL

FL

(1,2,3…n), and then calculate the brain transcriptome distance between each model, S

(1,2,3…n)，然后计算每个模型之间的脑转录组距离，S

CS/FL

计算机科学/功能语言

=

=

\(\sum_{1}^{n}|{FC}_{cs}n-{FC}_{FL}n|\)

\(\sum_{1}^{n}|{FC}_{cs}n-{FC}_{FL}n|\)

, S

，S

HU/FL

匈牙利/佛罗里达

=

=

\(\sum_{1}^{n}|{FC}_{HU}n-{FC}_{FL}n|\)

\(\sum_{1}^{n}|{FC}_{HU}n-{FC}_{FL}n|\)

, S

，S

CS/HU

中文：计算机科学/人机交互

=

=

\(\sum_{1}^{n}|{FC}_{cs}n-{FC}_{HU}n|\)

\(\sum_{1}^{n}|{FC}_{cs}n-{FC}_{HU}n|\)

. The similarity of the three models can be compared by the magnitude of the distance value S. By utilizing the multiple expression changes of each gene in different models relative to their respective control groups, a heatmap can be drawn to visually display and compare the differences in the brain transcriptome of each model..

三个模型的相似性可以通过距离值S的大小来比较。通过利用每个基因在不同模型中相对于各自对照组的多种表达变化，可以绘制热图以直观显示和比较每个模型的脑转录组差异。

Serum metabolomics

血清代谢组学

To extract metabolites, 400 μl of cold extraction solvent methanol/acetonitrile/H

为了提取代谢物，使用400微升的冷提取溶剂甲醇/乙腈/H

2

2

O (2:2:1) was added to 100 μl of serum sample and adequately vortexed. The samples were then incubated on ice for 20 min and centrifuged at 14,000

O (2:2:1) 被加入到 100 μl 的血清样品中并充分涡旋。然后将样品在冰上孵育 20 分钟，并在 14,000 下离心。

g

g

for 20 min at 4 °C. The supernatant was dried in a vacuum centrifuge. For LC–MS analysis, the samples were re-dissolved in 100 μL acetonitrile/water (1:1, v/v) solvent and centrifuged at 14,000

在4°C下进行20分钟。上清液在真空离心机中干燥。对于LC-MS分析，样品重新溶解在100 μL乙腈/水（1:1，v/v）溶剂中，并在14,000转速下离心。

g

g

at 4 °C for 15 min, then the supernatant was injected. 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.

在4°C下进行15分钟，然后注入上清液。对于极性代谢物的非靶向代谢组学分析，提取物通过亲水相互作用色谱与电喷雾电离联用，使用四极杆飞行时间质谱仪（Sciex Triple TOF 6600）进行分析，由上海应用蛋白质技术有限公司完成。

Ltd. Preparation of QC samples by mixing aliquots of all samples. The raw LC–MS data obtained were processed using Proqenesis QI Software (Waters Corporation Milford, USA). For statistical analysis, we made use of both univariate (Student’s t-test and fold change analysis) and multivariate (principal component analysis and orthogonal partial least squares discriminant analysis) method.

有限公司。通过混合所有样品的等分试样来制备QC样品。使用Proqenesis QI软件（美国沃特世公司米尔福德）处理获得的原始LC-MS数据。在统计分析中，我们同时使用了单变量（学生t检验和倍数变化分析）和多变量（主成分分析和正交偏最小二乘判别分析）方法。

The variable importance in projection (VIP) scores > 1 and .

投影中的变量重要性（VIP）得分 > 1 且 。

P

P

values < 0.05, and fold change > 2 or < 0.5 were used to filter the differential metabolites. For the KEGG annotation of metabolic pathways, the metabolites were compared against the online KEGG database using BLAST to retrieve their COs, and then mapped to the corresponding KEGG pathways

值 < 0.05，且倍数变化 > 2 或 < 0.5 被用于筛选差异代谢物。对于代谢通路的KEGG注释，代谢物通过BLAST与在线KEGG数据库进行比对以获取其COs，然后映射到相应的KEGG通路。

16

16

,

， ```

17

17

,

， ```

18

18

.

。

Analysis of the diversity of gut microbiota in feces

粪便中肠道微生物群多样性的分析

OE biotech Co., Ltd. (Shanghai, China) conducted the 16S amplicon sequencing and analysis. Briefly, genomic DNA was extracted from fecal samples using the DNeasy PowerSoil Kit (QIAGEN, Germany) and its concentration and purity were assessed by NanoDrop 2000 (Agilent, USA). The extracted DNA was used as template for PCR amplification of bacterial 16S rRNA with universal primers for V3-V4 variable regions.

OE生物技术有限公司（中国上海）进行了16S扩增子测序和分析。简而言之，使用DNeasy PowerSoil试剂盒（QIAGEN，德国）从粪便样本中提取基因组DNA，并使用NanoDrop 2000（安捷伦，美国）评估其浓度和纯度。提取的DNA用作PCR扩增细菌16S rRNA的模板，并使用V3-V4可变区的通用引物进行扩增。

Sequencing was performed by Illuminaa NovaSeq 6000 with 250 bp paired-end reads. The raw sequencing data, in FASTQ format, were preprocessed using Cutadapt software to remove adapters. Following trimming, low-quality sequences were filtered out, and the remaining reads were denoised, merged, and chimera-checked using DADA2 with default QIIME2 parameters.

测序使用Illumina NovaSeq 6000进行，读取250 bp的双端序列。原始测序数据为FASTQ格式，使用Cutadapt软件进行预处理以去除接头。修剪后，过滤掉低质量序列，并使用DADA2对剩余读段进行去噪、合并和嵌合体检查，默认使用QIIME2参数。

The software ultimately output representative reads and an ASV abundance table. The representative read of each ASV was selected using the QIIME2 package, and all representative reads were annotated against the Silva database using q2-feature-classifier..

该软件最终输出代表性读段和ASV丰度表。每个ASV的代表性读段使用QIIME2包进行选择，并使用q2-feature-classifier将所有代表性读段与Silva数据库进行注释。

Micro-CT imaging of bone

骨骼的微型CT成像

The femoral bone was dissected of soft tissues, fixed in formalin and the distal metaphysis scanned with a Siemens INVEON scanner using the following settings: tube voltage, 60 kV; tube current, 400 µA; and exposure time, 800 ms over 360° rotation. The Feldkamp filtered back-projection algorithm was used to reconstruct the images to generate 3D reconstructions.

股骨被剥离软组织，固定于甲醛中，并使用西门子INVEON扫描仪以下列设置对远端干骺端进行扫描：管电压60 kV；管电流400 µA；曝光时间800 ms，360°旋转。使用Feldkamp滤波反投影算法重建图像以生成三维重建。

Images acquired from the scanner were viewed and calculated morphometric parameters with INVEON Workplace software..

使用INVEON Workplace软件查看从扫描仪获取的图像并计算形态计量参数。

Statistical analysis

统计分析

The data are shown as means ± SD. GraphPad Prism 10.0 software was used for statistical analysis. Two-way ANOVA followed by post-hoc test evaluated the significance of differences between groups. A difference was considered significant if the

数据显示为平均值±标准差。使用GraphPad Prism 10.0软件进行统计分析。通过双因素方差分析及后续检验评估组间差异的显著性。如果差异的P值小于0.05，则认为差异具有显著性。

P

P

value < 0.05.

值 < 0.05。

Results

结果

Effects of 3D clinostat on the body weight and general status in mice

三维回转器对小鼠体重和整体状态的影响

Figure

图

1

1

b showed the daily changes in weight of mice over the first 4 weeks. The SB group mice exhibited a weight profile similar to that of the MC group, indicating good adaptability to survival box. In contrast, the CS group mice undergoing 3D clinostat initially experienced a significant weight loss during the first 4 days.

b展示了小鼠在前4周内的体重每日变化。SB组小鼠的体重变化与MC组相似，表明其对生存盒有良好的适应性。相比之下，经历3D回转器的CS组小鼠在前4天内最初出现了显著的体重下降。

However, these mice gradually adapted to the 3D clinostat and their weight stabilized, followed by a slow recovery to levels similar to those of the SB control group and MC control group. Overall, the mice appeared healthy throughout the study period, indicating good adaptability to the 3D clinostat instrument..

然而，这些小鼠逐渐适应了3D回转器，体重趋于稳定，并缓慢恢复到与SB对照组和MC对照组相似的水平。总体而言，小鼠在整个研究期间表现健康，表明对3D回转器仪器的良好适应性。

The impact of 3D clinostat on cognitive and motor related behavior

3D旋转器对认知和运动相关行为的影响

We conducted a series of behavioral experiments to observe the 3D clinostat simulated microgravity effect on cognitive and motor related behaviors in mice. The results showed 3D clinostat treatment significantly reduced mice activity and exploratory desire, indicated by the significant reduction in movement distance and exercise time in the open field test as shown in Fig. .

我们进行了一系列行为实验，以观察三维回转器模拟微重力对小鼠认知和运动相关行为的影响。结果显示，三维回转器处理显著降低了小鼠的活动量和探索欲望，表现为旷场试验中移动距离和运动时间的显著减少，如图所示。

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c–d, as well as in other behavioral experiments, including Y Maze, elevated plus maze and novel object recognition test (data not shown). In terms of learning and memory, there were no statistically significant differences in the recognition index in novel object recognition test (Fig.

c–d，以及其他行为实验，包括Y迷宫、高架十字迷宫和新物体识别测试（数据未显示）。在学习和记忆方面，新物体识别测试中的识别指数没有统计学上的显著差异（图。

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e), the spontaneous alternations index in the Y maze (Fig.

e)，Y迷宫中的自发交替指数（图。

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f), and the latency to the platform in the Morris water maze (Fig.

f)，以及在莫里斯水迷宫中到达平台的延迟（图。

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g) between CS and SB group, suggesting that 3D clinostat did not have a significant impact on the learning and memory. Regarding motor ability, a series of tests including grip strength test, rotarod test, and gait analysis were conducted. The results of the grip strength test revealed no notable differences among the three groups of mice (Fig. .

g) CS组与SB组之间，表明3D回转器对学习和记忆没有显著影响。在运动能力方面，进行了一系列测试，包括握力测试、转棒测试和步态分析。握力测试的结果显示，三组小鼠之间没有显著差异（图。

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a). In the rotarod test, mice in the SB group exhibited a significantly shorter latency to fall compared to those in the MC group. This shorter latency may be attributed to the restricted activity space within the survival box for the SB group mice. Conversely, the CS group mice displayed a significantly longer latency to fall compared to the SB group, potentially due to their gradually acquired balancing ability through the 3D clinostat process (Fig. .

a). 在转棒测试中，SB组小鼠的跌落潜伏期明显短于MC组小鼠。这种较短的潜伏期可能是由于SB组小鼠在生存盒内的活动空间受限所致。相反，CS组小鼠的跌落潜伏期显著长于SB组，这可能是因为它们通过3D回转器过程逐渐获得了平衡能力（图 。

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b). Besides, gait analysis revealed significant changes in certain gait parameters in the CS group compared to the SB group, indicating that prolonged exposure to the 3D clinostat had a notable impact on the vestibular balance system (Table

此外，步态分析显示，与SB组相比，CS组的某些步态参数发生了显著变化，表明长时间暴露于3D回转器对前庭平衡系统产生了显著影响（表

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, with the raw data shown in Supplementary Table S1). In terms of anxiety and depression, we conducted a series of experiments including the forced swim test, tail suspension test, and elevated plus maze. The results indicated that there were no statistically significant differences among the three groups of mice in terms of the duration of immobility in both the forced swim test and tail suspension test, as well as the percentage of open arm entries in the elevated plus maze.

，原始数据见补充表S1）。在焦虑和抑郁方面，我们进行了一系列实验，包括强迫游泳试验、尾悬挂试验和高架十字迷宫试验。结果表明，在强迫游泳试验和尾悬挂试验中，三组小鼠的不动时间以及在高架十字迷宫中进入开放臂的百分比均无统计学显著差异。

These findings suggest that exposure to the 3D clinostat did not result in significant depression or anxiety tendencies (Fig. .

这些结果表明，暴露于3D旋转器并没有导致显著的抑郁或焦虑倾向（图。

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, c-e). However, in the fear conditioning test, the 3D clinostat seemed to induce a trend towards increased sensitivity to fear behavior and enhanced fear memory. During the fear memory learning phase, the CS group mice exhibited a notable increase in freezing rates (Fig.

，在恐惧条件反射测试中，3D回转仪似乎会诱导对恐惧行为的敏感性增加以及恐惧记忆的增强。在恐惧记忆学习阶段，CS组小鼠表现出冻结率的显著增加（图。

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f), coupled with a tendency of increased freezing rates in the contextual memory phase (Fig.

f)，结合在上下文记忆阶段冻结率增加的趋势（图。

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g). Especially during the extinction phase, CS mice still demonstrated a significant increase in freezing rate, indicating enhanced fear memory and weakened extinction (Fig.

g). 特别是在消退阶段，CS小鼠仍然表现出冻结率的显著增加，表明恐惧记忆增强而消退减弱（图。

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h). Additionally, an increasing trend in freezing rate was also observed in the CS group mice during the subsequent sound stimulus phase (Fig.

h). 此外，在随后的声音刺激阶段，还观察到CS组小鼠的冻结率呈上升趋势（图。

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i). Upon comprehensive analysis of the behavioral research, it can be concluded that the 3D clinostat did not exert a notable influence on learning and memory processes. However, it potentially influenced the fear behavioral, along with subtle alterations related to motor balance.

i). 通过对行为研究的综合分析，可以得出结论，3D回转器对学习和记忆过程没有产生显著影响。然而，它可能影响了恐惧行为，并带来了与运动平衡相关的细微变化。

Fig. 2

图2

Behavioral test results. (

行为测试结果。 （

a

a

) Grip strength of limbs in the grip strength test; (

) 四肢在握力测试中的握力； (

b

b

) Fall time in the rotarod test; (

) 旋转棒测试中的跌落时间； (

c

c

) Inactive time in the forced swim test; (

) 强迫游泳试验中的不活跃时间；(

d

d

) Immobility time in the tail suspension test; (

) 尾悬挂试验中的不动时间； (

e

e

) Percentage of open arm entries in the elevated plus maze; (

) 高架十字迷宫中开放臂进入次数的百分比；(

f

f

–

–

i

i

) Freezing rates of mice in the fear conditioning test during the learning phase (

在恐惧条件反射测试中，学习阶段小鼠的冻结率（

f

f

), contextual memory phase (

），上下文记忆阶段（

g

g

), fear extinction phase (

），恐惧消退阶段（

h

h

), and the conditioned sound stimulus phase (i). MC: ordinary cage control group; SB: survival box control group; CS: 3D clinostat model group. The data are presented as the mean ± SD, *

），条件声音刺激阶段（i）。MC：普通笼子对照组；SB：生存箱对照组；CS：3D旋转器模型组。数据以均值±标准差表示，*

P

P

< 0.05, **

< 0.05, **

P

P

< 0.01, ***

< 0.01, ***

P

P

< 0.001, n = 6 for each group.

< 0.001，每组n = 6。

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Table 1 Gait Parameters with differences between the CS and SB groups.

表1 CS组和SB组之间的步态参数差异。

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The impact of 3D clinostat on femur parameter

3D回转器对股骨参数的影响

To investigate the effects of 3D clinostat on bone metabolism, we utilized micro-CT to scan the femurs of mice and analyzed changes in parameters such as bone volume, tissue volume, trabecular pattern factor, trabecular number, and trabecular separation. The results revealed that both SB and CS group of mice exhibited significant bone loss compared to the MC group, with a notable decrease in bone volume and trabecular number, as well as a significant increase in trabecular pattern factor and trabecular separation.

为了研究3D回转器对骨代谢的影响，我们利用微型CT扫描小鼠股骨，并分析了骨体积、组织体积、骨小梁模式因子、骨小梁数量和骨小梁分离度等参数的变化。结果表明，与MC组相比，SB组和CS组小鼠均出现明显的骨质流失，骨体积和骨小梁数量显著减少，而骨小梁模式因子和骨小梁分离度则显著增加。

Furthermore, compared to the SB group, the CS group displayed a trend of further aggravating bone loss (Fig. .

此外，与SB组相比，CS组显示出进一步加重骨质流失的趋势（图。

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3

).

）。

Fig. 3

图3

Changes in femoral bone parameters by micro-CT scanning. (

微CT扫描股骨参数的变化。

a

a

) Typical micro-CT cross-sectional images of femoral bone; (

典型的股骨微CT横截面图像；

b

b

–

–

g

g

) Changes in bone parameters including Tissue volume (

) 骨参数的变化，包括组织体积 (

b

b

), Bone volume (

），骨体积（

c

c

), Percent bone volume (

），骨体积百分比（

d

d

), Trabecular number (

），骨小梁数量（

e

e

), Trabecular separation (

），骨小梁分离（

f

f

), and Trabecular pattern factor (

），以及小梁模式因子（

g

g

) measured after 3D reconstruction. MC: ordinary cage control group; SB: survival box control group; CS: 3D clinostat model group. The data are presented as the mean ± SD, *

) 在3D重建后测量。MC：普通笼子对照组；SB：生存箱对照组；CS：3D旋转器模型组。数据以均值±标准差表示，*

P

P

< 0.05, **

< 0.05, **

P

P

< 0.01, n = 6 for each group.

< 0.01，每组n = 6。

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The impact of 3D clinostat on brain transcriptome

3D回转器对大脑转录组的影响

To evaluate the impact of 3D clinostat on the central nervous system, we conducted whole brain transcriptome sequencing. The results showed that there were 568, 320, and 426 significantly different genes between each group respectively, as shown in Fig.

为了评估三维回转器对中枢神经系统的影响，我们进行了全脑转录组测序。结果显示，各组之间分别有568、320和426个显著差异基因，如图所示。

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a. Figure

图

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b displays a heatmap of gene expression among the three groups for 320 genes that exhibited significant differences between the CS and SB groups. To further analyze the impact of 3D clinostat on the brain, we conducted KEGG pathway analysis of the differentially expressed genes, as shown in Fig.

b 显示了在 CS 和 SB 组之间表现出显著差异的 320 个基因在三组中的基因表达热图。为了进一步分析 3D 回转器对大脑的影响，我们对差异表达基因进行了 KEGG 通路分析，如图所示。

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c. 3D clinostat may primarily affect immune and endocrine-related signaling pathways, such as those associated with human diseases like Malaria, African trypanosomiasis, Autoimmune thyroid disease, Rheumatoid arthritis, and Staphylococcus aureus infection, as well as GnRH secretion and signaling pathway, Ovarian steroidogenesis, IL17 signaling pathway, Prolactin signaling pathway, Leukocyte transendothelial migration, PPAR signaling pathway, and Neutrophil extracellular trap formation..

c. 3D旋转器可能主要影响免疫和内分泌相关的信号通路，例如与人类疾病相关的通路，如疟疾、非洲锥虫病、自身免疫性甲状腺疾病、类风湿性关节炎和金黄色葡萄球菌感染，以及GnRH分泌和信号通路、卵巢类固醇生成、IL17信号通路、催乳素信号通路、白细胞跨内皮迁移、PPAR信号通路和中性粒细胞胞外陷阱形成。

Fig. 4

图4

Alterations in brain transcriptome and serum metabolome of mice subjected to 3D clinostat. (

3D回转器对小鼠脑转录组和血清代谢组的影响。

a

a

) Venn diagram illustrating differentially expressed genes between pairwise comparisons; (

) 显示两两比较之间差异表达基因的维恩图；(

b

b

) Heatmap of differentially expressed genes exhibiting significant differences between the CS and SB groups; (

）CS组和SB组之间差异表达基因的热图，显示出显著差异；（

c

c

) Main KEGG pathways affected between the CS and SB groups; (

主要影响CS组和SB组之间的KEGG通路；

d

d

) OPLS-DA analysis of serum metabolites between groups; (

) 组间血清代谢物的OPLS-DA分析；(

e

e

) Upregulated and downregulated differential serum metabolites between the CS and SB groups; (

）CS组和SB组之间上调和下调的血清差异代谢物；（

f

f

) KEGG analysis of serum metabolites revealing the main pathways affected between the CS and SB groups. MC: ordinary cage control group; SB: survival box control group; CS: 3D clinostat model group.

）KEGG分析血清代谢物揭示CS组和SB组之间受影响的主要通路。MC：普通笼子对照组；SB：生存箱对照组；CS：3D旋转器模型组。

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The impact of 3D clinostat on serum metabolomics

三维旋转仪对血清代谢组学的影响

To investigate the systemic effects of the 3D clinostat on mice, we conducted a non-targeted metabolomics analysis of serum samples. This experiment yielded a total of 15,470 detected substances, among which 1431 were annotated as known metabolic molecules. OPLS-DA analysis revealed distinct differentiation among the three groups (Fig. .

为了研究3D回转器对小鼠的系统性影响，我们对血清样本进行了非靶向代谢组学分析。该实验共检测到15,470种物质，其中1,431种被注释为已知的代谢分子。OPLS-DA分析显示三组之间存在明显差异（图）。

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d). Notably, in comparison to the SB group, the CS group exhibited significant upregulation of serum metabolites such as Lys-Leu, 2-keto-D-Gluconic acid, Thyroxine 4’-o-.beta.-d-glucuronide, Beta-octylglucoside, Demissidine, Pro-Trp, and Pentobarbital. Conversely, serum metabolites including Poricoic acid a, L-Lysine, Oleic acid, Ile-Pro, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-n,n-dimethyl, Niacinamide, and 1-(1z-octadecenyl)-2-(4z,7z,10z,13z,16z,19z-docosahexaenoyl)-sn-glycero-3-phosphocholine were significantly downregulated (Fig. .

d). 值得注意的是，与SB组相比，CS组表现出血清代谢物如Lys-Leu、2-酮-D-葡萄糖酸、甲状腺素4'-O-β-D-葡萄糖醛酸苷、β-辛基葡萄糖苷、Demissidine、Pro-Trp和戊巴比妥的显著上调。相反，包括Poricoic acid a、L-赖氨酸、油酸、Ile-Pro、1,2-二油酰-sn-甘油-3-磷酸乙醇胺-N,N-二甲基、烟酰胺和1-(1Z-十八烯基)-2-(4Z,7Z,10Z,13Z,16Z,19Z-二十二碳六烯酰基)-sn-甘油-3-磷酸胆碱在内的血清代谢物显著下调（图.）。

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e). These alterations primarily implicate pathways such as ABC transporters, Sphingolipid signaling pathway and metabolism, Biotin metabolism, and the Pentose phosphate pathway (Fig.

这些改变主要涉及以下通路：ABC转运蛋白、鞘脂信号通路与代谢、生物素代谢以及磷酸戊糖途径（图）。

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f).

f).

The impact of 3D clinostat on microbiome

三维旋转器对微生物组的影响

To investigate the impact of 3D clinostat on the gastrointestinal microbiota, 16S rRNA sequencing was performed on mouse feces samples, revealing 333 ± 23, 273 ± 36, and 267 ± 43 ASVs detected in each group, respectively. In terms of alpha diversity, the fecal microbiota richness was observed to be higher in the MC group compared to the SB and CS groups (Fig. .

为了研究3D回转器对胃肠道微生物群的影响，对小鼠粪便样本进行了16S rRNA测序，分别在各组中检测到333±23、273±36和267±43个ASVs。在α多样性方面，观察到MC组的粪便微生物群丰富度高于SB组和CS组（图。

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b). However, no significant differences were observed in the Shannon index among the three groups (Fig.

b). 然而，三组之间的香农指数没有观察到显著差异（图。

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5

c). Regarding beta diversity, PCA analysis demonstrated distinct segregation of the fecal microbiota profiles among the three groups (Fig.

c). 关于β多样性，主成分分析（PCA）显示三组之间的粪便微生物群落结构存在明显分离（图。

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a). At the genus level, Fig.

a). 在属水平上，图。

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d illustrated the top 10 differentially abundant bacterial genera among the three groups. Notably, the

d展示了三组中排名前十的差异丰富的细菌属。值得注意的是，

Bacteroides

拟杆菌属

and

和

Turicibacter

图里西巴菌属

were significantly reduced in the CS group compared to both the SB and MC groups (Fig.

与SB组和MC组相比，CS组显著减少（图。

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5

d). Functional analysis of the microbial community was conducted using the PICRUSt2 software. The results indicated significant differences in KEGG signaling pathways between the CS and SB groups, specifically in Ferroptosis, PPAR signaling pathway, Lysosome, Glycosaminoglycan degradation, Lipopolysaccharide biosynthesis, and Other glycan degradation (Fig. .

d). 使用PICRUSt2软件对微生物群落进行了功能分析。结果表明，CS组和SB组在KEGG信号通路中存在显著差异，特别是在铁死亡、PPAR信号通路、溶酶体、糖胺聚糖降解、脂多糖生物合成和其他聚糖降解方面（图 。

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e). These findings provide valuable insights into the effects of 3D clinostat on the composition and functional properties of the gastrointestinal microbiota, suggesting potential implications for host health.

这些发现为3D旋转器对胃肠道微生物群组成和功能特性的影响提供了宝贵的见解，暗示了对宿主健康的潜在影响。

Fig. 5

图5

Changes in the fecal microbiome of of mice subjected to 3D clinostat. (

暴露于3D回转器的小鼠粪便微生物组的变化。(

a

a

) PCA analysis of the fecal microbiome among the three groups of mice; (

) 三组小鼠粪便微生物组的PCA分析；(

b

b

) Alpha diversity analysis using Chao1 index; (

) 使用Chao1指数进行Alpha多样性分析；(

c

c

) Alpha diversity analysis using Shannon index; (

) 使用香农指数进行阿尔法多样性分析；(

d

d

) Changes in the top 10 differentially abundant bacteria at the species level; (

）在物种水平上排名前十的差异丰富细菌的变化；（

e

e

) KEGG functional analysis of the microbial community differences between the CS and SB groups. MC: ordinary cage control group; SB: survival box control group; CS: 3D clinostat model group. The data are presented as the mean ± SD, *

) CS组和SB组之间微生物群落差异的KEGG功能分析。MC：普通笼子对照组；SB：生存箱对照组；CS：3D旋转器模型组。数据以平均值±标准差表示，*

P

P

< 0.05, n = 6 for each group.

< 0.05，每组n = 6。

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Fig. 6

图6

Comparison of the 3D clinostat simulated microgravity effect model with the classical hindlimb unloading model and spaceflight mice. (

3D旋转器模拟微重力效应模型与经典后肢卸载模型和太空飞行小鼠的比较。(

a

a

) Venn diagram of differentially expressed genes in brain transcriptomes among the three models; (

) 三个模型中脑转录组差异表达基因的维恩图；(

b

b

) Main KEGG pathways affected in the hindlimb unloading model; (

）后肢卸载模型中受影响的主要KEGG通路；（

c

c

) Main KEGG pathways affected in the spaceflight model; (

) 空间飞行模型中主要受影响的KEGG通路；(

d

d

) Heatmap visualizing the expression levels of 131 genes commonly detected in all three models and fulfilling the criteria of differential expression (

热图可视化了所有三个模型中常见检测到的131个基因的表达水平，并满足差异表达的标准 (

P

P

value < 0.05 and fold change > 2 or fold change < 0.5) in at least one model. Red markers: KEGG pathways commonly affected by all three models; Green markers: KEGG pathways commonly affected by the HU and FL models; Blue markers: KEGG pathways commonly affected by the CS and FL models. CS: 3D clinostat model; HU: hindlimb unloading model; FL: spaceflight model..

在至少一个模型中，value < 0.05 且 fold change > 2 或 fold change < 0.5。红色标记：所有三个模型共同影响的KEGG通路；绿色标记：HU和FL模型共同影响的KEGG通路；蓝色标记：CS和FL模型共同影响的KEGG通路。CS：3D旋转器模型；HU：后肢卸载模型；FL：太空飞行模型。

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Comparison of 3D clinostat simulated microgravity effect model with classical hindlimb unloading model and spaceflight mice.

三维回转器模拟微重力效应模型与经典后肢卸载模型及太空飞行小鼠的比较。

To comprehensively assess the validity of the 3D clinostat simulated microgravity effect model, we conducted a comparative analysis of mRNA expression profiles in brain tissue, juxtaposing our findings with those derived from literature on hindlimb unloading (HU) mice and spaceflight (FL) mice models.

为了全面评估3D旋转器模拟微重力效应模型的有效性，我们对脑组织中的mRNA表达谱进行了对比分析，将我们的研究结果与文献中关于后肢卸载（HU）小鼠和太空飞行（FL）小鼠模型的数据进行了比较。

The study revealed minimal overlap among the three models, underscoring substantial variations. Specifically, eight differentially expressed genes (Ibsp, Adam3, Xlr3b, G530011O06Rik, Wdr62, Cd22, Gm4675, and Nusap1) were shared between the CS and FL models, whereas only two genes (Serpinc1 and Ccl19) overlapped between the HU and FL models.

研究揭示了这三个模型之间的重叠极少，突显了显著的差异。具体而言，CS 和 FL 模型共享八个差异表达基因（Ibsp、Adam3、Xlr3b、G530011O06Rik、Wdr62、Cd22、Gm4675 和 Nusap1），而 HU 和 FL 模型之间仅有两个基因（Serpinc1 和 Ccl19）重叠。

Notably, no shared differentially expressed genes were identified between the CS and HU models, highlighting notable disparities between these two models (Fig. .

值得注意的是，在CS和HU模型之间未发现共享的差异表达基因，突显了这两个模型之间的显著差异（图。

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6

a). These findings imply that neither the CS nor the HU model perfectly recapitulates the complex effects of actual space microgravity on the brain, albeit the CS model demonstrates relatively greater fidelity compared to the HU model.

这些发现表明，CS 模型和 HU 模型都不能完美地概括实际太空微重力对大脑的复杂影响，尽管与 HU 模型相比，CS 模型显示出相对更高的保真度。

With respect to the functional disparities among genes, the HU model predominantly features genes tied to neurodegenerative disorders, whereas the FL model encompassed a diverse array of genes related to immunity, endocrinology, cardiovascular functions, and beyond. Among the top 20 KEGG pathways, the GnRH signaling pathway stands out as a significantly altered pathway across all three models, highlighted in red.

关于基因之间的功能差异，HU模型主要包含与神经退行性疾病相关的基因，而FL模型则涵盖了与免疫、内分泌、心血管功能等相关的多种基因。在前20个KEGG通路中，GnRH信号通路在所有三个模型中均显著改变，并以红色突出显示。

Notably, the CS and FL models share additional two commonly altered pathways: Neuroactive ligand-receptor interaction and Ovarian steroidogenesis, both denoted with blue. Furthermore, Diabetic cardiomyopathy, indicated with green, emerges as a shared altered pathway between the CS and HU models. These observations imply that the CS model exhibited a greater degree of similarity to the FL model in comparison to the HU model (Fig. .

值得注意的是，CS模型和FL模型共享另外两个常见的改变通路：神经活性配体-受体相互作用和卵巢甾体生成，均用蓝色表示。此外，糖尿病心肌病，用绿色表示，是CS模型和HU模型之间共同的改变通路。这些观察结果表明，与HU模型相比，CS模型与FL模型表现出更高程度的相似性（图）。

6

6

b–c).

b–c)。

To compare the differences in brain gene expression profiles across these three models, we performed a comprehensive distance comparison involving 5687 genes that were consistently detected in all models. The results of this analysis are as follows: S

为了比较这三种模型之间大脑基因表达谱的差异，我们对所有模型中一致检测到的5687个基因进行了全面的距离比较。该分析的结果如下：S

CS/FL

计算机科学/功能语言

=

=

\(\sum_{1}^{n}\left|{FC}_{cs}n-{FC}_{FL}n\right|=747.25\)

\(\sum_{1}^{n}\left|{FC}_{cs}n-{FC}_{FL}n\right|=747.25\)

, S

，S

HU/FL

匈牙利/佛罗里达州

=

=

\(\sum_{1}^{n}|{FC}_{HU}n-{FC}_{FL}n|=1756.26\)

\(\sum_{1}^{n}|{FC}_{HU}n-{FC}_{FL}n|=1756.26\)

, and S

，以及 S

CS/HU

中文：计算机科学/人机交互

=

=

\(\sum_{1}^{n}|{FC}_{cs}n-{FC}_{HU}n|=1575.88\)

\(\sum_{1}^{n}|{FC}_{cs}n-{FC}_{HU}n|=1575.88\)

. Notably, the S

。特别是，S

CS/FL

计算机科学/功能语言

distance emerges as significantly smaller than the S

距离明显小于S

HU/FL

匈牙利/佛罗里达

distance, underscoring the closer resemblance of the CS model to the FL model in terms of gene expression patterns. Additionally, by generating a heatmap that visualizes the expression levels of 131 genes commonly detected in all three models and fulfilling the criteria of differential expression (.

距离，强调了在基因表达模式上，CS模型与FL模型更为相似。此外，通过生成热图来可视化在所有三个模型中均检测到的131个基因的表达水平，并满足差异表达的标准（。

P

P

value < 0.05 and and foldchange > 2 or foldchange < 0.5) in at least one model, it becomes strikingly apparent that the CS model demonstrates a superior performance compared to the HU model (Fig.

在至少一个模型中，当value < 0.05且foldchange > 2或foldchange < 0.5时，可以明显看出CS模型的表现优于HU模型（图。

6

6

d).

d).

Discussion

讨论

This study explored the impact of simulated microgravity effect using a 3D clinostat on mice, with a particular focus on behavioral, metabolic, gut microbiome and brain transcriptome changes. Our findings revealed that the simulated microgravity effect induced by the 3D clinostat triggered noteworthy transformations within the central nervous system.

本研究探讨了使用3D回转器模拟微重力效应对小鼠的影响，特别关注行为、代谢、肠道微生物组和大脑转录组的变化。我们的研究结果表明，由3D回转器诱导的模拟微重力效应引发了中枢神经系统内的显著变化。

This model has the potential to serve as a valuable tool for investigating cognitive changes associated with microgravity environments, while also offering new insights into the potential mechanisms underlying spaceflight-related health risks..

该模型有潜力成为研究微重力环境下认知变化的宝贵工具，同时为与太空飞行相关的健康风险潜在机制提供了新的见解。

In this study, the behavioral experiments demonstrated that the 3D clinostat had a significant impact on the physical activity and exploratory behavior. The observed reduction in movement distance and exercise time in the open field test, combined with similar findings in the Y Maze, elevated plus maze, and novel object recognition tests, suggests a marked decrease in the overall motivation and exploratory drive.

本研究的行为实验表明，三维回转器对身体活动和探索行为有显著影响。在旷场试验中观察到的移动距离和运动时间减少，结合Y迷宫、高架十字迷宫和新物体识别测试中的类似发现，表明整体动机和探索驱动力显著下降。

These results are consistent with existing literature on the effects of microgravity, where diminished activity levels and altered behavioral patterns have been reported.

这些结果与现有的关于微重力影响的文献一致，其中已经报道了活动水平降低和行为模式改变的情况。

19

19

,

， ```

20

20

,

， ```

21

21

. For instance, mice displayed similar declines in exploratory behavior after a 30-day spaceflight on Bion-M1 satellite

例如，在Bion-M1卫星上进行30天太空飞行后，小鼠的探索行为出现了类似的下降。

21

21

. Different from past results, we did not find obvious anxiety or depression in mice

与过去的结果不同，我们没有在小鼠中发现明显的焦虑或抑郁。

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,

， ```

22

22

,

， ```

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23

. It is known that chronic restraint can lead to anxiety and depression

慢性束缚会导致焦虑和抑郁，这一点早已为人所知。

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24

. We speculate that the anxiety or depression emotion of animals found in the HU model might be caused by tail suspension and restraint, rather than the direct effect of microgravity.

我们推测在HU模型中发现的动物的焦虑或抑郁情绪可能是由尾部悬挂和束缚引起的，而不是微重力的直接影响。

The absence of notable disparities in learning and memory between the CS and SB groups, as assessed by the Morris water maze and other memory related tests, might initially seem surprising. One would expect that the reduced physical activity and altered emotional states might extend to cognitive functions.

通过Morris水迷宫和其他记忆相关测试评估，CS组和SB组在学习和记忆方面没有显著差异，这一结果初看可能令人惊讶。人们可能会预期，身体活动的减少和情绪状态的改变会延伸到认知功能。

However, the results suggest that the impact of microgravity on cognition might be more complex, potentially involving compensatory mechanisms that preserve cognitive abilities despite changes in other behavioral domains. This finding aligns with studies in which astronauts, despite experiencing emotional and physical stressors, often maintain cognitive performance during space missions, possibly due to intensive training and adaptation processes.

然而，结果表明微重力对认知的影响可能更为复杂，可能涉及补偿机制，即使在其他行为领域发生变化的情况下，也能保持认知能力。这一发现与一些研究相符，即宇航员尽管经历情绪和身体的压力源，但在太空任务期间通常能保持认知表现，这可能是由于高强度的训练和适应过程。

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25

. In fact, there are indeed some controversies about the effects of microgravity on cognitive function

事实上，关于微重力对认知功能的影响确实存在一些争议。

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25

,

， ```

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26

,

， ```

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. During spaceflights, the overall cognitive performance of astronauts does not show significant decline, but their cognitive function declines notably after returning to Earth

在太空飞行期间，宇航员的整体认知表现没有显著下降，但返回地球后，他们的认知功能显著下降。

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. Wollseiffen et al. found that neurocognitive performance can even be improved during short-term microgravity, thus suspecting that cognitive impairment is caused by the combined effects of complex spaceflight stressors rather than microgravity itself

沃尔赛芬等人发现，即使在短期微重力环境下，神经认知能力也可以得到提升，因此他们怀疑认知障碍是由复杂太空飞行应激源的综合影响造成的，而非微重力本身。

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. The adaptive changes such as changes in brain blood supply caused by re-adaptation to the gravitational environment after the flight, rather than microgravity itself, may be the key factors that lead to changes in cognitive function. Besides, it is also possible that the short duration of microgravity effect exposure in our study was insufficient to produce detectable cognitive deficits.

重新适应飞行后的重力环境所引起的脑供血等适应性变化，而不是微重力本身，可能是导致认知功能改变的关键因素。此外，我们研究中微重力效应暴露的持续时间较短，可能不足以产生可检测到的认知缺陷。

Future studies with prolonged exposure and more sensitive cognitive assessments might reveal subtle impairments not captured in this study..

未来的研究如果延长暴露时间并采用更敏感的认知评估方法，可能会揭示出本研究未捕捉到的细微损伤。

The motor coordination and balance tests, such as the rotarod and gait analysis, indicated subtle yet significant changes in the vestibular system of the mice. These alterations are particularly relevant given the well-documented vestibular dysfunctions reported by astronauts during and after space missions.

电机协调性和平衡测试，如转棒实验和步态分析，显示小鼠的前庭系统发生了细微但显著的变化。鉴于宇航员在太空任务期间和之后报告的前庭功能障碍已有充分记录，这些变化尤其相关。

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. The role of vestibular system in spatial orientation and balance is crucial, and its disruption in microgravity can lead to disorientation, dizziness, and impaired motor function

前庭系统在空间定向和平衡中起着至关重要的作用，其在微重力环境中的紊乱可能导致迷失方向、头晕和运动功能受损。

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. Under the premise that there was no significant change in grip strength, our findings indicated that the latency to fall in the rotarod test for CS group was increased compared to SB group, suggesting an adaptation or learning process that may mimic the vestibular compensation observed in astronauts.

在握力没有显著变化的前提下，我们的研究结果表明，与SB组相比，CS组在转棒实验中的跌落潜伏期增加，这提示可能存在一种适应或学习过程，可能模拟了宇航员中观察到的前庭代偿。

This adaptive response could be due to the continuous 3D clinostat environment, which might induce a reorganization of neural circuits involved in balance and coordination..

这种适应性反应可能是由于持续的3D回转器环境，这可能诱导了参与平衡和协调的神经回路的重组。

Interestingly, the fear conditioning test showed that the 3D clinostat treated mice exhibited an increased sensitivity to fear stimuli and enhanced fear memory. This observation might be linked to changes in the amygdala and related brain structures, which are critical for processing fear and stress responses.

有趣的是，恐惧条件反射测试显示，经过3D旋转器处理的小鼠对恐惧刺激的敏感性增加，恐惧记忆增强。这一观察结果可能与杏仁核和相关脑结构的变化有关，这些结构对处理恐惧和压力反应至关重要。

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. Research on rats has revealed significant alterations in amygdala neural activity after landing, marked by Fos-positive cells, suggesting a potential impact on fear-related emotional responses

对大鼠的研究表明，着陆后杏仁核神经活动发生了显著变化，Fos阳性细胞增多，这可能影响与恐惧相关的情绪反应。

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,

， ```

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. In 15 days of –6° head-down bed rest, volunteers showed temporal overestimation of the fear stimuli in the middle phase

在15天的-6°头低脚高卧床休息中，志愿者在中期阶段对恐惧刺激表现出时间上的高估。

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. This finding has important implications for understanding the psychological and emotional challenges faced by astronauts during long-term space missions, where prolonged exposure to microgravity could exacerbate stress and anxiety, potentially impairing mission performance and well-being. In the conditioned fear experiment, the most significant difference between the CS and SB groups occurred during the fear extinction phase, suggesting an attenuation of fear memory extinction.

这一发现对于理解宇航员在长期太空任务中面临的心里和情感挑战具有重要意义，因为在微重力环境下长时间暴露可能会加剧压力和焦虑，可能损害任务表现和幸福感。在条件性恐惧实验中，CS组和SB组之间最显著的差异出现在恐惧消退阶段，表明恐惧记忆的消退有所减弱。

The specific neural circuit mechanisms underlying this phenomenon require further in-depth research..

这种现象背后的特定神经回路机制需要进一步深入研究。

In terms of bone density, mice in the CS group exhibited a decrease in bone volume and trabecular number, accompanied by a notable increase in trabecular pattern factor and trabecular separation, indicating bone loss changes, when compared to the MC control group. However, despite a seemingly more pronounced tendency towards bone loss in the CS group compared to the SB group, no statistically significant difference was observed.

在骨密度方面，与MC对照组相比，CS组小鼠的骨体积和骨小梁数量减少，同时骨小梁模式因子和骨小梁分离显著增加，表明骨质流失的变化。然而，尽管CS组相较于SB组似乎表现出更明显的骨质流失趋势，但未观察到统计学上的显著差异。

This may be attributed to the enhanced gripping and limb loading during the 3D clinostat process, as the mice attempted to counteract the body repositioning caused by the rotation. In the rotarod test, the prolonged fall latency observed in mice from the CS group, as well as subtle gait alterations detected in gait analysis, may also be associated with the similar underlying reasons.

这可能是因为在三维回转器过程中，小鼠试图抵抗旋转引起的身体重新定位，从而增强了抓握和肢体负重。在滚轮测试中，CS组小鼠表现出的跌落潜伏期延长，以及步态分析中检测到的细微步态改变，也可能与类似的根本原因有关。

On the other hand, the restricted space within the survival box likely constrained the motor abilities of the SB group mice, and simultaneously induced bone loss in these mice. This could potentially account for the reduced difference between the CS and SB groups, representing another plausible explanation..

另一方面，生存盒内的有限空间可能限制了SB组小鼠的运动能力，同时诱导了这些小鼠的骨质流失。这可能潜在地解释了CS组和SB组之间差异的减少，提供了另一个合理的解释。

The transcriptomic analysis of the brain tissues revealed significant changes in gene expression associated with immune response, endocrine signaling, and nervous system functions. These alterations suggested that the simulated microgravity effect triggered a broad and complex biological response, affecting multiple systems that are crucial for maintaining homeostasis and health.

脑组织的转录组分析显示，与免疫反应、内分泌信号传导和神经系统功能相关的基因表达发生了显著变化。这些改变表明，模拟微重力效应引发了一种广泛而复杂的生物反应，影响了多个对维持体内平衡和健康至关重要的系统。

These findings are consistent with previous studies that have reported immune dysregulation and hormonal imbalances in response to microgravity in spaceflight animal models.

这些发现与之前的研究一致，这些研究报道了在太空飞行动物模型中微重力导致的免疫失调和激素失衡。

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,

， ```

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. Immune dysregulation and hormonal imbalances could lead to a range of health problems, including increased susceptibility to infections, metabolic disorders, and mental health issues. Effective monitoring and the development of targeted interventions could help mitigate these risks and improve the health of astronauts..

免疫失调和激素失衡可能导致一系列健康问题，包括感染易感性增加、代谢紊乱和心理健康问题。有效的监测和针对性干预措施的开发可能有助于减轻这些风险，并改善宇航员的健康状况。

One of the key findings of this study is the differential gene expression pattern observed in the CS model compared to the HU and FL models. The minimal overlap in differentially expressed genes between these models highlights the distinct biological effects induced by each type of simulated microgravity effect.

本研究的关键发现之一是，在CS模型中观察到的差异基因表达模式与HU和FL模型相比有所不同。这些模型之间差异表达基因的最小重叠突显了每种模拟微重力效应所诱导的不同生物学效应。

Specifically, the CS model appears to more closely mimic the gene expression changes seen in real spaceflight, particularly in terms of immune and endocrine-related pathways. This suggests that the 3D clinostat might be a more accurate and reliable model for studying the effects of microgravity on the central nervous system.

具体来说，CS 模型似乎更接近于模拟真实太空飞行中看到的基因表达变化，特别是在免疫和内分泌相关途径方面。这表明三维回转器可能是研究微重力对中枢神经系统影响的更准确和可靠的模型。

The CS and FL models demonstrated a convergent alteration in three KEGG signaling pathways: GnRH signaling pathway, Neuroactive ligand-receptor interaction, and Ovarian steroidogenesis, as well as eight common differential genes, including Ibsp, Adam3, Xlr3b, G530011O06Rik, Wdr62, Cd22, Gm4675, and Nusap1.

CS和FL模型在三个KEGG信号通路中显示出收敛性改变：GnRH信号通路、神经活性配体-受体相互作用和卵巢类固醇生成，以及八个共同的差异基因，包括Ibsp、Adam3、Xlr3b、G530011O06Rik、Wdr62、Cd22、Gm4675和Nusap1。

The GnRH signaling pathway plays a pivotal role in the hypothalamus, regulating the function of the hypothalamic–pituitary–gonadal (HPG) axis. Under microgravity conditions, significant changes occur in the endocrine system, which may impact the release of GnRH and the activity of the signaling pathway.

GnRH信号通路在下丘脑中起着关键作用，调节下丘脑-垂体-性腺（HPG）轴的功能。在微重力条件下，内分泌系统会发生显著变化，这可能影响GnRH的释放和信号通路的活性。

This alteration in the GnRH signaling pathway may subsequently lead to fluctuations in sex hormone levels and participate in the regulation of cognition, emotion, and behavior.

GnRH信号通路的这种改变可能会随后导致性激素水平的波动，并参与认知、情绪和行为的调节。

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. The Neuroactive Ligand-Receptor Interaction pathway encompasses the intricate interplay between numerous neurotransmitters and hormones (such as dopamine, serotonin, GABA, glutamate, and others) and their respective receptors. This interaction is vital for neural transmission, emotional regulation, cognitive functions, and neural plasticity.

神经活性配体-受体相互作用通路涵盖了众多神经递质和激素（如多巴胺、血清素、GABA、谷氨酸等）与其各自受体之间的复杂相互作用。这种相互作用对于神经传递、情绪调节、认知功能和神经可塑性至关重要。

Microgravity and spaceflight can potentially induce alterations in neural adaptability, as well as changes in the levels and sensitivity of neurotransmitters, which may be linked to the Neuroactive Ligand-Receptor Interaction.

微重力和太空飞行可能会引起神经适应性的改变，以及神经递质水平和敏感性的变化，这些可能与神经活性配体-受体相互作用有关。

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. The Ovarian Steroidogenesis signaling pathway is concerned with the production of estrogen and progesterone. Although ovarian steroidogenesis primarily occurs within the reproductive system, it also exerts significant influence on the brain, particularly in regulating emotions, cognition, and neural plasticity.

卵巢类固醇生成信号通路与雌激素和孕激素的产生有关。尽管卵巢类固醇生成主要发生在生殖系统内，但它也对大脑产生显著影响，特别是在调节情绪、认知和神经可塑性方面。

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. Furthermore, estrogen possesses neuroprotective properties, contributing to the maintenance of neuronal health and function

此外，雌激素具有神经保护特性，有助于维持神经元的健康和功能。

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. Under microgravity conditions, a decrease in estrogen levels may diminish its neuroprotective effects, potentially increasing the risk of neurodegenerative diseases or cognitive decline. The greater similarity in gene expression profiles between the CS and FL models supports the use of this method as a representative model for spaceflight-induced neurobiological changes..

在微重力条件下，雌激素水平的下降可能会削弱其神经保护作用，从而增加神经退行性疾病或认知能力下降的风险。CS和FL模型之间基因表达谱的更高相似性支持了将该方法用作代表航天诱导神经生物学变化的模型。

The lack of overlap in differentially expressed genes between the HU and FL models further underscores the limitations of the HU model in replicating the effects of actual spaceflight. While the HU model has been widely used to simulate microgravity effect, particularly for studying muscle atrophy and bone loss, it appears to fall short in accurately modeling the complex changes in brain function induced by real spaceflight.

不同ially表达基因在HU模型和FL模型之间缺乏重叠，进一步凸显了HU模型在复制实际太空飞行效果方面的局限性。虽然HU模型已被广泛用于模拟微重力效应，特别是用于研究肌肉萎缩和骨质流失，但其在准确模拟真实太空飞行引起的脑功能复杂变化方面似乎存在不足。

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. This discrepancy may be due to the localized nature of the HU model, which primarily affects the hindlimbs and may not adequately capture the whole-body effects of microgravity. In contrast, the 3D clinostat model provides a more holistic simulation of microgravity effect, potentially leading to more relevant insights into how spaceflight affects the brain.

这种差异可能是由于HU模型的局部特性，其主要影响后肢，可能无法充分捕捉微重力对全身的影响。相比之下，3D旋转器模型提供了更全面的微重力效应模拟，可能带来与太空飞行如何影响大脑更相关的见解。

The HU model demonstrates a heightened sensitivity to alterations in cerebral blood flow, rendering it an appropriate choice for investigating notable cognitive shifts following return from spaceflight, while the 3D clinostat model is more likely to focus on subtle cognitive changes caused by adaptation to microgravity in space, which may be related to vestibular function and spatial orientation..

HU模型对脑血流变化表现出更高的敏感性，因此它是研究从太空飞行返回后显著认知变化的合适选择，而3D回转器模型更可能聚焦于由适应太空微重力引起的细微认知变化，这可能与前庭功能和空间定向有关。

The metabolomic analysis of serum samples provided additional insights into the systemic effects of 3D clinostat model. The observed alterations in various metabolic pathways, including ABC transporters, Sphingolipid signaling, and the Pentose phosphate pathway, suggest that microgravity effect disrupts lipid metabolism, energy production, and cellular signaling processes.

血清样本的代谢组学分析为3D回转器模型的系统效应提供了更多见解。观察到的多种代谢通路的改变，包括ABC转运蛋白、鞘脂信号传导以及磷酸戊糖途径，表明微重力效应破坏了脂质代谢、能量产生和细胞信号传导过程。

The downregulation of key metabolites such as L-Lysine, oleic acid, and niacinamide points to potential deficiencies in essential nutrients, which could exacerbate the stress response and impair overall health. L-Lysine is an essential amino acid involved in protein synthesis, while oleic acid is a major component of membrane phospholipids and has anti-inflammatory properties.

关键代谢物如L-赖氨酸、油酸和烟酰胺的下调表明可能存在必需营养素的缺乏，这可能会加剧应激反应并损害整体健康。L-赖氨酸是一种参与蛋白质合成的必需氨基酸，而油酸是膜磷脂的主要成分，并具有抗炎特性。

43

43

. Niacinamide, a form of vitamin B3, is crucial for energy production and DNA repair

烟酰胺，一种维生素B3的形式，对能量产生和DNA修复至关重要。

44

44

,

， ```

45

45

. The reduction in these metabolites suggests that microgravity might lead to nutritional imbalances that could have far-reaching effects on health.

这些代谢物的减少表明，微重力可能会导致营养失衡，从而对健康产生深远的影响。

The microbiome analysis further supports these findings, showing significant alterations in gut microbiota composition, particularly the reduction in

微生物组分析进一步支持了这些发现，显示出肠道微生物群组成的重大变化，特别是减少方面。

Bacteroides

拟杆菌属

and

和

Turicibacter

图里西巴菌

. The reduction in

. 减少

Bacteroides

拟杆菌属

is consistent with the reported elevation of the

与报告中提到的海拔高度一致

Firmicutes

厚壁菌门

/

/

Bacteroidetes

拟杆菌门

ratio in the intestinal flora of astronauts during spaceflights

宇航员在太空飞行期间肠道菌群的比例

25

25

. The gut microbiota plays a critical role in regulating immune function, metabolism, and overall health, and its disruption could have serious consequences during spaceflight. Previous studies have shown that changes in the gut microbiota can lead to a range of health issues, including immune dysregulation, metabolic disorders, and even mental health problems.

肠道微生物群在调节免疫功能、代谢和整体健康方面发挥着关键作用，其失调在太空飞行期间可能会带来严重后果。以往的研究表明，肠道微生物群的变化可能导致一系列健康问题，包括免疫失调、代谢紊乱，甚至心理健康问题。

45

45

. The observed changes in the gut microbiota in this study suggest that simulated microgravity effect may lead to similar health challenges, emphasizing the need for strategies to maintain gut health during long-term space missions. The functional analysis of the microbial community revealed significant differences in KEGG signaling pathways between the CS and SB groups.

本研究中观察到的肠道菌群变化表明，模拟微重力效应可能导致类似的健康挑战，突显了在长期太空任务中维持肠道健康的策略需求。对微生物群落的功能分析显示，CS组和SB组之间的KEGG信号通路存在显著差异。

Pathways such as Ferroptosis, PPAR signaling, and Glycosaminoglycan degradation were notably affected, indicating potential alterations in cellular death processes, lipid metabolism, and extracellular matrix remodeling. These changes could have implications for tissue integrity and function, particularly in the gastrointestinal tract, which is highly sensitive to environmental stressors.

铁死亡、PPAR 信号传导和糖胺聚糖降解等通路受到显著影响，表明细胞死亡过程、脂质代谢和细胞外基质重塑可能发生潜在变化。这些变化可能对组织的完整性和功能产生影响，尤其是在对环境压力高度敏感的胃肠道中。

46

46

. The disruption of these pathways could contribute to the gastrointestinal problems of astronauts, such as increased gut permeability, altered bowel habits and increased susceptibility to infections

这些途径的中断可能会导致航天员出现胃肠道问题，例如肠道通透性增加、排便习惯改变以及感染易感性增加。

47

47

. The alterations in gut microbiota composition observed in this study also suggest that dietary interventions or probiotics could be explored as potential strategies to maintain gut health during space missions.

本研究中观察到的肠道菌群组成的变化还表明，饮食干预或益生菌可作为在太空任务期间维持肠道健康的潜在策略进行探索。

The findings of this study have significant implications for understanding the health risks associated with long-term spaceflight. The 3D clinostat model offers a practical and cost-effective method for simulating microgravity effect on Earth, allowing for the systematic study of its effects on various physiological systems.

这项研究的发现对于理解长期太空飞行相关的健康风险具有重要意义。三维回转器模型提供了一种实用且经济高效的方法，可以在地球上模拟微重力效应，从而系统地研究其对各种生理系统的影响。

The results suggest that microgravity may have a more pronounced impact on emotional and motivational states than on cognitive functions, highlighting the need for further research into the psychological well-being of astronauts during extended missions..

结果表明，微重力对情绪和动机状态的影响可能比对认知功能的影响更为显著，这突显了在长期任务中进一步研究宇航员心理健康的重要性。

While our study provided important insights into the 3D clinostat simulated microgravity effect, it is essential to acknowledge certain limitations. Firstly, although some behavioral changes were observed, many of them did not reach statistical significance, potentially due to the relatively small sample size in the study, leading to the possibility that subtle behavioral changes might have been overlooked.

虽然我们的研究为3D旋转器模拟微重力效应提供了重要的见解，但必须承认某些局限性。首先，尽管观察到一些行为变化，但许多变化未达到统计学显著性，这可能是由于研究中样本量相对较小，导致可能忽略了一些细微的行为变化。

Secondly, in space environments, males and females exhibit gender-specific differences in the responses of various bodily systems. This study exclusively used male mice; however, considering the impact of gender factors, it is a scientifically valuable question worth further investigation to determine whether female mice exhibit different responses to the 3D clinostat.

其次，在空间环境中，男性和女性在各身体系统的反应上表现出性别特异性差异。本研究仅使用了雄性小鼠；然而，考虑到性别因素的影响，雌性小鼠对3D回转器是否表现出不同反应，是一个值得进一步研究的、具有科学价值的问题。

Furthermore, we noted significant differences in multiple behavioral indicators, as well as in the transcriptome and metabolome, between mice in the survival box control (SB) group compared to ordinary cage control (MC) group, highlighting the crucial impact of social isolation and spatial confinement on the organism.

此外，我们注意到生存箱对照组（SB）与普通笼子对照组（MC）小鼠在多种行为指标以及转录组和代谢组方面存在显著差异，突显了社会隔离和空间限制对机体的关键影响。

Further analysis of the synergistic effects of microgravity, social isolation, and spatial constraints is a scientific issue worthy of further attention in the next step. Moreover, it is important to acknowledge that variability in transcriptome data may be influenced by differences in time points, exposure durations, and collection methodologies employed across clinostat studies, hindlimb unloading experiments, and spaceflight missions.

进一步分析微重力、社会隔离和空间限制的协同效应是下一步值得关注的科学问题。此外，必须承认，转录组数据的变异性可能受到回转器研究、后肢卸载实验和太空飞行任务中所采用的时间点、暴露时长和收集方法差异的影响。

Additionally, although mice spend most of their time inactive in the resting chamber of the survival box, they may still be forced to move or perform actions against gravity-direction changes. It is acknowledged that the 3D clinostat and hindlimb unloadin.

此外，尽管小鼠在生存箱的休息室中大部分时间都不活动，但它们仍可能被迫移动或执行对抗重力方向变化的动作。人们认识到，3D旋转器和后肢卸载装置...

Conclusion

结论

In summary, our research underscored that 3D clinostat model exerted profound effects on the behavior, metabolism, microbiome, and brain transcriptome. Compared to hindlimb unloading model, the 3D clinostat model demonstrates a closer resemblance to the brain transcriptome expression profiles observed in spaceflight animals, thereby offering a novel and invaluable research tool for simulating microgravity effect on Earth..

总之，我们的研究强调了三维回转器模型对行为、代谢、微生物组和大脑转录组有深远的影响。与后肢卸载模型相比，三维回转器模型表现出与太空飞行动物观察到的大脑转录组表达谱更为相似的特征，从而为在地球上模拟微重力效应提供了一种新颖且宝贵的研究工具。

Data availability

数据可用性

The data that support the findings of this study have been deposited into CNGB Sequence Archive (CNSA) of China National GeneBank DataBase (CNGBdb) with accession number CNP0006448 (

本研究结果所依赖的数据已存入中国国家基因库数据库（CNGBdb）的CNGB序列档案库（CNSA），登录号为CNP0006448（

https://db.cngb.org/search/project/CNP0006448

https://db.cngb.org/search/project/CNP0006448

). Additionally, the corresponding author is available to provide data upon request (guojianguo@cnilas.org).

此外，通讯作者可应要求提供数据 (guojianguo@cnilas.org)。

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Acknowledgements

致谢

We are grateful to the Shanghai OE Biotech Co., Ltd. for transcriptome & microbiome analysis and to the Shanghai Applied Protein Technology Co., Ltd. for metabolome analysis.

我们感谢上海欧易生物医学科技有限公司进行的转录组和微生物组分析，以及上海应用蛋白质科技有限公司进行的代谢组分析。

Funding

资金筹集

This research was supported by the Aerospace Science and Technology Collaborative Innovation Center Project (BSAUEA5740600223), the National Key R&D Program of China (2022YFF0710803, 2021YFF0703400), the National Natural Science Foundation of China (General Program, 82070103), CAMS Innovation Fund for Medical Science (CIFMS, 2021-I2M-1-036) and the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2023-PT180-01)..

本研究得到了航空航天科学技术协同创新中心项目（BSAUEA5740600223）、国家重点研发计划（2022YFF0710803、2021YFF0703400）、国家自然科学基金（面上项目，82070103）、中国医学科学院医学科学创新基金（CIFMS，2021-I2M-1-036）和中国医学科学院非营利性中央研究院基金（2023-PT180-01）的支持。

Author information

作者信息

Authors and Affiliations

作者与所属机构

Key Laboratory of Human Disease Comparative Medicine, Chinese Ministry of Health, Key Laboratory for Animal Models of Emerging and Reemerging Infectious Diseases, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences and Comparative Medicine Center, Peking Union Medical College, Beijing, China.

中国卫生部人类疾病比较医学重点实验室，中国医学科学院实验动物研究所新发再发传染病动物模型研究重点实验室，北京协和医学院比较医学中心，北京，中国。

Li Zhou, Chenchen Song, Hu Yang, Lianlian Zhao, Xianglei Li, Xiuping Sun, Kai Gao & Jianguo Guo

李周，宋晨晨，杨虎，赵连连，李向前，孙秀萍，高凯，郭建国

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Li Zhou

李周

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Chenchen Song

宋晨晨

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胡杨

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连莲 赵

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郭建国

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Contributions

贡献

L.Z. performed the majority of the experiments, analyzed the data, and wrote the original manuscript; C.S. participated in animal management and dissection for sampling; H.Y. participated in manuscript arrangement; L.Z. participated in data analysis; X.L. participated in behavioral testing; X.S. contributed behavioral testing tools; K.G.

L.Z.进行了大部分实验，分析了数据，并撰写了原始稿件；C.S.参与了动物管理和取样解剖；H.Y.参与了稿件整理；L.Z.参与了数据分析；X.L.参与了行为测试；X.S.提供了行为测试工具；K.G.

contributed micro-CT analysis tools; J.G. designed the study, supervised the project, contributed experiment materials and reviewed the manuscript. All authors reviewed and approved the final manuscript..

贡献了微CT分析工具；J.G.设计了研究，监督了项目，提供了实验材料并审阅了手稿。所有作者审阅并批准了最终手稿。

Corresponding author

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Correspondence to

致信

Jianguo Guo

建国郭

.

。

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.

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Zhou, L., Song, C., Yang, H.

周，L.，宋，C.，杨，H.

et al.

等人

Behavioral and multiomics analysis of 3D clinostat simulated microgravity effect in mice focusing on the central nervous system.

行为和多组学分析三维回转器模拟微重力对小鼠中枢神经系统的影响。

Sci Rep

科学报告

15

15

, 5731 (2025). https://doi.org/10.1038/s41598-025-90212-y

，5731（2025）。https://doi.org/10.1038/s41598-025-90212-y

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12 October 2024

2024年10月12日

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2025年2月11日

Published

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17 February 2025

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DOI

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https://doi.org/10.1038/s41598-025-90212-y

https://doi.org/10.1038/s41598-025-90212-y

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Keywords

关键词

Microgravity

微重力

Three-dimensional clinostat

三维回转器

Brain

大脑

Transcriptomics

转录组学

Microbiomics

微生物组学

Serum metabolomics

血清代谢组学

Animal model

动物模型

Subjects

主题

Environmental impact

环境影响

Neuroscience

神经科学

Physiology

生理学

Risk factors

风险因素

Transcriptomics

转录组学

Zoology

动物学