MA Hong ZHANG Chen LI Yujuan LÜ Xuefei LI Xiaoqiong ZHANG Ying YANG Chunhua LIU Huayan FAN Yunlong DENG Yulin

1(School of Life Science, Beijing Institute of Technology, Beijing 100081)

2(School of Medical Technology, Beijing Institute of Technology, Beijing 100081)

Abstract With the further advancement of China’s major manned spaceflight project,the national space laboratory was successfully built.China has also made considerable progress and breakthroughs in the field of space life sciences.This paper reviews the related biological effects under space flight conditions,mainly including epigenetic effects,skeleton remodeling and peripheral body fluid circulation effects,as well as the research and application of space life science related biotechnology in the field of microbial culture and biological regeneration life support system.

Key words Space life sciences,Space biology technology,Microgravity,Ionizing radiation

1 Introduction

With the entry of human beings and other life forms into outer space and the exploration of life outside the Earth,the new interdisciplinary subject of space life science was born.The basic problems of space life science include:how do Earth life bodies (including astronauts)perceive,respond to and adapt to the space environment,and what are their basic laws? How to further support human space exploration for a longer time in space?How does life originate and evolve? Is there any other life in the universe? How to use the special space environment to understand the essence of life on the Earth and play a role in improving human life on the Earth? In the past two years,China’s space life science has made remarkable breakthroughs in many aspects.

2 Spaceflight-associated Biology Effects

2.1 Cell Adhesion Effects

Microgravity induces a number of significant physiological changes in the cardiovascular,nervous,immune systems,as well as the bone tissue of astronauts.Changes in cell adhesion properties are one aspect affected during long-term spaceflights in mammalian cells.Cellular adhesion behaviors can be divided into cell-cell and cellmatrix adhesion.These behaviors trigger cell-cell recognition,conjugation,migration,cytoskeletal rearrangement,and signal transduction.Cellular Adhesion Molecule (CAM) is a general term for macromolecules that mediate the contact and binding between cells or between cells and the Extracellular Matrix (ECM).The four major classes of adhesion molecules that regulate cell adhesion,including integrins,Immunoglobulin Superfamily (Ig-SF),cadherins,and selectin,which induced the effects of spaceflight and simulated microgravity on the adhesion of endothelial cells,immune cells,tumor cells,stem cells,osteoblasts,muscle cells,and other types of cells. Moreover,these adhesion molecules can activate the following signal pathway and biological effects.Spaceflight-associated immune system weakening ultimately limits the ability of humans to expand their presence beyond the Earth’s orbit.A mechanistic study of microgravity-regulated immune cell function is necessary to overcome this challenge.Qianet al.demonstrate that both spaceflight and simulated microgravity significantly reduce macrophage differentiation,decrease macrophage quantity and functional polarization,and lead to metabolic reprogramming,as demonstrated by changes in gene expression profiles.Moreover,they identified RAS/ERK/NFκB as a major microgravity-regulated pathway.Exogenous ERK and NFκB activators significantly counteracted the effect of microgravity on macrophage differentiation.In addition,microgravity also affects the p53 pathway,which might reveal a new mechanism for the effects of microgravity on macrophage development and provide potential molecular targets for the prevention or treatment of macrophage differentiation deficiency in spaceflight[1–3].Further studies on the effects of microgravity on cell adhesion and the corresponding physiological behaviors may help increase the safety and improve the health of astronauts in space.

2.2 Peripheral System Effect

2.2.1 Intestinal Injury and Protection

The space environment mainly includes microgravity,strong radiation,and high noise.Recent studies show that Microgravity (MG) could cause injury of the digestive system.It has been reported that spaceflight significantly decreased mucin production of Intestinal Epithelial Cells (IECs) in rats.SMG damaged intestinal homeostasisviaincreasing intestinal permeability,impairing barrier function,and increasing the susceptibility to colitis and the risk of intestinal infection.The above evidence suggests that MG or SMG damages Intestinal Epithelial Barrier (IEB) function.

The small intestine is an important organ in the human body.The intestine mucosal structure forms an essential barrier between the external environment and internal milieu,restricting the passage of harmful substances,infectious agents,and microorganisms into circulation in human body IEB dysfunction could lead to malnutrition,diarrhea,and Inflammatory Bowel Diseases (IBDs),which may increase the intestinal infection risk of astronauts during long-term space travel.Thus,it is critical to investigate the underlying mechanism of EIB damage under SMG and seek for protection strategy against IEB dysfunction.

Based on histomorphology,TEM,permeability of intestine,inflammatory factors in rat plasma and intestine,expression of tight and adherens junction proteins,and the proteomic approach with the tail-suspended rat model to simulate microgravity for 21 days,it has been found that IEB has been damaged with histomorphology injury,increased intestinal permeability,down-regulated adhesion molecules.416 Differentially Expressed Proteins (DEPs) were identified and clustered into pathways for metabolism,focal adhesion,regulation of actin cytoskeleton,drug metabolism enzymes and so on.It has been found that MLCK dependent up-regulation MLC phosphorylation mediates intestinal barrier dysfunction during simulated microgravity injury.This may indicate that the regulation of epithelial MLCK is a potential target for the therapeutic treatment of microgravity injury.SMG could damage IEB also through the formation of focal adhesions mediated by the Rac1-WAVE2-Arp2/3 pathway,which benefits intestinal epithelial cell migration and barrier repair.

The study has been carried out on the alteration of Intestinal Drug Metabolizing Enzymes (IDMEs) following 14-day simulated microgravity in rat intestinal mucosa.Totally 335 DEPs were identified,190 DEPs were up-regulated and 145 DEPs were down-regulated.Most of DEPs exhibited hydrolase,oxidoreductase,transferase,ligase or lyase catalytic activity.DEPs were mainly enriched in metabolic pathways,including the metabolism of amino acid,glucose,and carbon.11 of DEPs were involved in exogenous drug and xenobiotics metabolism.Because the IDMEs are important for the efficacy and safety of oral drugs the expression of cytochrome P1 A2 (CYP1 A2),CYP2 D1,CYP3 A2,CYP2 E1,Alcohol Dehydrogenase 1 (ADH1) and Glutathione S-Transferase Mu 5 (GSTM5) in rat intestine mucosa was determined by Western-blot.The activity of ADH,Aldehyde Dehydrogenase (ALDH) and GST was evaluated.SMG led to dramatically decreased expression of CYP1 A2,CYP2 D1,CYP3 A2 and ADH1,while the GSTM5 was significantly up-regulated.ADH activity was reduced,and ALDH and GST activities did not alter remarkably.It could be concluded that SMG dramatically affected the expression and activity of some IDMEs,which might alter the efficacy or safety of their substrate drugs under microgravity[4].

A traditional Chinese medicinal herb,Dragon’s Blood (DB),was used to prevent IEB damage induced by SMG.It has been found that DB could protect histomorphology,reduce permeability and increase the expression of junction proteins in SMG-rat ileum.Proteomic analysis showed that DB regulated 1080 DEPs in rat ileum mucosa,including proteins for cell-cell adhesion,focal adhesion and cytoskeleton regulation.DB increased the expression of Rac1-WAVE2-Arp2/3 pathway proteins and F-actin to G-actin ratio,which promoted the formation of focal adhesions and finally benefits intestinal epithelial cell migration and barrier repair.The present study firstly provided some preliminary information on IDMEs under microgravity.It may be helpful to understand the intestinal health of astronauts,and supply a scientific basis for medication use during space travel[5].

2.2.2 Peripheral Metabolic Effect

In order to screen the biomarkers of neurochemicals in peripheral blood of rats after nerve injury induced by whole brain irradiation,Menget al. established a method of chemical derivation of neurochemicals in serum-ultra high performance liquid chromatography tandem mass spectrometry was used to analyze different whole brain irradiation doses (0,10,30 Gy) and fractional cumulative irradiation (10 Gy × 3).The concentrations of 42 neurochemicals in rat serum were measured and statistically analyzed based on the targeted metabonomics strategy.The change degree of neurochemical content in irradiation groups was lower than the control group,indicating that acetylcholine,glutamate,tyramine and melatonin can be used as potential biomarkers of nerve injury induced by whole brain irradiation.Denget al.used the similar established rat model of nerve radiation injury to evaluate the characteristic changes of neurochemicals in rat serum,including neurotransmitters,amino acids and biogenic amines.The researchers combined reversed-phase liquid chromatography tandem mass spectrometry with chemical derivatization to establish an efficient and sensitive method for the detection of 42 polar neurochemicals.The optimized benzoyl chloride derivatization reaction can be easily carried out in one-pot reaction,and stable neurochemical derivatives (except acetylcholine and melatonin) can be obtained under mild conditions within 5 min.Derivatization can also realize rapid chromatographic separation on the HSS T3 column by gradient elution by re-labeling the analyte with labeled derivatization reagent.The multiple reaction monitoring acquisition mode can quantify the neurochemicals in rat serum,with the detection limit of 0.05 nm to 11.63 nm and the lower limit of quantification of 0.09 nm to 46.50 nm.The method has been well verified in terms of linearity and extraction recovery.This method is also effective for the extensive targeted analysis of 42 neurochemicals in serum[6].

2.3 Epigenetic Effects

Plants grown in spaceflight exhibited differential methylation responses and this is important because plants are sessile,they are constantly exposed to a variety of environmental pressures and respond to them in many ways.Xuet al.previously showed that the Arabidopsis genome exhibited a lower methylation level after spaceflight for 60 h in orbit.Here,using the offspring of the seedlings grown in microgravity environment in the SJ-10 satellite for 11 days and returned to Earth,they systematically studied the potential effects of spaceflight on DNA methylation,transcriptome,and phenotype in the offspring. Whole-genome methylation analysis in the first generation of offspring (F1) showed that,although there was no significant difference in methylation level as had previously been observed in the parent plants,some residual imprints of DNA methylation differences were detected.Combined DNA methylation and RNAsequencing analysis indicated that the expression of many pathways,such as the abscisic acid-activated pathway,protein phosphorylation,and nitrate signaling pathway,etc.were enriched in the F1 population.As some phenotypic differences still existed in the F2 generation,it was suggested that these epigenetic DNA methylation modifications were partially retained,resulting in phenotypic differences in the offspring.Furthermore,some of the spaceflight-induced heritable Differentially Methylated Regions (DMRs) were retained.Changes in epigenetic modifications caused by spaceflight affected the growth of two future seed generations.Altogether,their research is helpful in better understanding the adaptation mechanism of plants to the spaceflight environment[7].

Moreover,the epigenetic effects of mammalian cells also involve some important physiological systems.The Central Nervous System (CNS) is one of the most important systems in the human brain.During spaceflight,radiation and microgravity have different biological effects on the human brain.The inflammatory activation of glial cells is the main sign of impaired neural function.By establishing anin vitromodel of radiation,simulated microgravity and a combination of the two conditions,Maet al.explored the biological changes in human glial cells,including the release of inflammatory factors,the changes of autophagy and the transcriptional expression of the key regulator of histone methyltransferase enzyme Enhancer of Zeste Homolog 2 (EZH2),a potential dual regulator of inflammation and autophagy.Their results showed that the simulated space environment significantly affected the growth and morphological changes of nerve cells,neurons released cytokines,recruited monocytes,triggered inflammatory response,and both radiation and microgravity could activate autophagy.In a simulated space environment,the transcriptional level of EZH2 decreased,and the down-regulation of EZH2 induced autophagy by activating the mTOR pathway.The change of autophagy level can activate NF-kB and induce the release of inflammatory factor IL-6.At the same time,the interaction between NF-κB and EZH2 may in turn affect the expression of EZH2,suggesting that EZH2 may be a dual regulator regulating inflammatory activation and autophagy in the space environment,which helps to explain the biological damage of the central nervous system caused by neuroinflammation observed in the space environment and provide a molecular target for the health protection of astronauts in long-term space flight[8].

2.4 Cytoskeleton Reorganization Effects

Decades of spaceflight studies have provided abundant evidence that individual cellsin vitroare capable of sensing space microgravity and responding to cellular changes both structurally and functionally. However,how microgravity is perceived,transmitted,and converted to biochemical signals by single cells remains unrevealed.Studies on cells of the musculoskeletal system,cardiovascular system,and immune system were covered.Among all the reported cellular changes in response to space microgravity,Cytoskeleton (CSK) reorganization emerges as a key indicator.The CSK network is a complex and elaborate system.With the MFs,IFs,and MTs constituting the backbone of the CSK,other structures,like the spectrin network,primary cilium,septin,and Lamin A/C nucleoskeleton have also been demonstrated to be involved in cell mechanotransduction.Based on the evidence of CSK reorganization from space flight research,a possible mechanism from the standpoint of “cellular mechanical equilibrium” is proposed for the explanation of cellular response to space microgravity.Cytoskeletal equilibrium is broken by the gravitational change from ground to space and is followed by cellular morphological changes,cell mechanical properties changes,extracellular matrix reorganization,as well as signaling pathway activation/inactivation,all of which ultimately lead to the cell functional changes in space microgravity.The polymerization/depolymerization of CSK filaments is dynamic.Cytoskeletal reorganization might be different at the beginning of entering space with accommodation after long-term spaceflight,their role in gravitational sensation still needs further investigation[9–11].

3 Space Biology Technology

3.1 Microfluidic Chip-based Long-term Preservation and Culture of Engineering Bacteria for DNA Damage Evaluation

Understanding the effects of long-term exposure to space environment is paramount to maintaining the safety and health of astronauts.The physical dosimeters currently used on the space station cannot be used to assess the physiological effects of radiation.Moreover,some developed biological methods are time-consuming and passive,and cannot be used for active and real-time detection of the physiological effects of radiation in space environment.Here,the SOS promoter:recA-eGFP genetic engineering bacteria was constructed and characterized,and DNA damage effects of some chemical reagents and radiation were evaluated.The results indicated the constructed engineering bacteria can distinguish DNA damage reagents from non-damage reagents,and have a good dose-fluorescence effect against Co-60 radiation with the detection limit of 0.64 Gy (Fig.1andFig.2).To overcome the restriction of long-term preservation of bacteria in space environment,the bacteria were freeze-dried,and the protectants were optimized,the storage time of bacteria under dry conditions was explored by accelerated storage experiments.Finally,a microfluidic chip was designed and fabricated for freeze-drying genetic engineering bacteria recovery,culture,and analysis in space environment (Fig.3).This study can provide support for the establishment of on-orbit radiation damage risk monitoring and early warning,and can provide basic data for maintaining the health and performance of astronauts on long-term space flight missions.Moreover,the technique developed herein has a great potential to be used as a powerful tool for efficiently screening of various radioactive substances,toxic chemicals,drugs,etc.

Fig.1 Damage and fluorescence effect of engineering bacteria induced by chemical reagents

Fig.2 Response of genetic engineering bacterial strain to different doses of Co-60 radiation

Fig.3 Microfluidic chip structure and culture system

3.2 Microorganism Culture and Detection Payload in the China Space Station

The research project on the prevention and control technology of microorganisms in extraterrestrial habitats undertaken by Denget al.of Beijing Institute of Technology has completed most of the work since the project started in 2020.The project aims to carry out on-orbit cultivation and analysis of specific microorganisms for the needs of microorganism prevention and control in the China Space Station,as well as in the extraterrestrial habitation facilities for future lunar exploration projects and deep space exploration.The project uses microorganisms that have the ability to degrade certain aviation materials collected in the final assembly workshop of the space station as the research object,and develops a special culture chip to study the corrosion process of microorganisms on aviation materials in the space environment.

Based on the results of ground-based experiments,the project developed a microorganism culture and detection payload,and two chip cartridges that support quick installation (Fig.4).Among the two chip cartridges,the culture chip cartridge is used to provide solution environment and temperature control for microorganism culture,and the microbes are photographed by the payload.The detection chip cartridge is used for quantitative detection of specific microorganisms.The detection principle is based on Q-LAMP technology.A microfluidic chip is used to lyse microorganisms and amplify nucleic acids,and the payload is responsible for fluorescence collection and quantitative analysis.The entire detection process takes about 60 min.In the future,this payload will be upgraded to support on-orbit sampling and detection of microorganisms.These two chip cartridges contain all the reagents,chips and liquid drive systems,and have high air tightness,which is convenient for astronauts to operate in orbit and has high biological safety.The payload has a power supply interface and a communication port that are compatible with the relevant cabinet platform of the Mengtian experimental module of the China Space Station,and supports the downloading of on-orbit experimental data to the ground.The payload will be launched with the Mengtian experimental module to dock with the China Space Station in 2022,and follow-up on-orbit tests will be carried out as planned.

Fig.4 Microorganism culture and detection payload.(a) Payload appearance,(b) detection chip cartridge

During a spaceflight,astronauts need to live in a spacecraft on-orbit for a long time,and the relationship between humans and microorganisms in the closed environment of space is not as same as on the ground.The dynamic study of microorganisms in confined space shows that with the extension of the isolation time,harmful bacteria gradually accumulate.Monitoring and controlling microbial pollution in a confined environment system are very important for crew health and the sustainable operation of a space life support system.Culture-based assays have been used traditionally to assess the microbial loads in a spacecraft,and uncultured-based techniques are already underway according to the NASA global exploration roadmap.High-throughput sequencing technology has been used generally to study the communities of the environment and humans on the ground,and shows its broad prospects applied onboard.Chenet al.[12]reviewed the recent application of highthroughput sequencing on space microbiology and analyze its feasibility and potential as an on-orbit detection technology.

3.3 Bioregenerative Life Support Systems Which Can Support Humans Living in Space

In order to travel outside the Earth and achieve longterm survival in the deep space,humans need to build a Biological system-regenerative Life Support System(BLSS),which reduces the demand for Earth supply by regenerating the oxygen,water and food required by astronauts,and prevents astronauts from polluting outer stars by recycling waste.In 2016,Yuegong No.1 completed the technical upgrade,and the “Yuegong 365”experiment began on 10 May 2017 and was completed on 15 May 2018,lasting 370 days.The influence of unit shift change and electromechanical failure on the stability of BLSS under long-term operation conditions is studied,the regulation technology of long-term operation is established,two groups of units with different metabolic levels are established,and three shift change stages are formed.The results show that Yuegong BLSS has good stability in the long-term operation process,eliminates the influence of gas interference caused by shift and electromechanical fault through self-feedback adjustment,and has strong robustness.The crew planted 35 kinds of plants,including grains,vegetables and berries.The production of the factory fully meets the crew’s demand for plant food.The purification effect of domestic sewage has reached the standard of plant irrigation,and urine and solid waste have also been recycled.Under the load of four crew members,the experiment achieved 100% oxygen and water regeneration,83% food regeneration and 98.2% overall material closure.However,the overall impact of space environment on BLSS is not clear,and research work is still needed.Lunar exploration projects such as lunar village and lunar research station are being carried out one after another.Therefore,the future BLSS research will focus on the payload carrying experiment of lunar probe,so as to study the mechanism of space closed ecosystem without covering the moon,clarify the impact of space environmental conditions on the ecosystem,and correct the design and operation parameters of ground-based BLSS. These studies will provide technical support for the application of BLSS in manned deep space exploration[13–14].

4 Conclusion

Since 1992,with the continuous development and deepening of China’s manned spaceflight and space exploration activities,especially the start-up and construction of China’s manned space station project,the next 20 years will be a golden period in the development history of China’s space life science.Chinese scientists will continue to develop and obtain innovative achievements belonging to China on the basis of the achievements inherited from their predecessors,so as to serve human space exploration and benefit human life on the ground.