Simulated Microgravity Group Research Articles

Evaluation of osteoclast-derived exosomal miRNA under simulated microgravity conditions using next-generation sequencing

The important role of osteoclast-derived exosomes in bone remodeling of microgravity-induced osteoporosis has been validated. However, the underlying mechanism remains unclear. A set of total miRNAs expressed in osteoclast-derived exosomes may configure the specific signature of the bone tissue in the microgravity condition. This study aimed to investigate the miRNA expression profile of osteoclast-derived exosomes in simulated microgravity using next-generation sequencing. Cell culture supernatant of osteoclasts under normal gravity conditions and simulated microgravity conditions were harvested for the extraction of exosomes and exosomal miRNAs. Our results revealed that 116 aberrantly expressed exosomal miRNAs including 29 up-regulated miRNAs and 87 down-regulated miRNAs were found in the simulated microgravity group compared to the normal gravity control group. The candidate target genes and signaling pathways of 15 selected differentially expressed miRNAs were identified via target genes prediction, GO and KEGG analyses. Furthermore, miRNA qPCR assay was performed to validate the RNA sequencing results. Collectively, these analyses provide insight regarding the effects of microgravity in modulating the extracellular miRNA profile through exosomes released from osteoclasts. The selective packaging of miRNA cargo in exosomes under microgravity could facilitate the development of promising and effective targets for the diagnosis, prevention, and treatment of microgravity-induced osteoporosis.

Effects of RCCS simulated microgravity on proliferation and cell cytoskeleton of human HaCaT keratinocyte

Objective To investigate the effects of simulated microgravity by RCCS on proliferation and cell cytoskeleton of human HaCaT keratinocyte. Methods The rotary cell culture system (RCCS) was used to simulate the microgravity environment, and human HaCaT keratinocytes were divided randomly(random number) into the simulated microgravity group (SMG) and normal gravity group (NG). HaCaT cells in the two groups were harvested respectively after 32, 36 and 42 h culture. The HaCaT cells proliferation and cycles were detected by flow cytometry, the concentration of hb-EGF in supernatant was detected by ELISA, and the cell cytoskeleton was observed after 42 hours’ culture under laser confocal microscope with FITC-labeled technique. SPSS 23.0 statistical software was used for statistical analysis, and P <0.05 was considered statistically significant. Results The flow cytometry showed that the proportions of human HaCaT keratinocytes in G1 and G2/M phases were increased while the proportion of HaCaT cells in S stage was decreased significantly after 32, 36 and 42 h RCCS culture compared with those in the normal gravity group. The HaCaT cells in G1 stage were declined along with incubation time. ELISA results showed that the hb-EGF concentration in HaCaT supernatant under simulated microgravity culture for 24 and 36 h was lower than that in the normal control group (P<0.01). The laser confocal microscope revealed that the HaCaT fluorescence intensity was decreased, and there were disordered microfilaments, structural ambiguity, pseudopodia reduction and irregular shape among FITC-labeled HaCaT cells cultured 42 h in RSSC compared with the normal gravity group. Conclusions RCCS simulated microgravity environment could inhibit the cell cycle transformation and proliferation of human HaCaT keratinocyte, affect the keratinocyte-secreting function, and induce alterations of the cell cytoskeleton. Key words: Simulated microgravity; RCCS; Keratinocyte; HaCaT cell line; Proliferation; Cell cycle; hb-EGF; Cytoskeleton

Effects of the rotary cell culture system-simulated microgravity on growth features of thyroid follicular epithelial cells in rats

The rotary cell culture system(RCCS)was used to simulate the microgravity environment, and FRTL-5 cells were divided into simulated microgravity group(SMG)and normal gravity group(NG). FRTL-5 cells were harvested after treatment for 6, 12, 24, and 36 h, the cell viability was measured by MTT assay, and the cells cycles were detected by flow cytometry. The ultrastructure of FRTL-5 cells was observed under laser confocal microscope with FITC-labeled technique. The MTT assay showed that the proliferation of FRTL-5 cells was significantly inhibited after RCCS treatment for 6, 12, 24, and 36h compared with NG(P<0.05), in which the most obvious effect was observed at 24h. The flow cytometry showed that the proportion of FRTL-5 cells at G1 stage in RCCS group was increased significantly after 6, 12, 24, and 36h compared with NG(P<0.05), while the proportion of FRTL-5 cells at S stage was decreased significantly(P<0.05)except that cultured with RCCS for 6 h. The proportion of FRTL-5 cells at G2/M stage was decreased in early phase(6-12 hours)of RCCS culture, with the lowest at 12h and transient increase at 24h of RCCS culture. The laser confocal microscope revealed that there were local microfilament depolymerization, tension fibers decrease, structure disorder, cellular pseudopodia reduction, and irregular shape among FITC-labeled FRTL-5 cells cultured with RCCS for 36h. (Chin J Endocrinol Metab, 2018, 34: 598-601) Key words: Simulated microgravity; Thyroid follicular epithelial cells; FRTL-5 cell line; Proliferation; Microfilament

Microarray Profiling and Bioinformatic Analysis of Circular RNAs in RAW264.7 Macrophages Under Simulated Microgravity

Macrophages play a vital role in the innate immune system. Lots of studies have showed that spaceflight can change the function of macrophage and induce the immune disorder, however, the mechanisms remain to be illustrated. circular RNAs (circRNAs) can act as miRNA sponges and play an important role in regulating gene expression, here we tested the hypothesis that simulated microgravity can induce the differential expression of circRNAs in RAW 264.7 macrophages under simulated microgravity. Arraystar Mouse circRNA Array (8x15K, Arraystar) was used to identify the circRNA expression profiles in RAW 264.7 macrophages from Control(Con) and simulated microgravity(SM) groups, the cells in SM group were treated by clinostat for 72 hours. Total RNA was quantified using the NanoDrop ND‐1000. The sample preparation and microarray hybridization were performed based on the Arraystar's standard protocols, Agilent Feature Extraction software was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the R software package. Differentially expressed circRNAs with statistical significance between two groups were identified through Volcano Plot filtering with a threshold of fold change ≥1.5 and P&lt;0.05. Quantitative RT‐PCR was performed in the Real‐time PCR System (Applied Biosystems) using SYBR Green qPCR master mix(Arraystar). Bioinformatic analyses including gene ontology (GO) and KEGG pathway were applied to predict the potential functions of circRNAs. The results from circRNA microarrays revealed that there were 60 differentially expressed circRNAs in SM group compared with Con group, in which 34 circRNAs were found to be up‐regulated and 26 circRNAs were down‐regulated. Furthermore, the up‐regulated expression of circR003780, circR015248 and circR015947 were verified by qRT‐PCR. GO analysis indicated that the top20 up‐regulated enriched GO terms included biological process “positive regulation of chronic inflammatory response”. KEGG analysis showed that the top 30 pathway enrichment included Wnt and mTOR signaling pathway, protein processing in endoplasmic reticulum, apoptosis, et al. These results indicated that simulated microgravity can change the circRNAs expression pattern in RAW 264.7 macrophages, the differentially‐expressed circRNAs may participate in the function changes of macrophage in spaceflight.Support or Funding InformationFunded by National Basic Research Programs of China, No.2014CB744404This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neural oscillations as a bridge between glutamatergic system and emotional behaviors in simulated microgravity-induced mice

This study aims to investigate if neural oscillations can play a role as a bridge between the alteration of glutamatergic system and emotional behaviors in simulated microgravity (SM) mice. Adult male C57BL/6J mice were randomly divided into two groups: SM and control groups. The animal model was established by hindlimb unloading (HU). The mice were exposed to HU continued for 14days. Weight and sucrose consumption were measured. The degree of anxious and depressive was evaluated by Open field test and Elevated plus maze test. Local field potentials were recorded in the hippocampal perforant path (PP) and dentate gyrus (DG) regions. The NMDAR2A/2B (NR2A/2B) subunits expression and glutamate level were measured by Western and high performance liquid chromatography (HPLC), respectively. After 14days, SM mice exhibited depressive-like and anxiety-like behaviors, while the expression of NR2A/2B subunits and the glutamate level were significantly decreased in the SM group. Moreover, the power distribution of theta (3–8Hz) was decreased by HU, which further significantly attenuated the identical-frequency strength of phase synchronization and the neural information flow at theta rhythm on the PP-DG pathway. The theta-gamma phase synchronization strength was also significantly reduced by HU. The data imply that the neural oscillations measurements is a sign of the emotional behaviors impairment and the glutamatergic system change induced by HU.

Simulated microgravity affects the proliferation and related genes expression in murine liver Kupffer cells

Objective To investigate the effects of simulated microgravity on the proliferation and expression of related genes in murine liver Kupffer cells. Methods The rotary cell culture system (RCCS) was used to simulate the microgravity environment and culture the murine liver Kupffer cells, which were divided into simulated microgravity group (SMG) and normal gravity group (NG) at random, and were harvested after being cultured for 3, 5 and 7 days, respectively. The cell count and cell cycle were detected by hemocytometer and flow cytometry. The Kupffer cell’s expression of PCNA, Ki-67, ERK, CDK2 and Cyclin B were assayed by real-time fluorescent quantitative PCR (qPCR). Results After being cultured for 3 days, the number of Kupffer cells in SMG group were less than that in NG group (2.6±0.1 vs. 3.1±0.2, P<0.05), while after being cultured for 5 and 7 days, the number of Kupffer cells in SMG group were increased significantly compared with that in NG group (6.9±0.4 vs. 5.9±0.2, P<0.05; 8.4±0.3 vs. 6.5±0.3, P<0.05). The flow cytometry also revealed that at 3rd days of culture, the G0/G1 phase of Kupffer cells in SMG group was significantly higher than that in NG group (78.1±0.2 vs. 59.7±1.2, P<0.05), while the S phase and the G2/M phase were significantly lower than that in NG group (12.0±0.4 vs. 27.6±0.9, P<0.05; 9.9±0.3 vs. 12.7±0.4, P<0.05). In contrary, the G0/G1 phase of Kupffer cells being cultured for 5 days in SMG group was decreased (69.5±1.5 vs. 74.8±0.7, P<0.05), and the S phase and G2/M phase were increased (21.2±1.5 vs. 17.3±0.4, P<0.05; 9.3±0.4 vs. 7.9±0.4, P<0.05) significantly compared with those in NG group. After 7 days of culture, the G0/G1 phase of the SMG group was still decreased (73.9±1.9 vs. 81.7±1.3, P<0.05) while the S phase and the G2/M phase were also increased (18.9±1.9 vs. 12.1±0.8, P<0.05; 7.3±0.2 vs. 6.2±0.6) compared with the control. The qPCR assay showed that the expressions of PCNA, ki-67, ERK, CDK2 and Cyclin B in SMG were down-regulated at 3rd days of culture, and up-regulated from 5th to 7th days of culture compared with those of NG group. Conclusion The proliferation of murine liver Kupffer cells was depressed during microgravity stress period, and were then activated and enhanced through certain genes mediated modulation. Key words: Rotary cell culture system; Simulated microgravity; Liver; Kupffer cells; Proliferation; Genes

Increased biofilm formation ability in Klebsiella pneumoniae after short-term exposure to a simulated microgravity environment.

Biofilm formation is closely related to the pathogenetic processes of Klebsiella pneumoniae, which frequently causes infections in immunocompromised individuals. The immune system of astronauts is compromised in spaceflight. Accordingly, K. pneumoniae, which used to be isolated from orbiting spacecraft and astronauts, poses potential threats to the health of astronauts and mission security. Microgravity is a key environmental cue during spaceflight. Therefore, determining its effects on bacterial biofilm formation is necessary. In this study, K. pneumoniae ATCC BAA‐1705 was exposed to a simulated microgravity (SMG) environment. K. pneumoniae grown under SMG formed thicker biofilms compared with those under normal gravity (NG) control after 2 weeks of subculture. Two indicative dyes (i.e., Congo red and calcofluor) specifically binding to cellulose fibers and/or fimbriae were utilized to reconfirm the enhanced biofilm formation ability of K. pneumoniae grown under SMG. Further analysis showed that the biofilms formed by SMG‐treated K. pneumoniae were susceptible to cellulase digestion. Yeast cells mannose‐resistant agglutination by K. pneumoniae type 3 fimbriae was more obvious in the SMG group, which suggests that cellulose production and type 3 fimbriae expression in K. pneumoniae were both enhanced under the SMG condition. Transcriptomic analysis showed that 171 genes belonging to 15 functional categories were dysregulated in this organism exposed to the SMG conditions compared with those in the NG group, where the genes responsible for the type 3 fimbriae (mrkABCDF) and its regulator (mrkH) were upregulated.

Cytomorphometric Changes in Hippocampal CA1 Neurons Exposed to Simulated Microgravity Using Rats as Model

Microgravity and sleep loss lead to cognitive and learning deficits. These behavioral alterations are likely to be associated with cytomorphological changes and loss of neurons. To understand the phenomenon, we exposed rats (225–275 g) to 14 days simulated microgravity (SMg) and compared its effects on CA1 hippocampal neuronal plasticity, with that of normal cage control rats. We observed that the mean area, perimeter, synaptic cleft, and length of active zone of CA1 hippocampal neurons significantly decreased while dendritic arborization and number of spines significantly increased in SMg group as compared with controls. The mean thickness of the postsynaptic density and total dendritic length remained unaltered. The changes may be a compensatory effect induced by exposure to microgravity; however, the effects may be transient or permanent, which need further study. These findings may be useful for designing effective prevention for those, including the astronauts, exposed to microgravity. Further, subject to confirmation, we propose that SMg exposure might be useful for recovery of stroke patients.

Morphologic Characteristics and Proliferation of Rabbit Corneal Stromal Cells Onto Complexes of Collagen–Chitosan–Sodium Hyaluronate Under Simulated Microgravity

We investigated the morphologic characteristics and proliferation of rabbit corneal stromal cells (CSCs) onto scaffolds under simulated microgravity. Rabbit CSCs were cultured under simulated microgravity (SMG) and static condition. Complexes of collagen-chitosan-sodium hyaluronate with pores were used as scaffolds. Rotational speed was set at 15, 20, and 30 rpm in the first, second, and third weeks of culture, respectively. Histology, immunofluorescence staining, atomic force microscope (AFM), and scanning electron microscope (SEM) examinations were performed. The cell proliferation was analyzed by cell counting kit-8 (CCK-8) assay. In the SMG group, more CSCs adhered to the carriers in 24 hours. Confocal microscopic evaluation showed aggregated cells positively immunostained with vimentin. The SEM displayed the complex network of triangular or polygonal dendritic morphology of the cell bodies with many fine and long processes, which adhered to the scaffolds tightly. After 18 days of SMG culture, keratocyte-like CSCs with rich cell interconnections not only grew on the surface, but also into the interior of scaffolds. There were degradation phenomena in scaffolds in the SMG condition. Under static condition, cells just grew on scaffolds forming a monolayer. Cells showed elongating spindle shape and developed less processes. The absorbance values of the CCK-8 assay in the SMG group were significantly higher (P < 0.01) than in the conventional group. The condition of SMG and porous collagen-chitosan-sodium hyaluronate scaffolds facilitate the proliferation of CSCs. Cells showed robust growing characteristics and morphologic properties of keratocytes. The techniques for microgravity culture of keratocyte-like CSCs on scaffolds can yield cell aggregates or cell sheets that are favorable to the reconstruction of tissue engineering of the corneal stromal layer.

The simulated microgravity enhances multipotential differentiation capacity of bone marrow mesenchymal stem cells

Multi-differentiation capability is an essential characteristic of bone marrow mesenchymal stem cells (BMSCs). Method on obtaining higher-quality stem cells with an improved differentiation potential has gained significant attention for the treatment of clinical diseases and developmental biology. In our study, we investigated the multipotential differentiation capacity of BMSCs under simulated microgravity (SMG) condition. F-actin staining found that cytoskeleton took on a time-dependent change under SMG condition, which caused spindle to round morphological change of the cultured cells. Quantitative PCR and Western Blotting showed the pluripotency marker OCT4 was up-regulated in the SMG condition especially after SMG of 72h, which we observed would be the most appropriate SMG duration for enhancing pluripotency of BMSCs. After dividing BMSCs into normal gravity (NG) group and SMG group, we induced them respectively in endothelium oriented, adipogenic and neuronal induction media. Immunostaining and Western Blotting found that endothelium oriented differentiated BMSCs expressed higher VWF and CD31 in the SMG group than in the NG group. The neuron-like cells derived from BMSCs in the SMG group also expressed higher level of MAP2 and NF-H. Furthermore, the quantity of induced adipocytes increased in the SMG group compared to the NG group shown by Oil Red O staining, The expression of PPARγ2 increased significantly under SMG condition. Therefore, we demonstrated that SMG could promote BMSCs to differentiate into many kinds of cells and predicted that enhanced multi-potential differentiation capacity response in BMSCs following SMG might be relevant to the changes of cytoskeleton and the stem cell marker OCT4.

Simulated Microgravity Exposure Modulates the Phenotype of Cultured Vascular Smooth Muscle Cells

Evidence from ground-based animal studies using tail-suspended hindlimb unloaded rats model has clearly demonstrated that simulated microgravity-induced smooth muscle cell phenotype conversion, a characteristic vascular structural and functional remodeling, may be one of the key contributors to postspaceflight orthostatic intolerance. However, the rats model involves multiple collective effects of microgravity including cephalic fluid shift and postural muscle unloading on smooth muscle cells (SMCs). It cannot isolate a single factor from the collective ones and therefore is not ideal to study the effects of gravitational vector alteration alone on SMCs. To test the hypothesis that gravitational vector alteration per se might affect smooth muscle cell phenotype, a roller culture apparatus was employed to expose cultured rat aortic smooth muscle cells (RASMCs) to simulated microgravity. Cell proliferation, cell cycle distribution, apoptosis, migration, and nitric oxide production rates were measured and compared between the control and the simulated microgravity groups. Cell cytoskeleton reorganization induced by simulated microgravity was observed by confocal microscopy. Specific contractile and synthetic Gene expression at the mRNA level was quantified by reverse transcriptional polymerase chain reaction. It was observed that simulated microgravity suppressed RASMC proliferation and migration, enhanced cell apoptosis, stimulated NO release, and destroyed the original well-organized cytoskeleton. Moreover, at the mRNA level, long-time exposure (≥ 72 h) to simulated microgravity induced a contractile phenotype tendency by up-regulating smMHC expression. All these findings suggest that the phenotype modulation of vascular smooth muscle cells may be gravity dependent.

The simulated microgravity enhances the differentiation of mesenchymal stem cells into neurons

Growing evidence shows that physical microenvironments and mechanical stresses, independent of soluble factors, help influence mesenchymal-stem-cell fate. rMSCs (rat mesenchymal stem cells) present spread, spindle shape when cultured in normal gravity (NG) while in simulated microgravity (SMG) they become unspread, round shape. Here we demonstrate that simulated microgravity can enhance the differentiation of mesenchymal stem cells into neurons, which might be a new strategy for the treatment of central nervous system diseases. rMSCs were cultured respectively in normal gravity and in a clinostat to simulate microgravity, followed with neuronal differentiated medium. The neuronal cells derived from rMSCs in SMG express higher microtubule-associated protein-2 (MAP-2), tyrosine hydroxylase (TH) and choline acetyltransferase (CHAT). Furthermore, as rMSCs are subjected to SMG, they excrete more neurotrophins like nerve growth factor (NGF), brain derived neurophic factor (BDNF) and ciliary neurotrophic factor (CNTF). Neuronal cells from SMG group generated more mature action potentials and displayed repetitive action potentials by comparison to cells from NG group. We conclude that simulated microgravity can enhance the differentiation of mesenchymal stem cells into neurons.

Nitric oxide synthase activity in the abdominal aorta of rats is decreased after 4 weeks of simulated microgravity

1. The aim of the present study was to investigate the effects of simulated microgravity on the arterial dilatory responsiveness and L-arginine (L-Arg)-nitric oxide (NO)-cGMP pathway in the abdominal aorta of rats. 2. Twenty healthy male Sprague-Dawley were randomly divided into control and simulated microgravity groups. Rats in the simulated microgravity group were subjected to hindlimb unweighting (HU). After 4 weeks, arterial dilatory responsiveness was examined in vitro in isolated abdominal aortic rings. Western blotting was used to measure endothelial (e) and inducible (i) NO synthase (NOS) protein content. Total concentrations of nitrate and nitrite (NO(x)), the stable metabolites of NO, were determined by the chemiluminescence method. Nitric oxide synthase activity in the abdominal aorta was determined through the conversion of [(3)H]-L-Arg to [(3)H]-L-citrulline. 3. The data showed that the dilatory responses of the arterial rings to L-Arg and acetylcholine decreased in rats exposed to simulated microgravity, but the dilatory responses to sodium nitroprusside and 8-bromo-cGMP were similar in both simulated microgravity and control rats. The expression of eNOS and iNOS did not differ significantly between the two groups. The NO(x) concentration in the abdominal aorta of HU rats was significantly less than that in control rats. Nitric oxide synthase activity in the aorta decreased after 4 weeks of HU. 4. The data indicate that endothelium-dependent vasorelaxation in the abdominal aorta decreased due to 4 weeks of simulated microgravity in rats and that this impaired dilatory responsiveness may result from decreased NOS activity.

Morphological Characteristics and Proliferation of Keratocytes Cultured Under Simulated Microgravity

This study probed the changes of keratocytes cultured under simulated microgravity. Keratocytes were isolated from rabbit corneas using collagenase digestion method. Cells were seeded in a 55-mL capacity high-aspect-ratio vessel (HARV) of rotary cell culture system (RCCS) at a density of 1 x 10(4) cells/mL. Dehydrated bovine acellular corneal stroma (5 x 5 x 1 mm, n = 30) was used as a carrier for keratocyte culture. Rotational speed was set at 15, 20, and 30 rpm in the first, second, and third week of culture, respectively. Histological evaluation showed that keratocytes in simulated microgravity culture grew into carriers, but those under conventional gravity grew on the surface of carriers. Scanning electron microscopic evaluation showed that after 19 days in culture, keratocytes on the carriers were spherical and spread in the spaces among the collagen fibers. Cells were dendritic or spindle shaped, and they developed many foot processes linked with surrounding cells. The absorbance values of the simulated microgravity group were significantly higher (P < 0.01) than that of the conventional group from 10 to 19 days of culture. The RCCS obviously enhanced the proliferation of rabbit keratocytes and facilitated the cells' growth into or on the dehydrated bovine acellular corneal stroma. Cells showed more natural morphology.

Effects of simulated microgravity on arterial nitric oxide synthase and nitrate and nitrite content.

The aim of the present work was to investigate the alterations in nitric oxide synthase (NOS) expression and nitrate and nitrite (NOx) content of different arteries from simulated microgravity rats. Male Wistar rats were randomly assigned to either a control group or simulated microgravity group. For simulating microgravity, animals were subjected to hindlimb unweighting (HU) for 20 days. Different arterial tissues were removed for determination of NOS expression and NOx. Western blotting was used to measure endothelial NOS (eNOS) and inducible NOS (iNOS) protein content. Total concentrations of NOx, stable metabolites of nitric oxide, were determined by the chemiluminescence method. Compared with controls, isolated vessels from simulated microgravity rats showed a significant increase in both eNOS and iNOS expression in carotid arteries and thoracic aorta and a significant decrease in eNOS and iNOS expression of mesenteric arteries. The eNOS and iNOS content of cerebral arteries, as well as that of femoral arteries, showed no differences between the two groups. Concerning NOx, vessels from HU rats showed an increase in cerebral arteries, a decrease in mesenteric arteries, and no change in carotid artery, femoral artery and thoracic aorta. These data indicated that there were differential alterations in NOS expression and NOx of different arteries after hindlimb unweighting. We suggest that these changes might represent both localized adaptations to differential body fluid redistribution and other factors independent of hemodynamic shifts during simulated microgravity.