(1) Background: Many studies have shown that microgravity experienced by astronauts or long-term bedridden patients results in increased oxidative stress and bone loss. Low-molecular-weight ...chondroitin sulfates (LMWCSs) prepared from intact chondroitin sulfate (CS) have been demonstrated to possess good antioxidant and osteogenic activities in vitro. This study aimed to assess the antioxidant activity of the LMWCSs in vivo and evaluate their potential in preventing microgravity-induced bone loss. (2) Methods: we used hind limb suspension (HLS) mice to simulate microgravity in vivo. We investigated the effects of LMWCSs against oxidative stress damage and bone loss in HLS mice and compared the findings with those of CS and a non-treatment group. (3) Results: LMWCSs reduced the HLS-induced oxidative stress level, prevented HLS-induced alterations in bone microstructure and mechanical strength, and reversed changes in bone metabolism indicators in HLS mice. Additionally, LMWCSs downregulated the mRNA expression levels of antioxidant enzyme- and osteogenic-related genes in HLS mice. The results showed that overall effect of LMWCSs was better than that of CS. (4) Conclusions: LMWCSs protect against the bone loss caused by simulated microgravity, which may be related to their ability to reduce oxidative stress. LMWCSs can be envisaged as potential antioxidants and bone loss protective agents in microgravity.
Microgravity is known to affect the organization of the cytoskeleton, cell and nuclear morphology and to elicit differential expression of genes associated with the cytoskeleton, focal adhesions and ...the extracellular matrix. Although the nucleus is mechanically connected to the cytoskeleton through the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, the role of this group of proteins in these responses to microgravity has yet to be defined. In our study, we used a simulated microgravity device, a 3-D clinostat (Gravite), to investigate whether the LINC complex mediates cellular responses to the simulated microgravity environment. We show that nuclear shape and differential gene expression are both responsive to simulated microgravity in a LINC-dependent manner and that this response changes with the duration of exposure to simulated microgravity. These LINC-dependent genes likely represent elements normally regulated by the mechanical forces imposed by gravity on Earth.
The effects of weightlessness on enteric microorganisms have been extensively studied, but have mainly been focused on pathogens. As a major component of the microbiome of the human intestinal tract, ...probiotics are important to keep the host healthy. Accordingly, understanding their changes under weightlessness conditions has substantial value. This study was carried out to investigate the characteristics of
Lactobacillus acidophilus
, a typical probiotic for humans, under simulated microgravity (SMG) conditions. The results revealed that SMG had no significant impact on the morphology of
L. acidophilus
, but markedly shortened its lag phase, enhanced its growth rate, acid tolerance ability up to pH < 2.5, and the bile resistance at the bile concentration of <0.05%. SMG also decreased the sensitivity of
L. acidophilus
to cefalexin, sulfur gentamicin, and sodium penicillin. No obvious effect of SMG was observed on the adhesion ability of
L. acidophilus
to Caco-2 cells. Moreover, after SMG treatment, both the culture of
L. acidophilus
and its liquid phase exhibited higher antibacterial activity against
S. typhimurium
and
S. aureus
in a time-dependent manner. The SMG treatment also increased the in vitro cholesterol-lowering ability of
L. acidophilus
by regulating the expression of the key cholesterol metabolism genes
CYP7A1, ABCB11, LDLR
, and
HMGCR
in the HepG2 cell line. Thus, the SMG treatment did have considerable influence on some biological activities and characteristics of
L. acidophilus
related to human health. These findings provided valuable information for understanding the influence of probiotics on human health under simulated microgravity conditions, at least.
Cellular processes are influenced in many ways by changes in gravitational force. In previous studies, we were able to demonstrate, in various cellular systems and research platforms that reactions ...and adaptation processes occur very rapidly after the onset of altered gravity. In this study we systematically compared differentially expressed gene transcript clusters (TCs) in human Jurkat T cells in microgravity provided by a suborbital ballistic rocket with vector-averaged gravity (vag) provided by a 2D clinostat. Additionally, we included 9×
centrifuge experiments and rigorous controls for excluding other factors of influence than gravity. We found that 11 TCs were significantly altered in 5 min of flight-induced and vector-averaged gravity. Among the annotated clusters were
,
,
,
, and
. Our results revealed that less than 1% of all examined TCs show the same response in vag and flight-induced microgravity, while 38% of differentially regulated TCs identified during the hypergravity phase of the suborbital ballistic rocket flight could be verified with a 9×
ground centrifuge. In the 2D clinostat system, doing one full rotation per second, vector effects of the gravitational force are only nullified if the sensing mechanism requires 1 s or longer. Due to the fact that vag with an integration period of 1 s was not able to reproduce the results obtained in flight-induced microgravity, we conclude that the initial trigger of gene expression response to microgravity requires less than 1 s reaction time. Additionally, we discovered extensive gene expression differences caused by simple handling of the cell suspension in control experiments, which underlines the need for rigorous standardization regarding mechanical forces during cell culture experiments in general.
With the increasing interest in exploring the deep-space environment, the problems of health and safety of astronauts' bone induced by microgravity warrants an investigation. Recent studies have ...discovered that several microRNAs (miRNAs) have played a vital role in osteoblast differentiation and bone formation under microgravity, whereas the in-depth signaling pathway mechanisms are not yet completely understood. Here, we performed the hind limb unloading (HLU) mice model and 3D clinostat-random position machine (RPM) to simulate the effects of microgravity on bone at animal and cellular level, respectively. Firstly, we screened the different expressed miRNAs under simulated microgravity by miRNA sequencing, we then identified the highest different expressed miRNA (miR-138–5p) that was up-regulated under simulated microgravity and inhibited osteoblast differentiation in MC3T3-E1 cells. Moreover, we analyzed miR-138's targets during osteogenic differentiation and we found that these targets regulated osteogenic differentiation through Wnt/β-catenin signaling, a key signaling pathway in controlling osteoblast differentiation. The association between miR-138–5p and β-catenin activity was determined by a luciferase reporter assay. Further experiments confirmed that miR-138–5p suppressed β-catenin expression and β-catenin activity. Moreover, β-catenin overexpression attenuated osteoblast differentiation reduction induced by increased miR-138–5p level in miR-138–5p overexpression osteoblastic cells. In addition, inhibition of miR-138–5p counteracted the negative effects of simulated microgravity on osteoblast differentiation and β-catenin expression in MC3T3-E1 cells. Our findings demonstrated that a signaling pathway mechanism of miR-138–5p in regulating osteoblast differentiation under simulated microgravity, which may reveal a novel mechanism for astronauts' bone loss induced by microgravity and provide a potential therapeutic target for disused osteoprosis.
•MiR-138–5p is a microgravity-sensitive miRNA.•MiR-138–5p inhibits osteoblast differentiation in MC3T3-E1 cells.•MiR-138–5p inhibits β-catenin expression and β-catenin activity.•Inhibition of miR-138–5p counteracts decrease of osteoblast differentiation induced bysimulated microgravity.•Providing a novel mechanism and a potential therapeutic target for disused osteoprosis.
Cultured mammalian cells have been shown to respond to microgravity (μG), but the molecular mechanism is still unknown. The study we report here is focused on molecular and cellular events that occur ...within a short period of time, which may be related to gravity sensing by cells. Our assumption is that the gravity-sensing mechanism is activated as soon as cells are exposed to any new gravitational environment. To study the molecular events, we exposed cells to simulated μG (SμG) for 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h using a three-dimensional clinostat and made cell lysates, which were then analyzed by reverse phase protein arrays (RPPAs) using a panel of 453 different antibodies. By comparing the RPPA data from cells cultured at 1G with those of cells under SμG, we identified a total of 35 proteomic changes in the SμG samples and found that 20 of these changes took place, mostly transiently, within 30 min. In the 4 h and 8 h samples, there were only two RPPA changes, suggesting that the physiology of these cells is practically indistinguishable from that of cells cultured at 1 G. Among the proteins involved in the early proteomic changes were those that regulate cell motility and cytoskeletal organization. To see whether changes in gravitational environment indeed activate cell motility, we flipped the culture dish upside down (directional change in gravity vector) and studied cell migration and actin cytoskeletal organization. We found that compared with cells grown right-side up, upside-down cells transiently lost stress fibers and rapidly developed lamellipodia, which was supported by increased activity of Ras-related C3 botulinum toxin substrate 1 (Rac1). The upside-down cells also increased their migratory activity. It is possible that these early molecular and cellular events play roles in gravity sensing by mammalian cells. Our study also indicated that these early responses are transient, suggesting that cells appear to adapt physiologically to a new gravitational environment.
Spaceflight and ground-based microgravity analog experiments have suggested that microgravity can affect microbial growth and metabolism. Although the effects of microgravity and its analogs on ...microorganisms have been studied for more than 50 years, plausible conflicting and diverse results have frequently been reported in different experiments, especially regarding microbial growth and secondary metabolism. Until now, only the responses of a few typical microbes to microgravity have been investigated; systematic studies of the genetic and phenotypic responses of these microorganisms to microgravity in space are still insufficient due to technological and logistical hurdles. The use of different test strains and secondary metabolites in these studies appears to have caused diverse and conflicting results. Moreover, subtle changes in the extracellular microenvironments around microbial cells play a key role in the diverse responses of microbial growth and secondary metabolisms. Therefore, "indirect" effects represent a reasonable pathway to explain the occurrence of these phenomena in microorganisms. This review summarizes current knowledge on the changes in microbial growth and secondary metabolism in response to spaceflight and its analogs and discusses the diverse and conflicting results. In addition, recommendations are given for future studies on the effects of microgravity in space on microbial growth and secondary metabolism.
Bone loss remains a major health concern for astronauts during space flight. Increased osteoclast activity is one of the main causes of bone loss in astronauts subjected to microgravity conditions. ...However, the underlying molecular mechanisms remain unclear. Microtubule actin crosslinking factor 1 (MACF1) has been implicated in the regulation of cytoskeletal distribution, cell migration, and cell differentiation. Our previous studies have shown that MACF1 promotes the differentiation of pre-osteoclasts. However, whether MACF1 regulates the migration and cytoskeletal arrangement of pre-osteoclasts under microgravity conditions has not yet been elucidated. In this study, we used a hind-limb unloading (HLU) mouse model and a random positioning machine (RPM) to simulate the effects of microgravity on pre-osteoclasts. In the HLU mouse model, the expression of MACF1 was upregulated in primary pre-osteoclasts of mice, accompanied by enhanced migration in vivo. Moreover, simulated microgravity using the RPM also promoted MACF1 expression and migration of pre-osteoclasts in vitro. Additionally, knockdown of MACF1 disrupted cytoskeletal arrangement (F-actin and microtubules) and further inhibited the migration of pre-osteoclasts via the RhoA/ROCK1 signaling pathway. We further demonstrated that knockdown of MACF1 disrupted the enhanced migration and cytoskeleton arrangement of pre-osteoclasts induced by simulated microgravity. These data demonstrate that MACF1 positively regulates the migration and cytoskeletal organization of pre-osteoclasts under simulated microgravity, suggesting that MACF1 may be a therapeutic target for the treatment of bone loss induced by microgravity.
•MACF1 is a microgravity sensitive gene.•Simulated microgravity induced MACF1 expression and migration of pre-osteoclasts.•MACF1 knockdown attenuated the cytoskeleton arrangement of pre-osteoclasts.•MACF1 knockdown inhibited the migration via the RhoA/Rock1 pathway.•MACF1 knockdown prevented the migration and cytoskeleton arrangement of pre-osteoclasts induced by simulated microgravity.
As the number of manned space flights increase, studies on the effects of microgravity on the human body are becoming more important. Due to the high expense and complexity of sending samples into ...space, simulated microgravity platforms have become a popular way to study these effects on earth. In addition, simulated microgravity has recently drawn the attention of regenerative medicine by increasing cell differentiation capability. These platforms come with many advantages as well as limitations. A main limitation for usage of these platforms is the lack of high-throughput capability due to the use of large cell culture vessels. Therefore, there is a requirement for microvessels for microgravity platforms that limit waste and increase throughput. In this work, a microvessel for commercial cell culture plates was designed. Four 3D printable (polycarbonate (PC), polylactic acid (PLA) and resin) and castable (polydimethylsiloxane (PDMS)) materials were assessed for biocompatibility with adherent and suspension cell types. PDMS was found to be the most suitable material for microvessel fabrication, long-term cell viability and proliferation. It also allows for efficient gas exchange, has no effect on cell culture media pH and does not induce hypoxic conditions. Overall, the designed microvessel can be used on simulated microgravity platforms as a method for long-term high-throughput biomedical studies.
Human cells, when exposed to both real and simulated microgravity (s-µ
), form 3D tissue constructs mirroring in vivo architectures (e.g., cartilage, intima constructs, cancer spheroids and others). ...In this study, we exposed human foetal osteoblast (hFOB 1.19) cells to a Random Positioning Machine (RPM) for 7 days and 14 days, with the purpose of investigating the effects of s-µ
on biological processes and to engineer 3D bone constructs. RPM exposure of the hFOB 1.19 cells induces alterations in the cytoskeleton, cell adhesion, extra cellular matrix (ECM) and the 3D multicellular spheroid (MCS) formation. In addition, after 7 days, it influences the morphological appearance of these cells, as it forces adherent cells to detach from the surface and assemble into 3D structures. The RPM-exposed hFOB 1.19 cells exhibited a differential gene expression of the following genes: transforming growth factor beta 1 (
, bone morphogenic protein 2 (
), SRY-Box 9 (
), actin beta (
), beta tubulin (
), vimentin (
), laminin subunit alpha 1 (
), collagen type 1 alpha 1 (
), phosphoprotein 1 (
) and fibronectin 1 (
). RPM exposure also induced a significantly altered release of the cytokines and bone biomarkers sclerostin (SOST), osteocalcin (OC), osteoprotegerin (OPG), osteopontin (OPN), interleukin 1 beta (IL-1β) and tumour necrosis factor 1 alpha (TNF-1α). After the two-week RPM exposure, the spheroids presented a bone-specific morphology. In conclusion, culturing cells in s-µ
under gravitational unloading represents a novel technology for tissue-engineering of bone constructs and it can be used for investigating the mechanisms behind spaceflight-related bone loss as well as bone diseases such as osteonecrosis or bone injuries.
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