Through the history of human development, the vastness of space has always been an infinite attraction for humans. Although magnificent, space is rife with various environmental changes that can adversely physically affect those who enter it to explore. More specifically, during the execution of space missions, astronauts are not only affected by changes in the space environment which mainly include weightlessness, high radiation and vacuum, but are also restricted inside a narrow and closed cabin environment. Under these conditions, changes in the symbiotic intestinal flora of the intestines of astronauts may occur and further affect their physical health. The health problem of astronauts is a factor that could hinder the success of a space mission.
The development of complex molecular biology technology has allowed us to conduct different types of research studies. In order to further explore the influence of complex space environments on intestinal flora, we used the ground-based simulated module and a TS model to explore the influence of a combined effects on changes in species composition, function and resistance genes of intestinal flora in mice. For the experiment, nine C57BL/6 mice with the same genetic background were randomly divided into the NC group, TS group, and TS + SM group to observe changes in intestinal flora caused by different environments, and fecal samples were collected for further analysis after the end of the 45-day experiment. Then, DNA extraction, quality inspection, bank building and other processes were performed on the qualified feces samples to obtain effective Unigenes for species annotation, functional analysis and resistance gene detection.
We first found that different environments could cause changes in the species composition of intestinal flora in mice, and that these changes were distributed at different levels. This is consistent with the results of the species analysis of the intestinal flora of short-flying astronauts in study conducted by Turroni(28). Especially at the phylum and genus levels, the effects of a simulated microgravity environment and combined effects environment can be observed on species composition. Specifically, Firmicutes and Bacteroidetes showed an increasing and decreasing trend, respectively, these changes were significant in a combined effects environment. Under normal circumstances, Firmicutes and Bacteroidetes are the main components of intestinal flora(29). A variety of Firmicutes bacteria can metabolize carbohydrates to produce butyrate, which is not only beneficial for the metabolism of energy substances, but can also protect the intestinal mucosa and improve immune function(30). Firmicutes is a class of gram-positive bacteria and one of the largest families in the domain bacteria. Most Firmicutes bacterial walls contain high levels of peptidoglycans (50%-80%) and are thick enough to form spores, resulting in them being highly resistant to dehydration or extreme environments. In addition, several studies have shown that a large number of Firmicutes bacteria in the gut are associated with obesity(31–33). Although obesity does not affect the performance of space missions, its associated metabolic changes require further attention due to its metabolic dysfunction. Several studies have demonstrated that many changes in the lipid metabolism of gastric cancer cells and epidermal stem cells have been observed under simulated microgravity(34, 35). Bacteroidetes, a core flora of the human intestinal tract, contains a powerful polysaccharide degrading system to digest dietary fibers consumed by the body and convert it into short-chain fatty acids(36). Relying on its powerful metabolic capacity, Bacteroidetes has the main high levels of stability for human health(37). However, this stability depends on the balance between nutrient absorption and consumption in the body(38). In addition, the main source of Vitamin K in humans is also synthesized by Bacteroides(39). Vitamin K deficieny affects various systems in the body, such as the coagulation system and the musculoskeletal system(40, 41). Due to these important functions, Bacteroidetes are widely regarded as beneficial bacteria that can decrease intestinal inflammation, immune dysfunction and metabolic disorders, and may even function in preventing the occurrence of cancer(42). Moreover, the metabolites of Bacteroides, such as propionate and acetate, can also block the absorption of enteric endotoxin(43) and induce the apoptosis of colon cancer cells(44), playing a preventive and therapeutic role. Zitomersky et al. found that the specific mechanism by which the protective role of Bacteroides is exerted may be through recruitment of intraepithelial lymphocytes to produce IL-6(45). Overall, Bacteroides is a protective barrier of the host intestinal tract(46). However, in certain cases Bacteroidetes can also become opportunistic pathogens that can cause diseases(47). Although there was a decrease in the number of Bacteroidetes in our study, these changes need to be addressed due as they indicate changes in immune function in space extreme environment. In addition, we screened biomarkers that could be used at the species level for the effects caused by simulated weightlessness or complex environmental factors. The increase of Bacteroidetes in the NC group could be used as biomarkers, which suggested that the relative abundance of Bacteroidetes decreased under weightlessness environment and combined effects environment. This is consistent with the results obtained through species annotation, and also indicates that the changes in Bacteroidetes abundance in combined effects environment are very important for health.
The KEGG enrichment analysis showed that compared with the NC group, the proportion of metabolic pathways under the two different environments decreased, while the decrease was most obvious in the combined effects environment. The changes in these metabolic pathways were highly correlated with changes in intestinal floral species composition. To respond to environmental changes, the functional genes involved in environmental information processing of the gut microbiota were increased significantly in the combined effects environment, which may be related to the adaptation of the body and intestinal flora under changes in the environment. It is worth noting that changes in intestinal floral species composition and functional genes in mice were not consistent with that of weightlessness and combined effects environment, which needs to be confirmed through further studies.
The abuse of antibiotics leads to irreversible changes in the human body and microbial communities in the environment, which poses risks to human health and the ecological environment. Therefore, the study of resistance genes has attracted extensive attention from researchers. We expect to study the changes in resistance genes in intestinal flora to provide new insights for the application of antibiotics and the prevention of diseases. The weightlessness environment and combined effects environment both significantly affected the expression of resistance genes in the intestinal flora of the mice. Although the total number of resistance genes were decreased in the TS and TS + SM groups, the fluctuation in the expression of the resistance genes was more meaningful. For example, the multidrug efflux pump is capable of transporting structurally varied molecules, including antibiotics, out of the bacterial cell. This efflux lowers the intracellular antibiotic concentrations, allowing bacteria to survive at higher antibiotic concentrations(48).
The manned spaceflight environment is a complex environment that is subjected to multiple physical changes. In this study, we focused on the effects of combined effects environment on species composition and function of intestinal flora in mice, but more studies on the effect of complex factors on intestinal flora are needed to confirm the applicability of these results. In addition, the specific reasons for the changes in intestinal floral species composition and functions in mice under weightlessness or complex factors need to be further confirmed. Based on previously reported results, it remains to be determined whether there is a correlation between the changes in immune function and the changes in intestinal flora microecology under microgravity environment, while mutual influences induced by the two groups, as well as the recovery of intestinal flora after exposure to a space environment are also worthy of attention.