Transcriptional landscape in rat intestines under hypobaric hypoxia

Oxygen metabolism is closely related to the intestinal homeostasis environment, and the occurrence of many intestinal diseases is as a result of the destruction of oxygen gradients. The hypobaric hypoxic environment of the plateau can cause dysfunction of the intestine for humans, such as inflammation. The compensatory response of the small intestine cells to the harsh environment definitely changes their gene expression. How the small intestine cells response the hypobaric hypoxic environment is still unclear. We studied the rat small intestine under hypobaric hypoxic conditions to explore the transcriptional changes in rats under acute/chronic hypobaric hypoxic conditions. We randomly divided rats into three groups: normal control group (S), acute hypobaric hypoxia group, exposing to hypobaric hypoxic condition for 2 weeks (W2S) and chronic hypobaric hypoxia group, exposing to hypobaric hypoxic condition for 4 weeks (W4S). The RNA sequencing was performed on the small intestine tissues of the three groups of rats. The results of principal component analysis showed that the W4S and W2S groups were quite different from the control group. We identified a total of 636 differentially expressed genes, such as ATP binding cassette, Ace2 and Fabp. KEGG pathway analysis identified several metabolic and digestive pathways, such as PPAR signaling pathway, glycerolipid metabolism, fat metabolism, mineral absorption and vitamin metabolism. Cogena analysis found that up-regulation of digestive and metabolic functions began from the second week of high altitude exposure. Our study highlights the critical role of metabolic and digestive pathways of the intestine in response to the hypobaric hypoxic environment, provides new aspects for the molecular effects of hypobaric hypoxic environment on intestine, and raises further questions about between the lipid metabolism disorders and inflammation.


INTRODUCTION
Quick entry into the hypoxic environment of the medical plateau, defined as an area located 3000 m above sea level, can lead to serious damage to the organs of the body (Taino et al., 2019). In high-altitude areas, oxygen content and oxygen partial pressure decrease, forming a hypobaric hypoxic environment. Hypobaric hypoxia can lead to insufficient arterial blood oxygen content; this phenomenon is called systemic hypoxemia and causes cell and mitochondrial oxygen utilization disorders (Murray, 2016). The hypobaric hypoxic environment tends to increase the severity of inflammation, which can lead to brain damage (Li et al., 2017), pulmonary edema, and multiple organ dysfunction syndrome (MODS) which is the most complicated and difficult to cure (Swank & Deitch, 1996).
When entering high-altitude areas residents living at low altitudes will experience physiological reactions including changes in respiration and cardiovascular function, erythropoiesis factors, and gastrointestinal symptoms. The mechanism of the gastrointestinal symptoms of acute mountain sickness (AMS) caused by rising to high altitude is unclear (Fruehauf et al., 2020). Individuals with sufficient adaptability to highaltitude can maintain homeostasis under hypoxic conditions, but the metabolism and physiological functions will be affected, such as changes in energy metabolism in the heart and skeletal muscles. In other studies, the levels of Adenosine Triphosphate (ATP) and phosphocreatine (PCr) in skeletal muscle decreased with the altitude increases and this decrease continued over time. This indicates that decreasing ATP demand in high-altitude areas cannot meet the requirement of suppressing ATP supply (Hochachka et al., 1996).
In recent years, while excluding the symptoms of AMS, the incidence of gastrointestinal symptoms is extremely high, mainly manifested as abdominal pain, diarrhea, nausea, vomiting, and even hematochezia and melena for symptoms of gastrointestinal bleeding (Frühauf, 2014). Studies have shown that gastrointestinal mucosal barrier function damage caused by severe trauma, infection, hemorrhagic shock, and other factors can activate the inflammatory response pathway (Dempsey, Little & Cui, 2019). The intestine has essential biological functions at different oxygen concentrations (Sengupta, Ray & Chowdhury, 2014). Due to the existence of numerous gut microbiome, the intestine is in a hypoxic environment which maintains nutrient absorption, immune defense, and intestinal barrier functions (Singhal & Shah, 2020).
At present, various studies have shown that oxygen metabolism is closely related to the intestinal homeostasis environment and the occurrence of many intestinal diseases is a result of the destruction of oxygen gradients (Ramakrishnan & Shah, 2016). Under hypoxic conditions, cells adapt to the hypoxic environment by adjusting gene expression. Many metabolic changes are regulated by hypoxia inducible factors (HIFs). The encoding of enzymes regulated by HIFs involves glycolytic metabolism, lipid metabolism, nutrient absorption, peroxisomal metabolism, and mitochondrial function (Xie & Simon, 2017). Furthermore, it has been shown that hypoxia can increase leukocyte adhesion, stimulate mucosal tissues inflammation, and destroy the tissue barrier function (Deng et al., 2018;Saeedi et al., 2015;Seys et al., 2013). Regulating HIFs promote the expression of transcription factors in hypobaric conditions and through hypoxia, but how HIFs are regulated in hypobaric hypoxia and the downstream response is not clear (Wang et al., 2018). We conducted a more in-depth study through RNA-Seq analysis to explore the effects of 2 to 4 weeks of hypobaric hypoxic conditions on intestinal gene expression in in vivo studies.

Animals and treatment
Adult male Sprague Dawley (SD) rats were randomly divided into three groups with four rats in each group, but during modeling both the S and W2S groups lost one rat. According to the study by Qian Ni et al. (2014), we divided the experimental rats into three groups: the normal control group (S), the acute hypobaric hypoxia group (W2S) which was exposed to hypobaric hypoxic conditions for 2 weeks, and the chronic hypobaric hypoxia group (W4S) which was exposed to hypobaric hypoxic conditions for 4 weeks. The hypobaric hypoxic environment was constructed to simulate a 5,500-meter-high atmospheric environment using a FLYDWC50-1C hypobaric hypoxia cabin (Guizhou Fenglei Air Ordnance LTD, Guizhou, China). The rats were anesthetized with 10% chloral hydrate (0.4 ml/100g), the small intestine tissue was taken out, washed quickly, and stored in liquid nitrogen. The study was approved by the Animal Ethics Committee of the Chinese PLA General Hospital (2017-X13-05).

RNA extraction
The small intestine sample was chopped and mixed with liquid nitrogen to form a powder. Add Trizol reagent to dissociate nucleoprotein; add 0.3ml chloroform, shake for 15 s, and wait at room temperature for 2 min; 12000 rpm centrifuge (4 • C for 15 min), add 0.5 ml isopropanol and wait for 15 min at room temperature; 12000 rpm (4 • C for 15 min), aspirate the supernatant; add one mL 4 • C pre-cooled 75% ethanol to the RNA at the bottom; centrifuge (10,000 rpm, 5 min, 4 • C), and aspirate the supernatant; centrifuge (10,000 rpm, 4 • C, 5 min), aspirate the remaining liquid, and ventilate for 10 min; RNA concentration was determined in a Qubit2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) following the Qubit RNA Assay Kit operating instruction.

Transcriptome library construction and Sequence
Reverse transcription synthesis of the first strand cDNA, recombination into the second strand of DNA; then according to the above template in vitro transcription synthesis of cDNA, cDNA purification and reverse recording, purification, and quantification of the reverse transcription cDNA, and finally the library quality was assessed. The small intestine samples were sequenced with TruSeq PE Cluster Kit v3-cBot-HS (Illumina). All analyses are based on clean data.

Reference genome alignment
Reference genome was obtained from the relevant genome analysis website. The required gene annotation files were taken from the database as well. Using the HISAT2 v 2.0.4 comparison tool, the paired end readings were aligned with the reference genome (Langmead & Salzberg, 2012).

Differential expression analysis
The analysis of differentially expressed genes (DEGs) was finished through the Limma package in R language. Differentially expressed genes are based on the standard of |logFC|>1, P value<0.05. Genes with logFC >1 are defined as an up-regulated gene. Genes with LogFC <−1 are defined as a down-regulated gene.

Functional analysis and co-expression analysis
Gene ontology (GO) was implemented using the DEGs in three functional categories: biological process (BP), molecular function (MF), and cellular component (CC). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database was used to annotate DEGs on the pathway in the database for statistical analysis (Mao et al., 2005). The co-expression gene set analysis was finished using the Cogena software package (Jia et al., 2016).

qRT-PCR analysis
Total RNA was collected using the TRIzol reagent (TaKaRa, Japan) following the manufacturer's instructions. Then RNA was reverse transcribed to cDNA using reverse transcriptase (Promega GoTaq qPCR Master Mix, China). qRT-PCR analysis was actualized by Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA).
The hierarchical clustering of the DEGs is shown on the heat map in Fig. 1. W2S represented acute hypobaric hypoxia, while the W4S group represented chronic hypobaric hypoxia. The expression patterns of DEGs were vastly different from each other. After analysis, we found that the relationship between the W4S and the S groups were more closely related when compared with the W2S groups.
There are 195/255 up-regulated genes in the W2S/W4S groups, respectively, compared with the S group. There are 120 up-regulated genes and 52 down-regulated genes in both the W2S and W4S groups compared with the S group and four genes up-regulated in the W2S but down-regulated in the W4S. Conversely, there are 19 genes down-regulated in the W2S but up-regulated in the W4S (Fig. 2
Full-size DOI: 10.7717/peerj.11823/ fig-1 associated with identical protein binding, transmembrane transport, and oxygen carrier activity. These up-regulated gene expressions may be associated with hypobaric hypoxia. Further principal component analyses suggested similar results as the observation above (Fig. 3). The three groups are separated, but the members in the same group are clustered together; this indicated that the model construction was successful.

Biological functional analysis of DEGs
To explore the impact of hypobaric hypoxia on gene function, we used Gene Ontology (GO) analysis. DEGs are classified into three functional categories. For biological processes, the GO analysis indicated that nutrient transport ontologies, especially anion, fatty, and acid were enriched. For molecular function, some transmembrane transporter ontologies, such as metal ion and active, were enriched. For cellular components, the DEGs were active in the plasma membrane (Fig. 4).
We used KEGG pathway enrichment analysis to further explore the biological functions of DEGs in response to hypobaric hypoxia. Several metabolic and digestive pathways, such as PPAR signaling pathway, glycerolipid metabolism, fat metabolism, mineral absorption, ferroptosis, and vitamin metabolism were identified (Fig. 5).

Co-expression analysis
Co-expression analysis can identify genes with similar functions; in this co-expression analysis, we observed three patterns. Most of the genes in the first cluster were downregulated in the 2nd week and up-regulated in the 4th week. In the second cluster, 126 genes were down-regulated until the 4th week. In the third cluster, 244 genes were up-regulated from the 2nd week until the 4th week (Fig. 6). The Cogena Bioconductor software package was used for pathway analysis of each co-expression cluster. According to the manual, we selected three clusters for analysis and used the k-means clustering method. Interestingly, metabolic and digestive related pathways, such as PPAR and the renin angiotensin pathway, and fatty acid metabolism, are only enriched in the third cluster (Fig. 7). In the third cluster, we found that the metabolic and digestive pathways start to work in the 2nd week.

Gene transcription
The most changed genes, including the top 20 genes with increased expression and top 20 with decreased expression, have been listed (Fig. 8). The top five differentially expressed genes for each group, including the up-regulated genes and down-regulated genes, were detected through RT-PCR (Fig. 9). The trend of genes differentially expressed between each group is mostly matched with the transcriptional landscape.

DISCUSSION
The gastrointestinal tract is the key organ involved in the digestion of food and providing nutrients to the body for proper maintenance. However, the hypobaric hypoxic environment of the medical plateau can cause dysfunction of the gastrointestinal system. A previous study found that hypobaric hypoxia can effectively regulate immune/inflammatory processes and energy metabolism (Gangwar et al., 2020), and the degree of change has a clear relationship with time (Padhy et al., 2016). The high-altitude hypoxic environment destroys lipid metabolism and activates inflammatory processes, which causes abnormal metabolic function. Related studies have shown that the liver X receptor (LXR) plays an essential role in cholesterol, fatty acid, and glucose metabolism (Li & Glass, 2004). The physiological functions of LXRs have a close relationship with nuclear receptors (such as PPAR) (Hong & Tontonoz, 2014). The main role of the LXR pathway is based on energy homeostasis of lipid metabolism. In the high-altitude hypoxia model, the LXR pathway is strongly activated in the later stage (Tang et al., 2014). However, its mechanism of action in a hypoxic environment is still unclear, and the changes in the pathway may affect lipid metabolism. Several groups are currently conducting drug research and development based on LXR. Some studies have found that LXR activates macrophages through stearoyl-CoA desaturase (Wang, Kurdi-Haidar & Oram, 2004). The latest findings indicate that inflammation regulation is closely related to lipid metabolism. This study provides the first basis for investigating the changes in transcription and signaling pathways in rat small intestine tissues under acute and chronic hypobaric hypoxic conditions and found that the hypobaric hypotension environment affects fatty acid metabolism in the small intestine.
These findings indicate that metabolic and digestive related pathways play a key role in the differences in the mechanism between hypobaric hypoxia and acute/chronic mountain sickness. Studies have shown that oxygen metabolism is closely related to the intestinal homeostasis environment (Ramakrishnan & Shah, 2016). Under hypoxic conditions, many metabolic changes are regulated by hypoxia inducible factors (HIFs). The encoding of enzymes regulated by HIFs involves glycolytic metabolism, lipid metabolism, and nutrient absorption (Xie & Simon, 2017). Hypoxia can increase leukocyte adhesion, stimulate mucosal tissues inflammation, and destroy the tissue barrier function (Deng et al., 2018;Saeedi et al., 2015;Seys et al., 2013). By studying the changes of gene expression in small intestine tissues exposed to hypobaric hypoxia for 2 to 4 weeks, these results provide a basis for further research on the molecular pathogenesis of diseases under hypobaric hypoxic conditions. Through genome-wide transcription analysis, we found that hypoxia increased pro-inflammatory cytokines release, causing damage to the small intestine metabolism from the 2nd week. This study plays an important part in the research of the molecular mechanism of small intestine injury in high altitude areas and sets the stage for further genetic and functional research. There is a clear correlation between systemic inflammation and AMS (Boos et al., 2016;Li et al., 2015). A likely mechanism is that hypoxia changes cellular immunity and regulates the release of cytokines. Studies have shown that intermittent hypoxia has links with metabolic dysfunction (including abnormal lipid metabolism and insulin resistance). Intermittent hypoxia may induce sympathetic activation, increased inflammation, regulate hormone metabolism, and directly cause pancreatic β-cell damage to interfere with glucose metabolism (Drager, Jun & Polotsky, 2010). At the same time, in another study, scholars found that intermittent hypoxia conditions can increase the rate of lipid oxidation and suppress carbohydrate oxidation (Kelly & Basset, 2017). Previous reports have indicated that this severe intermittent hypoxia brings about a series of changes, such as low-grade inflammation, oxidative stress, and endoplasmic reticulum stress (Chacaroun et al., 2020). These adverse reactions are harmful to many systems, such as cardiovascular, respiratory, and metabolism, as well as cognition etc. (Chacaroun et al., 2020;Gabryelska et al., 2020;Gauda et al., 2020;Vermeulen et al., 2020).
In a previous study, Sweet R et al. found that hypoxia can up-regulate the expression of S100a8, the Retn gene, and Slc19a3 (Jiao, Wang & Zhang, 2019;Sweet, Paul & Zastre, 2010;Uchiyama et al., 2019). The transcriptional landscape and RT-PCR results of this study align with the trends of other researchers, indicating that this study will be a useful reference for future studies on intestinal diseases under hypobaric hypoxia. The angiotensin I converting enzyme 2 (Ace2) gene, which regulates systemic arterial blood pressure through the renin-angiotensin system, and the membrane metallo-endopeptidase (Mme) gene, that influences kidney development, are the DEGs found in this study, they play an important role in renin-angiotensin system and metabolic disorders related to the renin-angiotensin pathway were also observed. Revera M et al. found that during acute and chronic exposure to high altitude, the aortic pulse wave velocity increased significantly with altitude and the subendocardial oxygen supply/demand index significantly decreased (Revera et al., 2017). Acute exposure to hypobaric hypoxia can cause aortic stiffness and reduce subendocardial blood supply. A randomized clinical trial found that as the altitude increases, blood pressure will gradually increase, and will remain elevated for 3 weeks after entering the high-altitude area. Angiotensin receptor blockers can reduce blood pressure at 3400 m, but the effect is not apparent at 5400 m (Parati et al., 2014). A significant feature of hypoxic pulmonary hypertension is the remodeling of the pulmonary artery (Mikami et al., 1996). In another study by Mikami O et al. 1996, it was found that the angiotensin II (Ang II) receptors increased in the lung tissue of rats with hypoxic pulmonary hypertension, the Ang II level rose, and the renin-angiotensin system activated. Then, it induced hypertrophy and proliferation of aortic smooth muscle cells and cardiomyocytes (Parati et al., 2014).
Our study found that mineral absorption is overexpressed in a hypobaric hypoxic environment. Hypoxic environments and HIFs play an important role in the absorption of iron and minerals in the small intestine (Das et al., 2015;Das et al., 2020). The absorption of bones is affected by the hypobaric hypoxic environment (Guner et al., 2013) which causes abnormal absorption of minerals. This indicates that the hypobaric hypoxic environment will interfere with the metabolism of rats. This metabolic change may be the body's adaptation to a hypobaric hypoxic environment. This study has a few limitations including an insufficient number of subjects, which generated an accurate cut-off value for the diagnosis of AMS in rats. Also, since no blood samples were obtained in this experiment, we were unable to investigate plasma-related indicators.

CONCLUSION
This study found 636 DEGs associated with ATPase activity, fatty acid metabolic process, NADP metabolic process, cholesterol and lipid transport, thiamine (Vitamin B1) transmembrane transporter activity, and the renin-angiotensin system. KEGG pathway