Label-free quantitative proteomics of rat liver exposed to simulated microgravity

Acta Astronautica 170 (2020) 251–260

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Research paper

Label-free quantitative proteomics of rat liver exposed to simulated microgravity

T

Bo Chena, George Q. Lib,c, Yongzhi Lid, Jun-Lae Choe,f, Jiaping Wangd, Jianyi Gaod, Yulin Denga, Yujuan Lia,∗ a

School of Life Science, Beijing Institute of Technology, No. 5 Zhongguancun South Street, Haidian District, Beijing, 100081, China NICM Health Research Institute, Western Sydney University, Penrith, NSW 2751, Australia c Institute of Natural Products and Metabolomics, Chengdu University of Traditional Chinese Medicine, Chengdu, 610075, China d China Astronaut Research and Training Centre, No. 26 Beiqing Road, Haidian District, Beijing, 100094, China e Faculty of Pharmacy, The University of Sydney, Sydney, NSW 2006, Australia f Centres for Excellence in Advanced Food Enginomics, Faculty of Engineering and Information Technologies, The University of Sydney, Sydney, NSW 2006, Australia b

A R T I C LE I N FO

A B S T R A C T

Keywords: Simulated microgravity Liver Proteomics Metabolism Oxidative stress

Microgravity affects the content of carbohydrates, lipids and proteins in liver. Since liver is the major metabolic organ of endogenous and exogenous compounds, a deep understanding of microgravity-induced effects on liver could be of great importance. Therefore, livers from tail-suspension simulated microgravity (SM) rat model were analyzed using a label-free quantitative proteomic method in the present study. The results indicated that hepatic metabolic functions were obviously disrupted by SM. After short-term SM, enzymes for oxidizing nutrients were greatly increased and thus glucose, non-ester fatty acids and amino acids in liver were heavily consumed. Meanwhile, proteins for oxidative phosphorylation were also remarkably regulated. Therefore, the vast electrons generated from oxygenolysis could not be fully transferred to dioxygen and subsequently strong oxidative stress was triggered in liver. On the other hand, antioxidants were increased compensatorily. With the restoration of most energy-metabolic proteins and the continuous up-regulation of antioxidants, hepatic oxidative status returned to physiological level after long-term SM. Moreover, several enzymes involved in xenobiotic metabolism, such as cytochrome P450s and UDP-glucuronosyltransferases were also regulated by SM in a duration-dependent way. Our data might be helpful in understanding the effects of microgravity on liver and would support the design of protective measures for manned space missions.

1. Introduction Microgravity has been shown to obviously impact several mammalian organs, inducing biochemical and physiological dysfunctions including fluid shifts, bone demineralization, muscle atrophy and cardiovascular issues [1]. In addition, spaceflight studies, especially the ‘COSMOS’ missions, have demonstrated that the content of carbohydrates, lipids, proteins and some specific enzymes involved in the metabolism of these compounds in liver are also affected by microgravity [2–9]. Since liver is the major metabolic organ of endogenous and exogenous compounds, a deep understanding of microgravity-induced effects on liver could be of great importance for manned spaceflight. However, systematic investigations on this purpose are relatively few up to now.

The high cost of spaceflight, low frequencies of missions and difficulties in performing experiments in spacecraft have severely limited the study of microgravity-related biological effects. Hence, several ground-based models have been developed and validated to mimic the influence of microgravity on organism, such as human head-down bed rest, cell rotatory culture and murine tail-suspension (TS) [10–14]. Using the TS model, multiple kinds of hepatic pathological alterations were suggested during exposure to simulated microgravity (SM), including portal endotoxemia, neutrophil infiltration, acute-phase response, fibrosis, granular degeneration, apoptosis and inhibited cell proliferation [15–19]. However, the underlying mechanism is largely unknown. Meanwhile, the proteomic technology, a powerful tool to uncover thousands of differentially regulated proteins in biological matrix, has

Abbreviations: TS, tail-suspension; SM, simulated microgravity; NEFAs, non-ester fatty acids; TAAs, total amino acids; H2O2, hydrogen peroxide; MDA, malondialdehyde; SOD, superoxide dismutase; GSH, glutathione; OXPHOS, oxidative phosphorylation; ROS, reactive oxygen species; CYP450, cytochrome P450 ∗ Corresponding author. E-mail address: [email protected] (Y. Li). https://doi.org/10.1016/j.actaastro.2020.02.007 Received 29 August 2019; Received in revised form 27 January 2020; Accepted 3 February 2020 Available online 05 February 2020 0094-5765/ © 2020 IAA. Published by Elsevier Ltd. All rights reserved.

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centrifuge tubes as previously described [25] with some modification. Detailed protocol was described in Supplemental Protocol 1. The dried peptides were stored at −20 °C before use.

been introduced into space medicine. Various investigations utilizing proteomic methods have been performed to explore the effects of SM. For instance, Zhang identified 22 differentially abundant proteins related to microfilament network in SH-SY5Y cells after 24-h rotatory culture [20]; Iqbal demonstrated the differential expression of 159 proteins involved in the homeostatic cascades like sleep/wake cycle, drinking behavior and cellular defense in rat hypothalamus using the TS model [21,22]; Wang revealed that, in rat hippocampus, 21-day TS differentially regulated 53 proteins involved in cognitive functions and 42/67 metabolic proteins showed significant alterations after 7/21-day TS [23,24]. However, for now, investigations on liver proteome in SM have barely been reported. In the present study, 3 and 21-day TS rat models were established to mimic short and long-term microgravity. Livers were then analyzed using an LC-MS/MS proteomic method combined with label-free quantification to identify altered proteins. The results might help to understand the response of liver to SM and give further insight into the design of protective measures for manned space missions.

2.3. LC-MS/MS The peptides were dissolved in 30 μL of acetonitrile/H2O/formic acid (2%: 98%: 0.1%) and 5 μL of the peptide solution was injected into a C18 reverse-phase column (15 cm × 75 μm, 3 μm, Eksigent Technologies, Waltham, USA). The column was operated at a controlled temperature of 40 °C. The LC system was an Eksigent nano2D LC (Eksigent Technologies, Waltham, USA) and set at a flow rate of 300 nL/min. Mobile phase A was 0.1% formic acid-water and mobile phase B was 0.1% formic acid-acetonitrile. A linear gradient from 5% of mobile phase B to 35% of mobile phase B over 60 min was used to separate the peptides. The LC system was coupled with a TripleTOF™ 4600 mass spectrometer (AB SCIEX, Redwood City, USA) through a nano-electrospray source (AB SCIEX, Redwood City, USA). The mass spectrometer was operated in the data-dependent mode and survey scans were acquired at a resolution of 30 K at m/z 800 for MS1 (mass range 350–1800 m/z) while 15 K at m/z 700 for MS2 (mass range 100–1000 m/z). The transient time was set at 256 ms. Top 10 most abundant precursor ions with charge ≥2 were selected and fragmented by higher energy collisional dissociation. The maximum ion injection times for the survey scan and the MS/MS scan were 20 and 60 ms, respectively, and the ion intensity threshold value for both scans was set at 106.

2. Material and methods 2.1. Animals The present study complied with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication no. 85-23, revised in 1985), and all animal experiments were approved by Beijing Institute of Technology Animal Care and Use Committee (Beijing, China). 18 male SD rats (240 ± 20 g, 10 weeks old) were purchased from Academy of Military Medical Sciences (Beijing, China). The rats were kept in a temperature, humidity and illumination-controlled room (temperature: 25 °C, humidity: 55%, illumination: 12-h light-dark cycle) with free access to water and normal standard chow diet. All animals were housed for one week prior to the study. The rats were then randomly divided into 3 groups (6 rats per group): control group, rats were freely raised in normal cages; 2 TS groups, rats were tail-suspended for 3 and 21 days (termed 3-day and 21-day group, respectively). 3 and 21-day TS were used to simulate short and long-term microgravity. TS was performed according to Morey-Holton's protocol [12]. Briefly, disinfected rat tail was attached with surgical tape and then connected to a pulley by a metal bar. The hindlimbs of rat were elevated through the suspended tail to produce a −30° tilt angle in relation to the horizontal. The rat could freely move to any directions with its forelimbs and had free access to water and food. After TS, rats were sacrificed under deep anesthesia by cardio perfusion with ice-cold normal saline. The livers were harvested and stored at −80 °C for further study.

2.4. Protein identification and quantification The MS data was searched against the 2015 Swissprot rat database (7965 entries) using software environment MaxQuant 1.5.2.8. Search settings were specified below: mass tolerance of precursor was 0.07 Da for the first search and 0.006 Da for the main search, respectively, while of MS/MS was 40 ppm; the minimum number of peptides to consider a protein as identified was 1 and unique + razor peptides were used for protein grouping and quantification; based on the reversed database, FDR < 0.01 was set at both peptide and protein level; carbamidomethyl cysteines was set as fixed modification while oxidation of methionines was set as variable modification; trypsin was selected as the digestive enzyme with 2 missed cleavages at most. Label-free quantitative method was carried out in the current study and the protein content was calculated as the sum of peptide intensities integrated over the elution profile [26].

2.2. Protein extraction and in-gel digestion

2.5. Bioinformatics

0.5 g of liver tissue was put into 2 mL of ice-cold 10 mM PBS buffer (containing cocktail protease inhibitor, Roche Diagnostics, Indianapolis, USA). The mixture was homogenized thoroughly using a Teflon-glass homogenizer, followed by 1-min sonication (1-s sonication and 2-s pause in ice-bath). The homogenate was then centrifuged at 12,000 g for 15 min at 4 °C to collect the supernatant. The protein concentration was determined by Bradford (Bio-Rad, Hercules, USA) method. Later, equal amounts of proteins from each individual were mixed for each group. After denatured by protein loading buffer (Solarbio, Beijing, China), 50 μg of proteins was loaded onto an SDS-PAGE system (5% stacking gel plus 12% separating gel) in triplicate. Electrophoresis was performed at 80 V for 30 min and 110 V for 80 min. The separating gel was stained by Coomassie blue dye and destained by 20% methanol. Each lane was evenly cut into 4 parts and each part was then cut into approximately 1 mm3 pieces. In-gel digestion was performed in 2-mL

Proteins confidently identified in at least 2 of the 3 technical replicates were used for comparative statistical analysis. The median value of protein abundance was used to calculate fold change. Differentially abundant proteins were selected using conditions as follows: P-value of t-test was lower than 0.05 and fold change cutoff was set at > 1.5 and < 0.5 for up and down-regulation, respectively [27,28]. Then, the list of altered proteins was input into online software DAVID (version 6.8, https://david.ncifcrf.gov/home.jsp), which performed GO and KEGG analysis automatically with 2 threshold parameters (count: 2, EASE: 0.1) to reveal remarkably affected molecular functions, biological processes and pathways. Annotation terms with Pvalue < 0.05 were considered as significant ones. Then, the parameter “fold enrichment” (the ratio of the percentage a term appearing in the input list to that in the whole genome) was used to rank the significant terms. Terms with < 0.05 P-values and top 5 enrichment values were selected for further study. 252

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metabolic process, glutamate catabolic process to aspartate, glutamate catabolic process to 2-oxoglutarate, arginine biosynthetic process via ornithine, and protein oxidation (Fig. 2C, all biological process terms were shown in Supplemental Table 4). Similarly, biological processes related to nutrient metabolism were significantly clustered for 21-day group (Fig. 2D, all biological process terms were shown in Supplemental Table 4). Therefore, the present study mainly centered on hepatic metabolism. Furthermore, based on the results of KEGG pathway analysis (all KEGG terms were shown in Supplemental Table 5), we primarily classified the varying proteins into carbohydrate metabolism (Table 1), fatty acid metabolism (Table 2), amino acid metabolism (Table 3), oxidative phosphorylation (OXPHOS) and antioxidant metabolism (Table 4), and exogenous compound metabolism (Table 5).

2.6. Western blot and biochemical tests Protein samples of 3 rats from control and 3-day groups were used in western blot to validate the results obtained from proteomic tests. Proteins were separated using a 12% SDS-PAGE and transferred onto a 0.22-μm PVDF membrane (Millipore, Burlington, USA). After blocked in 5% skim milk (BD, Franklin Lakes, USA) for 2 h at room temperature, the membrane was incubated with primary antibodies (rabbit antiIDHP monoclonal antibody, rabbit anti-COX5A monoclonal antibody and rabbit anti-PARK7 monoclonal antibody were from Abcam, Cambridge, UK; mouse anti-GAPDH monoclonal antibody was from Beyotime Biotechnology, Shanghai, China) at 4 °C overnight. The membrane was washed 4 times in TBST buffer and then incubated with respective secondary antibodies labeled with HRP (ZSGB-Bio, Beijing, China) at room temperature for 2 h. The membrane was washed in TBST buffer 4 times again and developed using ECL reagents (Millipore, Burlington, USA). GAPDH was used as the loading control. Hepatic concentration of glucose, non-ester fatty acids (NEFAs) and total amino acids (TAAs) was tested using assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the supplier's protocol. Levels of oxidative stress markers hydrogen peroxide (H2O2) and malondialdehyde (MDA); endogenous antioxidants superoxide dismutase (SOD) and glutathione (GSH) in rat liver were detected using biochemical reagent kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the supplier's instructions.

3.1. Carbohydrate metabolism For glycolysis, ATP-dependent 6-phosphofructokinase liver type (PFKAL), phosphoglycerate mutase 1 (PGAM1), pyruvate kinase PKM (KPYM) and dihydrolipoyllysine-residue acetyltransferase (ODP2) were increased in liver after 3-day TS (Table 1). Most of these enzymes returned to normal level after 21-day TS except PGAM1, which was still at higher abundance (Table 1). Similar variations were observed in TCA cycle enzymes. Citrate synthase (CISY), isocitrate dehydrogenase (NADP) (IDHP, the higher amount was validated by western blot as shown in Fig. 3), 2-oxoglutarate dehydrogenase (ODO1), succinate dehydrogenase iron-sulfur subunit (SDHB) and malate dehydrogenase (MDHM) had their abundance increased by 3-day TS, whereas after 21day TS, only CISY and IDHP were up-regulated (Table 1). Investigations concerning carbohydrate metabolism during spaceflight have suggested that glycogen level in rat liver was elevated while that in mouse liver was reduced [5,8,9,30]. However, to our knowledge, no assessment has been performed on hepatic enzymes of glycolysis or TCA cycle in microgravity or SM. Although no proteins involved in glycogen metabolism were identified as altered ones by our data, the significant increase of PFKAL, PGAM1 and KPYM by 3-day TS might suggest an enhanced glycolysis since PFKAL and KPYM catalyze the rate-limiting reactions [31]. Accordingly, 5 enzymes in total of TCA cycle showed an increased abundance, which might be an adaption to the enhanced glycolysis. As shown in Fig. 4A, the test of hepatic glucose showed that liver of 3-day (39.91 ± 2.51 μmol/gprot, P < 0.01) and 21-day group (43.10 ± 0.99 μmol/gprot, P < 0.01) had much less glucose when compared to control group (173.28 ± 6.80 μmol/gprot). The depletion of hepatic glucose after 3-day TS might be associated with the up-regulation of 9 enzymes (PFKAL, PGAM1, KPYM, ODP2, CISY, IDHP, ODO1, SDHB and MDHM), while after 21-day TS with the up-regulation of 3 enzymes (PGAM1, CISY and IDHP). Meanwhile, with the restoration of 6 enzymes (PFKAL, KPYM, ODP2, ODO1, SDHB and MDHM), hepatic glucose of 21-day group showed a slight (but not significant) increase relative to 3-day group. Our results were consistent with those in Costantini's study, in which highly differentiated hepatocytes HepG2 were shown to consume much more glucose when cultured in SM [32]. Yet, the results about carbohydrate metabolism obtained in the present study should be cautiously extrapolated to real microgravity, because at least tests of hepatic glycogen level using spaceflight and TS rats have showed contradictory results [9].

2.7. Statistical analysis Statistical analysis for western blot and biochemical tests was performed using SPSS 20.0 software (IBM, Armonk, USA). Data was expressed as mean ± SEM. Difference between groups was determined by Independent-Samples t-test (western blot) or one-way ANOVA (biochemical tests). P-values less than 0.05 were considered statistically significant. 3. Results and discussion Using the LC-MS/MS method, 1220 proteins in total were identified (FDR < 0.01, Supplemental Table 1) to profile rat liver proteome. Subsequently, label-free quantitative strategy was employed to reveal the differentially abundant proteins. Volcano plots illustrated the Pvalues (transferred to -lgP) and fold changes (transferred to log2(fold change)) of all identified proteins in the 2 TS groups, as compared with control group (Fig. 1A and B). Venn diagram was used to show common and specific altered proteins between TS groups (Fig. 1C). The filter condition (P-value < 0.05, fold change > 1.5 or < 0.5) led to 213 (198 proteins were up-regulated while 15 proteins were down-regulated) and 94 (63 proteins were up-regulated while 31 proteins were down-regulated) varying proteins for 3 and 21-day group, respectively (Supplemental Table 2). Among them, 44 proteins were identified as the common ones. The varying proteins were then analyzed using DAVID software for molecular function and biological process annotation. Since liver is a key metabolic organ [29], it was unsurprising that the top 5 most affected molecular functions for 3-day group were all related to metabolism (L-aspartate: 2-oxoglutarate aminotransferase activity, L-phenylalanine: 2-oxoglutarate aminotransferase activity, N-acyltransferase activity, long-chain-acyl-CoA dehydrogenase activity, and peroxiredoxin activity, Fig. 2A, all molecular function terms were shown in Supplemental Table 3). For 21-day group, although protein processing and degradation, such as proteasome-activating ATPase activity and dolichyl-diphosphooligosaccharide-protein glycotransferase activity became significant, metabolic functions also appeared to be most clustered ones (Fig. 2B, all molecular function terms were shown in Supplemental Table 3). In biological process annotation, altered proteins of 3-day group were remarkably enriched in argininosuccinate

3.2. Fatty acid metabolism Regarding fatty acid degradation, although peroxisomal carnitine O-octanoyltranserase (OCTC) and long-chain fatty acid-CoA ligase 5 (ACSL5) were decreased by 3 and 21-day TS, respectively, a series of enzymes involved in the activation, transport across the mitochondrial membrane and β-oxidation of fatty acids were increased by either 3 or 21-day TS (Table 2). Furthermore, on the other hand, the content of acetyl-CoA carboxylase 1 (ACACA), the key enzyme initiating the 253

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Fig. 1. Differentially abundant proteins of SM rat liver. (A) and (B), volcano plots depicted all identified proteins of 3 and 21-day SM rat liver, respectively. The red lines showed differentially regulated ones (P < 0.05 and fold change was > 1.5 or < 0.5 for up or down-regulation, respectively). (C) Venn diagram indicated common and specific altered proteins between groups. SM, simulated microgravity. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

mentioned outcomes of fatty acid metabolism might be compensatory effects to avoid lipotoxicity [34].

biosynthesis of long-chain fatty acids from acetyl-CoA showed significant decrease at both time points (Table 2). Regulation of fatty acid metabolism is of great importance for energy homoeostasis in organs such as liver, heart and skeletal muscles [33]. Our findings that acyl-CoA synthetase (ACSL5, ACSM3 and ACSM5), gamma-butyrobetaine dioxygenase (BODG) and multiple coworking enzymes of β-oxidation were up-regulated were consistent with several previous studies [8,30,34]. The variations of enzyme content might suggest that TS obviously promote the oxygenolysis of fatty acids and simultaneously suppressed the synthesis of them as well. This hypothesis could be partially verified by hepatic concentration of NEFAs (Fig. 4B), which sharply decreased in both 3-day (103.93 ± 1.81 μmol/gprot, P < 0.01) and 21-day TS rat liver (147.16 ± 12.16 μmol/gprot, P < 0.01), as compared with control group (273.66 ± 20.01 μmol/gprot). Moreover, with some enzymes returning to normal level (for instance, ACSM3, ACSM5, ACADV, ACADS and THIL), NEFAs in 21-day TS rat liver showed a significant increase relative to 3-day group (P < 0.05). Since lipid droplets were observed to be accumulated in mouse liver after spaceflight, the up-

3.3. Amino acid metabolism Amino acid metabolism is a much complicated metabolic pathway, and many proteins participate in the process. In 3-day group, 15 enzymes were up-regulated; and in 21-day group, 11 enzymes were upregulated (Table 3). In addition, several enzymes participating in the urea cycle were increased (Table 3). The most dramatic alterations were the up-regulation of aspartate aminotransferase (also known as glutamic-oxaloacetic transaminase) and especially alanine aminotransferase 1 (also known as glutamic-pyruvic transaminase). It is indubitable that muscle atrophy occurs during both spaceflight and TS treatment, which generates abundant free amino acids [35]. Therefore, as the executor of the deamination step of amino acid metabolism, aminotransferases increased accordingly. Free amino acids can act as raw materials of hepatic gluconeogenesis. Indeed, the rate of gluconeogenesis in TS rat liver has been suggested to be augmented

Fig. 2. GO annotation of altered proteins. (A) and (B) were molecular function enrichment results of 3 and 21-day group, respectively. (C) and (D) were biological process enrichment results of 3 and 21-day group, respectively. Top 5 terms were shown. 254

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Table 1 Varying proteins involved in carbohydrate metabolism. Uniprot Acc. No.

Glycolysis P30835 P25113 P11980 P08461 TCA cycle Q8VHF5 P56574 Q5XI78 P21913 P04636

Protein name

3-day group

21-day group

Fold change

P-value

Fold change

ATP-dependent 6-phosphofructokinase, liver type (PFKAL) Phosphoglycerate mutase 1 (PGAM1) Pyruvate kinase PKM (KPYM) Dihydrolipoyllysine-residue acetyltransferase (ODP2)

2.1587 2.4587 6.5512 1.6313

0.0111 0.0082 0.0310 0.0113

Not significant 2.2028 Not significant Not significant

Citrate synthase, mitochondrial (CISY) Isocitrate dehydrogenase [NADP], mitochondrial (IDHP) 2-oxoglutarate dehydrogenase, mitochondrial (ODO1) Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial (SDHB) Malate dehydrogenase, mitochondrial (MDHM)

2.7222 1.9772 1.6500 1.9425 1.6104

0.0091 0.0089 0.0038 0.0190 0.0273

2.2695 1.5076 Not significant Not significant Not significant

P-value

0.0159

0.0331 0.0330

Table 2 Varying proteins involved in lipid metabolism. Uniprot Acc. No.

Protein name

3-day group Fold change

Fatty acid degradation O88813 Long-chain fatty acid-CoA ligase 5 (ACSL5) Q6SKG1 Acyl-coenzyme A synthetase ACSM3, mitochondrial (ACSM3) Q6AYT9 Acyl-coenzyme A synthetase ACSM5, mitochondrial (ACSM5) Q9QZU7 Gamma-butyrobetaine dioxygenase (BODG) P11466 Peroxisomal carnitine O-octanoyltransferase (OCTC) P45953 Very long-chain specific acyl-CoA dehydrogenase, mitochondrial (ACADV) P15650 Long-chain specific acyl-CoA dehydrogenase, mitochondrial (ACADL) P14604 Enoyl-CoA hydratase, mitochondrial (ECHM) P15651 Short-chain specific acyl-CoA dehydrogenase, mitochondrial (ACADS) P17764 Acetyl-CoA acetyltransferase, mitochondrial (THIL) P13437 3-ketoacyl-CoA thiolase, mitochondrial (THIM) P21775 3-ketoacyl-CoA thiolase A, peroxisomal (THIKA) P23965 Enoyl-CoA delta isomerase 1, mitochondrial (ECI1) Q62651 Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, mitochondrial (ECH1) Q64591 2,4-dienoyl-CoA reductase, mitochondrial (DECR) Q920F5 Malonyl-CoA decarboxylase, mitochondrial (DCMC) Fatty acid biosynthesis P11497 Acetyl-CoA carboxylase 1 (ACACA)

Not significant 6.3537 1.9237 2.6802 0.2099 1.5122 2.4126 1.8084 2.5287 1.5471 Not significant Not significant 1.6831 1.8438 2.0111 2.1363 0.4507

21-day group P-value

Fold change

P-value

0.0004

0.0010 0.0113 0.0163 0.0131

0.4673 Not significant Not significant 2.6999 1.8898 Not significant 1.7179 1.5541 Not significant Not significant 1.8560 1.6513 Not significant Not significant Not significant Not significant

0.0026

0.3011

0.0011

0.0045 0.0164 0.0482 0.0020 0.0033 0.0071 0.0004 0.0053 0.0412

0.0478 0.0055 0.0345 0.0473

0.0059 0.0236

lower amount was validated by western blot as shown in Fig. 3) and cytochrome c oxidase subunit 5B (COX5B) decreased in either 3 or 21day group, while cytochrome c oxidase subunit 4 isoform 1 (COX41) and cytochrome c oxidase subunit 2 (COX2) was at sharply higher level in 3-day group (Table 4). Regarding the disruption of OXPHOS proteins, we proposed the possibility of oxidative stress in 3-day TS rat liver (Fig. 5): through the enhanced glycolysis, β-oxidation and TCA cycle, large amount of NADH were imported into electron transport chain. Accordingly, as the contributor of catalytic core in Complex I [37], NDUS2 was up-regulated, which might stimulate the activity of ubiquinone reductase and favour the excessive production of reduced ubiquinone [38]. Meanwhile, the increase of SDHB might also benefit the process of ubiquinone reduction [39]. In contrast, COX5B, the essential element to the stability of whole cytochrome c oxidase [40], was sharply down-regulated. Additionally, COX41 inhibits the activity of cytochrome c oxidase through its binding with ATP [41,42] while COX5A reverses this negative effect [43]. COX41 and COX5A were obviously up and down-regulated in 3day TS rat liver, respectively. Thus, the transport of electrons might be decelerated in Complex IV. The overproduction of reduced ubiquinone and the decelerated transport might force electrons back into Complex I to generate reactive oxygen species (ROS) [38–40,44]. To verify this hypothesis, hepatic levels of H2O2 and MDA were tested. Results indicated that 3-day TS resulted in the explosion of H2O2 (compared with 1.61 ± 0.24 mmol/gprot of control group, P < 0.05, Fig. 6A) and thus led to the obvious generation of MDA (compared with

[36]. However, our data showed no obvious up-regulation of gluconeogenic enzymes, but several enzymes for amino acid oxidation, such as isovaleryl-CoA dehydrogenase, mitochondrial (IVD), methylcrotonoyl-CoA carboxylase subunit alpha, mitochondrial (MCCA) and hydroxymethylglutaryl-CoA lyase, mitochondrial (HMGCL) were at higher level. Hence, were free amino acids driven into gluconeogenesis or oxidation remained an open question. In any case, urea cycle was enhanced for the excretion of ammonia. Fig. 4C illustrated that significant difference (P < 0.05) of TAAs content only existed between control (0.54 ± 0.05 μmol/mgprot) and 3-day group (0.32 ± 0.05 μmol/ mgprot). The obvious decrease of amino acids was also reported in Costantini's work with HepG2 cells in SM [32]. The recovery of TAAs level after 21-day TS might result from the aggravated atrophy of muscles [36]. 3.4. OXPHOS and antioxidant metabolism In relation to OXPHOS, the content of NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 5 (NDUA5), NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 9 (NDUA9) and NADH dehydrogenase (ubiquinone) iron-sulfur protein 2 (NDUS2) in Complex I was increased by 3-day TS (Table 4). SDHB in Complex II was also up-regulated by 3-day TS as mentioned above (Table 4). Additionally, ATP synthase subunit beta (ATPB) showed greater abundance in 3-day group (Table 4). However, for Complex IV (cytochrome c oxidase), levels of cytochrome c oxidase subunit 5A (COX5A, the 255

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Table 3 Varying proteins involved in amino acid metabolism. Uniprot Acc. No.

Protein name

3-day group

Amino acid metabolism D-amino-acid oxidase (OXDA) O35078 P28492 Glutaminase liver isoform, mitochondrial (GLSL) P13221 Aspartate aminotransferase, cytoplasmic (AATC) P00507 Aspartate aminotransferase, mitochondrial (AATM) Q64565 Alanine-glyoxylate aminotransferase 2, mitochondrial (AGT2) P50554 4-aminobutyrate aminotransferase, mitochondrial (GABT) P25409 Alanine aminotransferase 1 (ALAT1) P09367 L-serine dehydratase/L-threonine deaminase (SDHL) P97519 Hydroxymethylglutaryl-CoA lyase, mitochondrial (HMGCL) Q5I0C3 Methylcrotonoyl-CoA carboxylase subunit alpha, mitochondrial (MCCA) P15651 Short-chain specific acyl-CoA dehydrogenase, mitochondrial (ACADS) O70351 3-hydroxyacyl-CoA dehydrogenase type-2 (HCD2) P14604 Enoyl-CoA hydratase, mitochondrial (ECHM) P17764 Acetyl-CoA acetyltransferase, mitochondrial (THIL) P12007 Isovaleryl-CoA dehydrogenase, mitochondrial (IVD) P13437 3-ketoacyl-CoA thiolase, mitochondrial (THIM) P51650 Succinate-semialdehyde dehydrogenase, mitochondrial (SSDH) P50554 4-aminobutyrate aminotransferase, mitochondrial (GABT) Q64602 Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial (AADAT) Q5XI78 2-oxoglutarate dehydrogenase, mitochondrial (ODO1) Q6AYQ8 Acylpyruvase FAHD1, mitochondrial (FAHD1) Urea cycle B0BNN3 Carbonic anhydrase 1 (CAH1) P27139 Carbonic anhydrase 2 (CAH2) P14141 Carbonic anhydrase 3 (CAH3) P07756 Carbamoyl-phosphate synthase [ammonia], mitochondrial (CPSM) P09034 Argininosuccinate synthase (ASSY) P20673 Argininosuccinate lyase (ARLY) P00481 Ornithine carbamoyltransferase, mitochondrial (OTC)

21-day group

Fold change

P-value

Fold change

6.4274 Not significant 1.9775 1.5652 Not significant Not significant Not significant 2.5849 2.8921 2.7887 2.5287 2.0495 1.8084 1.5471 1.6607 Not significant 2.6413 Not significant 1.5330 1.6500 2.0415

0.0044

Not significant 1.6688 1.6600 Not significant 1.7654 1.5710 12.6695 3.2086 Not significant Not significant Not significant 1.5726 1.5541 Not significant Not significant 1.8560 Not significant 1.5710 Not significant Not significant 1.8275

3.1593 2.1886 1.6695 Not significant 2.4522 2.1054 1.6299

0.0172 0.0291

0.0043 0.0325 0.0393 0.0053 0.0022 0.0004 0.0412 0.0489 0.0445 0.0402 0.0038 0.0120 0.0080 0.0070 0.0092 0.0019 0.0193 0.0248

1.6437 Not significant 1.7854 1.8824 2.3495 1.9699 Not significant

P-value

0.0214 0.0280 0.0160 0.0394 0.0010 0.0003

0.0257 0.0473

0.0059 0.0394

0.0106 0.0191 0.0089 0.0361 0.0022 0.0222

Table 4 Varying proteins involved in oxidative phosphorylation and antioxidant metabolism. Uniprot Acc. No.

Protein name

3-day group

Oxidative phosphorylation Q63362 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 5 (NDUA5) Q5BK63 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 (NDUA9) Q641Y2 NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 (NDUS2) P21913 Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial (SDHB) P00406 Cytochrome c oxidase subunit 2 (COX2) P10888 Cytochrome c oxidase subunit 4 isoform 1 (COX41) P11240 Cytochrome c oxidase subunit 5A (COX5A) P12075 Cytochrome c oxidase subunit 5B (COX5B) P10719 ATP synthase subunit beta (ATPB) Antioxidant metabolism P07895 Superoxide dismutase [Mn], mitochondrial (SODM) O88767 Protein deglycase DJ-1 (PARK7) P46413 Glutathione synthetase (GSHB) P04041 Glutathione peroxidase 1 (GPX1)

21-day group

Fold change

P-value

Fold change

3.1674 14.0084 13.4542 1.9425 45.1375 2.0394 0.3970 0.4888 1.5669

0.0440 0.0255 0.0048 0.0190 0.0134 0.0044 0.0365 0.0057 0.0127

Not significant Not significant Not significant Not significant Not significant Not significant 0.2210 Not significant Not significant

2.2766 20.2319 2.5889 1.5985

0.0009 0.0114 0.0468 0.0060

Not Not Not Not

P-value

0.0419

significant significant significant significant

several liver diseases, such as chronic viral hepatitis, alcoholic liver diseases and non-alcoholic steatohepatitis [52,53]. Moreover, it has been suggested that oxidative stress not only damages biomacromolecules, but also modulates essential biological functions in liver. For instance, Kupffer cells, hepatic stellate cells and endothelial cells can be activated by ROS. When activated, Kupffer cells secrete cytokines like TNF-α increasing inflammation and apoptosis [51]; hepatic stellate cells start to proliferate and synthesize collagen (the deposition of collagen causes fibrosis) [54]. The diabetogenic phenotype of astronauts and inflammatory cell infiltration/steatosis of mouse liver after spaceflight were considered to be associated with ROS-induced injury [55–57]. Hence, to explore the inducements of oxidative stress in liver exposed to SM could be of great importance. In addition, it has been reported that spaceflight stimulated

0.80 ± 0.08 nmol/mgprot of control group, P < 0.01, Fig. 6B). Interestingly, MDA concentration returned to physiological level at 21 day (compared with 3-day group, P < 0.05, Fig. 6B). It has been suggested that oxidative stress occurs in various extrahepatic tissue when humans or animals are exposed to microgravity [45–48]. However, investigations directly regarding the oxidative damage of liver during spaceflight are extremely scarce. To date, only Hollander has reported the accumulation of MDA in the liver of rats flown aboard Space Shuttle STS-63 for 8 days [49], but the underlying mechanism is largely unknown. Our data might present an explanation for the occurrence of hepatic oxidative stress. Liver is a major target of ROS attack [50]. Many exogenous factors are responsible for the induction of ROS in liver, such as alcohol, drugs, toxins, virus and UV light [51]. Oxidative stress is believed to be a significant factor of 256

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Table 5 Varying proteins involved in exogenous compound metabolism. Uniprot Acc. No.

Protein name

3-day group

Cytochrome P450 family P04799 Cytochrome P450 1A2 (CYP1A2) P10633 Cytochrome P450 2D1 (CYP2D1) P12938 Cytochrome P450 2D3 (CYP2D3) P12939 Cytochrome P450 2D10 (CYP2D10) P05182 Cytochrome P450 2E1 (CYP2E1) P05183 Cytochrome P450 3A2 (CYP3A2) P08516 Cytochrome P450 4A10 (CYP4A10) UDP-glucuronosyltransferase family Q64550 UDP-glucuronosyltransferase 1-1 (UD11) P08430 UDP-glucuronosyltransferase 1–6 (UD16) P08541 UDP-glucuronosyltransferase 2B2 (UD2B2) Other enzymes Q9Z339 Glutathione S-transferase omega-1 (GSTO1) Q01579 Glutathione S-transferase theta-1 (GSTT1) P30713 Glutathione S-transferase theta-2 (GSTT2) Q66HF8 Aldehyde dehydrogenase X, mitochondrial (AL1B1) P08011 Microsomal glutathione S-transferase 1 (MGST1) P22734 Catechol O-methyltransferase (COMT) P21396 Amine oxidase [flavin-containing] A (AOFA) D3ZW55 Inosine triphosphate pyrophosphatase (ITPA) Q9Z0T0 Thiopurine S-methyltransferase (TPMT) P27605 Hypoxanthine-guanine phosphoribosyltransferase (HPRT) P10959 Carboxylesterase 1C (EST1C) Q03248 Beta-ureidopropionase (BUP1) P17988 Sulfotransferase 1A1 (ST1A1)

21-day group

Fold change

P-value

Fold change

P-value

Not significant 1.5182 1.6527 1.7238 3.9382 2.1112 15.1711

0.3439 Not significant Not significant Not significant 1.8177 Not significant Not significant

0.0434

0.0113 0.0219 0.0020 0.0011 0.0074 0.0210

0.0139 0.0069

0.0313

0.4638 0.1342 Not significant

Not significant Not significant 0.0086 1.5516 2.3922 1.6559 2.4228 Not significant 1.6935 Not significant 4.7892 1.5846 1.5755 0.4699 Not significant Not significant

0.0226 0.0245 0.0007 0.0306 0.0051 0.0052 0.0169 0.0275 0.0139

Not significant Not significant Not significant Not significant 0.4898 Not significant 0.4158 2.8722 Not significant Not significant 0.4604 1.9609 1.5161

0.0461

0.0083 0.0292 0.0334

0.0102 0.0476 0.0282

Fig. 3. Western blot analysis. (A) was representative western blot bands of IDHP, COX5A, PARK7 and the loading control GAPDH. (B) was the gray intensity results of target proteins. *P < 0.05, **P < 0.01, compared with control group.

Fig. 4. Results of hepatic glucose, NEFAs and TAAs test. (A) and (B), levels of hepatic glucose and NEFAs sharply decreased in both 3 and 21-day group, as compared with control group. (C), levels of hepatic TAAs only showed obvious decrease in 3-day group, as compared with control group. *P < 0.05, **P < 0.01. NEFAs, non-ester fatty acids; TAAs, total amino acids; NS, not significant.

(99.23 ± 2.85 U/mgprot) was much higher than that of control group (81.90 ± 4.49 U/mgprot, P < 0.05). Regarding GSH, the 2 TS groups both held obviously larger amount relative to control group (P < 0.05 or 0.01, Fig. 6D). In addition, the content of another ROS-sensitive protein, protein deglycase DJ-1 (PARK7) [24], was sharply increased by 3-day TS (the enhanced expression was validated by western blot as shown in Fig. 3). PARK7 belongs to the DJ-1/Thi/PfpI protein superfamily and possesses the capacity to scavenge H2O2 through the formation of sulfinic acid of its Cys-106 [62]. These antioxidants were

antioxidant activities of liver [58–60]. The current proteomic data also revealed that some proteins involved in the metabolism of antioxidants were significantly up-regulated by 3-day TS (Table 4). The increased superoxide dismutase [Mn], mitochondrial (SODM) might eliminate superoxide right after its generation [61]. Meanwhile, glutathione synthetase (GSHB) and glutathione peroxidase 1 (GPX1) both showed higher abundance, which might enhance the synthesis and function of GSH, respectively. To verify this assumption, SOD and GSH in rat liver were quantified. As shown in Fig. 6C, SOD level in 3-day TS rat liver

257

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Fig. 5. The disrupted OXPHOS in liver of 3-day group might induce the explosion of ROS. The overproduction of reduced ubiquinone and the decelerated transport of electrons might force electrons back into Complex I to generate ROS. The transport of electrons was illustrated by black arrows. The enhanced and inhibited processes were indicated by red star and green cross, respectively. OXPHOS, oxidative phosphorylation; ROS, reactive oxygen species. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 6. Results of hepatic oxidative stress markers and endogenous antioxidants test. (A) and (B), the high levels of H2O2 and MDA demonstrated that strong oxidative stress was induced in liver of 3-day group, which was attenuated in liver of 21-day group. (C) and (D), the synthesis of SOD and GSH was up-regulated compensatorily. *P < 0.05, **P < 0.01. H2O2, hydrogen peroxide; MDA, malondialdehyde; SOD, superoxide dismutase; GSH, glutathione; NS, not significant.

activities of CYP450s were suggested to decrease in rat liver by ‘COSMOS’ and SLS-2 studies [8,69]. Differential expression of specific CYP450 isoforms has also been investigated. For instance, Baba reported that CYP4A1 was induced in rat liver after short-term spaceflight [70]. Moskaleva used a targeted proteomic method to measure CYP450 isoforms in mouse liver and demonstrated that long-term spaceflight differentially regulated several CYP450s [71]. Our results indicated that the regulatory effects of SM on CYP450s depended obviously on durations, although they were not quite consistent with previous ones due to different duration or animal species. Furthermore, the altered content of UDP-glucuronosyltransferase (a superfamily essential to phase II drug metabolism and detoxification [72–74]) and several other nonCYP450 xenobiotic metabolic enzymes has barely been evaluated in microgravity or SM (to our knowledge, only Talbot has reported the decrease of phase II activity using PICM-19 cells [75]). Thus, the present study might give some new insight into drug metabolism and detoxification of toxins for manned spaceflight.

possibly stimulated by innate physiological cascades trying to avoid oxidative damage [63–65]. Coupled with the restoration of most enzymes, the up-regulated antioxidants normalized hepatic oxidative status after 21-day TS. Some negative results about hepatic antioxidants in microgravity also exist in the literature. Pecaut reported the decrease of GSH in mouse liver after 13-day spaceflight [30]; Hollander also suggested the suppression of catalase, SOD and GSH in rat liver after 8-day spaceflight [49]. However, Markin demonstrated that the inhibition of antioxidants could be due to reentry to gravity field, but not microgravity per se [59]. 3.5. Exogenous compound metabolism Some isoforms of hepatic cytochrome P450 (CYP450) were suggested as significant ones as listed in Table 5, most of which had their abundance increased by 3-day TS. The increase of CYP450s was attenuated by 21-day TS (CYP1A2 even decreased). Considering UDP-glucuronosyltransferases, 1-1 (UD11), 1–6 (UD16) and 2B2 (UD2B2) showed obviously lower amount after either 3 or 21-day TS (Table 5). Meanwhile, many other enzymes involved in xenobiotic metabolism exhibited dramatic regulation (Table 5). Biotransformation of foreign compounds to their hydrophilic forms is vital to elimination. CYP450 is a superfamily of heme-containing proteins that catalyze the metabolism of most xenobiotics including drugs, pesticides, environmental pollutants and toxins. Commonly used drugs for manned spaceflight are substrates of CYP450 [66–68]. Therefore, the variations of hepatic CYP450s have been taken into consideration since inchoate missions. The overall content and

4. Conclusions Collectively, based on the proteomic data, we proposed that shortterm SM enhanced the decomposition of nutrients in rat liver and meanwhile disrupted the OXPHOS pathway. Hence, electrons could not be fully transferred to dioxygen, which triggered the burst of ROS. On the other hand, antioxidative cascades were initiated compensatorily. Coupled with the restoration of most energy-metabolic enzymes, the up-regulated antioxidants normalized hepatic oxidative status after long-term SM. Meanwhile, several enzymes involved in xenobiotic 258

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metabolism, such as CYP450s and UDP-glucuronosyltransferases were also regulated by SM in a duration-dependent way. Our results might be helpful in understanding hepatic response to microgravity, especially giving further insight into the effects of microgravity on endogenous and exogenous compound metabolism. Future work could be carried out using samples from real spaceflight and adjustments of nutrients/ drugs might be taken into consideration for astronauts.

[18]

[19] [20]

[21]

Data statement The raw mass spectrometry data has been deposited to the ProteomeXchange Consortium via the iProX partner repository (dataset identifier PXD014494).

[22]

Funding

[23]

This work was supported by 1226 Major Project [grant number AWS16J018] and the National Natural Science Foundation of China [grant number 81973572 and 81573693].

[24]

[25]

Declaration of competing interest [26]

None. Appendix A. Supplementary data

[27]

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.actaastro.2020.02.007.

[28]

References

[29] [30]

[1] Y. Honda, S. Honda, M. Narici, N.J. Szewczyk, Spaceflight and ageing: reflecting on Caenorhabditis elegans in space, Gerontology 60 (2014) 138–142. [2] I. Ahlers, E. Misurova, M. Praslicka, R.A. Tigranyan, Biochemical changes in rats flown on board the Cosmos 690 biosatellite, Life Sci. Space Res. 14 (1976) 185–188. [3] R.A. Belitskaya, Content of carbohydrates and lipids in rat-liver tissue after 22-day space-flight, Kosm. Biol. Aviak. Med. 11 (1977) 75–76. [4] S. Abraham, C.Y. Lin, H.P. Klein, C. Volkmann, R.A. Tigranyan, E.G. Vetrova, Studies of specific hepatic enzymes involved in the conversion of carbohydrates to lipids in rats exposed to prolonged spaceflight aboard Cosmos 1129, Physiologist 23 (1980) S55–S58. [5] S. Nemeth, L. Macho, M. Palkovic, N. Skottova, R.A. Tigranyan, Metabolic changes in rats subjected to space flight for 18.5 days in the biosatellite Cosmos 936, Adv. Space Res. 1 (1981) 219–224. [6] S. Abraham, C.Y. Lin, C.M. Volkmann, H.P. Klein, Biochemical changes in rat liver after 18.5 days of spaceflight, Proc. Soc. Exp. Biol. Med. 172 (1983) 334–339. [7] A.H. Merrill Jr., E. Wang, D.P. Jones, J.L. Hargrove, Hepatic function in rats after spaceflight: effects on lipids, glycogen, and enzymes, Am. J. Physiol. 252 (1987) R222–R226. [8] A.H. Merrill Jr., M. Hoel, E. Wang, R.E. Mullins, J.L. Hargrove, D.P. Jones, I.A. Popova, Altered carbohydrate, lipid, and xenobiotic metabolism by liver from rats flown on Cosmos 1887, Faseb. J. 4 (1990) 95–100. [9] A.H. Merrill Jr., E. Wang, R. LaRocque, R.E. Mullins, E.T. Morgan, J.L. Hargrove, H.L. Bonkovsky, I.A. Popova, Differences in glycogen, lipids, and enzymes in livers from rats flown on COSMOS 2044, J. Appl. Physiol. 73 (1992) (1985) 142S–147S. [10] S.K. Chapes, A.M. Mastro, G. Sonnenfeld, W.D. Berry, Antiorthostatic suspension as a model for the effects of spaceflight on the immune system, J. Leukoc. Biol. 54 (1993) 227–235. [11] E.R. Morey-Holton, R.K. Globus, Hindlimb unloading of growing rats: a model for predicting skeletal changes during space flight, Bone 22 (1998) 83S–88S. [12] E.R. Morey-Holton, R.K. Globus, Hindlimb unloading rodent model: technical aspects, J Appl Physiol 92 (2002) (1985) 1367–1377. [13] M.E. Tischler, E.J. Henriksen, K.A. Munoz, C.S. Stump, C.R. Woodman, C.R. Kirby, Spaceflight on STS-48 and earth-based unweighting produce similar effects on skeletal muscle of young rats, J. Appl. Physiol. 74 (1993) (1985) 2161–2165. [14] L. Vico, V.E. Novikov, J.M. Very, C. Alexandre, Bone histomorphometric comparison of rat tibial metaphysis after 7-day tail suspension vs. 7-day spaceflight, Aviat Space Environ. Med. 62 (1991) 26–31. [15] Y. Chen, J. Xu, C. Yang, H. Zhang, F. Wu, J. Chen, K. Li, H. Wang, Y. Li, Z. Dai, Upregulation of miR-223 in the rat liver inhibits proliferation of hepatocytes under simulated microgravity, Exp. Mol. Med. 49 (2017) e348. [16] C.A. Rivera, M.H. Tcharmtchi, L. Mendoza, C.W. Smith, Endotoxemia and hepatic injury in a rodent model of hindlimb unloading, J. Appl. Physiol. 95 (2003) (1985) 1656–1663. [17] F. Du, Y. Ding, J. Zou, Z. Li, J. Tian, R. She, D. Wang, H. Wang, D. Lv, L. Chang, Morphology and molecular mechanisms of hepatic injury in rats under simulated

[31] [32]

[33] [34]

[35] [36]

[37]

[38]

[39] [40]

[41] [42] [43] [44]

[45] [46]

259

weightlessness and the protective effects of resistance training, PloS One 10 (2015) e0127047. E.S. Miller, G. Sonnenfeld, Influence of suspension on the expression of protective immunological memory to murine Listeria monocytogenes infection, J. Leukoc. Biol. 54 (1993) 578–583. E.S. Miller, G. Sonnenfeld, Influence of antiorthostatic suspension on resistance to murine Listeria monocytogenes infection, J. Leukoc. Biol. 55 (1994) 371–378. Y. Zhang, H. Wang, C. Lai, L. Wang, Y. Deng, Comparative proteomic analysis of human SH-SY5Y neuroblastoma cells under simulated microgravity, Astrobiology 13 (2013) 143–150. J. Iqbal, W. Li, M. Hasan, Y. Juan Li, K. Ullah, W. Yun, U. Awan, H. Qing, Y. Deng, Distortion of homeostatic signaling proteins by simulated microgravity in rat hypothalamus: A(16) O/(18) O-labeled comparative integrated proteomic approach, Proteomics 14 (2014) 262–273. J. Iqbal, W. Li, M. Hasan, K. Liu, U. Awan, Y. Saeed, Y. Zhang, A. Muhammad Khan, A. Shah, H. Qing, Y. Deng, Differential expression of specific cellular defense proteins in rat hypothalamus under simulated microgravity induced conditions: comparative proteomics, Proteomics 14 (2014) 1424–1433. Y. Wang, J. Iqbal, Y. Liu, R. Su, S. Lu, G. Peng, Y. Zhang, H. Qing, Y. Deng, Effects of simulated microgravity on the expression of presynaptic proteins distorting the GABA/glutamate equilibrium–A proteomics approach, Proteomics 15 (2015) 3883–3891. Y. Wang, I. Javed, Y. Liu, S. Lu, G. Peng, Y. Zhang, H. Qing, Y. Deng, Effect of prolonged simulated microgravity on metabolic proteins in rat Hippocampus: steps toward safe space travel, J. Proteome Res. 15 (2016) 29–37. S.V. Saveliev, C.C. Woodroofe, G. Sabat, C.M. Adams, D. Klaubert, K. Wood, M. Urh, Mass spectrometry compatible surfactant for optimized in-gel protein digestion, Anal. Chem. 85 (2013) 907–914. J. Cox, M.Y. Hein, C.A. Luber, I. Paron, N. Nagaraj, M. Mann, Accurate proteomewide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ, Mol. Cell. Proteomics 13 (2014) 2513–2526. X. Liu, P.J. Pai, W. Zhang, Y. Hu, X. Dong, P.Y. Qian, D. Chen, H. Lam, Proteomic response of methicillin-resistant S. aureus to a synergistic antibacterial drug combination: a novel erythromycin derivative and oxacillin, Sci. Rep. 6 (2016) 19841. J. Qiao, S.J. Cui, L.L. Xu, S.J. Chen, J. Yao, Y.H. Jiang, G. Peng, C.Y. Fang, P.Y. Yang, F. Liu, C. Filamin, A dysregulated protein in cancer revealed by label-free quantitative proteomic analyses of human gastric cancer cells, Oncotarget 6 (2015) 1171–1189. L. Rui, Energy metabolism in the liver, Comp. Physiol. 4 (2014) 177–197. M.J. Pecaut, X.W. Mao, D.L. Bellinger, K.R. Jonscher, L.S. Stodieck, V.L. Ferguson, T.A. Bateman, R.P. Mohney, D.S. Gridley, Is spaceflight-induced immune dysfunction linked to systemic changes in metabolism? PloS One 12 (2017) e0174174. H.S. Han, G. Kang, J.S. Kim, B.H. Choi, S.H. Koo, Regulation of glucose metabolism from a liver-centric perspective, Exp. Mol. Med. 48 (2016) e218. D. Costantini, D. Overi, L. Casadei, V. Cardinale, L. Nevi, G. Carpino, S. Di Matteo, S. Safarikia, M. Valerio, F. Melandro, M. Bizzarri, C. Manetti, P.B. Berloco, E. Gaudio, D. Alvaro, Simulated microgravity promotes the formation of tridimensional cultures and stimulates pluripotency and a glycolytic metabolism in human hepatic and biliary tree stem/progenitor cells, Sci. Rep. 9 (2019) 5559. S.M. Houten, R.J. Wanders, A general introduction to the biochemistry of mitochondrial fatty acid beta-oxidation, J. Inherit. Metab. Dis. 33 (2010) 469–477. K.R. Jonscher, A. Alfonso-Garcia, J.L. Suhalim, D.J. Orlicky, E.O. Potma, V.L. Ferguson, M.L. Bouxsein, T.A. Bateman, L.S. Stodieck, M. Levi, J.E. Friedman, D.S. Gridley, M.J. Pecaut, Spaceflight activates lipotoxic pathways in mouse liver, PloS One 11 (2016) e0152877. T.P. Stein, C.E. Wade, Metabolic consequences of muscle disuse atrophy, J. Nutr. 135 (2005) 1824S–1828S. I.R. Bederman, V. Chandramouli, Y. Sandlers, L. Henderson, M.E. Cabrera, Time course of hepatic gluconeogenesis during hindlimb suspension unloading, Exp. Physiol. 98 (2013) 278–289. M.C. Fernandez-Aguera, L. Gao, P. Gonzalez-Rodriguez, C.O. Pintado, I. AriasMayenco, P. Garcia-Flores, A. Garcia-Perganeda, A. Pascual, P. Ortega-Saenz, J. Lopez-Barneo, Oxygen sensing by arterial chemoreceptors depends on mitochondrial complex I signaling, Cell Metabol. 22 (2015) 825–837. B. Chance, G. Hollunger, The interaction of energy and electron transfer reactions in mitochondria. I. General properties and nature of the products of succinate-linked reduction of pyridine nucleotide, J. Biol. Chem. 236 (1961) 1534–1543. M.P. Murphy, How mitochondria produce reactive oxygen species, Biochem. J. 417 (2009) 1–13. D. Galati, S. Srinivasan, H. Raza, S.K. Prabu, M. Hardy, K. Chandran, M. Lopez, B. Kalyanaraman, N.G. Avadhani, Role of nuclear-encoded subunit Vb in the assembly and stability of cytochrome c oxidase complex: implications in mitochondrial dysfunction and ROS production, Biochem. J. 420 (2009) 439–449. S. Arnold, B. Kadenbach, The intramitochondrial ATP/ADP-ratio controls cytochrome c oxidase activity allosterically, FEBS Lett. 443 (1999) 105–108. J. Napiwotzki, K. Shinzawa-Itoh, S. Yoshikawa, B. Kadenbach, ATP and ADP bind to cytochrome c oxidase and regulate its activity, Biol. Chem. 378 (1997) 1013–1021. B. Kadenbach, M. Huttemann, The subunit composition and function of mammalian cytochrome c oxidase, Mitochondrion 24 (2015) 64–76. B.B. Seo, M. Marella, T. Yagi, A. Matsuno-Yagi, The single subunit NADH dehydrogenase reduces generation of reactive oxygen species from complex I, FEBS Lett. 580 (2006) 6105–6108. T.P. Stein, M.J. Leskiw, Oxidant damage during and after spaceflight, Am. J. Physiol. Endocrinol. Metab. 278 (2000) E375–E382. C. De Luca, I. Deeva, S. Mariani, G. Maiani, A. Stancato, L. Korkina, Monitoring antioxidant defenses and free radical production in space-flight, aviation and

Acta Astronautica 170 (2020) 251–260

B. Chen, et al.

[47]

[48]

[49]

[50]

[51]

[52] [53]

[54] [55]

[56] [57] [58]

[59]

[60]

[61] [62]

[63]

[64]

[65] F. Nogales, R.M. Rua, M.L. Ojeda, M.L. Murillo, O. Carreras, Oral or intraperitoneal binge drinking and oxidative balance in adolescent rats, Chem. Res. Toxicol. 27 (2014) 1926–1933. [66] L.M. Hesse, L.L. von Moltke, D.J. Greenblatt, Clinically important drug interactions with zopiclone, zolpidem and zaleplon, CNS Drugs 17 (2003) 513–532. [67] M. Litzenburger, F. Kern, Y. Khatri, R. Bernhardt, Conversions of tricyclic antidepressants and antipsychotics with selected P450s from Sorangium cellulosum So ce56, Drug Metab. Dispos. 43 (2015) 392–399. [68] H. Ikuta, A. Kawase, M. Iwaki, Stereoselective pharmacokinetics and chiral inversion of ibuprofen in adjuvant-induced arthritic rats, Drug Metab. Dispos. 45 (2017) 316–324. [69] S. Rabot, O. Szylit, L. Nugon-Baudon, J.C. Meslin, P. Vaissade, F. Popot, M. Viso, Variations in digestive physiology of rats after short duration flights aboard the US space shuttle, Dig. Dis. Sci. 45 (2000) 1687–1695. [70] T. Baba, M. Nishimura, Y. Kuwahara, N. Ueda, S. Naitoh, M. Kume, Y. Yamamoto, J. Fujita, Y. Funae, M. Fukumoto, Analysis of gene and protein expression of cytochrome P450 and stress-associated molecules in rat liver after spaceflight, Pathol. Int. 58 (2008) 589–595. [71] N. Moskaleva, A. Moysa, S. Novikova, O. Tikhonova, V. Zgoda, A. Archakov, Spaceflight effects on cytochrome P450 content in mouse liver, PloS One 10 (2015) e0142374. [72] N. Yang, R. Sun, X. Liao, J. Aa, G. Wang, UDP-glucuronosyltransferases (UGTs) and their related metabolic cross-talk with internal homeostasis: a systematic review of UGT isoforms for precision medicine, Pharmacol. Res. 121 (2017) 169–183. [73] A. Rowland, J.O. Miners, P.I. Mackenzie, The UDP-glucuronosyltransferases: their role in drug metabolism and detoxification, Int. J. Biochem. Cell Biol. 45 (2013) 1121–1132. [74] E.C.C. Mano, A.L. Scott, K.M. Honorio, UDP-glucuronosyltransferases: structure, function and drug design studies, Curr. Med. Chem. 25 (2018) 3247–3255. [75] N.C. Talbot, T.J. Caperna, L. Blomberg, P.G. Graninger, L.S. Stodieck, The effects of space flight and microgravity on the growth and differentiation of PICM-19 pig liver stem cells, In Vitro Cell. Dev. Biol. Anim. 46 (2010) 502–515.

railway engine operators, for the prevention and treatment of oxidative stress, immunological impairment, and pre-mature cell aging, Toxicol. Ind. Health 25 (2009) 259–267. S.M. Smith, J.E. Davis-Street, B.L. Rice, J.L. Nillen, P.L. Gillman, G. Block, Nutritional status assessment in semiclosed environments: ground-based and space flight studies in humans, J. Nutr. 131 (2001) 2053–2061. S.M. Smith, S.R. Zwart, G. Block, B.L. Rice, J.E. Davis-Street, The nutritional status of astronauts is altered after long-term space flight aboard the International Space Station, J. Nutr. 135 (2005) 437–443. J. Hollander, M. Gore, R. Fiebig, R. Mazzeo, S. Ohishi, H. Ohno, L.L. Ji, Spaceflight downregulates antioxidant defense systems in rat liver, Free Radic. Biol. Med. 24 (1998) 385–390. V. Sanchez-Valle, N.C. Chavez-Tapia, M. Uribe, N. Mendez-Sanchez, Role of oxidative stress and molecular changes in liver fibrosis: a review, Curr. Med. Chem. 19 (2012) 4850–4860. S. Li, H.Y. Tan, N. Wang, Z.J. Zhang, L. Lao, C.W. Wong, Y. Feng, The role of oxidative stress and antioxidants in liver diseases, Int. J. Mol. Sci. 16 (2015) 26087–26124. A.K. Singal, S.C. Jampana, S.A. Weinman, Antioxidants as therapeutic agents for liver disease, Liver Int. 31 (2011) 1432–1448. Y. Feng, N. Wang, X. Ye, H. Li, F. Cheung, T. Nagamatsu, Hepatoprotective effect and its possible mechanism of Coptidis rhizoma aqueous extract on carbon tetrachloride-induced chronic liver hepatotoxicity in rats, J. Ethnopharmacol. 138 (2011) 683–690. H. Cichoz-Lach, A. Michalak, Oxidative stress as a crucial factor in liver diseases, World J. Gastroenterol. 20 (2014) 8082–8091. M.S. Da Silva, P.M. Zimmerman, M.M. Meguid, J. Nandi, K. Ohinata, Y. Xu, C. Chen, T. Tada, A. Inui, Anorexia in space and possible etiologies: an overview, Nutrition 18 (2002) 805–813. B.W. Tobin, P.N. Uchakin, S.K. Leeper-Woodford, Insulin secretion and sensitivity in space flight: diabetogenic effects, Nutrition 18 (2002) 842–848. E.A. Blaber, M.J. Pecaut, K.R. Jonscher, Spaceflight activates autophagy programs and the proteasome in mouse liver, Int. J. Mol. Sci. 18 (2017). A.A. Markin, N.V. Delenian, [Lipid peroxidation and the system of antioxidant protection in rats following a 13-day space flight on the Kosmos-1887 biosatellite], Aviakosm. Ekolog. Med 26 (1992) 43–45. A.A. Markin, O.A. Zhuravleva, [Lipid peroxidation and antioxidant defense system in rats after a 14-day space flight in the "Space-2044" spacecraft], Aviakosm. Ekolog. Med 27 (1993) 47–50. F.P. Baqai, D.S. Gridley, J.M. Slater, X. Luo-Owen, L.S. Stodieck, V. Ferguson, S.K. Chapes, M.J. Pecaut, Effects of spaceflight on innate immune function and antioxidant gene expression, J. Appl. Physiol. 106 (2009) (1985) 1935–1942. J.F. Turrens, Mitochondrial formation of reactive oxygen species, J. Physiol. 552 (2003) 335–344. E. Andres-Mateos, C. Perier, L. Zhang, B. Blanchard-Fillion, T.M. Greco, B. Thomas, H.S. Ko, M. Sasaki, H. Ischiropoulos, S. Przedborski, T.M. Dawson, V.L. Dawson, DJ1 gene deletion reveals that DJ-1 is an atypical peroxiredoxin-like peroxidase, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 14807–14812. A.R. Kammer, J.I. Orczewska, K.M. O'Brien, Oxidative stress is transient and tissue specific during cold acclimation of threespine stickleback, J. Exp. Biol. 214 (2011) 1248–1256. D. Mukhopadhyay, A. Chattopadhyay, Induction of oxidative stress and related transcriptional effects of sodium fluoride in female zebrafish liver, Bull. Environ. Contam. Toxicol. 93 (2014) 64–70.

Bo Chen, male, Ph. D, majoring in space life sciences George Q Li, male, Ph. D, majoring in the quality control, chemistry and pharmacology of popular botanical medicines Yongzhi Li, female, researcher, majoring in medical research in space Jun-Lae Cho, male, Ph. D, majoring in hormonal physiology in cancer and immunology, pharmacology of medicinal plants, cardiovascular diseases and senescence model Jiaping Wang, female, Ph. D, majoring in space medicine Jianyi Gao, male, majoring in aerospace pharmacy Yulin Deng, male, Ph. D, majoring in space life sciences Yujuan Li, female, Ph. D, majoring in space life sciences

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