{"paper_id":"24ca2f5e-9f18-4cd6-aaba-c39ffc318b79","body_text":"The effect of training on the expression of protein and metabolites in plasma exosomes of Yili Horse | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 9 January 2025 V1 Latest version Share on The effect of training on the expression of protein and metabolites in plasma exosomes of Yili Horse Authors : Xinxin Yuan 0009-0007-3270-532X , Xinkui YAO , Yaqi ZENG , Jianwen Wang , Wanlu Ren , Tongliang Wang , Xueyan Li , Lipin Yang , Xixi Yang , and Jun Meng [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.173639314.49142001/v1 Published Animals Version of record Peer review timeline 157 views 94 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Objective: Multimodal omics approaches have propelled the study of mechanisms related to exercise, yet the effects of physical training on protein expression and metabolites in the plasma exosomes of Yili horses remain unclear. This study aims to elucidate the impact of exercise training on protein expression and metabolites of Yili horse plasma exosomes through a comprehensive omics analysis, providing reference indicators for the training and evaluation of athletic performance in Yili horses. Methods: Eight three-year-old Yili horses of similar age were selected for the study, with four untrained horses serving as the control group and four trained horses comprising the training group. All participating horses were drug-free, had no history of illness, and were in a healthy condition. After extracting and identifying the plasma exosomes, we conducted proteomics and metabolomics analyses to detect and analyze differences in exosomal proteins and metabolites. Results: There was no significant difference in the particle size of plasma exosomes between the two groups. However, significant changes were observed in the proteomics and metabolomics profiles of the training group. Notably, the primary cellular composition differences in were related to the cytoplasm and nucleus, with significant alterations in transcription and transcription regulation processes. Proteomic subcellular localization differences were mainly concentrated in the cytoplasm and nucleus, with enhanced cell signal transduction functions. Additionally, there was a significant reduction in carbohydrates and their metabolic products within the metabolites. Conclusion: Training significantly alters the expression, protein expression, and metabolite composition of Yili horse blood exosomes, with these changes primarily associated with enhanced metabolic capacity. Introduction: The horse industry holds significant economic value 1 , with horse racing being one of the sectors that demand high athletic performance from horses 2 . Enhancing athletic ability is beneficial for increasing the value of horses 3 . Currently, the assessment of a horse’s athletic capability is subjectively conducted through experience and professional observation 4 . Finding more objective evaluation standards is of positive significance for optimizing the assessment criteria for equine athletic performance. During exercise, a series of bioactive substances are released into the circulatory system 5 , including cell-secreted vesicles with biological activity 6 . Exosomes are one type of these vesicles, containing RNA and proteins 7 , and are present in bodily fluids such as plasma 8 , breast milk 9 , and cerebrospinal fluid 10 . They can promote corneal endothelial regeneration 11 , improve bone microstructure and the accumulation of bone marrow fat 12 , participate in the development of cardiovascular health and disease 13 , and serve as potential biomarkers for disease diagnosis 14, 15 and preclinical and clinical assessments 16 . There are abundant exosomes in plasma 17 , and it has been found that training has a significant impact on human plasma exosome levels 18 , however, the impact of training on the plasma exosomes of Yili horses is not yet clear. Exercise can significantly change the protein expression in male exosomes 19 , and studies on mice have shown that exercise can significantly affect the protein expression of plasma exosomes 20 . Research on horses has indicated that prolonged aerobic exercise induces significant changes in plasma protein modifications 21 . However, the impact of exercise on the protein expression of equine plasma exosomes is still unclear. The development of metabolomics has made the study of metabolic mechanisms popular in human exercise and has provided important insights for analyzing the mechanisms of athletic ability 22 . Various exosomes are hypothesized to transport exercise-related metabolites 23 and may be involved in exercise-induced adaptive intercellular communication 24 . Yet, the changes in metabolites in equine plasma exosomes due to exercise are not well understood. Multimodal omics analyses have advanced research related to exercise 25 , but the effects of exercise training on protein expression, and metabolomics of Yili horse plasma exosomes are still not clear. Through systematic training, the exercise ability of horses can be effectively improved 26 , which may be related to changes in plasma exosomes. We hypothesize that exercise training has a significant impact on the plasma exosomes of Yili horses, by analyzing the impact of training on plasma exosomes, provides a reference basis for further searching for plasma exosome factors that may improve the exercise ability of Yili horses, and provides objective indicators for analyzing the exercise ability of untrained Ili horses, is positively meaningful for improving the breeding work of Yili horses and selecting horses with different athletic abilities. In this study, we investigate the differences in protein expression, and metabolites in the plasma exosomes of Yili horses subjected to exercise training using proteomics and metabolomics approaches. Our goal is to explore the effects of exercise on protein expression and metabolites in Yili horse plasma exosomes, aiming to identify potential exosome-related biomarkers that could enhance the athletic performance of Yili horses and provide reference for the training and evaluation system of their athletic capabilities. Method: Experimental animals and grouping: In this study, eight adult male Yili horses, each three years old, were selected as research subjects. Four untrained horses served as the control group, while four horses that had completed a year of training formed the training group. Plasma exosomes were extracted after a year of uninterrupted training, with no breaks in their training regimen in the week preceding sampling. The trained horses began their training at two years old and were mature enough to be registered for the 2023 “China Horse Club Yueyang Tower Silk Road Cup” Yili Horse Speed Performance Test, covering a distance of 5000 meters. These horses were healthy, having successfully passed equine and anti-doping tests. The control group horses had not undergone any formal training and were kept under grazing conditions, without engaging in any exercise or physical labor beyond their daily activities in the recent week. All horses involved were medication-free, had no history of illness, and were in good health. Ethical statement: All procedures involving animal experiments were established according to Chinese animal welfare legislation, and all animal care and usage procedures adhered to the guidelines set by the Institutional Animal Care and Use Committee of [masked for review]. Blood collection: Blood samples were drawn from the horses’ jugular veins in a resting state before their daily exercise at 12:00 PM. Each horse contributed a 20 mL blood sample, collected using EDTA-K2 anticoagulant tubes, followed immediately by the extraction of exosomes. Exosome Extraction This study used ultracentrifugation to extract exosomes. In general, after separating horse plasma, it was stored in liquid nitrogen. Before extraction, the plasma was thawed at 37 ℃. It was then centrifuged at 2,000 × g for 30 min at 4 ℃, and the supernatant was transferred to a new centrifuge tube. It was then centrifuged at 10,000 × g for 45 min at 4 ℃ to remove larger vesicles. The supernatant was filtered through a 0.45 μm filter, and the filtrate was centrifuged at 100,000 × g for 70 min at 4 ℃. The supernatant was removed, and the pellet was resuspended in 10 ml of pre-cooled 1×PBS. It was then centrifuged again at 100,000 × g for 70 min at 4 ℃. The supernatant was removed, and the pellet was resuspended in 200μL of pre-cooled 1×PBS, and then stored at -80℃. Transmission Electron Microscopy Observation Observation was conducted using a Hitachi HT-7700 model projection electron microscope. 10 μL of exosomes were taken out, and 10 μL of the sample was drawn and dropped onto the copper mesh to precipitate for 1 minute, the supernatant was absorbed with filter paper, 10 μL of uranyl acetate was dropped onto the copper mesh to precipitate for 1 minute, the supernatant was absorbed with filter paper, and after drying at room temperature for a few minutes, electron microscope detection and imaging were carried out at 100 kv to obtain the results of transmission electron microscopy imaging(×20,000). Quantitative proteomics This study employs the diaPASEF acquisition mode of the timsTOF Pro2 series mass spectrometer for exosome differential quantitative proteomic analysis. The overall procedure involves adding a lysis buffer (8M urea) containing 1mM PMSF and 2mM EDTA (final concentration) to the sample, followed by incubation for 5 minutes and ultrasonic lysis for another 5 minutes. The lysate is then centrifuged at 4°C, 15,000g for 10 minutes, and the supernatant is collected. The total protein concentration is determined through BCA protein quantification analysis. Based on the protein concentration, an equal volume of protein solution is taken, and the volume is made up to 200ul with 8M urea. Then, it is reduced with 10 mM DTT at 37°C for 45 minutes, and alkylated with 50 mM iodoacetamide (IAM) in a dark room at room temperature for 15 minutes. Four times the volume of pre-cooled acetone is added to the protein solution, and it is precipitated at -20°C for 2 hours. After centrifugation, the protein precipitate is dried and resuspended in 200ul of 25mM bicarbonate solution and 3ul of trypsin (Promega), and digested overnight at 37°C. After digestion, the peptides of each sample are desalted on a C18 column, concentrated by vacuum centrifugation, and re-dissolved in 0.1% (v/v) formic acid for machine analysis. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) This study conducts a full-spectrum metabolome detection through liquid chromatography-tandem mass spectrometry. The overall procedure is as follows: Exosome samples are taken out from a -80°C freezer and thawed on ice (all subsequent operations are required to be performed on ice). Then, 500 uL of 80% methanol aqueous internal standard extractant is added, followed by vortexing for 3 minutes. The centrifuge tube is quickly frozen in liquid nitrogen for 5 minutes, then thawed on ice for 5 minutes, followed by another 5 minutes of thawing on ice and vortexing for 2 minutes. After repeating the freezing, thawing, and vortexing process three times, the sample is centrifuged at 12,000 r/min for 10 minutes at 4°C. The supernatant (450uL) is transferred to a new centrifuge tube and concentrated until completely dry. Then, it is re-dissolved in 100 uL of 70% methanol water, vortexed for 3 minutes, and sonicated in an ice-water bath for 10 minutes. After centrifugation at 12,000 r/min for 3 minutes at 4°C, the supernatant (80uL) is transferred to the corresponding sample vial for machine analysis. Bioinformatics analysis and statistical analysis Gene Ontology(GO) analysis using http://geneontology.org/ conduct analysis, Cluster of Orthologous Groups of proteins(COG) analysis using http://www.ncbi.nlm.nih.gov/COG conduct analysis, Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis using https://www.genome.jp/kegg/ conduct analysis. The data are presented as mean values with standard errors. The Student’s t-test was utilized to evaluate the differences between groups. A P-value of less than 0.05 was deemed statistically significant. Results : Extraction and Identification of Exosomes from Yili Horse Plasma Figure 1A presents the research pathway employed in this study. After isolating plasma exosomes from both groups of Yili horses, we utilized scanning electron microscopy (SEM) for identification purposes, as depicted in Figure 1B. Subsequent analysis of particle sizes indicated that there was no significant disparity in the exosome sizes between the two groups, as demonstrated in Figure 1C. Figure 1. Characteristics of exosomes isolated from Yili horse blood. (A) Roadmap for this study. (B) Representative images of blood-derived exosomes of (Up) control group and (Down) training group obtained by transmission electron microscopy. (C) Comparison of exosome particle size analysis results. Error bar = SEM, ns = not significant. Training significantly altered protein expression of plasma exosomes in Yili Horse In an effort to delve deeper into the effects of training on exosomal content, we employed proteomics to analyze the protein expression within the exosomes. Our findings demonstrated that training notably increased the expression of 484 proteins and decreased the expression of 234 proteins (Figure 2A). The majority of these proteins were predominantly localized in the cytoplasm and nucleus (Figure 2B). Further KOG analysis revealed that the principal functional alterations in these proteins pertained to signal transduction processes (Figure 2C). Further significance analysis was conducted on the differentially expressed proteins, shows the top 20 proteins with significant differences (Figure 3). Figure 2. Proteomics to analyze the protein expression within the exosomes. (A) Volcano plot of differential exosome proteins. The horizontal axis represents log 2 of the difference multiple, the vertical axis represents -log l0 P-value, red and green dots represent up-regulated and down-regulated differential proteins respectively. Gray dots represent proteins with no significant difference. (B) Comparison bar chart of subcellular localization results of up and down regulation, the horizontal axis represents the subcellular, the vertical axis represents the number of differentially expressed proteins annotated to this subcellular, red and blue colors represent up-regulated and down-regulated differentially expressed proteins respectively. (C) Bar chart of Cluster of Orthologous Groups of proteins, the horizontal axis represents KOG functional classification, the vertical axis represents the number of differentially expressed proteins annotated to the corresponding function, the legend on the right represents the description of functional classification. Figure 3. Perform significant difference analysis on the top 20 proteins with different expression levels. Statistical analysis was conducted using a double tailed t-test, p -value <0.05 was considered statistically significant. Training significantly altered metabolome of plasma exosomes in Yili Horse After preliminarily determining the impact of training on the metabolic pathways of exosomes, we used LC-MS/MS to detect and compare metabolites. The data showed that training significantly changed the composition of metabolites (Figure 4A), KEGG analysis showed a significant increase in metabolites related to the pentose phosphate pathway, nucleotide metabolism, and fructose and mannose metabolism in the training group (Figure 4B), and reduced carbohydrates and their metabolites, while increasing glycerol phospholipid and organic acids and their derivatives (Figure 5). Further significance analysis was conducted on the differentially metabolites, shows the top 20 proteins with significant differences (Figure 6). Figure 4. (A) OPLS-DA score plot, note: the horizontal axis represents the predictive principal component, the horizontal axis direction can see the difference between groups; the vertical axis represents the orthogonal principal component, the vertical axis direction can see the difference within the group; the percentage represents the explanation rate of this component to the data set. Each point in the figure represents a sample, samples from the same group are represented by the same color, group is the grouping. (B) KEGG enrichment plot of differential metabolites. The horizontal axis represents the Rich Factor corresponding to each pathway, the vertical axis is the pathway name (sorted by P-value), the color of the point reflects the size of P-value, the redder it is, the more significant the enrichment. The size of the point represents the number of differential metabolites enriched. Figure 5. Cluster heatmap of differential metabolites, horizontally for sample names, vertically for differential metabolite information, group is the grouping, different colors are filled with different values obtained after normalization of different relative contents (red represents high content, green represents low content). Among them, heatmap class: heatmap by substance classification, class is the primary classification of substances. Figure 6. Perform significant difference analysis on the top 20 metabolites with different expression levels. Statistical analysis was conducted using a double tailed t-test, p -value <0.05 was considered statistically significant. Discussion : Exercise is increasingly recognized as a non-pharmacological intervention for disease management 27 , and equine-assisted interventions have been shown to ameliorate certain health conditions 28 . Consequently, promoting equine sports has positive social implications, and objectively assessing the athletic capabilities of horses contributes positively to this field. This study seeks to identify potential biomarkers for evaluating the athletic performance of Yili horses by comparing protein expression and metabolite changes in plasma exosomes between trained and untrained horses, thereby analyzing the impact of training on these parameters. Several studies have linked the health benefits of exercise to exosomes, with exercise improving vascular formation in type 2 diabetes through exosomal mechanisms 29 . Our electron microscopy images indicate that while exercise training does not significantly alter the size of exosomes in Yili horses, it does significantly change their plasma exosomal protein expression, and metabolite composition. The development of proteomics and metabolomics technologies has simplified the detection of proteomes and metabolomes 30 . Proteomics and metabolomics analyses have identified metabolic abnormalities in equine follicles matured both in vitro and in vivo 31 , which can help optimize breeding strategies. It has been found that different types of exercise lead to significant differences in the proteomics and metabolomics profiles of athletes’ blood 32 . Our study reveals that exercise induces significant changes in the protein expression and metabolites of equine plasma exosomes, with the most numerous differences in proteins associated with the cytoplasm and nucleus, consistent with miRNA changes, suggesting that miRNAs regulate the expression of related proteins. There is a significant difference in the metabolic products of exosomes between control and trained groups, with a notable reduction in carbohydrates and their metabolites in the trained group, presumably due to the consumption of these substances during exercise training. The pentose phosphate pathway, nucleotide metabolism, and fructose and mannose metabolism are the metabolic pathways with the most significant differences, suggesting that training induces changes in cellular gene expression in Yili horses, leading to differences in protein expression and thus altering metabolic capacity, making these metabolism-related metabolites the most significantly altered. Although significance analysis showed no significant differences in protein expression, there were trend changes in the expression of multiple proteins. Among them, CYP2D50 with high expression trend may be related to horse drug metabolism 33 . Adhesion G protein coupled receptor L2 (ADGRL2) is associated with developmental delay 34 , and regulates cellular metabolism 35 , participates in immune regulation 36, 37 , and is a determinant of endothelial cell adhesion and barrier function 38 . The NPEPL1 gene is involved in tumor growth and metastasis 39 , and its mutations may cause cancer 40 , it can be used as a gene for renal cell carcinoma risk scoring and is involved in pigmentation 41 . PTER is an N-acetyltaurine hydrolase involved in the regulation of obesity 42 , which may be related to its involvement in energy balance regulation. The expression of Nedd41 has a focal ischemic protective effect 43 , which may be related to its neuroprotective effect during endoplasmic reticulum stress 44 . Pum2 can regulate cell cycle, cell migration, adhesion, and morphology 45 , promoting the growth of mature neurons 46 . Overexpression of NOP58 can promote the formation of cell colonies 47 . SLC2A9 can regulate fat deposition and skeletal muscle function 48 . The low expression trend of NHSL2 regulates cell morphology 49 and bone homeostasis 50 , and the genotype of the MSTN gene encoding myostatin can predict the genetic potential of exercise traits 51 . This study suggests that these upregulated proteins in plasma exosomes after training may be related to cell growth, stress, and metabolism. Unlike the insignificant trend in protein expression, there was a significant difference in the metabolic products of the two groups of extracellular vesicles, and the top 20 metabolites were all elevated in the training group. Among them, phosphatic acid has antiviral effects 52 , previous studies have found that training increases the concentration of plasma lysophosphatidylethanolamine 53 . The data from this study also shows this change in plasma exosomes. Furthermore, the increase in phosphatidylethanolamine levels may be related to the increase in lysophosphatidylethanolamine levels 54 , HexCr may be involved in mediating apoptosis, necrosis, and inflammation levels 55 , which may be one of the potential factors leading to patient fatigue, chronic pain, and cognitive difficulties 56 , speculated to be related to injury after exercise. Multiple factors can affect a horse’s athletic ability 57 , and genetic characteristics can predict this 51 , Systematic training can also alter protein expression, thereby affecting its athletic ability 58 , therefore, by analyzing the impact of training on horse plasma exosomes, it is possible to provide objective indicators for evaluating horse sports ability and provide reference for the training and breeding of Yili horses. Studies have shown that moderate exercise enhances the release and function of endothelial progenitor cell exosomes 59 , suggesting that training may also affect the overall level of plasma exosomes in Yili horses, warranting further investigation. Studies have indicated that exercise-induced changes in plasma proteins are related to gender 60 , and whether exercise-induced changes in exosomal proteins are also gender-related merits further study. Purebred and non-purebred Jeju horses exhibit different plasma metabolites after exercise 61 , and we believe it is necessary to further compare the plasma metabolites of purebred and crossbred Yili horses post-exercise to obtain more biological reference indicators. In conclusion, this study finds that training significantly alters protein expression and metabolomics of plasma exosomes in Yili horses, with these changes primarily concentrated in biological functions related to gene expression, signal transduction, and metabolism, providing new insights into the training and assessment of athletic abilities in Yili horses. References : 1. Chiaradia E, Miller I. In slow pace towards the proteome of equine body fluids. Journal of Proteomics . 2020;2252. Huybers S, Apostolaki M, van der Eerden BC, Kollias G, Naber TH, Bindels RJ, et al. Murine tnf(deltaare) crohn’s disease model displays diminished expression of intestinal ca2+ transporters. Inflamm Bowel Dis . 2008;14:803-8113. 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Authors Affiliations Xinxin Yuan 0009-0007-3270-532X Xinjiang Agricultural University College of Animal Science View all articles by this author Xinkui YAO Xinjiang Agricultural University College of Animal Science View all articles by this author Yaqi ZENG Xinjiang Agricultural University College of Animal Science View all articles by this author Jianwen Wang Xinjiang Agricultural University College of Animal Science View all articles by this author Wanlu Ren Xinjiang Agricultural University College of Animal Science View all articles by this author Tongliang Wang Xinjiang Agricultural University College of Animal Science View all articles by this author Xueyan Li Xinjiang Agricultural University College of Animal Science View all articles by this author Lipin Yang Xinjiang Agricultural University College of Animal Science View all articles by this author Xixi Yang Xinjiang Agricultural University College of Animal Science View all articles by this author Jun Meng [email protected] Xinjiang Agricultural University College of Animal Science View all articles by this author Metrics & Citations Metrics Article Usage 157 views 94 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Xinxin Yuan, Xinkui YAO, Yaqi ZENG, et al. 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