Rhodiola polysaccharides affect the antioxidant capacity and testosterone secretion of PLCs in hypoxia environment through the metabolism of unsaturated fatty acids and glutathione

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Rhodiola polysaccharide (RDP), a distinguished bioactive compound o f Rhodiola rosea L., demonstrates strong antioxidant activity. Whether the RDP have protective effect on hypoxia injury of porcine Leydig cells (PLCs) merits further investigation. Our research showed that when RDP was introduced to PLCs under hypoxia condition, both the antioxidant capacity and testosterone (T) secretion of PLCs were enhanced. Notably, this treatment revealed a significant correlation between T levels and specific metabolites, suggesting RDP's role in diminishing reactive oxygen species and fortifying antioxidant defenses. Moreover, RDP promoted the synthesis of antioxidant metabolites and modulated pathways involved in unsaturated fatty acids and glutathione metabolism, mitigating oxidative stress. These results suggested that RDP could improve the cellular antioxidant capacity and stimulate T secretion of PLCs in hypoxia environment through multiple pathways. Biological sciences/Cell biology/Glycobiology Biological sciences/Cell biology RDP PLCs testosterone antioxidant Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction As people’s quality of life has improved, higher requirements have been placed on the meat quality of porcine. Introduced species is an effective way to improve the production performance of local porcine. Oxygen is essential for all animals’ life, and hypoxia, a prominent feature of plateau environments, poses significant challenges to introduced species. When animals are relocated to plateaus, they must regulate their physiological functions to adapt to reduced oxygen levels. Hypoxia occurs when oxygen is inadequate to supply to tissue cells, which disrupts normal physiological functions [ 1 ] . Hypoxia usually causes stress reactions in clinical practice, affecting animal reproductive performance [ 2 ] . For example, hypoxia can impair sperm production, libido, testicular function, and reproductive capacity in livestock [ 3 ] . It is also associated with a reduction in the secretion of testosterone (T) [ 4 ] . As an important regulatory hormone for male reproductive function, T can affect libido, sexual organ development, and spermatogenesis [ 5 ] . Natural antioxidant such as natural polysaccharides plant origin popular due to its non-toxic nature. Rhodiola rosea L., a traditional anti-altitude stress Chinese medicinal material, contains the active substance Rhodiola Polysaccharides (RDP), which is known for the anti-hypoxia, anti-stress and hypoglycemic effects [ 6 ] . Previous studies have shown that adding RDP to the culture medium can significantly enhance the proliferation of PLCs [ 7 ] . This experiment aims to explore the effect of RDP on T secretion and antioxidant capacity of PLCs under hypoxia environment. And through the metabolomic study, we preliminarily revealed its effects and protective mechanism. This study provides scientific basis for RDP as a natural medicine that can relieve the stress of introduced animals. Results Effect of RDP on Antioxidant capacity of PLCs under hypoxia condition Table 1 illustrated notable differences in antioxidant markers among the groups studied. Specifically, compared to the normal (N) group, the hypoxia (H) group showed significantly lower levels of SOD and GSH-Px, and significantly higher levels of MDA ( P < 0.05). In contrast, the content of SOD and GSH-Px in the hypoxia + RDP (HR) group was increased, and the MDA content was decreased ( P < 0.05) compared with the H group, and the antioxidant indicators of HR returned nearly to the levels of the N group. Table 1 Effect of RDP on Antioxidant capacity of PLCs under hypoxia condition Group N H HR SOD (U/L) 69.4 ± 2.54 a 49.43 ± 5.45 c 59.42 ± 5.77 b MDA (nmoL/mL) 49.7 ± 0.32 b 55.82 ± 1.25 a 50.19 ± 0.28 b GSH-Px (U/mL) 676.31 ± 24 a 631.38 ± 11.34 b 670.87 ± 32.17 a Note: Different letters indicate significant difference ( P 0.05). MDA: malondialdehyde; GSH-Px: glutathione peroxidase; SOD: superoxide dismutase. N: normal group; H: hypoxia group; HR: hypoxia + RDP group. Effect of RDP on T s ecretion from PLCs under hypoxia condition Figure 1 showed that the T secretion level of the H group was significantly reduced compared to that in the N group. Conversely, in the HR group, T secretion was notably increased compared to the H group ( P < 0.05). Multivariate statistical analysis of metabolomics Figure 2 showed that all samples fell within the 95% confidence interval, with QC samples tightly clustered together, demonstrating a stable and repeatable experimental condition. Under a positive model, PC1 and PC2 accounted for 27.96% and 22.01% of the variance, respectively, while under a negative model, they accounted for 29.21% and 16.70% accordingly. All the points in the group N were clearly separated from the group H, thus indicating the occurrence of metabolic disorders in PLCs. The group ellipse of the group H partly coincided with that of the group HR, which also tended to approach the N group, suggesting the beneficial effect of RDP on PLCs. The abscissa PC1 and ordinate PC2 represent the scores of the first and second principal components, respectively; the scatter points of different colors represent samples in different experimental groups; and the ellipse represents the 95% confidence interval ((A) positive ion; (B) negative ion). To enhance classification accuracy, we employed PLS-DA. The PLS-DA score plots (Figs. 3 A, C) clearly demonstrated significant separation between normal and hypoxia conditions, underscoring substantial metabolic differences. The PLS-DA score chart showed that there was partial overlap between group H and group HR ( Figs. 3 E, G ). This observation aligns with our earlier findings from PCA. Score plots were crucial in supervising the PLS-DA analysis. Further, in order to ensure the reliability of the results, a permutation test (n = 200) was used to verify the PLS-DA model of metabolomics analysis. The R2-intercepts for groups N and H were 0.78 and 0.76, respectively, whereas the Q2-intercepts were 0.228 and 0.234, respectively (Fig. 3 B, D). Additionally, the positive-ion mode showed R2 and Q2 intercepts for the group H and HR were at 0.85 and 0.78, respectively, while in the negative-ion mode they were at 0.80 and 1.07, respectively (Fig. 3 F, H). These results collectively confirm the absence of overfitting in our model. Effects of different oxygen condition on PLCs metabolism The results of untargeted metabolomics technology showed that a total of 596 positive-ion-mode metabolites and 204 negative-ion-mode metabolites were identified in 18 samples. Differential metabolites were selected based on the variable importance in projection (VIP) scores greater than 1.0, fold change (FC) values exceeding 1.2 or less than 0.883, and a significance level of P < 0.05 [ 6 – 8 ] . Subsequently, hierarchical clustering analysis was conducted on these differentially expressed metabolites between the compared pairs. The relative quantitative values were normalized and converted for clustering. Figures S1 A and S1B depicted the results of this clustering analysis for positive and negative ion modes, respectively. The volcanic map results showed that 178 positive-ion-mode metabolites were significantly different in the H vs. N group, among which 116 metabolites were upregulated and 62 metabolites were downregulated (Fig. 4 A). Meanwhile, in negative-ion mode, 73 metabolites were significantly different, comprising 26 upregulated and 47 downregulated metabolites (Fig. 4 B). Our findings indicated that in positive ion mode, metabolites involved in metabolic pathways such as retinol metabolism, beta-alanine metabolism, and purine metabolism were predominantly affected. And in negative ion mode, biosynthesis of unsaturated fatty acids emerged as a key pathway (Fig. 4 C for positive ion mode and Fig. 4 D for negative ion mode). Effect of RDP on PLCs metabolism under hypoxia condition A total of 77 metabolites were annotated and selected as potential biomarkers between the hypoxia and HR groups under two ESI modes (Fig. 5 A, B). Notably, 9 of these metabolites were found to participate in various KEGG pathways, such as Cys-Gly, Riboflavin-5-phosphate, and others (Table 2 ). These metabolites were involved in about 20 KEGG pathways, including the biosynthesis of unsaturated fatty acids, glutathione metabolism, and riboflavin metabolism, etc. Further comparison between the H group and the HR group showed that five metabolites in the HR group showed levels close to those of the N group. Table 2 The metabolites involved in KEGG pathways Metabolite H vs. N HR vs. N HR vs. H KEGG Pathway Cys-Gly ↓** ↑& Glutathione metabolism 4-Nitrophenol ↓** ↑& Aminobenzoate degradation Riboflavin-5-phosphate ↑& Riboflavin metabolism, Biosynthesis of type II polyketide products, Oxidative phosphorylation, Vitamin digestion and absorption, Metabolic pathways Cytidine-5'-monophosphate ↓* ↑& Pyrimidine metabolism, Metabolic pathways Orotic Acid ↑## ↑& Pyrimidine metabolism, Metabolic pathways Docosapentaenoic acid ↑** ↑## ↓& Biosynthesis of unsaturated fatty acids L-Adrenaline ↓## ↓& Two-component system, Quorum sensing, Adrenergic signaling in cardiomyocytes, Tyrosine metabolism, cAMP signaling pathway, Regulation of lipolysis in adipocytes, Renin secretion, Neuroactive ligand-receptor interaction L-Homocystine ↓# ↓& Cysteine and methionine metabolism Phenylacetylglutamine ↑** ↓& Phenylalanine metabolism Note: ↑ and ↓ mean the metabolites up- and downregulated in the hypoxia (H) vs. normal (N) groups and the hypoxia + RDP (HR) vs. H groups; * indicates significant difference between N and H groups, indicates significant difference between H and HR groups, & indicates significant difference between N and HR groups. *, #, & p < 0.05, **, ##, && p < 0.01, ***, ###, &&& p < 0.001; Student’s t test was used to compare the significance of the metabolite level differences among different groups. Correlation analysis between Top19 differential metabolites and antioxidant indicators and T between N group and NR group Pearson correlation analysis was employed to investigate the relationship between differential metabolites of TOP19 and both T levels and antioxidant indicators in the HR and H groups (Fig. 6 ). The analysis revealed that, among the TOP19 differential metabolites identified in positive and negative ion modes, all metabolites except Cytidine-5'-monophosphate exhibited either positive or negative correlations with at least one antioxidant index. Among the 38 metabolites analyzed, those linked to T levels included Lysops 22:5, tert-Butyl N-[1-(aminocarbonyl)-3-methylbutyl] carbamate, LPC 18:1, PC O-16:1, Thymopentin, TP-5, Arginin, Riboflavin-5-phosphate, 10-Hydroxydecanoic acid, 5-(tert-butyl)-2-methyl-N-(5-methyl-3-isoxazolyl)-3-furamide, Orotic Acid, and Cys-Gly. Discussion With increasing altitude, atmospheric pressure and thus oxygen pressure decreases upon altitude [ 8 ] . The extreme environment of plateau regions can lead to hypoxia in animals, representing a considerable challenge for introduced livestock [ 9 ] . This condition is particularly prominent at high altitudes. Research indicates that the hypoxia environment reduces peroxidative activity and testosterone (T) secretion in male animals, thereby compromising reproductive system integrity [ 4 ] . Considering that T is mainly secreted by Porcine Leydig cells (PLCs), this study focuses on PLCs as the research object. Hypoxia induces cells to generate excessive reactive oxygen species (ROS), resulting in oxidative stress and metabolic disturbances [ 10 ] . Mitochondria are the main sources of ROS, and damaged mitochondria produce higher ROS levels [ 11 ] . Increased ROS levels from hypoxia can damage the structural integrity of cellular mitochondria and endoplasmic reticulum, thereby impairing their normal functions [ 12 , 13 ] . T is produced by Leydig cells through the coordinated function of mitochondria and smooth endoplasmic reticulum [ 14 ] . Hypoxia can lead to the upregulation of Hypoxia-Inducible Factor 1-alpha (HIF-1α), which can trigger cellular autophagy and apoptosis while inhibiting genes involved in T synthesis [ 15 ] . Studies have demonstrated that various plant polysaccharides possess antioxidant properties, potentially mitigating oxidative stress by reducing reactive oxygen species [ 16 ] . For instance, Astragalus polysaccharide enhances T synthesis by modulating the vitamin D system in human adrenocortical cells [ 17 ] . Malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) content is a vital indicator of the potential antioxidant capacity of the cells [ 18 – 20 ] . In our research, we observed a significant increase in MDA levels and decrease in GSH-Px, SOD, and T levels in porcine Leydig cells (PLCs) under hypoxia conditions. However, the administration of Rhodiola polysaccharide (RDP) mitigated these adverse effects of hypoxia, boosted the levels of GSH-Px, SOD, and T, while suppressed MDA levels. Through Enzyme-Linked Immunosorbent Assay (ELISA), we found that hypoxia reduced the antioxidant capacity of cells, causing cell damage and resulting in decreased T secretion. RDP can alleviate these detrimental effects by enhancing their antioxidant capacity and improving their adaptability to hypoxia conditions. This indicates that RDP can effectively alleviate oxidative stress resulting from hypoxia, thereby exerting antioxidant effects and promoting T secretion. Based on untargeted metabolomic sequencing, we focused on identifying significant small molecule metabolites that differed between the Hypoxia (H) and Hypoxia with RDP treatment (HR) groups. In order to clarify the relationship between differential metabolites and cellular antioxidants and T, we conducted Pearson correlation analysis for differential metabolites and antioxidant indicators in the H group and HR group. This analysis revealed that metabolites exhibiting positive correlations with antioxidant markers also positively correlated with T. Importantly, these metabolites exhibit antioxidant properties, suggesting that RDP could enhance both antioxidant metabolite levels and enzyme offering a potential defense against hypoxia oxidative stress. Key metabolites include Lysops 22:5, 10-Hydroxydecanoic acid (10-HDA), Arginine, and Thymopentin (TP-5). Lysops 22:5, an active cellular membrane substance, possesses powerful antioxidant abilities, mitigating cellular oxidative stress damage by neutralizing free radicals and protecting cells from damage and inflammation [ 21 ] . Studies have demonstrated that supplementing Lysops 22:5 can suppress the production of adrenocorticotropic hormone and cortisol post-exercise, while enhancing the T to cortisol ratio [ 22 ] . 10-HDA, a unique long-chain fatty acid, offers an array of biological and pharmacological benefits, including the ability to neutralize free radicals and reduce oxidative cellular stress [ 23 ] . Research by Chen has shown that in a polycystic ovary syndrome model, demonstrate the potential of 10-HDA in promoting the expression of anti-oxidative stress genes such as Nrf-2 and Forkhead Box O1 (FOXO1), thereby reducing oxidative stress levels [ 24 ] . Arginine, a fundamental amino acid with electron-carrying properties, interacts with free radicals through its guanidine group, thereby terminating free radical chain reactions and exhibiting strong scavenging abilities against DPPH (1,1-Diphenyl-2-picrylhydrazyl radical) and superoxide radicals [ 25 ] . These property underscores the antioxidant capacity and reducing power in vitro. TP-5, a polypeptide composed of five amino acids, has been documented to increase antioxidant enzyme activity in the immune organs of broilers, consequently reducing oxygen free radical levels [ 26 ] . In comparison, the HR group exhibited increased expression of antioxidant metabolites to the H group as featured in Table S1 , implying that RDP administration may enhance antioxidant defenses, thereby protecting cellular functionality under hypoxia stress condition. The study investigated how RDP mitigate hypoxia-induced damage to PLCs by elucidating the role of specific metabolites and their metabolic pathways. There is a close relationship between the metabolism of glutathione metabolism and oxidative stress. Within the glutathione metabolism system, glutathione peroxidase reducing oxidative damage by catalyzing reactions between glutathione and hydrogen peroxide, maintaining the structure and function of cell membrane [ 27 , 28 ] . Cysteinylglycine (Cys-Gly), a crucial precursors for glutathione synthesis [ 29 ] . The increase of Cys-Gly can directly promote the synthesis process of glutathione, thereby enhancing the antioxidant capacity of cells and protecting cells from oxidative damage [ 30 ] . Riboflavin-5-phosphate, a widely involved coenzyme, plays a crucial role in intracellular redox reactions and serves as a coenzyme for glutathione reductase within cells, facilitating the conversion of oxidized glutathione (GSSG) to its reduced form (GSH) [ 31 ] . In addition, the biosynthesis of unsaturated fatty acids was evaluated as important in metabolic pathway analysis between groups H and N. Docosapentaenoic acid (DPA) is significantly enriched in this pathway. DPA levels were notably higher in group N compared to group H. Following treatment with RDP, DPA levels decreased but remained elevated relative to group N. DPA is well-documented as a constituent of phospholipids present in all animal cell membranes, pivotal for cell membrane integrity, repair, and regeneration [ 32 ] . DPA is renowned for its antioxidative and anti-inflammatory properties, crucial in mitigating oxidative stress and inflammatory responses, thereby shielding cells from oxidative damage [ 33 ] . We hypothesize that the hypoxia conditions induce alterations in intracellular redox balance, prompting cells to enhance their antioxidant defenses, potentially through lipid modulation involving DPA. Moreover, the addition of Rhodiola polysaccharide demonstrated a reduction in cellular oxidative stress compared to group H, implying a protective role of RDP in cellular homeostasis. In summary, we speculated that RDP promotes the synthesis of antioxidant metabolites, such as Lysops 22:5 and riboflavin in cells and relieves oxidative stress caused through hypoxia via regulating the unsaturated fatty acids metabolism and the glutathione metabolic pathway. RDP maintains the ability of PLCs to secrete T by protecting cells from ROS attack. Conclusions Hypoxia significantly suppressed T secretion and reduced antioxidant capacity, while RDP had a positive impact on intracellular antioxidant activity and T secretion. In addition, the study revealed two signaling pathways through which RDP improves cellular antioxidant capacity. Notably, RDP can improve the antioxidant capacity of cells and resist hypoxia stress by increasing the expression of Cys-Gly and riboflavin in cellular glutathione metabolism. These results suggested that RDP enhanced both antioxidant capacity and T secretion in porcine testicular Leydig cells under hypoxia condition through modulation of cellular metabolism. Materials and Methods Cell Culture PLCs were purchased from Otwo Biotech Inc (Guangzhou, China). The cells were cultured in DMEM-F12 medium containing 10% fetal bovine serum (FBS; GIBCO, Shanghai, China), 1% penicillin (100 units/mL)/streptomycin (100 mg/mL) at 37°C in an atmosphere of 5% CO 2 with the medium replaced every 24 h. When the confluence reached 80%, the cells were digested with 0.25% trypsin (Solarbio, Beijing, China) for generation or subsequent experiments. Cells were divided into four groups: the normal group (N), hypoxia group (H), and hypoxia + RDP group (HR). The N group was cultured under normal condition without hypoxia and RDP (Xi'an Qingzhi Biotechnology Co., Ltd., Xi'an, China). The cells were exposed to hypoxia stimulation in a Modular Incubator Chamber (Embrient Inc., USA) under an atmosphere of 1% oxygen (O 2 ), 5% carbon dioxide (CO 2 ), and 94% nitrogen (N 2 ) for 18 h. The HR group was stimulated with RDP (12.5 µg/mL) for 18 h before hypoxia, while the H group was simulated with medium. Assays of SOD Activity, MDA, GSH-Px Level and T Level Testosterone (T), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activity, Malondialdehyde (MDA) level and were detected according to SOD, GSH-Px activity, MDA and T (Enzyme linked Biotechnology Co., Ltd, Shanghai, China) detection assay kit instructions, respectively. In brief, action buffers were added to the supernatants of cellular samples. The mixtures were incubated at 37°C and the optical densities were measured at 450 nm using visible spectrophotometer for SOD, GSH-PX activities and MDA. Assay kits for MDA, SOD, and GSH-Px were purchased from Jiangsu Enzyme Label Biotechnology Co., Ltd. (Jiangsu, China). Cell Metabolite Extraction After incubation, each cell pellet sample was suspended into 300 µL of 80% aqueous methanol in an EP tube. And snap-frozen in liquid nitrogen. After centrifugation at 4 ℃ for 1 min at 5,000 rpm, the supernatant was transferred into a new centrifuge tube and freeze-dried. Then, methanol was used to dissolve the residue and the solution was used for LC-MS/MS [ 34 , 35 ] . Metabolite extraction The chromatography column and condition are as follows: Chromatographic column: Hyperil Gold column (C18); column temperature: 40°C; flow rate: 0.2 mL/min; positive mode: mobile phase A: 0.1% formic acid; mobile phase B: methanol; negative mode: mobile phase A: 5 mM ammonium acetate, pH 9.0; mobile phase B: methanol (2) Elution gradient: 98:2 (V/V) at 0 min, 98:2 (V/V) at 1.5 min, 15:85 (V/V) at 3.0 min, 0:100 (V/V) at 10 min, 98:2 (V/V) at 10.1 min, 98:2 (V/V) at 11.0 min, and 12.0 min for 98:2 (V/V). Mass spectrometry condition Mass spectrometry conditions were as follows: electrospray ion source (ESI), scanning range of m/z 100–1,500. Spray Voltage: 3.5kV; Sheath gas flow rate: 35psi; Aux Gas flow rate: 10 L/min; Capillary Temp: 320°C; S-lens RF level: 60; Aux gas heater temp: 350°C; Polarity: positive, negative; MS/MS secondary scan is data-dependent scans [ 36 , 37 ] . Data processing The raw data files were imported into the CD 3.3 search software to perform simple screening of retention time, mass-to-charge ratio, Peak extraction was performed according to the set mass deviation of 5 ppm, signal intensity deviation of 30%, minimum signal intensity of 100,000 and at the same time the peak area was quantified. The molecular formula of peak and fragment ions was predicted and compared with mzCloud, mzVault and MassList databases. The blank sample was used to remove background ions. Data Analysis These metabolites were annotated using the KEGG database ( https://www.genome.jp/kegg/pathway.html ), HMDB database ( https://hmdb.ca/metabolites ) and LIPIDMaps database ( http://www.lipidmaps.org/ ). For multivariate statistical analysis, MetaX was used to transform the data and then perform the principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). The VIP value, obtained in the PLS-DA model, indicates the importance of each metabolite in the discrimination between groups. The statistical significance (p-value) and fold change (FC) of each metabolite between the two groups were calculated based on univariate analysis (t-test). The screening criteria for differential metabolites were VIP > 1.0, fold change (FC) > 1.2 or FC and P value 0.05. The metabolic pathway enrichment of differential metabolites was performed; when the ratios were satisfied by x/n > y/N, the metabolic pathways were considered enriched, and when the p-value of the metabolic pathway was the metabolic pathway was considered to have experienced statistically significant enrichment. Declarations Acknowledgments Not applicable. Author Contributions Jinting Luo: investigation, data curation, formal analysis, methodology, writing—original draft. Lei Wang: methodology, writing. Xuan Luo: methodology, writing. Jianbo Zhang: investigation, methodology. Tian Tian: writing—review and editing. Youli Yao: writing—review and editing. Dandan Luo: writing—review and editing. Guofang Wu: conceptualization, writing—review and editing, supervision. All authors have read and agreed to the published version of the manuscript. Funding This research was funded by the basic research project of Qinghai, grant number 2022-ZJ-752. Data availability Data is provided within the manuscript or supplementary information files. Competing interests The authors declare that they have no competing interests. References Wu, D. D. et al. Convergent genomic signatures of high-altitude adaptation among domestic mammals. Natl. Sci. Rev. 7 (6), 952–963 (2020). Rao, F. Z., Tian, H., Li, W. Q., Hung, H. L. & Sun, F. Potential role of punicalagin against oxidative stress induced testicular damage. Asian J. Androl. 18 (4), 627–632 (2016). Parraguez, V. H. & Gonzalez-Bulnes, A. Endocrinology of reproductive function and pregnancy at high altitudes - ScienceDirect. Curr. 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Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry. Nat. Protoc. 6 (7), 1060–1083 (2011). Wilson, I. D. et al. Global metabolic profiling procedures for urine using UPLC-MS. Nat. Protoc. 5 (6), 1005–1018 (2010). Additional Declarations No competing interests reported. Supplementary Files image1.png Supplementarymaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5056340","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":379347730,"identity":"b4f99fdd-c545-45ba-accc-f92f32f58ea3","order_by":0,"name":"Jinting Luo","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Jinting","middleName":"","lastName":"Luo","suffix":""},{"id":379347731,"identity":"b0dae42f-1fe1-403d-a2ff-46687e271e37","order_by":1,"name":"Lei Wang","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Wang","suffix":""},{"id":379347735,"identity":"4d31718a-73f5-4632-8475-59c1a6160612","order_by":2,"name":"Youli Yao","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Youli","middleName":"","lastName":"Yao","suffix":""},{"id":379347737,"identity":"4aba3980-a44b-485e-af54-6118ca20fe21","order_by":3,"name":"Xuan Luo","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Xuan","middleName":"","lastName":"Luo","suffix":""},{"id":379347738,"identity":"1f5ff9cd-44c0-4f1b-bc8e-abd48dbc2cc1","order_by":4,"name":"Jianbo Zhang","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Jianbo","middleName":"","lastName":"Zhang","suffix":""},{"id":379347739,"identity":"edfe9738-cf76-428a-8688-29adfe5c3098","order_by":5,"name":"Dandan Luo","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Dandan","middleName":"","lastName":"Luo","suffix":""},{"id":379347740,"identity":"e12c0f95-8c1f-4d1b-87e6-dc645f5961cb","order_by":6,"name":"Tian Tian","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Tian","middleName":"","lastName":"Tian","suffix":""},{"id":379347742,"identity":"b352b7ab-c972-4bb4-b4ad-6781746185f1","order_by":7,"name":"Guofang Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYDACCQYDCIP5AJCokJDjJ14LWwKQOGNhLNlAkhbGtorEDYS08N1u3ibN21ab2M/G/Ozh13kSjBsYmB8+uoFHi+SdY8XGvG3HE2e2sZkby26TYDZnYDM2zsGjxeBGjuHj3LZjiRvuN5hJS26TYLNs4GGTJqDF4DBYyzH2b9KScyR4DA4Q1gKypQaohcdM8mODhARBLZI30oqN/5w7YDyzjadMmuGYhIFkMwG/8N1I3iY5o6xOtp+NfZvkj5q6+n725oeP8WlhOAAmD4NJZh4wiU85QksdmGT8QUj1KBgFo2AUjEgAAJfxTeOpgnD6AAAAAElFTkSuQmCC","orcid":"","institution":"Qinghai University","correspondingAuthor":true,"prefix":"","firstName":"Guofang","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2024-09-09 08:14:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5056340/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5056340/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71035353,"identity":"05443834-6dd4-46be-b2b8-ae885261e953","added_by":"auto","created_at":"2024-12-10 12:42:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":28335,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of RDP on T secretion in PLCs under hypoxia\u003c/p\u003e\n\u003cp\u003eDifferent letters indicate significant difference (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05), while the same letters indicate no significant difference (\u003cem\u003eP \u003c/em\u003e\u0026gt; 0.05). N: normal group; H: hypoxia group; HR: hypoxia+RDP group.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5056340/v1/b5f75ea066486be386d33ba0.png"},{"id":71035586,"identity":"ac0a2f03-da85-46e0-b57f-e68629e5af3e","added_by":"auto","created_at":"2024-12-10 12:50:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":140189,"visible":true,"origin":"","legend":"\u003cp\u003eThe score plots of PCA for metabolite profiles in cell.\u003c/p\u003e\n\u003cp\u003eThe abscissa PC1 and ordinate PC2 represent the scores of the first and second principal components, respectively; the scatter points of different colors represent samples in different experimental groups; and the ellipse represents the 95% confidence interval ((A) positive ion; (B) negative ion).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5056340/v1/a01dfcbd7c66b57d5d4c7227.png"},{"id":71035360,"identity":"43da088b-290b-4930-8051-95e0fe1c0f79","added_by":"auto","created_at":"2024-12-10 12:42:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":185466,"visible":true,"origin":"","legend":"\u003cp\u003ePLS-DA score scatter plot and ranking verification chart.\u003c/p\u003e\n\u003cp\u003e(A, B, C, D)Hypoxia vs N group PLS-DA score plot and permutation test(A, B): positive ion; (C, D): negative ion). (E, F, G, H) Hypoxia vs HR group PLS-DA plot and permutation test in positive and negative modes ((E, F): positive ion; (G, H): negative ion).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5056340/v1/7a3a53cc7de88d3436f471b5.png"},{"id":71035357,"identity":"56eb72a3-617d-4c6c-b6bd-a0841bcafa53","added_by":"auto","created_at":"2024-12-10 12:42:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":755590,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of hypoxia on metabolism of PLCs\u003c/p\u003e\n\u003cp\u003e(A, B) Volcanic map of differential metabolites and bubble map of differential metabolite pathway enrichment in positive mode. (C, D) Volcanic map of differential metabolites and bubble map of differential metabolite pathway enrichment in negative mode.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5056340/v1/1733c4a8fd9780b8fcd5eec6.png"},{"id":71035588,"identity":"ca08fc7b-ca14-4afd-a0b6-e07f4a65fd28","added_by":"auto","created_at":"2024-12-10 12:50:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":424850,"visible":true,"origin":"","legend":"\u003cp\u003eMatchstick map of different metabolites between HR group and H group\u003c/p\u003e\n\u003cp\u003eMatchstick plots show fold changes in expression of differential metabolites between hypoxia and normal groups. (A) postive mode; (B) negative mode.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5056340/v1/ddc542c3f91435a73fc26407.png"},{"id":71035587,"identity":"67bde64a-dcfa-4bc2-902d-036bdea7296d","added_by":"auto","created_at":"2024-12-10 12:50:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":274768,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation analysis of different metabolites and T and antioxidant indexes of TOP19 in HR group vs H group\u003c/p\u003e\n\u003cp\u003e1 means completely positive correlation, 0 means no linear relationship, and -1 means completely negative correlation; (A) positive mode; (B) negative mode\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-5056340/v1/5496c9ce7d86a05320d3fc57.png"},{"id":72255257,"identity":"d4a5d3ca-3f56-4ea6-bfec-bd25825b8364","added_by":"auto","created_at":"2024-12-24 09:31:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2462725,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5056340/v1/e1f34a99-def4-4040-b3cc-6dedc10ca344.pdf"},{"id":71035355,"identity":"fe8b3de0-ade5-4d69-a195-a24e1dd9e2a1","added_by":"auto","created_at":"2024-12-10 12:42:14","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":215940,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5056340/v1/4b774a7295a6c67ea848f735.png"},{"id":71035356,"identity":"d690953a-b304-45bd-9bb9-93fe685c71c4","added_by":"auto","created_at":"2024-12-10 12:42:14","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":787297,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-5056340/v1/9c758979d1ff7fe61613a836.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Rhodiola polysaccharides affect the antioxidant capacity and testosterone secretion of PLCs in hypoxia environment through the metabolism of unsaturated fatty acids and glutathione","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAs people\u0026rsquo;s quality of life has improved, higher requirements have been placed on the meat quality of porcine. Introduced species is an effective way to improve the production performance of local porcine. Oxygen is essential for all animals\u0026rsquo; life, and hypoxia, a prominent feature of plateau environments, poses significant challenges to introduced species. When animals are relocated to plateaus, they must regulate their physiological functions to adapt to reduced oxygen levels. Hypoxia occurs when oxygen is inadequate to supply to tissue cells, which disrupts normal physiological functions\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Hypoxia usually causes stress reactions in clinical practice, affecting animal reproductive performance\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. For example, hypoxia can impair sperm production, libido, testicular function, and reproductive capacity in livestock\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. It is also associated with a reduction in the secretion of testosterone (T)\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. As an important regulatory hormone for male reproductive function, T can affect libido, sexual organ development, and spermatogenesis\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNatural antioxidant such as natural polysaccharides plant origin popular due to its non-toxic nature. \u003cem\u003eRhodiola rosea\u003c/em\u003e L., a traditional anti-altitude stress Chinese medicinal material, contains the active substance Rhodiola Polysaccharides (RDP), which is known for the anti-hypoxia, anti-stress and hypoglycemic effects\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Previous studies have shown that adding RDP to the culture medium can significantly enhance the proliferation of PLCs\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. This experiment aims to explore the effect of RDP on T secretion and antioxidant capacity of PLCs under hypoxia environment. And through the metabolomic study, we preliminarily revealed its effects and protective mechanism. This study provides scientific basis for RDP as a natural medicine that can relieve the stress of introduced animals.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEffect of RDP on Antioxidant capacity of PLCs under hypoxia condition\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrated notable differences in antioxidant markers among the groups studied. Specifically, compared to the normal (N) group, the hypoxia (H) group showed significantly lower levels of SOD and GSH-Px, and significantly higher levels of MDA (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, the content of SOD and GSH-Px in the hypoxia\u0026thinsp;+\u0026thinsp;RDP (HR) group was increased, and the MDA content was decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared with the H group, and the antioxidant indicators of HR returned nearly to the levels of the N group.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of RDP on Antioxidant capacity of PLCs under hypoxia condition\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHR\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSOD (U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e69.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.54\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e49.43\u0026thinsp;\u0026plusmn;\u0026thinsp;5.45\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e59.42\u0026thinsp;\u0026plusmn;\u0026thinsp;5.77\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMDA (nmoL/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e49.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGSH-Px (U/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e676.31\u0026thinsp;\u0026plusmn;\u0026thinsp;24\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e631.38\u0026thinsp;\u0026plusmn;\u0026thinsp;11.34\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e670.87\u0026thinsp;\u0026plusmn;\u0026thinsp;32.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eNote: Different letters indicate significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the same letters indicate no significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). MDA: malondialdehyde; GSH-Px: glutathione peroxidase; SOD: superoxide dismutase. N: normal group; H: hypoxia group; HR: hypoxia\u0026thinsp;+\u0026thinsp;RDP group.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of RDP on T\u003c/b\u003e s\u003cb\u003eecretion from PLCs under hypoxia condition\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e showed that the T secretion level of the H group was significantly reduced compared to that in the N group. Conversely, in the HR group, T secretion was notably increased compared to the H group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMultivariate statistical analysis of metabolomics\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e showed that all samples fell within the 95% confidence interval, with QC samples tightly clustered together, demonstrating a stable and repeatable experimental condition. Under a positive model, PC1 and PC2 accounted for 27.96% and 22.01% of the variance, respectively, while under a negative model, they accounted for 29.21% and 16.70% accordingly. All the points in the group N were clearly separated from the group H, thus indicating the occurrence of metabolic disorders in PLCs. The group ellipse of the group H partly coincided with that of the group HR, which also tended to approach the N group, suggesting the beneficial effect of RDP on PLCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe abscissa PC1 and ordinate PC2 represent the scores of the first and second principal components, respectively; the scatter points of different colors represent samples in different experimental groups; and the ellipse represents the 95% confidence interval ((A) positive ion; (B) negative ion).\u003c/p\u003e \u003cp\u003eTo enhance classification accuracy, we employed PLS-DA. The PLS-DA score plots (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, C) clearly demonstrated significant separation between normal and hypoxia conditions, underscoring substantial metabolic differences. The PLS-DA score chart showed that there was partial overlap between group H and group HR ( Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, G ). This observation aligns with our earlier findings from PCA. Score plots were crucial in supervising the PLS-DA analysis. Further, in order to ensure the reliability of the results, a permutation test (n\u0026thinsp;=\u0026thinsp;200) was used to verify the PLS-DA model of metabolomics analysis. The R2-intercepts for groups N and H were 0.78 and 0.76, respectively, whereas the Q2-intercepts were 0.228 and 0.234, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, D). Additionally, the positive-ion mode showed R2 and Q2 intercepts for the group H and HR were at 0.85 and 0.78, respectively, while in the negative-ion mode they were at 0.80 and 1.07, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, H). These results collectively confirm the absence of overfitting in our model.\u003c/p\u003e \n\u003ch3\u003eEffects of different oxygen condition on PLCs metabolism\u003c/h3\u003e\n\u003cp\u003eThe results of untargeted metabolomics technology showed that a total of 596 positive-ion-mode metabolites and 204 negative-ion-mode metabolites were identified in 18 samples. Differential metabolites were selected based on the variable importance in projection (VIP) scores greater than 1.0, fold change (FC) values exceeding 1.2 or less than 0.883, and a significance level of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003csup\u003e[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Subsequently, hierarchical clustering analysis was conducted on these differentially expressed metabolites between the compared pairs. The relative quantitative values were normalized and converted for clustering. Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA and S1B depicted the results of this clustering analysis for positive and negative ion modes, respectively.\u003c/p\u003e \u003cp\u003eThe volcanic map results showed that 178 positive-ion-mode metabolites were significantly different in the H vs. N group, among which 116 metabolites were upregulated and 62 metabolites were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Meanwhile, in negative-ion mode, 73 metabolites were significantly different, comprising 26 upregulated and 47 downregulated metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Our findings indicated that in positive ion mode, metabolites involved in metabolic pathways such as retinol metabolism, beta-alanine metabolism, and purine metabolism were predominantly affected. And in negative ion mode, biosynthesis of unsaturated fatty acids emerged as a key pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC for positive ion mode and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD for negative ion mode).\u003c/p\u003e\n\u003ch3\u003eEffect of RDP on PLCs metabolism under hypoxia condition\u003c/h3\u003e\n\u003cp\u003eA total of 77 metabolites were annotated and selected as potential biomarkers between the hypoxia and HR groups under two ESI modes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Notably, 9 of these metabolites were found to participate in various KEGG pathways, such as Cys-Gly, Riboflavin-5-phosphate, and others (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These metabolites were involved in about 20 KEGG pathways, including the biosynthesis of unsaturated fatty acids, glutathione metabolism, and riboflavin metabolism, etc. Further comparison between the H group and the HR group showed that five metabolites in the HR group showed levels close to those of the N group.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe metabolites involved in KEGG pathways\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetabolite\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH vs. N\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHR vs. N\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHR vs. H\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKEGG Pathway\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCys-Gly\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026darr;**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026uarr;\u0026amp;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGlutathione metabolism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4-Nitrophenol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026darr;**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026uarr;\u0026amp;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAminobenzoate degradation\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRiboflavin-5-phosphate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026uarr;\u0026amp;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRiboflavin metabolism, Biosynthesis of type II polyketide products, Oxidative phosphorylation, Vitamin digestion and absorption, Metabolic pathways\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCytidine-5'-monophosphate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026darr;*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026uarr;\u0026amp;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePyrimidine metabolism, Metabolic pathways\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOrotic Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026uarr;##\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026uarr;\u0026amp;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePyrimidine metabolism, Metabolic pathways\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDocosapentaenoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026uarr;**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026uarr;##\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026darr;\u0026amp;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBiosynthesis of unsaturated fatty acids\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Adrenaline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026darr;##\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026darr;\u0026amp;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTwo-component system, Quorum sensing, Adrenergic signaling in cardiomyocytes, Tyrosine metabolism, cAMP signaling pathway, Regulation of lipolysis in adipocytes, Renin secretion, Neuroactive ligand-receptor interaction\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Homocystine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026darr;#\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026darr;\u0026amp;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCysteine and methionine metabolism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhenylacetylglutamine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026uarr;**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026darr;\u0026amp;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePhenylalanine metabolism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eNote: \u0026uarr; and \u0026darr; mean the metabolites up- and downregulated in the hypoxia (H) vs. normal (N) groups and the hypoxia\u0026thinsp;+\u0026thinsp;RDP (HR) vs. H groups; * indicates significant difference between N and H groups, indicates significant difference between H and HR groups, \u0026amp; indicates significant difference between N and HR groups. *, #, \u0026amp; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **, ##, \u0026amp;\u0026amp; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***, ###, \u0026amp;\u0026amp;\u0026amp; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Student\u0026rsquo;s t test was used to compare the significance of the metabolite level differences among different groups.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCorrelation analysis between Top19 differential metabolites and antioxidant indicators and T between N group and NR group\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePearson correlation analysis was employed to investigate the relationship between differential metabolites of TOP19 and both T levels and antioxidant indicators in the HR and H groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The analysis revealed that, among the TOP19 differential metabolites identified in positive and negative ion modes, all metabolites except Cytidine-5'-monophosphate exhibited either positive or negative correlations with at least one antioxidant index. Among the 38 metabolites analyzed, those linked to T levels included Lysops 22:5, tert-Butyl N-[1-(aminocarbonyl)-3-methylbutyl] carbamate, LPC 18:1, PC O-16:1, Thymopentin, TP-5, Arginin, Riboflavin-5-phosphate, 10-Hydroxydecanoic acid, 5-(tert-butyl)-2-methyl-N-(5-methyl-3-isoxazolyl)-3-furamide, Orotic Acid, and Cys-Gly.\u003c/p\u003e "},{"header":"Discussion","content":"\u003cp\u003eWith increasing altitude, atmospheric pressure and thus oxygen pressure decreases upon altitude\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. The extreme environment of plateau regions can lead to hypoxia in animals, representing a considerable challenge for introduced livestock\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. This condition is particularly prominent at high altitudes. Research indicates that the hypoxia environment reduces peroxidative activity and testosterone (T) secretion in male animals, thereby compromising reproductive system integrity\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Considering that T is mainly secreted by Porcine Leydig cells (PLCs), this study focuses on PLCs as the research object. Hypoxia induces cells to generate excessive reactive oxygen species (ROS), resulting in oxidative stress and metabolic disturbances\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Mitochondria are the main sources of ROS, and damaged mitochondria produce higher ROS levels\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Increased ROS levels from hypoxia can damage the structural integrity of cellular mitochondria and endoplasmic reticulum, thereby impairing their normal functions\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. T is produced by Leydig cells through the coordinated function of mitochondria and smooth endoplasmic reticulum\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Hypoxia can lead to the upregulation of Hypoxia-Inducible Factor 1-alpha (HIF-1α), which can trigger cellular autophagy and apoptosis while inhibiting genes involved in T synthesis\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Studies have demonstrated that various plant polysaccharides possess antioxidant properties, potentially mitigating oxidative stress by reducing reactive oxygen species\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. For instance, Astragalus polysaccharide enhances T synthesis by modulating the vitamin D system in human adrenocortical cells\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) content is a vital indicator of the potential antioxidant capacity of the cells\u003csup\u003e[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. In our research, we observed a significant increase in MDA levels and decrease in GSH-Px, SOD, and T levels in porcine Leydig cells (PLCs) under hypoxia conditions. However, the administration of Rhodiola polysaccharide (RDP) mitigated these adverse effects of hypoxia, boosted the levels of GSH-Px, SOD, and T, while suppressed MDA levels. Through Enzyme-Linked Immunosorbent Assay (ELISA), we found that hypoxia reduced the antioxidant capacity of cells, causing cell damage and resulting in decreased T secretion. RDP can alleviate these detrimental effects by enhancing their antioxidant capacity and improving their adaptability to hypoxia conditions. This indicates that RDP can effectively alleviate oxidative stress resulting from hypoxia, thereby exerting antioxidant effects and promoting T secretion.\u003c/p\u003e \u003cp\u003eBased on untargeted metabolomic sequencing, we focused on identifying significant small molecule metabolites that differed between the Hypoxia (H) and Hypoxia with RDP treatment (HR) groups. In order to clarify the relationship between differential metabolites and cellular antioxidants and T, we conducted Pearson correlation analysis for differential metabolites and antioxidant indicators in the H group and HR group. This analysis revealed that metabolites exhibiting positive correlations with antioxidant markers also positively correlated with T. Importantly, these metabolites exhibit antioxidant properties, suggesting that RDP could enhance both antioxidant metabolite levels and enzyme offering a potential defense against hypoxia oxidative stress. Key metabolites include Lysops 22:5, 10-Hydroxydecanoic acid (10-HDA), Arginine, and Thymopentin (TP-5). Lysops 22:5, an active cellular membrane substance, possesses powerful antioxidant abilities, mitigating cellular oxidative stress damage by neutralizing free radicals and protecting cells from damage and inflammation\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Studies have demonstrated that supplementing Lysops 22:5 can suppress the production of adrenocorticotropic hormone and cortisol post-exercise, while enhancing the T to cortisol ratio\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. 10-HDA, a unique long-chain fatty acid, offers an array of biological and pharmacological benefits, including the ability to neutralize free radicals and reduce oxidative cellular stress \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Research by Chen has shown that in a polycystic ovary syndrome model, demonstrate the potential of 10-HDA in promoting the expression of anti-oxidative stress genes such as \u003cem\u003eNrf-2\u003c/em\u003e and Forkhead Box O1 (FOXO1), thereby reducing oxidative stress levels\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Arginine, a fundamental amino acid with electron-carrying properties, interacts with free radicals through its guanidine group, thereby terminating free radical chain reactions and exhibiting strong scavenging abilities against DPPH (1,1-Diphenyl-2-picrylhydrazyl radical) and superoxide radicals\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. These property underscores the antioxidant capacity and reducing power in vitro. TP-5, a polypeptide composed of five amino acids, has been documented to increase antioxidant enzyme activity in the immune organs of broilers, consequently reducing oxygen free radical levels\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. In comparison, the HR group exhibited increased expression of antioxidant metabolites to the H group as featured in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, implying that RDP administration may enhance antioxidant defenses, thereby protecting cellular functionality under hypoxia stress condition.\u003c/p\u003e \u003cp\u003eThe study investigated how RDP mitigate hypoxia-induced damage to PLCs by elucidating the role of specific metabolites and their metabolic pathways. There is a close relationship between the metabolism of glutathione metabolism and oxidative stress. Within the glutathione metabolism system, glutathione peroxidase reducing oxidative damage by catalyzing reactions between glutathione and hydrogen peroxide, maintaining the structure and function of cell membrane\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Cysteinylglycine (Cys-Gly), a crucial precursors for glutathione synthesis\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. The increase of Cys-Gly can directly promote the synthesis process of glutathione, thereby enhancing the antioxidant capacity of cells and protecting cells from oxidative damage\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Riboflavin-5-phosphate, a widely involved coenzyme, plays a crucial role in intracellular redox reactions and serves as a coenzyme for glutathione reductase within cells, facilitating the conversion of oxidized glutathione (GSSG) to its reduced form (GSH)\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. In addition, the biosynthesis of unsaturated fatty acids was evaluated as important in metabolic pathway analysis between groups H and N. Docosapentaenoic acid (DPA) is significantly enriched in this pathway. DPA levels were notably higher in group N compared to group H. Following treatment with RDP, DPA levels decreased but remained elevated relative to group N. DPA is well-documented as a constituent of phospholipids present in all animal cell membranes, pivotal for cell membrane integrity, repair, and regeneration\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. DPA is renowned for its antioxidative and anti-inflammatory properties, crucial in mitigating oxidative stress and inflammatory responses, thereby shielding cells from oxidative damage\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. We hypothesize that the hypoxia conditions induce alterations in intracellular redox balance, prompting cells to enhance their antioxidant defenses, potentially through lipid modulation involving DPA. Moreover, the addition of Rhodiola polysaccharide demonstrated a reduction in cellular oxidative stress compared to group H, implying a protective role of RDP in cellular homeostasis. In summary, we speculated that RDP promotes the synthesis of antioxidant metabolites, such as Lysops 22:5 and riboflavin in cells and relieves oxidative stress caused through hypoxia via regulating the unsaturated fatty acids metabolism and the glutathione metabolic pathway. RDP maintains the ability of PLCs to secrete T by protecting cells from ROS attack.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eHypoxia significantly suppressed T secretion and reduced antioxidant capacity, while RDP had a positive impact on intracellular antioxidant activity and T secretion. In addition, the study revealed two signaling pathways through which RDP improves cellular antioxidant capacity. Notably, RDP can improve the antioxidant capacity of cells and resist hypoxia stress by increasing the expression of Cys-Gly and riboflavin in cellular glutathione metabolism. These results suggested that RDP enhanced both antioxidant capacity and T secretion in porcine testicular Leydig cells under hypoxia condition through modulation of cellular metabolism.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture\u003c/h2\u003e \u003cp\u003ePLCs were purchased from Otwo Biotech Inc (Guangzhou, China). The cells were cultured in DMEM-F12 medium containing 10% fetal bovine serum (FBS; GIBCO, Shanghai, China), 1% penicillin (100 units/mL)/streptomycin (100 mg/mL) at 37\u0026deg;C in an atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e with the medium replaced every 24 h. When the confluence reached 80%, the cells were digested with 0.25% trypsin (Solarbio, Beijing, China) for generation or subsequent experiments. Cells were divided into four groups: the normal group (N), hypoxia group (H), and hypoxia\u0026thinsp;+\u0026thinsp;RDP group (HR). The N group was cultured under normal condition without hypoxia and RDP (Xi'an Qingzhi Biotechnology Co., Ltd., Xi'an, China). The cells were exposed to hypoxia stimulation in a Modular Incubator Chamber (Embrient Inc., USA) under an atmosphere of 1% oxygen (O\u003csub\u003e2\u003c/sub\u003e), 5% carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e), and 94% nitrogen (N\u003csub\u003e2\u003c/sub\u003e) for 18 h. The HR group was stimulated with RDP (12.5 \u0026micro;g/mL) for 18 h before hypoxia, while the H group was simulated with medium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAssays of SOD Activity, MDA, GSH-Px Level and T Level\u003c/h2\u003e \u003cp\u003eTestosterone (T), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activity, Malondialdehyde (MDA) level and were detected according to SOD, GSH-Px activity, MDA and T (Enzyme linked Biotechnology Co., Ltd, Shanghai, China) detection assay kit instructions, respectively. In brief, action buffers were added to the supernatants of cellular samples. The mixtures were incubated at 37\u0026deg;C and the optical densities were measured at 450 nm using visible spectrophotometer for SOD, GSH-PX activities and MDA. Assay kits for MDA, SOD, and GSH-Px were purchased from Jiangsu Enzyme Label Biotechnology Co., Ltd. (Jiangsu, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell Metabolite Extraction\u003c/h2\u003e \u003cp\u003eAfter incubation, each cell pellet sample was suspended into 300 \u0026micro;L of 80% aqueous methanol in an EP tube. And snap-frozen in liquid nitrogen. After centrifugation at 4 ℃ for 1 min at 5,000 rpm, the supernatant was transferred into a new centrifuge tube and freeze-dried. Then, methanol was used to dissolve the residue and the solution was used for LC-MS/MS\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMetabolite extraction\u003c/h2\u003e \u003cp\u003eThe chromatography column and condition are as follows: Chromatographic column: Hyperil Gold column (C18); column temperature: 40\u0026deg;C; flow rate: 0.2 mL/min; positive mode: mobile phase A: 0.1% formic acid; mobile phase B: methanol; negative mode: mobile phase A: 5 mM ammonium acetate, pH 9.0; mobile phase B: methanol (2) Elution gradient: 98:2 (V/V) at 0 min, 98:2 (V/V) at 1.5 min, 15:85 (V/V) at 3.0 min, 0:100 (V/V) at 10 min, 98:2 (V/V) at 10.1 min, 98:2 (V/V) at 11.0 min, and 12.0 min for 98:2 (V/V).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMass spectrometry condition\u003c/h2\u003e \u003cp\u003eMass spectrometry conditions were as follows: electrospray ion source (ESI), scanning range of m/z 100\u0026ndash;1,500. Spray Voltage: 3.5kV; Sheath gas flow rate: 35psi; Aux Gas flow rate: 10 L/min; Capillary Temp: 320\u0026deg;C; S-lens RF level: 60; Aux gas heater temp: 350\u0026deg;C; Polarity: positive, negative; MS/MS secondary scan is data-dependent scans\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eData processing\u003c/h2\u003e \u003cp\u003eThe raw data files were imported into the CD 3.3 search software to perform simple screening of retention time, mass-to-charge ratio, Peak extraction was performed according to the set mass deviation of 5 ppm, signal intensity deviation of 30%, minimum signal intensity of 100,000 and at the same time the peak area was quantified. The molecular formula of peak and fragment ions was predicted and compared with mzCloud, mzVault and MassList databases. The blank sample was used to remove background ions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis\u003c/h2\u003e \u003cp\u003eThese metabolites were annotated using the KEGG database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genome.jp/kegg/pathway.html\u003c/span\u003e\u003cspan address=\"https://www.genome.jp/kegg/pathway.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), HMDB database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://hmdb.ca/metabolites\u003c/span\u003e\u003cspan address=\"https://hmdb.ca/metabolites\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and LIPIDMaps database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.lipidmaps.org/\u003c/span\u003e\u003cspan address=\"http://www.lipidmaps.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). For multivariate statistical analysis, MetaX was used to transform the data and then perform the principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). The VIP value, obtained in the PLS-DA model, indicates the importance of each metabolite in the discrimination between groups. The statistical significance (p-value) and fold change (FC) of each metabolite between the two groups were calculated based on univariate analysis (t-test). The screening criteria for differential metabolites were VIP\u0026thinsp;\u0026gt;\u0026thinsp;1.0, fold change (FC)\u0026thinsp;\u0026gt;\u0026thinsp;1.2 or FC and P value 0.05. The metabolic pathway enrichment of differential metabolites was performed; when the ratios were satisfied by x/n\u0026thinsp;\u0026gt;\u0026thinsp;y/N, the metabolic pathways were considered enriched, and when the p-value of the metabolic pathway was the metabolic pathway was considered to have experienced statistically significant enrichment.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJinting Luo: investigation, data curation, formal analysis, methodology, writing—original draft. Lei Wang: methodology, writing. Xuan Luo: methodology, writing. Jianbo Zhang: investigation, methodology. Tian Tian: writing—review and editing. Youli Yao: writing—review and editing. Dandan Luo: writing—review and editing. Guofang Wu: conceptualization, writing—review and editing, supervision. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the basic research project of Qinghai, grant number 2022-ZJ-752.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWu, D. D. et al. Convergent genomic signatures of high-altitude adaptation among domestic mammals. \u003cem\u003eNatl. Sci. 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Protoc.\u003c/em\u003e \u003cb\u003e5\u003c/b\u003e (6), 1005\u0026ndash;1018 (2010).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"RDP, PLCs, testosterone, antioxidant","lastPublishedDoi":"10.21203/rs.3.rs-5056340/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5056340/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHypoxia induces oxidative stress and cellular dysfunction. Rhodiola polysaccharide (RDP), a distinguished bioactive compound o\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ef\u003c/span\u003e \u003cem\u003eRhodiola rosea\u003c/em\u003e L., demonstrates strong antioxidant activity. Whether the RDP have protective effect on hypoxia injury of porcine Leydig cells (PLCs) merits further investigation. Our research showed that when RDP was introduced to PLCs under hypoxia condition, both the antioxidant capacity and testosterone (T) secretion of PLCs were enhanced. Notably, this treatment revealed a significant correlation between T levels and specific metabolites, suggesting RDP's role in diminishing reactive oxygen species and fortifying antioxidant defenses. Moreover, RDP promoted the synthesis of antioxidant metabolites and modulated pathways involved in unsaturated fatty acids and glutathione metabolism, mitigating oxidative stress. These results suggested that RDP could improve the cellular antioxidant capacity and stimulate T secretion of PLCs in hypoxia environment through multiple pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Rhodiola polysaccharides affect the antioxidant capacity and testosterone secretion of PLCs in hypoxia environment through the metabolism of unsaturated fatty acids and glutathione","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-10 12:42:09","doi":"10.21203/rs.3.rs-5056340/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"69a2c779-3e5f-4ea4-8158-ec29b422c987","owner":[],"postedDate":"December 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":40391223,"name":"Biological sciences/Cell biology/Glycobiology"},{"id":40391224,"name":"Biological sciences/Cell biology"}],"tags":[],"updatedAt":"2024-12-24T09:23:43+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-10 12:42:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5056340","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5056340","identity":"rs-5056340","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}