Scutellarein-containing novel formula attenuates hypoxia through inhibiting apoptosis

Scutellarein-containing novel formula attenuates hypoxia through inhibiting apoptosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Scutellarein-containing novel formula attenuates hypoxia through inhibiting apoptosis Bo Liu, Wei Zheng, Cuiyao Tang, Jing Lu, Mengyang Long, Han Li, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7839138/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background High-altitude hypoxia limits human performance, and existing interventions face limitations in efficacy, safety, or personalization. Our team developed a novel formula to enhance hypoxic tolerance. Methods Anti-hypoxia effects were evaluated using normobaric and acute hypoxia models. Blood bioactive components were profiled via LC–MS non-targeted metabolomics. Network pharmacology and molecular docking identified potential targets and pathways, with cellular experiments validating the effects of the key component. Results The formula significantly prolonged survival time under normobaric hypoxia and increased survival rate dose-dependently in acute hypoxia. Twenty-three prototype blood components were identified, with scutellarein as the primary constituent. Network analysis revealed 88 potential targets, primarily involving the PI3K-Akt pathway. Scutellarein showed strong binding to Hsp90 (docking score: –9.560 kcal/mol). Cellular assays confirmed its dose-dependent anti-hypoxic effect via reduced LDH release and apoptosis. Conclusion The novel formula may alleviate acute mountain sickness by improving hypoxic tolerance, likely through flavonoid-mediated inhibition of neuronal apoptosis. Health sciences/Diseases Biological sciences/Drug discovery Scutellarein hypoxia non-targeted metabolomics network pharmacology molecular docking apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction The development of China's extensive plateau regions is challenged by severe hypoxia, a key trigger of acute high-altitude diseases. Acute mountain sickness (AMS), the most common of these conditions, can progress to life-threatening high-altitude cerebral oedema (HACE) or high-altitude pulmonary oedemacerebral (HAPE) 1 . Current preventive strategies, including gradual acclimatization 2 and prophylactic drugs 3,4 , are constrained by slow efficacy, significant side effects, or a lack of personalization, failing to meet the need for rapid, large-scale personnel deployment. Here, we developed a novel formula, Gao Yuan No. 2 (GY2H), based on the "individual constitution identification" theory. To evaluated novel formula's hypoxia tolerance effects and elucidate the mechanisms, this study integrated approach combining murine hypoxia models, liquid chromatography‒mass spectrometry (LC-MS) metabolomics, network pharmacology, molecular docking, and cellular assays. 2. Results 2.1. GY2H significantly prolonged the hypoxia time of mice and showed a trend of increased survival rate under hypoxic conditions In the normobaric hypoxia experiment, survival time of the medium and high-dose GY2H groups was significantly extended by 32.4% and 44.6% (F=33.75, p ＜0.0001) ( Fig.1a) compared to the blank group. In the acute hypoxia experiment, the survival rate of the high-dose GY2H group was 50% (vs. 10% in the blank group, χ 2 =4.090, p =0.462) ( Fig.1b) . 2.2. Identification of 23 prototype blood components from GY2H, with flavonoids being the most prevalent GY2H was identified to contain a total of 1,026 bioactive components, including 1,7-Dihydroxy-3,5-dimethoxyxanthone, 2-Linoleoyl Glycerol, and 6-Hydroxykaempferol etc.. These ingredients were categorized, and the content of the top 10 ingredient categories as well as their number were visualized. Among the categories, 19.18% were flavonoids, 16.06% were lipids, and 10.34% were carbohydrates and derivatives in terms of content ( Fig.2a ). In terms of number, 14.38% of the drug constituents were terpenoids and 13.63% were flavonoids ( Fig.2b ). Flavonoids accounted for more than 10% in both content and number within GY2H, suggesting that the preventive effect of GY2H against AMS may primarily be mediated by its flavonoid components. A total of 2,431 blood bioactive ingredients, including 1,7-Dihydroxy-3,5-dimethoxyxanthone and 2-Linoleoyl Glycerol etc., were identified in the drug-containing serum. These ingredients were also categorized, and the content of the top 10 ingredient categories and the number of top 10 ingredient categories were visualized. Among the categories, 43.35% of the content consisted of lipids, 33.33% of steroids and steroid derivatives, and 10.21% of amino acids ( Fig.2c ). In terms of number, 22.22% were terpenoids, 19.19% were flavonoids, and 11.11% were lipids ( Fig.2d ). As in GY2H, flavonoids exceeded 10% in both content and number within the drug-containing serum, further supporting the hypothesis that GY2H may exert its preventive effect against AMS through flavonoid components. A Venn diagram comparing the blank serum group, GY2H group, and drug-containing serum group revealed 23 prototype blood components from GY2H, with flavonoids being the most prevalent. Among these flavonoids, the content of scutellarein is the highest ( Table 1) . 2.3. Identification of 88 potential molecular targets and selection of the PI3K-Akt signalling pathway for preventing AMS Prototype blood bioactive components were predicted to target 470 proteins, with Gene Cards and OMIM databases identifying 1,783 and 529 targets respectively, yielding 1,334 shared targets. Subsequent filtering revealed 88 potential AMS-preventive targets, from which a PPI network was constructed (87 nodes, 123 edges; average degree=2.83; PPI enrichment: p1, Closeness>0.06689, Degree>3, Eigenvector>0.059474, LAC>0.666667, Network>1.5), generating a refined subnetwork of 15 nodes and 69 edges ( Fig.3c ). Ultimately, the top-ranked targets PIK3CA , PIK3CD , HSP90AA1 , HSP90AB1 , and AKT1 were identified as key targets of AMS prevention. KEGG enrichment analysis identified 131 significant pathways, with the top 20 pathways by count value and top 20 by p.adjust value sharing 15 common terms ( Fig.3d,e ). Key targets ( PIK3CA , HSP90AA1 , PIK3CD , HSP90AB1 , and AKT1 ) were enriched in disease-related pathways including Pathways in cancer, PI3K-Akt signalling, Estrogen signalling, Fluid shear stress and atherosclerosis, and Prostate cancer. The PI3K-Akt signalling pathway was prioritized as the key candidate mechanism for further investigation. GO enrichment analysis yielded 3,295 significant terms, comprising 2,821 biological process (BP), 311 molecular function (MF), and 63 cellular component (CC) terms. Intersection analysis revealed: the top 18 BP terms by count value and top 20 by p.adjust value shared 8 overlapping terms ( Fig.3f,g ); the top 19 MF terms by count value and top 20 by p.adjust value shared 9 common terms ( Fig.3h,i ); while the top 20 CC terms for both count value and p.adjust value shared 7 consensus terms ( Fig.3j,k ). 2.4. Hsp90 exhibited the best binding affinity with scutellarein Molecular docking of scutellarein with selected receptors—Hsp90 (isoforms encoded by HSP90AA1/AB1 ), PI3K (catalytic subunits from PIK3CA/CD ), and Akt kinase—revealed high-affinity binding modes at active sites: Akt formed hydrogen bonds with Lys158/Glu228/Ala230 ( Fig.4a ); Hsp90 showed hydrogen bonding with Asp93 and π-stacking with Phe138 ( Fig.4b ); while PI3K engaged residues Asp836/Asp841/Tyr867/Val882 through hydrogen bonds ( Fig.4c ). All docking scores were below -5.0 Kcal/mol, indicating strong binding affinity, with scores comparable to or better than those of positive ligands. Notably, Hsp90 exhibited the best binding affinity with scutellarein, with a docking score of -9.560 Kcal/mol (Akt: -6.172 Kcal/mol, PI3K: -8.711 Kcal/mol). 2.5. Scutellarein dose-dependently reduced lactate dehydrogenase levels in HT22 cells and decreased their apoptosis rate The concentrations used in this experiment, there was no statistically significant difference between the scutellarein dose group and the control group; however, statistically significant differences were observed between the CoCl 2 groups and the control group. The viability of scutellarein groups was above 96.23%, indicating that scutellarein at various concentrations had no toxic effect on HT22 cell activity ( Fig.5a ), while the viability of HT22 cells at 100 μM CoCl 2 was 86.54% and the viability at other concentrations was less than54.7% ( Fig.5b ). Compared to the model group, all groups treated with scutellarein significantly reduced LDH concentration in HT22 cells under hypoxic conditions. Additionally, as the concentration of scutellarein increased, the LDH concentration showed a gradual decreasing trend ( Fig.5c ). Similarly, the apoptosis rate in the group treated with scutellarein was significantly lower than that of the model group, and it showed a concentration-dependent relationship ( Fig.5d ). 3. Discussion Our findings reveal that GY2H produces a dose-dependent increase in survival time during normobaric hypoxia experiment. The results from the acute hypoxia experiment further imply a potential dose-dependent enhancement in survival rate. The lack of statistical significance in the latter may be attributable to the limited sample size, and a study with greater statistical power is warranted to validate this trend. We employed LC-MS-based non-targeted metabolomics, identifying 23 prototype blood bioactive compounds in circulation. Flavonoids were predominant, with scutellarein (derived from scutellaria baicalensis Georgi) exhibiting the largest peak area among them. Notably, scutellarein, the primary metabolite of scutellarin, achieves its highest concentration in brain tissue and demonstrates significant neuroprotective potential, exceeding that of its precursor Scutellarin 5-8 . Literature suggests this neuroprotection may involve anti-apoptotic mechanisms 9-12 , which is particularly relevant given evidence implicating apoptosis as a critical pathological feature of AMS, especially within the first three days of high-altitude exposure 13,14 . We used network pharmacology and predicted PI3K-Akt signalling pathway as signalling pathway, recognized for its anti-apoptotic role, through which GY2H anti-apoptotic effects. Molecular docking indicated scutellarein possessed the highest binding affinity for Hsp90 among the compounds studied. Therefore, we postulate that GY2H prevents hypoxia, which is the initiating factor of AMS, through flavonoid components like scutellarein interacting with targets such as Hsp90.As oxygen deficiency is the primary trigger for AMS and no universally accepted animal model exists, evaluating drug efficacy often relies on assessing hypoxia resistance improvement 15 . Our in vitro studies demonstrated scutellarein's protective effects under hypoxic conditions. In HT22 cells, scutellarein reduced LDH release, indicating anti-hypoxic properties. Flow cytometry further revealed that scutellarein decreased the apoptosis rate in hypoxic cells. Collectively, these data support the anti-apoptotic and anti-hypoxic effects of scutellarein, aligning with the proposed mechanism for GY2H in AMS prevention. In conclusion, our findings suggest that the novel formula may prevent AMS by enhancing hypoxic tolerance, likely through the inhibition of neuronal apoptosis by its flavonoid constituents. 4. Methods 4.1. Experimental materials 4.1.1. Experimental animals SPF-grade healthy male C57BL/6J mice (8 weeks old, n = 136) were provided by Sichuan Viton Lihua Laboratory Animal Technology Co. Ltd (Animal License No.: SCXK (Chuan) 2023-0040). The mice were housed in the animal facility of the Second Affiliated Hospital of the Army Medical University under controlled conditions (temperature: 21–26°C, relative humidity: 50 ± 5%, 12-hour light/dark cycle). All animal experimental protocols were approved by the Laboratory Animal Welfare and Ethics Committee of the Army Medical University.(AMUWEC20245294). 4.1.2. Experimental drugs The GY2H formulation comprised fried sour jujube kernel (batch no. 2307191301), codonopsis pilosula (batch no. 2403093301), morinda officinalis (batch no. 2403250301), poria cocos (batch no. 2405074301), rhizoma Ligustici (batch no. 2307291301), ophiopogon japonicus (batch no. 2409432301), radix rehmanniae (batch no. 2310108301), vinegar schisandra (batch no. 2409136301), astragalus (batch no. 2406227301), and rhodiola rosea (batch no. 2308239301). Rhodiola rosea oral solution (approval number: B20070002). All experimental drugs were provided by the Pharmacy Department of the Second Affiliated Hospital of the Army Medical University. 4.1.3. Main equipment The main equipment included ProOx-811L hypobaric chamber(Shanghai TOW Intelligent Technology Co., Ltd), vanquish horizon UHPLC system (Thermo Scientific), Q-Exactive mass spectrometer (Thermo Scientific), 5430 R and 5424 R freezing centrifuges (Eppendorf), SBL-10TD temperature-controlled ultrasonic cleaner (Ningbo Xinzhi Bio-Tech Co., Ltd.), LNG-T88 benchtop rapid centrifuge concentrator and dryer (Taicang Huamei Biochemical Instrumentation Co., Ltd.), JXDC-20 nitrogen purging apparatus (Shanghai Jingshun Industrial Development Co., Ltd.), new classic MF MS105DU analytical balance (Mettler Toledo), gallios flow cytometer (Beckman Coulter), and BK-EL10A microplate reader (Biobase). 4.1.4. Main reagents and consumables The reagents and consumables included: carbon dioxide absorbent (Shanghai NaHui Drying Reagent Factory), glass wide-mouth vials (JUNLIBO Bio), methanol (Fisher Chemical), acetonitrile (Fisher Chemical), formic acid (CNW), acetic acid (CNW), ultrapure water (Fisher Chemical), isopropanol (Merck), 2-chloro-L-phenylalanine (Adamas-beta), scutellarein (≥98%; Macklin), cobaltous chloride (CoCl₂, 99.7%; Macklin), dulbecco’s modified eagle medium (DMEM; high glucose; Sperikon), fetal bovine serum (FBS; Kelsciences), Penicillin-Streptomycin-Amphotericin B solution (Biosharp), CCK-8 cell viability kit (CCK-8, BIOGROUND), lactate dehydrogenasedetection kit (LDH, JONLNBIO), annexin V-FITC/PI apoptosis kit (Life-iLab). Cell lines: HT22 immortalized mouse hippocampal neuronal cell line (HT22, iCell Bioscience Inc.). 4.1.5. Statistical analysis Statistical analyses were performed using SPSS (version 26.0). Data were analyzed by analysis of variance, with a p-value of less than 0.05 considered statistically significant. Significance levels are denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Figures were generated using GraphPad Prism (version 9.5) 4.2. Experimental methods 4.2.1. Murine hypoxia models evaluated novel formula's hypoxia tolerance effects One hundred mice were randomly assigned to two primary experimental cohorts: normobaric hypoxia and acute hypoxia (50 mice per cohort). Within each cohort, mice were randomly distributed into five treatment groups (n=10/group): a blank control group, a positive control group receiving Rhodiola rosea oral solution (3.25 ml/kg), and three intervention groups receiving GY2H at low, medium, or high doses. The GY2H doses were set in a 0.5:1:2 ratio, with the medium dose defined as 13.1 g/kg/day. All interventions were administered by daily gavage over 21 days, preceded by a 7-day acclimatization period. The corresponding hypoxia challenges were carried out within 24 hours following the last gavage, in accordance with previously described methodologies 16,17 . 4.2.2. LC-MS-based non-targeted metabolomics profiling of prototype blood bioactive components GY2H powder (50 mg) was weighed into a 2 mL centrifuge tube, to which 6 mm grinding beads and 400 µL of a methanol-water extraction solution (4:1, v / v, containing 0.02 mg/mL L-2-chlorophenylalanine and four other internal standards) were added 18,19 . Samples underwent cryogenic grinding (-10 °C, 6 min, 50 Hz), then sequential processing including: solvent extraction (-20 °C, 30 min), static extraction (20 °C, 30 min), ultrasonic-assisted extraction (5 °C, 30 min, 40 kHz), and incubation (-20 °C, 30 min). Finally, the extracts were centrifuged (4 °C, 15 min, 13,000 × g), and supernatants were transferred to amber LC-MS vials for analysis. A total of 36 mice were randomly and equally divided into two groups: a blank serum group and a drug-containing serum group. The drug-containing serum group received twice the standard daily mouse dosage of GY2H (13.1 g/kg). After a 7-day acclimatization period, all mice were administered their respective treatments by daily gavage for 7 consecutive days. On day 7, following a 12-hour fast, the mice were anesthetized with sodium pentobarbital (70 mg/kg, i.p.) and euthanized by decapitation. Blood was collected from the abdominal aorta into EP tubes without anticoagulant 20 . After static stratification, the tubes were centrifuged (4 °C, 10 min, 3000 rpm). The supernatant was aspirated into EP tubes, with sera from three mice in each group pooled into one serum sample. Prior to testing, 100 µL of the serum samples was mixed with 400 µL of extraction solution in 1.5 mL centrifuge tubes, then sequential processing including: vortexing (30 s), ultrasonic extraction (5 °C, 30 min, 40 kHz), static placement( -20 °C, 30 min), centrifugation ( 4 °C and 13,000 × g, 15 min).The supernatant was dried under nitrogen. The sample was then reconstituted in 120 µL of acetonitrile-water (1:1, v / v), vortexed (30 s), ultrasonicated (5 °C, 5 min, 40 kHz), and centrifuged (4 °C, 10 min, 13,000 × g). The final supernatant was transferred to an LC-MS vial for analysis. Chromatographic conditions: an ACQUITY UPLC BEH C18 column was employed for chromatographic separation. Mobile phase A consisted of 2% acetonitrile in water with 0.1% formic acid, while mobile phase B was acetonitrile containing 0.1% formic acid. The flow rate was set at 0.40 mL/min, and the column temperature was maintained at 40°C. An injection volume of 3 μL was used for each analysis. Mass spectrometry conditions: mass spectrometric acquisition was performed in both positive and negative ion scanning modes. The mass-to-charge ratio (m / z) scanning range was 70-1050. The spray voltage was 3500 V in positive mode and -3000 V in negative mode. The sheath gas flow rate was set to 50 arbitrary units (arb), and the auxiliary gas flow rate was 13 arb. The heating temperature was maintained at 450°C, with a collision energy of 20%, 40%, and 60%. The resolution for Full MS was 70,000, and for MS2 it was 17,500. The capillary temperature was set at 320°C, and the S-Lens voltage was 40. The data was imported into progenesis QI v3.0 (Waters Corporation, Milford, USA), where it underwent baseline filtering, peak identification, integration, retention time correction, and peak alignment to produce a data matrix 18 .The data matrix was uploaded to the Meiji cloud platform 21 (https://cloud.majorbio.com/) for further analysis. The prototype blood bioactive components of GY2H was identified using the jvenn tool 22 (https://jvenn.soulouse.inrae.fr/app/example.html) 4.2.3. Network pharmacology-based prediction of molecular targets and signalling pathways Firstly, based on the prototype blood bioactive components, target prediction was performed using Swiss Target Prediction 23 (http: //www.swisstargetprediction.ch/), with screening based on a probability threshold of > 0. The resulting predicted targets were then standardized through the Uniprot database 24 (https: //www.uniprot.org/), followed by removing duplicate entries. Secondly, search terms including “acute high altitude disease,” “acute mountain sickness,” “altitude headache,” and “high altitude sickness” were used in the Gene Card 25 (https://www.genecards.org/) and OMIM 26 (https://mirror.omim.org/) databases. The predicted targets from both databases were merged and duplicates were removed. Finally, the Venn diagram illustrating the overlap between GY2H prototype blood bioactive components -related targets and AMS-related targets was created using the jvenn tool 22 (https://jvenn.soulouse.inrae.fr/app/example.html). The results were imported into the Multiple Proteins module of the online STRING database 27 (https://cn.string-db.org), selecting “Homo sapiens” for protein type, setting the minimum required interaction score to 0.9, and hiding disconnected nodes in the network. The interaction data were exported in .tsv format and imported into Cytoscape_v3.10.2 for visualization and analysis using the CytoNCA-2.1.6 plugin. Key molecular targets were identified by filtering nodes with values above the median for six centrality metrics: Betweenness, Closeness, Degree, Eigenvector, LAC, and Network. These targets inform the pathway analysis of GY2H's AMS prevention mechanism. KEGG and GO enrichment analyses were performed using the SangerBox Biomedical Data Analysis Box 28 (http://sangerbox.com/home.html). Significantly enriched terms were filtered based on count values and p.adjust value, with high-count/low-p.adjust terms prioritized as candidate pathways. 4.2.4. Molecular docking-based verification of primary blood bioactive component-target affinity Molecular docking was executed in Discovery Studio 2019 using primary blood bioactive components as ligands and key molecular targets as receptors. The target protein structures, retrieved from the PDB database 29 (https://www.rcsb.org/), underwent hydrogenation, structural optimization, and energy minimization after removal of redundant conformers. Ligands were prepared with OPLS3e force field parameters involving acid-base neutralization and generation of stereoisomeric conformations for 3D structural refinement. The C-Docker algorithm subsequently docked the processed ligands to receptors, with binding affinity quantified by the docking score of the optimal complex conformation. 4.2.5. Cellular-based validation of scutellarein’s anti-hypoxic effects and underlying mechanisms HT22 cells were grown in DMEM media supplemented with 10% FBS and 1% Penicillin- Streptomycin-Amphotericin B Solution at 37 ℃ and 5% CO 2 condition. The cells were sub-cultured in a rate of 1:3 with the culture medium being replaced every 2 days. After growing to 80% confluence, the cells were used for experiment. Firstly, cell viability were evaluated by treatment with CoCl 2 (100, 150, 200 μM ) or Scutellarein (20, 40, 80 μM) for 24 h. Cell viability was detected by CCK-8 assay. HT22 cells were inoculated with a density of 5 × 10 3 cells/well in 96-well plates 30 . After 24 h, CCK-8 (10 µl per well) was added and cells were cultured at 37 ℃ for 3 h. Microplate reader was used to detect cell viability at 450 nm. The lower cytotoxic concentration of scutellarein and CoCl 2 were selected for subsequent experiments. Secondly, HT22 cells were inoculated with a density of 5.0 × 10 3 cells/well in 96-well plates, cells were divided into blank group, scutellarein group, with model group treated with CoCl 2 (100μM) and scutellarein group treated with CoCl 2 (100μM) + scutellarein (20, 40 and 80 μM) for 24 h. After 24 h, added relevant reagents according to the manual's instructions, microplate reader was used to detect the LDH concentration at 450 nm or Gallios flow cytometer was used to detect the rate of apoptosis. Declarations Data availability statement The datasets generated during or analysed during the current study are available from the corresponding author on reasonable request Acknowledgements Not Applicable. Authors' contributions BL: Data curation, Formal analysis(equal), Investigation, Methodology, Visualization (equal) ; and Writing – original draft. WZ: Writing – review & editing(equal), Resources, Visualization(equal). CYT: Writing – review & editing(equal), Formal Analysis(equal), Visualization (equal). JL: Writing – review & editing (equal). MYL: Writing – review & editing (equal). HL: Writing – review & editing (equal). YQW: Project administration, Funding acquisition, Supervision and Writing – review & editing (equal). ZHX: Conceptualization, Funding acquisition, Supervision and Writing – review & editing (equal). Additional Information Competing interests The author(s) declare no competing interests. Consent to publish Not applicable. Funding This study was supported by the Chongqing Yingcai Master and Teacher Programme (Grant numbers [CQYC20220203145]) and the Youth Doctoral Incubation Program of the Second Affiliated Hospital of Army Medical University (Grant numbers [2024YQB007]). Clinical t rial n umber Not applicable. References Jin, J. Acute Mountain Sickness. Jama 318 , doi:10.1001/jama.2017.16077 (2017). 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Phytomedicine : international journal of phytotherapy and phytopharmac ology 109 , 154568, doi:10.1016/j.phymed.2022.154568. Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 15 Oct, 2025 Editor assigned by journal 14 Oct, 2025 Submission checks completed at journal 14 Oct, 2025 First submitted to journal 12 Oct, 2025 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-7839138","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":528264860,"identity":"9bf2bbfa-1068-4fca-8201-45113208b938","order_by":0,"name":"Bo Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYBACeWbm4x8+/rGp5+dvPkCcFsP2tjTGmQ1pCZIzjiUQac2ZM2bMvA2HEwwO5BgQp4NxRo7ZY94dzHkGB858vPGGwU5Ot4GAFnaJtHLDuWfYiiUP9262nMOQbGx2gKAtyRsk3rDxMPYdOLtNmofhQOI2QloYbiQYSPCwSTA2HMh5RqSWM0fMJHnbDBInHMhhI04LMJCTDWecSTAGBrKx5RwDIvwCjMqDDz5U/JcDRuXDG28q7OQIakEBEjxERg2yFlJ1jIJRMApGwYgAAEZiSU+5oO7XAAAAAElFTkSuQmCC","orcid":"","institution":"The Second Affiliated Hospital of Army Medical University","correspondingAuthor":true,"prefix":"","firstName":"Bo","middleName":"","lastName":"Liu","suffix":""},{"id":528264861,"identity":"d6abc44d-e377-4187-a1f8-11998c9e7ca6","order_by":1,"name":"Wei Zheng","email":"","orcid":"","institution":"The Second Affiliated Hospital of Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Zheng","suffix":""},{"id":528264862,"identity":"90b05878-b9c4-4704-9ecd-9b735bb8ece8","order_by":2,"name":"Cuiyao Tang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Cuiyao","middleName":"","lastName":"Tang","suffix":""},{"id":528264863,"identity":"c585ff7f-b801-4798-84ac-7efed2d4cbdd","order_by":3,"name":"Jing Lu","email":"","orcid":"","institution":"The Second Affiliated Hospital of Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Lu","suffix":""},{"id":528264864,"identity":"a3cb093c-b17d-434c-8a2c-9e550e22c5ed","order_by":4,"name":"Mengyang Long","email":"","orcid":"","institution":"The Second Affiliated Hospital of Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mengyang","middleName":"","lastName":"Long","suffix":""},{"id":528264865,"identity":"56ab80cc-0db3-476f-a7ec-41084bb34ce3","order_by":5,"name":"Han Li","email":"","orcid":"","institution":"The Second Affiliated Hospital of Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Han","middleName":"","lastName":"Li","suffix":""},{"id":528264866,"identity":"e92e6e0f-1cb1-4f40-a02e-9f045083407c","order_by":6,"name":"Yunqiao Wang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yunqiao","middleName":"","lastName":"Wang","suffix":""},{"id":528264869,"identity":"1667fdc9-6016-4cd0-9523-5057f8fa7a75","order_by":7,"name":"Zihui Xu","email":"","orcid":"","institution":"The Second Affiliated Hospital of Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zihui","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2025-10-12 07:53:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7839138/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7839138/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":93466932,"identity":"fb112ec0-713e-4e7d-a58c-68a625f494ad","added_by":"auto","created_at":"2025-10-14 07:35:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":21613,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of hypoxia tolerance in mice treated with GY2H. a Hypoxia tolerance time of mice exposed to normobaric hypoxi, the GY2H low-dose group increased survival time by 14.4% (*\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05), the GY2H medium-dose group by 32.4% (****\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001), and the GY2H high-dose group by 44.6% (****\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001); b Survival rates of mice following acute hypoxic, the survival rate of the blank group was 10%, the Rhodiola rosea group was 30%, the GY2H low-dose group was 30%, the GY2H medium-dose group was 40%, and the GY2H high-dose group was 50% (\u003cem\u003ep \u003c/em\u003e＞ 0.05)\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7839138/v1/e10772a9f0d8f4db830a24e7.png"},{"id":93467839,"identity":"b3b12276-8e6d-4d2c-927b-7672cf9ad9ff","added_by":"auto","created_at":"2025-10-14 07:43:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":85636,"visible":true,"origin":"","legend":"\u003cp\u003eThe results of LC-MS Analysis. a Top 10 chemical classes in GY2H ranked by relative abundance; b Top 10 chemical classes by GY2H component count; c Top 10 chemical classes in serum ranked by relative abundance ; d Top 10 chemical classes by serum component count\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7839138/v1/104aa23a89b7a071bedd608f.png"},{"id":93466937,"identity":"9f51b2c4-2b30-409e-8bca-3e0d8934ce0c","added_by":"auto","created_at":"2025-10-14 07:35:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1193206,"visible":true,"origin":"","legend":"\u003cp\u003eThe results of network pharmacology. a Candidate targets identified by GY2H screening; b Protein-protein interaction (PPI) network of candidate core targets; c Subnetwork of key targets; d KEGG: Top 20 pathways by gene count; e KEGG: Top 20 pathways by adjusted p-value; f GO-BP: Top 18 terms by gene count; g GO-BP: Top 20 terms by adjusted p-value; h GO-CC: Top 20 terms by gene count; i GO-CC: Top 20 terms by adjusted p-value; j GO-MF: Top 19 terms by gene count; k GO-MF: Top 20 terms by adjusted p-value.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7839138/v1/5fc28c0219879223fd95d510.png"},{"id":93466935,"identity":"15ac0768-45b4-4bcb-9d29-54c030253fd6","added_by":"auto","created_at":"2025-10-14 07:35:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1864879,"visible":true,"origin":"","legend":"\u003cp\u003eThe results of molecular docking analysis. a Scutellarein-Akt complex; b Scutellarein-Hsp90 complex; c Scutellarein-PI3K complex. (Left: 2D ligand-protein interaction diagram; Center: Ligand binding pose on protein surface; Right: 3D binding conformation within the protein binding pocket. Interactions: Hydrogen bonds (yellow dashed lines); π-Interactions (green dashed lines).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7839138/v1/f2fa82ab3c0d53452a954d1d.png"},{"id":93466933,"identity":"230c49e9-f601-4720-ae30-72d9cd7e647c","added_by":"auto","created_at":"2025-10-14 07:35:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":252710,"visible":true,"origin":"","legend":"\u003cp\u003eThe results of cellular experiment. a Treatment with Scutellarein (20, 40, 80 μM) did not significantly alter HT22 cell viability compared to control, n=4, P=0.1754; b Treatment with CoCl\u003csub\u003e2\u003c/sub\u003e (100, 150, 200 μM) significantly decrease HT22 cell viability compared to control, n=4, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. control group; ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 vs. control group. c Scutellarein attenuates hypoxia-induced LDH release in HT22 cells, n=3, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 vs. model group; D Scutellarein attenuates hypoxia-induced apoptosis in HT22 cells, n=3, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 and **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 vs. model group\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7839138/v1/636b6b199fc6b4e9116a716a.png"},{"id":93468097,"identity":"8708fa75-a559-49c0-a9b8-a5b57a7999ce","added_by":"auto","created_at":"2025-10-14 07:51:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4045955,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7839138/v1/37d5a93a-a4c6-4007-b4ab-d070e691b5e5.pdf"},{"id":93466936,"identity":"0b532157-3eec-46c0-94ff-cf381e5b585e","added_by":"auto","created_at":"2025-10-14 07:35:56","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":155877,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7839138/v1/7cb30c5a4ce40d18287e1547.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Scutellarein-containing novel formula attenuates hypoxia through inhibiting apoptosis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe development of China\u0026apos;s extensive plateau regions is challenged by severe hypoxia, a key trigger of acute high-altitude diseases. Acute mountain sickness (AMS), the most common of these conditions, can progress to life-threatening high-altitude cerebral oedema (HACE) or high-altitude pulmonary oedemacerebral (HAPE)\u003csup\u003e1\u003c/sup\u003e. Current preventive strategies, including gradual acclimatization\u003csup\u003e2\u003c/sup\u003e and prophylactic drugs\u003csup\u003e3,4\u003c/sup\u003e, are constrained by slow efficacy, significant side effects, or a lack of personalization, failing to meet the need for rapid, large-scale personnel deployment. Here, we developed a novel formula, Gao Yuan No. 2 (GY2H), based on the \u0026quot;individual constitution identification\u0026quot; theory. To evaluated novel formula\u0026apos;s hypoxia tolerance effects and elucidate the mechanisms, this study integrated approach combining murine hypoxia models, liquid chromatography‒mass spectrometry (LC-MS) metabolomics, network pharmacology, molecular docking, and cellular assays.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e2.1. GY2H significantly prolonged the hypoxia time of mice and showed a trend of increased survival rate under hypoxic conditions\u003c/p\u003e\n\u003cp\u003eIn the normobaric hypoxia experiment, survival time of the medium and high-dose GY2H groups was significantly extended by 32.4% and 44.6% (F=33.75, \u003cem\u003ep\u003c/em\u003e＜0.0001) (\u003cstrong\u003eFig.1a)\u003c/strong\u003e compared to the blank group. In the acute hypoxia experiment, the survival rate of the high-dose GY2H group was 50% (vs. 10% in the blank group,\u0026nbsp;\u0026chi;\u003csup\u003e2\u003c/sup\u003e=4.090, \u003cem\u003ep\u003c/em\u003e=0.462) (\u003cstrong\u003eFig.1b)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.2. Identification of 23 prototype blood components from GY2H, with flavonoids being the most prevalent\u003c/p\u003e\n\u003cp\u003eGY2H was identified to contain a total of 1,026 bioactive components, including 1,7-Dihydroxy-3,5-dimethoxyxanthone, 2-Linoleoyl Glycerol, and 6-Hydroxykaempferol etc.. These ingredients were categorized, and the content of the top 10 ingredient categories as well as their number were visualized. Among the categories, 19.18% were flavonoids, 16.06% were lipids, and 10.34% were carbohydrates and derivatives in terms of content (\u003cstrong\u003eFig.2a\u003c/strong\u003e). In terms of number, 14.38% of the drug constituents were terpenoids and 13.63% were flavonoids\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003eFig.2b\u003c/strong\u003e). Flavonoids accounted for more than 10% in both content and number within GY2H, suggesting that the preventive effect of GY2H against AMS may primarily be mediated by its flavonoid components.\u0026nbsp;A total of 2,431 blood bioactive ingredients, including 1,7-Dihydroxy-3,5-dimethoxyxanthone and 2-Linoleoyl Glycerol etc., were identified in the drug-containing serum. These ingredients were also categorized, and the content of the top 10 ingredient categories and the number of top 10 ingredient categories were visualized. Among the categories, 43.35% of the content consisted of lipids, 33.33% of steroids and steroid derivatives, and 10.21% of amino acids\u0026nbsp;(\u003cstrong\u003eFig.2c\u003c/strong\u003e). In terms of number, 22.22% were terpenoids, 19.19% were flavonoids, and 11.11% were lipids\u0026nbsp;(\u003cstrong\u003eFig.2d\u003c/strong\u003e). As in GY2H, flavonoids exceeded 10% in both content and number within the drug-containing serum, further supporting the hypothesis that GY2H may exert its preventive effect against AMS through flavonoid components.\u003c/p\u003e\n\u003cp\u003eA Venn diagram comparing the blank serum group, GY2H group, and drug-containing serum group revealed 23 prototype blood components from GY2H, with flavonoids being the most prevalent. Among these flavonoids, the content of scutellarein is the highest \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eTable\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;1)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e2.3. Identification of 88 potential molecular targets and selection of the PI3K-Akt signalling pathway for preventing AMS\u003c/p\u003e\n\u003cp\u003ePrototype blood bioactive components were predicted to target 470 proteins, with Gene Cards and OMIM databases identifying 1,783 and 529 targets respectively, yielding 1,334 shared targets. Subsequent filtering revealed 88 potential AMS-preventive targets, from which a PPI network was constructed (87 nodes, 123 edges; average degree=2.83; PPI enrichment: p\u0026lt;1.0\u0026times;10⁻\u0026sup1;⁶) (\u003cstrong\u003eFig.3a,b\u003c/strong\u003e). Core targets were extracted using multi-parameter thresholds (Betweenness\u0026gt;1, Closeness\u0026gt;0.06689, Degree\u0026gt;3, Eigenvector\u0026gt;0.059474, LAC\u0026gt;0.666667, Network\u0026gt;1.5), generating a refined subnetwork of 15 nodes and 69 edges\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003eFig.3c\u003c/strong\u003e). Ultimately, the top-ranked targets \u003cem\u003ePIK3CA\u003c/em\u003e, \u003cem\u003ePIK3CD\u003c/em\u003e, \u003cem\u003eHSP90AA1\u003c/em\u003e, \u003cem\u003eHSP90AB1\u003c/em\u003e, and \u003cem\u003eAKT1\u003c/em\u003e were identified as key targets of AMS prevention.\u003c/p\u003e\n\u003cp\u003eKEGG enrichment analysis identified 131 significant pathways, with the top 20 pathways by count value and top 20 by p.adjust value sharing 15 common terms (\u003cstrong\u003eFig.3d,e\u003c/strong\u003e). Key targets (\u003cem\u003ePIK3CA\u003c/em\u003e, \u003cem\u003eHSP90AA1\u003c/em\u003e, \u003cem\u003ePIK3CD\u003c/em\u003e, \u003cem\u003eHSP90AB1\u003c/em\u003e, and \u003cem\u003eAKT1\u003c/em\u003e) were enriched in disease-related pathways including Pathways in cancer, PI3K-Akt signalling, Estrogen signalling, Fluid shear stress and atherosclerosis, and Prostate cancer. The PI3K-Akt signalling pathway was prioritized as the key candidate mechanism for further investigation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGO enrichment analysis yielded 3,295 significant terms, comprising 2,821 biological process (BP), 311 molecular function (MF), and 63 cellular component (CC) terms. Intersection analysis revealed: the top 18 BP terms by count value and top 20 by p.adjust value shared 8 overlapping terms (\u003cstrong\u003eFig.3f,g\u003c/strong\u003e); the top 19 MF terms by count value and top 20 by p.adjust value shared 9 common terms (\u003cstrong\u003eFig.3h,i\u003c/strong\u003e); while the top 20 CC terms for both count value and p.adjust value shared 7 consensus terms (\u003cstrong\u003eFig.3j,k\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e2.4. Hsp90 exhibited the best binding affinity with scutellarein\u003c/p\u003e\n\u003cp\u003eMolecular docking of scutellarein with selected receptors\u0026mdash;Hsp90 (isoforms encoded by \u003cem\u003eHSP90AA1/AB1\u003c/em\u003e), PI3K (catalytic subunits from \u003cem\u003ePIK3CA/CD\u003c/em\u003e), and Akt kinase\u0026mdash;revealed high-affinity binding modes at active sites: Akt formed hydrogen bonds with Lys158/Glu228/Ala230 (\u003cstrong\u003eFig.4a\u003c/strong\u003e); Hsp90 showed hydrogen bonding with Asp93 and \u0026pi;-stacking with Phe138 (\u003cstrong\u003eFig.4b\u003c/strong\u003e); while PI3K engaged residues Asp836/Asp841/Tyr867/Val882 through hydrogen bonds (\u003cstrong\u003eFig.4c\u003c/strong\u003e). All docking scores were below -5.0 Kcal/mol, indicating strong binding affinity, with scores comparable to or better than those of positive ligands. Notably, Hsp90 exhibited the best binding affinity with scutellarein, with a docking score of -9.560 Kcal/mol (Akt: -6.172 Kcal/mol, PI3K: -8.711 Kcal/mol).\u003c/p\u003e\n\u003cp\u003e2.5. Scutellarein dose-dependently reduced lactate dehydrogenase levels in HT22 cells and decreased their apoptosis rate\u003c/p\u003e\n\u003cp\u003eThe concentrations used in this experiment, there was no statistically significant difference between the scutellarein dose group and the control group; however, statistically significant differences were observed between the CoCl\u003csub\u003e2\u003c/sub\u003e groups and the control group. The viability of scutellarein groups was above 96.23%, indicating that scutellarein at various concentrations had no toxic effect on HT22 cell activity (\u003cstrong\u003eFig.5a\u003c/strong\u003e), while the viability of HT22 cells at 100 \u0026mu;M CoCl\u003csub\u003e2\u003c/sub\u003e was 86.54% and the viability at other concentrations was less than54.7% (\u003cstrong\u003eFig.5b\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eCompared to the model group, all groups treated with scutellarein significantly reduced LDH concentration in HT22 cells under hypoxic conditions. Additionally, as the concentration of scutellarein increased, the LDH concentration showed a gradual decreasing trend (\u003cstrong\u003eFig.5c\u003c/strong\u003e). Similarly, the apoptosis rate in the group treated with scutellarein was significantly lower than that of the model group, and it showed a concentration-dependent relationship (\u003cstrong\u003eFig.5d\u003c/strong\u003e).\u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eOur findings reveal that GY2H produces a dose-dependent increase in survival time during normobaric hypoxia experiment. The results from the acute hypoxia experiment further imply a potential dose-dependent enhancement in survival rate. The lack of statistical significance in the latter may be attributable to the limited sample size, and a study with greater statistical power is warranted to validate this trend. We employed LC-MS-based non-targeted metabolomics, identifying 23 prototype blood bioactive compounds in circulation. Flavonoids were predominant, with scutellarein (derived from scutellaria baicalensis Georgi) exhibiting the largest peak area among them. Notably, scutellarein, the primary metabolite of scutellarin, achieves its highest concentration in brain tissue and demonstrates significant neuroprotective potential, exceeding that of its precursor Scutellarin\u003csup\u003e5-8\u003c/sup\u003e. Literature suggests this neuroprotection may involve anti-apoptotic mechanisms \u003csup\u003e9-12\u003c/sup\u003e, which is particularly relevant given evidence implicating apoptosis as a critical pathological feature of AMS, especially within the first three days of high-altitude exposure \u003csup\u003e13,14\u003c/sup\u003e. We used network pharmacology and predicted PI3K-Akt signalling pathway as signalling pathway, recognized for its anti-apoptotic role, through which GY2H anti-apoptotic effects. Molecular docking indicated scutellarein possessed the highest binding affinity for Hsp90 among the compounds studied. Therefore, we postulate that GY2H\u0026nbsp;prevents\u0026nbsp;hypoxia, which is\u0026nbsp;the initiating factor of\u0026nbsp;AMS, through flavonoid components like scutellarein interacting with targets such as Hsp90.As oxygen deficiency is the primary trigger for AMS and no universally accepted animal model exists, evaluating drug efficacy often relies on assessing hypoxia resistance improvement\u003csup\u003e15\u003c/sup\u003e. Our in vitro studies demonstrated scutellarein\u0026apos;s protective effects under hypoxic conditions. In HT22 cells, scutellarein reduced LDH release, indicating anti-hypoxic properties. Flow cytometry further revealed that scutellarein decreased the apoptosis rate in hypoxic cells. Collectively, these data support the anti-apoptotic and anti-hypoxic effects of scutellarein, aligning with the proposed mechanism for GY2H in AMS prevention.\u0026nbsp;In conclusion, our findings suggest that the novel formula may prevent AMS by enhancing hypoxic tolerance, likely through the inhibition of neuronal apoptosis by its flavonoid constituents.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"4. Methods","content":"\u003cp\u003e4.1. Experimental materials\u003c/p\u003e\n\u003cp\u003e4.1.1. Experimental animals\u003c/p\u003e\n\u003cp\u003eSPF-grade healthy male C57BL/6J mice (8 weeks old, n = 136) were provided by Sichuan Viton Lihua Laboratory Animal Technology Co. Ltd (Animal License No.: SCXK (Chuan) 2023-0040). The mice were housed in the animal facility of the Second Affiliated Hospital of the Army Medical University under controlled conditions (temperature: 21\u0026ndash;26\u0026deg;C, relative humidity: 50 \u0026plusmn; 5%, 12-hour light/dark cycle). All animal experimental protocols were approved by the Laboratory Animal Welfare and Ethics Committee of the Army Medical University.(AMUWEC20245294).\u003c/p\u003e\n\u003cp\u003e4.1.2. Experimental drugs\u003c/p\u003e\n\u003cp\u003eThe GY2H formulation comprised fried sour jujube kernel (batch no. 2307191301), codonopsis pilosula (batch no. 2403093301), morinda officinalis (batch no. 2403250301), poria cocos (batch no. 2405074301), rhizoma Ligustici (batch no. 2307291301), ophiopogon japonicus (batch no. 2409432301), radix rehmanniae (batch no. 2310108301), vinegar schisandra (batch no. 2409136301), astragalus (batch no. 2406227301), and rhodiola rosea (batch no. 2308239301). Rhodiola rosea oral solution (approval number: B20070002). All experimental drugs were provided by the Pharmacy Department of the Second Affiliated Hospital of the Army Medical University.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4.1.3. Main equipment\u003c/p\u003e\n\u003cp\u003eThe main equipment included ProOx-811L hypobaric chamber(Shanghai TOW Intelligent Technology Co., Ltd), vanquish\u0026nbsp;horizon UHPLC system (Thermo Scientific), Q-Exactive mass spectrometer (Thermo Scientific), 5430 R and 5424 R freezing centrifuges (Eppendorf), SBL-10TD temperature-controlled ultrasonic cleaner (Ningbo Xinzhi Bio-Tech Co., Ltd.), LNG-T88 benchtop rapid centrifuge concentrator and dryer (Taicang Huamei Biochemical Instrumentation Co., Ltd.), JXDC-20 nitrogen purging apparatus (Shanghai Jingshun Industrial Development Co., Ltd.),\u0026nbsp;new\u0026nbsp;classic MF MS105DU analytical balance (Mettler Toledo),\u0026nbsp;gallios flow cytometer (Beckman Coulter), and BK-EL10A microplate reader (Biobase).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4.1.4. Main reagents and consumables\u003c/p\u003e\n\u003cp\u003eThe reagents and consumables included: carbon dioxide absorbent (Shanghai NaHui Drying Reagent Factory), glass wide-mouth vials (JUNLIBO Bio), methanol (Fisher Chemical), acetonitrile (Fisher Chemical), formic acid (CNW), acetic acid (CNW), ultrapure water (Fisher Chemical), isopropanol (Merck), 2-chloro-L-phenylalanine (Adamas-beta), scutellarein (\u0026ge;98%; Macklin), cobaltous chloride (CoCl₂, 99.7%; Macklin),\u0026nbsp;dulbecco\u0026rsquo;s\u0026nbsp;modified\u0026nbsp;eagle\u0026nbsp;medium (DMEM;\u0026nbsp;high glucose; Sperikon), fetal bovine serum (FBS; Kelsciences), Penicillin-Streptomycin-Amphotericin B solution (Biosharp), CCK-8 cell viability kit (CCK-8,\u0026nbsp;BIOGROUND), lactate dehydrogenasedetection kit (LDH,\u0026nbsp;JONLNBIO),\u0026nbsp;annexin V-FITC/PI apoptosis kit (Life-iLab). Cell lines: HT22 immortalized mouse hippocampal neuronal cell line (HT22,\u0026nbsp;iCell Bioscience Inc.).\u003c/p\u003e\n\u003cp\u003e4.1.5. Statistical analysis\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using SPSS (version 26.0). Data were analyzed by analysis of variance, with a p-value of less than 0.05 considered statistically significant. Significance levels are denoted as follows: *\u0026nbsp;\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u0026nbsp;\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u0026nbsp;\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, and ****\u0026nbsp;\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. Figures were generated using GraphPad Prism (version 9.5)\u003c/p\u003e\n\u003cp\u003e4.2. Experimental methods\u003c/p\u003e\n\u003cp\u003e4.2.1. Murine hypoxia models evaluated novel formula\u0026apos;s hypoxia tolerance effects\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOne hundred mice were randomly assigned to two primary experimental cohorts: normobaric hypoxia and acute hypoxia (50 mice per cohort). Within each cohort, mice were randomly distributed into five treatment groups (n=10/group): a blank control group, a positive control group receiving Rhodiola rosea oral solution (3.25 ml/kg), and three intervention groups receiving GY2H at low, medium, or high doses. The GY2H doses were set in a 0.5:1:2 ratio, with the medium dose defined as 13.1 g/kg/day. All interventions were administered by daily gavage over 21 days, preceded by a 7-day acclimatization period. The corresponding hypoxia challenges were carried out within 24 hours following the last gavage, in accordance with previously described methodologies \u003csup\u003e16,17\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e4.2.2. LC-MS-based non-targeted metabolomics profiling of prototype blood bioactive components\u003c/p\u003e\n\u003cp\u003eGY2H powder (50 mg) was weighed into a 2 mL centrifuge tube, to which 6 mm grinding beads and 400 \u0026micro;L of a methanol-water extraction solution (4:1, v / v, containing 0.02 mg/mL L-2-chlorophenylalanine and four other internal standards) were added\u003csup\u003e18,19\u003c/sup\u003e. Samples underwent cryogenic grinding (-10 \u0026deg;C, 6 min, 50 Hz), then sequential processing including: solvent extraction (-20 \u0026deg;C, 30 min), static extraction (20 \u0026deg;C, 30 min), ultrasonic-assisted extraction (5 \u0026deg;C, 30 min, 40 kHz), and incubation (-20 \u0026deg;C, 30 min). Finally, the extracts were centrifuged (4 \u0026deg;C, 15 min, 13,000 \u0026times; g), and supernatants were transferred to amber LC-MS vials for analysis.\u003c/p\u003e\n\u003cp\u003eA total of 36 mice were randomly and equally divided into two groups: a blank serum group and a drug-containing serum group. The drug-containing serum group received twice the standard daily mouse dosage of GY2H (13.1 g/kg). After a 7-day acclimatization period, all mice were administered their respective treatments by daily gavage for 7 consecutive days. On day 7, following a 12-hour fast, the mice were anesthetized with sodium pentobarbital (70 mg/kg, i.p.) and euthanized by decapitation. Blood was collected from the abdominal aorta into EP tubes without anticoagulant\u003csup\u003e20\u003c/sup\u003e. After static stratification, the tubes were centrifuged (4 \u0026deg;C, 10 min, 3000 rpm). The supernatant was aspirated into EP tubes, with sera from three mice in each group pooled into one serum sample.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrior to testing, 100 \u0026micro;L of the serum samples was mixed with 400 \u0026micro;L of extraction solution in 1.5 mL centrifuge tubes, then sequential processing including: vortexing (30 s), ultrasonic extraction (5 \u0026deg;C, 30 min, 40 kHz), static placement( -20 \u0026deg;C, 30 min), centrifugation ( 4 \u0026deg;C and 13,000 \u0026times; g, 15 min).The supernatant was dried under nitrogen. The sample was then reconstituted in 120 \u0026micro;L of acetonitrile-water (1:1, v / v), vortexed (30 s), ultrasonicated (5 \u0026deg;C, 5 min, 40 kHz), and centrifuged (4 \u0026deg;C, 10 min, 13,000 \u0026times; g). The final supernatant was transferred to an LC-MS vial for analysis.\u003c/p\u003e\n\u003cp\u003eChromatographic conditions: an ACQUITY UPLC BEH C18 column was employed for chromatographic separation. Mobile phase A consisted of 2% acetonitrile in water with 0.1% formic acid, while mobile phase B was acetonitrile containing 0.1% formic acid. The flow rate was set at 0.40 mL/min, and the column temperature was maintained at 40\u0026deg;C. An injection volume of 3 \u0026mu;L was used for each analysis.\u003c/p\u003e\n\u003cp\u003eMass spectrometry conditions: mass spectrometric acquisition was performed in both positive and negative ion scanning modes. The mass-to-charge ratio (m / z) scanning range was 70-1050. The spray voltage was 3500 V in positive mode and -3000 V in negative mode. The sheath gas flow rate was set to 50 arbitrary units (arb), and the auxiliary gas flow rate was 13 arb. The heating temperature was maintained at 450\u0026deg;C, with a collision energy of 20%, 40%, and 60%. The resolution for Full MS was 70,000, and for MS2 it was 17,500. The capillary temperature was set at 320\u0026deg;C, and the S-Lens voltage was 40.\u003c/p\u003e\n\u003cp\u003eThe data was imported into progenesis QI v3.0 (Waters Corporation, Milford, USA), where it underwent baseline filtering, peak identification, integration, retention time correction, and peak alignment to produce a data matrix \u003csup\u003e18\u003c/sup\u003e.The data matrix was uploaded to the Meiji cloud platform \u003csup\u003e21\u003c/sup\u003e (https://cloud.majorbio.com/) for further analysis.\u003c/p\u003e\n\u003cp\u003eThe prototype blood bioactive components of GY2H was identified using the jvenn tool \u003csup\u003e22\u003c/sup\u003e (https://jvenn.soulouse.inrae.fr/app/example.html)\u003c/p\u003e\n\u003cp\u003e4.2.3. Network pharmacology-based prediction of molecular targets and signalling pathways\u003c/p\u003e\n\u003cp\u003eFirstly, based on the prototype blood bioactive components, target prediction was performed using Swiss Target Prediction \u003csup\u003e23\u003c/sup\u003e (http: //www.swisstargetprediction.ch/), with screening based on a probability threshold of \u0026gt; 0. The resulting predicted targets were then standardized through the Uniprot database \u003csup\u003e24\u003c/sup\u003e (https: //www.uniprot.org/), followed by removing duplicate entries. Secondly, search terms including \u0026ldquo;acute high altitude disease,\u0026rdquo; \u0026ldquo;acute mountain sickness,\u0026rdquo; \u0026ldquo;altitude headache,\u0026rdquo; and \u0026ldquo;high altitude sickness\u0026rdquo; were used in the Gene Card \u003csup\u003e25\u003c/sup\u003e(https://www.genecards.org/) and OMIM \u003csup\u003e26\u003c/sup\u003e (https://mirror.omim.org/) databases. The predicted targets from both databases were merged and duplicates were removed. Finally, the Venn diagram illustrating the overlap between GY2H prototype blood bioactive components -related targets and AMS-related targets was created using the jvenn tool \u003csup\u003e22\u003c/sup\u003e (https://jvenn.soulouse.inrae.fr/app/example.html).\u003c/p\u003e\n\u003cp\u003eThe results were imported into the Multiple Proteins module of the online STRING database\u003csup\u003e27\u003c/sup\u003e(https://cn.string-db.org), selecting \u0026ldquo;Homo sapiens\u0026rdquo; for protein type, setting the minimum required interaction score to 0.9, and hiding disconnected nodes in the network. The interaction data were exported in .tsv format and imported into Cytoscape_v3.10.2 for visualization and analysis using the CytoNCA-2.1.6 plugin. Key molecular targets were identified by filtering nodes with values above the median for six centrality metrics: Betweenness, Closeness, Degree, Eigenvector, LAC, and Network. These targets inform the pathway analysis of GY2H\u0026apos;s AMS prevention mechanism.\u003c/p\u003e\n\u003cp\u003eKEGG and GO enrichment analyses were performed using the SangerBox Biomedical Data Analysis Box \u003csup\u003e28\u003c/sup\u003e(http://sangerbox.com/home.html). Significantly enriched terms were filtered based on count values and p.adjust value, with high-count/low-p.adjust terms prioritized as candidate pathways.\u003c/p\u003e\n\u003cp\u003e4.2.4. Molecular docking-based verification of primary blood bioactive component-target affinity\u003c/p\u003e\n\u003cp\u003eMolecular docking was executed in Discovery Studio 2019 using primary blood bioactive components as ligands and key molecular targets as receptors. The target protein structures, retrieved from the PDB database\u003csup\u003e29\u003c/sup\u003e(https://www.rcsb.org/), underwent hydrogenation, structural optimization, and energy minimization after removal of redundant conformers. Ligands were prepared with OPLS3e force field parameters involving acid-base neutralization and generation of stereoisomeric conformations for 3D structural refinement. The C-Docker algorithm subsequently docked the processed ligands to receptors, with binding affinity quantified by the docking score of the optimal complex conformation.\u003c/p\u003e\n\u003cp\u003e4.2.5. Cellular-based validation of scutellarein\u0026rsquo;s anti-hypoxic effects and underlying mechanisms\u003c/p\u003e\n\u003cp\u003eHT22 cells were grown in DMEM media supplemented with 10% FBS and 1% Penicillin- Streptomycin-Amphotericin B Solution at 37 ℃ and 5% CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003econdition. The cells were sub-cultured in a rate of 1:3 with the culture medium being replaced every 2 days. After growing to 80% confluence, the cells were used for experiment.\u003c/p\u003e\n\u003cp\u003eFirstly, cell viability were evaluated by treatment with CoCl\u003csub\u003e2\u003c/sub\u003e (100, 150, 200 \u0026mu;M ) or Scutellarein (20, 40, 80 \u0026mu;M) for 24 h. Cell viability was detected by CCK-8 assay. HT22 cells were inoculated with a density of 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well in 96-well plates\u003csup\u003e30\u003c/sup\u003e. After 24 h, CCK-8 (10 \u0026micro;l per well) was added and cells were cultured at 37 ℃ for 3 h. Microplate reader was used to detect cell viability at 450 nm. The lower cytotoxic concentration of scutellarein and CoCl\u003csub\u003e2\u003c/sub\u003e were selected for subsequent experiments. Secondly,\u0026nbsp;HT22 cells were inoculated with a density of 5.0 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well in 96-well plates,\u0026nbsp;cells were divided into blank group, scutellarein group, with model group treated with CoCl\u003csub\u003e2\u003c/sub\u003e(100\u0026mu;M) and scutellarein group treated with CoCl\u003csub\u003e2\u003c/sub\u003e (100\u0026mu;M) + scutellarein (20, 40 and 80 \u0026mu;M) for 24 h. After 24 h, added relevant reagents according to the manual\u0026apos;s instructions, microplate reader was used to detect the LDH concentration at 450 nm or Gallios flow cytometer was used to detect the rate of apoptosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during or analysed during the current study are available from the corresponding author on reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBL: Data curation, Formal analysis(equal), Investigation, Methodology, Visualization (equal) ; and Writing\u0026nbsp;\u0026ndash;\u0026nbsp;original draft. WZ: Writing\u0026nbsp;\u0026ndash;\u0026nbsp;review \u0026amp; editing(equal), Resources, Visualization(equal). CYT: Writing\u0026nbsp;\u0026ndash;\u0026nbsp;review \u0026amp; editing(equal), Formal Analysis(equal), Visualization (equal). JL: Writing\u0026nbsp;\u0026ndash;\u0026nbsp;review \u0026amp; editing (equal). MYL: Writing\u0026nbsp;\u0026ndash;\u0026nbsp;review \u0026amp; editing (equal). HL: Writing\u0026nbsp;\u0026ndash;\u0026nbsp;review \u0026amp; editing (equal). YQW: Project administration, Funding acquisition, Supervision and Writing\u0026nbsp;\u0026ndash;\u0026nbsp;review \u0026amp; editing (equal). ZHX: Conceptualization, Funding acquisition, Supervision and Writing\u0026nbsp;\u0026ndash;\u0026nbsp;review \u0026amp; editing (equal).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Chongqing Yingcai Master and Teacher Programme (Grant numbers [CQYC20220203145]) and the Youth Doctoral Incubation Program of the Second Affiliated Hospital of Army Medical University (Grant numbers [2024YQB007]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003et\u003c/strong\u003e\u003cstrong\u003erial\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003en\u003c/strong\u003e\u003cstrong\u003eumber\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eJin, J. 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K.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e RCSB Protein Data Bank: powerful new tools for exploring 3D structures of biological macromolecules for basic and applied research and educa tion in fundamental biology, biomedicine, biotechnology, bioengineerin g and energy sciences. \u003cem\u003eNucleic Acids Res\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, D437-D451, doi:10.1093/nar/gkaa1038.\u003c/li\u003e\n \u003cli\u003eHou, Y.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Salidroside intensifies mitochondrial function of CoCl2-dam aged HT22 cells by stimulating PI3K-AKT-MAPK signaling pathway. \u003cem\u003ePhytomedicine : international journal of phytotherapy and phytopharmac ology\u003c/em\u003e \u003cstrong\u003e109\u003c/strong\u003e, 154568, doi:10.1016/j.phymed.2022.154568.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Scutellarein, hypoxia, non-targeted metabolomics, network pharmacology, molecular docking, apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-7839138/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7839138/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHigh-altitude hypoxia limits human performance, and existing interventions face limitations in efficacy, safety, or personalization. Our team developed a novel formula to enhance hypoxic tolerance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnti-hypoxia effects were evaluated using normobaric and acute hypoxia models. Blood bioactive components were profiled via LC–MS non-targeted metabolomics. Network pharmacology and molecular docking identified potential targets and pathways, with cellular experiments validating the effects of the key component.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe formula significantly prolonged survival time under normobaric hypoxia and increased survival rate dose-dependently in acute hypoxia. Twenty-three prototype blood components were identified, with scutellarein as the primary constituent. Network analysis revealed 88 potential targets, primarily involving the PI3K-Akt pathway. Scutellarein showed strong binding to Hsp90 (docking score: –9.560 kcal/mol). Cellular assays confirmed its dose-dependent anti-hypoxic effect via reduced LDH release and apoptosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe novel formula may alleviate acute mountain sickness by improving hypoxic tolerance, likely through flavonoid-mediated inhibition of neuronal apoptosis.\u003c/p\u003e","manuscriptTitle":"Scutellarein-containing novel formula attenuates hypoxia through inhibiting apoptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-14 07:35:51","doi":"10.21203/rs.3.rs-7839138/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-15T15:39:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-14T04:42:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-14T04:42:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-12T07:47:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"315e410d-b17e-49e5-b860-791828d519c0","owner":[],"postedDate":"October 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":56149494,"name":"Health sciences/Diseases"},{"id":56149495,"name":"Biological sciences/Drug discovery"}],"tags":[],"updatedAt":"2025-10-22T09:38:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-14 07:35:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7839138","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7839138","identity":"rs-7839138","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}