Bioinformatic Analysis of the Protective Effects of Dexmedetomidine and Thrombopoietin against Hypoxia/Reoxygenation-Induced Injury in AC16 Cells

Bioinformatic Analysis of the Protective Effects of Dexmedetomidine and Thrombopoietin against Hypoxia/Reoxygenation-Induced Injury in AC16 Cells | 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 Research Article Bioinformatic Analysis of the Protective Effects of Dexmedetomidine and Thrombopoietin against Hypoxia/Reoxygenation-Induced Injury in AC16 Cells Cuiyan Xing, Mingyi Wu, Xiaoyang Zhou, Benhang Gong This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5436669/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective: To investigate the protective mechanisms of dexmedetomidine (Dex) and thrombopoietin (TPO) against hypoxia/reoxygenation (H/R)-induced myocardial injury. Methods: Human cardiomyocyte AC16 cells were subjected to hypoxic conditions and treated with Dex and TPO. Cellular responses were assessed through proliferation, apoptosis, and autophagy assays. RNA sequencing and bioinformatic analyses were conducted to identify differentially expressed genes, followed by functional pathway enrichment analysis. Results: Dex and TPO significantly promoted cell proliferation, reduced apoptosis and autophagy, and inhibited caspase-3 activity and LC3B expression. Both agents induced changes in gene expression, with pathway enrichment analysis revealing involvement in MAPK, TGF-β, and TNF signaling pathways. While both treatments demonstrated overlapping effects, each exhibited unique gene regulation mechanisms. Conclusion: Dex and TPO mitigate H/R-induced myocardial injury through complex gene regulatory mechanisms, offering potential intervention strategies for myocardial ischemia-reperfusion injury (MIRI). dexmedetomidine thrombopoietin hypoxia/reoxygenation injury cardiomyocytes gene expression regulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Myocardial ischemia-reperfusion injury (MIRI) is a complex pathophysiological syndrome involving many risk factors, including calcium overload, oxygen free radicals, metabolic disorders, apoptosis, and inflammation[ 1 ]. MIRI causes organ damage and exacerbates tissue damage and is caused by blood flow restoration post-ischemia. MIRI is a serious clinical problem in patients undergoing coronary artery bypass grafting, percutaneous coronary intervention, cardiac transplantation, and thrombolysis therapy[ 2 ]. However, effective treatment of MIRI in the clinic remains challenging. Dexmedetomidine (Dex) is a selective α2-adrenergic receptor agonist that has been extensively clinically applied as an anesthetic and a sedative. Dex is effective for alleviating ischemia-reperfusion injury, although its mechanism of action is unclear[ 3 ]. Dex pre-conditioning significantly improved the symptoms of patients post-surgery[ 4 , 5 ] and exerted cardio-protective effects[ 6 ], indicating its potential for treating MIRI. Thrombopoietin (TPO) is a cytokine that stimulates the production of platelets and can alleviate thrombocytopenia[ 7 ]. Recent studies demonstrated that TPO can directly protect the brain and heart against ischemia-reperfusion injury[ 8 , 9 ].TPO can repair neurons and promote the formation of vascular endothelial cells[ 10 ]. However, the protective effects of TPO on cardiomyocytes and associated mechanisms are not completely clear, and its effects on myocardial hypoxia/reoxygenation (H/R) injury are also unknown. The present study evaluated the protective effects of Dex and TPO against myocardial H/R injury in an H/R injury model. The gene expression patterns of cardiomyocytes post H/R injury were examined by RNA-sequencing (RNA-seq) to identify Dex and TPO-associated genes and signaling pathways, and the mechanism through which Dex and TPO alleviate H/R injuries was investigated. Methods Cell culture and trial grouping The human cardiomyocyte cell line AC16 was purchased from Shanghai Cell Bank at the Chinese Academy of Science (Shanghai, China) and cultured in Dulbecco's modified Eagle’s medium (DMEM) supplemented with 1% (v/v) penicillin-streptomycin (Beyotime Biotech., Shanghai, China) and 10% fetal bovine serum (FBS, Gibco BRL, Grand Island, NY, USA), at a density of 1 × 10 5 cells/well, in 5% CO 2 at 37°C in a humidified incubator (Mode: MCO-15AC-SC, Sanyo, Tokyo, Japan) for 24 h, and then treated with Dex as the vehicle (Jiangsu Hengrui Medicine Co., Ltd., Jiangsu, China), or TPO (PeproTech, Rocky Hill, NJ, USA) for 30 min. The cells were divided into three groups: vehicle (CNTL), Dex, and TPO. The Dex group was divided into six sub-groups treated with increasing concentrations of Dex at 0.1, 0.3, 1.0, 3.0, 10.0, and 30 nM. The TPO group was similarly divided into six subgroups treated with 0.1, 0.3, 1.0, 3.0, 10.0, and 30 ng/mL of TPO. Three replicates were evaluated for each group. For H/R injury, AC16 cells were cultured in glucose-free DMEM supplemented with vehicle, Dex, or TPO for 30 min and subjected to hypoxic conditions under 95% nitrogen and 5% CO 2 at 37°C for 5 h. Cell counting assay The proliferative rates of AC16 cells were evaluated using Cell Counting Kit 8 (Cat. No. 96992, Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Briefly, AC16 cells were cultured in 96-well plates (Corning-Costar, Corning, NY, USA) at a density of 6000 cells/well and treated with different concentrations of Dex or TPO. AC16 cells undergoing H/R were continuously cultured for 0, 12, and 24 h. CCK-8 solution was added to the 96-well plates and cultured for 4 h. The optical density was examined with a microplate reader (Mode: MK3, Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm. The inhibition rate (%) was calculated according to Eq. 1 . Apoptosis assay The rate of AC16 cell apoptosis during H/R injury was evaluated by flow cytometry, Hoechst33342 assay, and caspase-3 analysis. The Hoechst33342 assay was conducted to observe the morphological characteristics of the apoptotic AC16 cells according the manufacturer's instructions (Hoechst33342 staining kit, Cat. No. C1022, Beyotime Biotech.). Briefly, AC16 cells were fixed in 4% formaldehyde (Sangon Biotech Co., Ltd., Shanghai, China) for 10 min at 4°C and then incubated with Hoechst33342 at a final concentration of 5 µg/mL in the dark for 10 min. The stained AC16 cells were observed using a laser-scanning confocal microscope (Bio-Tek, Winooski, VT, USA) at emission and excitation wavelengths of 460 and 350 nm, respectively. The Annexin V-PE/7-AAD Flow Cytometry Kit (Cat. No. 559763, BD Biosciences, Franklin Lakes, NJ, USA) was used to evaluate the rate of apoptosis according to the manufacturer's instructions. AC16 cells were washed three times with D-Hanks buffer (Beyotime Biotech) at 4°C for 5 min, and the adjusted to 10 6 cells/mL with DMEM. Next, 7-AAD (5 µL) and Annexin V-PE (1 µL) were added to 100 µL of cell suspension per well and incubated at 24°C for 10 min in the dark. Binding buffer (450 µL) was added to each well prior to analysis using a FACSCalibur flow cytometer (BD Biosciences). The stained AC16 cells were detected at excitation and emission wavelengths of 488 and 578 nm, respectively, for Annexin V-PE and 546 and 647 nm for 7-AAD, respectively. Caspase-3 activity was evaluated using the Caspase-3 Fluorometric Assay Kit (Cat. No. K105-25, BioVision, Inc. Mountain View, CA, USA) according to the manufacturer’s instructions. The cells were removed from the plates by digestion with trypsin (Cat. No. C0201, Beyotime Biotech) and collected by centrifugation at 600 × g for 5 min. The cells were lysed in an ice bath for 15 min, and cell debris was pelleted by centrifugation at 20,000 × g for 15 min at 4°C. The supernatants were collected to evaluate caspase-3 activity according to the manufacturer’s instructions. Measurement of light chain 3B (LC3B) expression AC16 cells were seeded onto cover slides at a density of 1 × 10 5 cells/slide, cultured for 24 h, treated with vehicle, Dex, or TPO, washed with phosphate-buffered saline (PBS, Beyotime Biotech), pre-fixed with 4% paraformaldehyde (Beyotime Biotech) for 15 s, and fixed and permeabilized with ice-cold methanol (100% concentration, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) at −20°C for 10 min. The prepared slides were washed with PBS and air-dried at 24°C. The slides were incubated with rabbit anti-human LC3B (1:1000, Cat. No. ab168831, Abcam, Cambridge, UK) overnight at 4°C. The slides were continuously washed three times with PBS for 5 min and incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit antibody (1:800, Cat. No. A0562, Beyotime Biotech) at 37°C for 30 min. The stained slides were observed under a fluorescence microscope (Mode: H600L, Nikon, Tokyo, Japan). The number of LC3B-positive dots was evaluated and quantified using the FV10-ASW 3.0 Image software (Olympus, Tokyo, Japan). Total RNA isolation AC16 cells were initially cultured in T25 flasks with Ready-to-Use Myocyte Growth Medium (PromoCell GmbH, Heidelberg, Germany) supplemented with 20% FBS (PromoCell GmbH) at 37°C under 5% CO 2 for 24 h. The cell medium was replaced with DMEM containing 17% medium 199 (Cat. No. 11150059, Gibco BRL), 10% horse serum (Gibco BRL), 5% FBS (Gibco BRL), and 0.5% penicillin-streptomycin (Beyotime Biotech). After pretreatment with vehicle, Dex, or TPO for 30 min, AC16 cells were subjected to hypoxic conditions under 95% nitrogen and 5% CO 2 at 37°C for 5 h, and then reoxygenated in 5% CO 2 for 16 h. Total RNA was extracted using the TRIzol reagent (Beyotime Biotech) according to the manufacturer's instructions. Total RNA was quantified using a spectrophotometer (Mode: UV754N, Shanghai Aucy Sci. Ins. Ltd., Shanghai, China). RNA integrity and purity were measured with an RNA 6000 Nano LabChip Kit (Cat. No. 5067 − 1511) and Bioanalyzer (Mode: Agilent 2100, Agilent Technologies, Santa Clara, CA, USA). The RNA integrity number was ≥ 9 and 260/280 ratio was ≥ 2.0 for all samples. RNA-sequencing RNA-sequencing was performed as previously described 11 with some modifications. The first- and second-strand cDNA were synthesized using 6-base random primers, dNTPs, RNase H, and DNA polymerase I. The cDNA was purified and washed using a QIAQuick PCR Purification Kit (Cat. No. 28104, Qiagen, Hilden, Germany) before end-repair, 3′ adenylation, and ligation of a sequencing adapter using a TruSeq RNA Sample Preparation Kit (Cat. No. RS-122-2503, Illumina, San Diego, CA, USA). Purified cDNA was recovered by agarose gel electrophoresis, and PCR was performed to obtain the whole-transcriptome libraries. Finally, the samples were sequenced using the PE150 protocol. Computational analysis and gene functional annotation Low-quality sequence and base joints were removed from raw files obtained using the Illumina sequencing platform to obtain high-quality clean reads. Differential expression analysis was performed on the filtered high-quality reads using the Edge approach, which depends on a Poisson model to assess RNA-seq data variance. Functional analysis of the differential genes was performed using the Database for the Annotation, Visualization and integrated Discovery (DAVID functional annotation bioinformatic microarray analysis, version: 6.7). Detailed computational analysis was performed as previously reported[ 11 ]. For differentially expressed genes, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was used to identify dysregulated functions in the H/R injury model[ 12 ]. For GO functional analysis, differential gene expression GO analysis and GO enrichment analysis[ 13 ] were conducted. GO terms were considered as enriched when their q value was < 0.05. Alternative splicing analysis Alternative splicing is a process that transforms mRNA precursors into multiple mRNA isoforms by differential splicing or different splice sites[ 14 ]. The OLego mapping method 15 and Quantas pipelines were applied to the RNA-seq data to identify potential alternative splicing events according to a previously published workflow[ 15 ]. Gene variation analysis Using Samtools[ 16 ], the sorted blast files with PCR repeats removed were compared to a reference sequence to identify treatment-specific gene variants, including single-nucleotide polymorphisms (SNPs) and insertions/deletions (InDels). The distribution and frequency of these events were determined based on a previously published study[ 17 ]. Statistical analysis Data are represented as mean ± standard deviation and were analyzed using SPSS software 20.0 (SPSS, Inc., Chicago, IL, USA). Analysis of variance was performed to compare groups using Tukey’s post-hoc test to identify significantly dysregulated genes. Student's t- test was used to analyze differences between two groups. A p < 0.05 was considered as statistically significant. Results Dex and TPO promote proliferation in H/R-treated AC16 cells AC16 cells were successfully cultured, and the effects of Dex or TPO were evaluated following H/R injury by using the CCK-8 assay. Compared with the vehicle group, the proliferation rate of AC16 cells treated with TPO increased significantly in a dose-dependent manner (Fig. 1 a, p < 0.05). At the indicated concentration of TPO, the rate of proliferation decreased with increasing culture time ranging from 0 to 24 h (Fig. 1 a, p < 0.05). The proliferation rate of AC16 cells treated with Dex similarly increased in a dose-dependent manner (Fig. 1 a, p < 0.05) and decreased with increasing culture time (Fig. 1 a, p < 0.05). The proliferation rates of H/R-treated AC16 cells in the TPO group were significantly higher than those in the Dex group (Fig. 1 a, p < 0.05). This suggests that TPO and Dex can protect AC16 cells against H/R injury. Based on the results of the CCK-8 assays, 10 ng/mL TPO and 10 nM Dex were used in subsequent experiments (Fig. 1 b). Dex and TPO inhibit apoptosis and caspase-3 activity in H/R-treated AC16 cells Dex and TPO significantly improved the morphology of AC16 cells treated with H/R (Fig. 1 b). The percentage of necrotic cells, early apoptotic cells, and late apoptotic cells were evaluated by flow cytometry. The percentage of necrotic cells, early apoptotic cells, late apoptotic cells, and total apoptotic cells in the Dex and TPO groups were significantly decreased as compared to those in the CNTL group (Fig. 1 c, all p < 0.05). The proportion of necrotic and apoptotic cells in the Dex group was significantly higher than that in the TPO group (Fig. 1 c, p < 0.05). Hoechst33342 staining in the Dex and TPO group decreased significantly compared to that in the CNTL group (Fig. 2 a, p < 0.05). The caspase-3 activity indicated by the concentration of pNA (p-nitroaniline) in the Dex and TPO groups was also significantly reduced as compared to that in the CNTL group (Fig. 2 b, p < 0.05). Therefore, TPO and Dex can ameliorate cell death caused by H/R injury. Dex and TPO reduce autophagy in H/R-treated AC16 cells Autophagy is a type of programmed cell death, the specific biomarker of which is LC3B[ 18 ]. Compared with the CNTL group, the numbers of LC3B-positive cells in the Dex and TPO groups were significantly decreased (Fig. 2 c and 2 d, p < 0.05). This result suggests that Dex and TPO can inhibit autophagy caused by H/R. Dex and TPO induce differential gene expression in AC16 cells Compared with the CNTL group, there were 31 upregulated genes and 134 downregulated genes in the Dex group and 19 upregulated genes and 118 downregulated genes in the TPO group (Fig. 3 a). However, there were no differentially expressed genes between the Dex and TPO groups. The treatment-specific shared and unique differentially expressed genes (DEGs) were identified. There were 120-shared DEGs between the Dex and TPO groups, 17 unique DEGs in the TPO group, and 45 unique DEGs in the Dex group (Fig. 3 b). The volcano plots and gene Wayne map (Fig. 3 c) showed a similar distribution of gene expression levels and significance for the Dex and TPO groups. GO functional analysis The results showed that there were 12 genes for cellular component (CC), 19 genes for biological process (BP), and 9 genes for molecular function (MF) in the TPO group (Fig. 3 d), and 13 genes for CC, 20 genes for BP, and 9 genes for MF in the Dex group (Fig. 3 e). The results illustrated similar GO terms, gene counts, and gene percentages between the TPO group and Dex group. Among the differentially expressed genes, the most differentially expressed genes were cell part genes in the CC category, cellular process genes in the BP category, and binding genes in the MF category (Fig. 3 d and 3 e). GO enrichment analysis GO enrichment analysis identified terms enriched for the differentially regulated proteins unique to the TPO group and to the Dex group. The enriched terms for the TPO group included negative regulation of cellular metabolic process, cell differentiation, and negative regulation of metabolic process. The enriched terms for the Dex group included regulation of multicellular organismal development, developmental process, and negative regulation of multicellular organismal process. This suggests that TPO treatment causes changes in cell differentiation and metabolic processes, whereas Dex treatment causes changes in cell growth and development. KEGG pathway enrichment analysis We performed KEGG pathway enrichment analysis to identify biological pathways associated with TPO and Dex treatment. We identified five enriched KEGG pathways based on the unique dysregulated genes in the TPO group, including systemic lupus erythematosus, TGF-beta signaling pathway, alcoholism, and signaling pathways (Fig. 4 a). Four KEGG pathways showed significant enrichment of differentially expressed genes in the Dex group, including MAPK signaling pathway, alcoholism, systemic lupus erythematosus, and TGF-beta signaling pathway (Fig. 4 a). Systemic lupus erythematosus, TGF-beta signaling pathway, and alcoholism were common KEGG pathways. The signaling pathways regulating pluripotency of stem cells and TNF signaling pathway were unique KEGG pathways in the TPO group. Notably, the Dex group uniquely involved the MAPK signaling pathway (Fig. 4 a). Effects of Dex and TPO on alternative splicing of AC16 cells exposed to H/R There were no differences in the alternative splicing events and alternative splicing counts between the CNTL, Dex, and TPO groups (Fig. 4 b, p > 0.05). Therefore, Dex and TPO treatment did not result in alternative splicing in AC16 cells. Effects of Dex and TPO on SNP, InDel, and total mutations in AC16 cells exposed to H/R There were no significant differences in the frequency of gene variants, including SNP, InDel, and total mutations, among the CNTL, Dex, and TPO groups (Fig. 4 c, p > 0.05). Neither Dex nor TPO treatment affected the frequency distribution of SNP mutations (Fig. 5 a, p > 0.05). There were no significant differences in the InDel length between the CNTL, Dex, and TPO groups (Fig. 5 b, p > 0.05). Taken together, Dex and TPO did not cause changes in the frequency of gene mutations in AC16 cells exposed to H/R. Discussion In the present study, we demonstrated that Dex and TPO promoted the proliferation of AC16 cells undergoing H/R in a concentration-dependent manner, and protected the cells from H/R injury, which is consistent with previous reports on the cardio-protective effect of Dex[ 19 – 21 ] and TPO[ 7 , 22 ]. Dex and TPO treatment significantly suppressed apoptosis and autophagy in AC16 cells exposed to H/R. The mechanism of action of these drugs may be related to the inhibition of caspase-3 activities. Dex is a novel, highly selective α2-adrenergic receptor agonist 3 that can inhibit inflammation, reduce the incidence of encephalitis, decrease IL-1 and TNF-α levels, and suppress ischemia-reperfusion injury and cerebral injury[ 23 – 26 ]. TPO plays important roles in regulating the production of hemopoietic monocyte macrophages and platelets and modulating the early hematopoiesis of hematopoietic stem cells, which is mediated by the PI3K/Akt, JAK/STAT, and MAPK signaling pathways[ 27 , 28 ]. To further investigate the genes involved in the protective effect of Dex and TPO in H/R injury of AC16 cells, we performed bioinformatics analysis with RNA-seq. There were no DEGs between the Dex and TPO groups. Compared with the CNTL group, multiple genes were differentially expressed (upregulated or downregulated), including 120 common DEGs between the Dex and TPO groups. These common genes, combined with the results of GO function analysis, suggested that Dex and TPO have similar mechanisms at the DNA level in protecting against H/R-induced injury in AC16 cells, from modulating CC, BP, and MF. However, the uniquely expressed genes in Dex- and TPO-treated AC16 cells suggested that they also have unique mechanisms, which was supported by the results of GO enrichment analysis. According to GO enrichment analysis of the treatment-specific differentially expressed genes, cells treated with Dex expressed genes enriched for the GO term ‘multicellular organismal development’, which is consistent with previously published studies[ 29 , 30 ]. TPO regulated the transcription of human platelet glycoprotein 6, forkhead-related family of mammalian transcription factor 1, and GATA-family transcription factors by activating demethylation processes[ 31 , 32 ]. The modulation of gene transcription and nuclear or chromatin signaling by Dex and TPO requires further exploration. To further clarify the functional role of Dex and TPO in H/R-associated injury, we performed functional enrichment of differentially regulated genes that were unique to each treatment using KEGG pathway enrichment analysis. Systemic lupus erythematosus, TGF-beta signaling pathway, and alcoholism were enriched in both the Dex and TPO gene sets. The signaling pathway regulating the pluripotency of stem cells and TNF signaling were uniquely enriched in the TPO group, whereas the MAPK signaling pathway was uniquely enriched in the Dex group. Systemic lupus erythematosus is an autoimmune disease involved in heart failure and multiple non-cardiac disorders and seriously influences the quality of life of patients[ 33 – 35 ]. TGF-β (containing TGF-β1 and TGF-β2) is a cytokine with multiple effects in the processes of embryonic development, wound healing, cell proliferation, and differentiation[ 36 , 37 ]. Alcoholism-related signaling pathways are involved in cardiovascular diseases through inflammation and MAPK signaling[ 38 , 39 ].The signaling pathways regulating the pluripotency of stem cells and TNF signaling pathways were uniquely enriched in TPO-treated AC16 cells. These signaling pathways include signal transducers and activators of transcription 3, octamer-binding transcription factor 3/4, Kruppel-like factor 4, extracellular regulated protein kinase, fibroblast growth factor, Wnt, nuclear factor κB (NF-κB), and phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt), which have been implicated in cardiovascular diseases and stem cell regeneration[ 40 – 45 ]. The MAPK signaling pathway was uniquely enriched in Dex-treated AC16 cells after H/R injury. Previous studies indicated that Dex can reduce H/R-induced cardiomyocyte injury through the MAPK signaling pathway[ 46 ]. These results suggest that Dex and TPO modulate H/R-induced injury through multiple common and specific signaling pathways. However, Dex or TPO did not significantly affect the rate of alternative splicing and gene mutations in AC16 cells undergoing H/R injury, suggesting that they do not cause damage at the DNA level. The phenomenon requires further study. In conclusion, Dex and TPO protected AC16 cells against the injury induced by H/R by promoting proliferation and inhibiting apoptosis, autophagy, and caspase-3 activities. Bioinformatics analysis indicated that the protective effect of Dex and TPO are related, with a complicated mechanism including upregulation and downregulation of expression of multiple genes. Further investigations are required to validate the results of the present study and establish whether the effects observed in this study occur in other cell lines and cardiac injury models. These results are promising and offer insight into a potential treatment strategy for cardiac injury induced by H/R. Furthermore, these findings reveal the advantages of using Dex in clinical anesthesia, particularly in cardiac surgery. Declarations Acknowledgments Not applicable. Authors’ contributions Conceptualization,cuiyan Xing and mingyi Wu; methodology,cuiyan Xing; software,xiaoyang Zhou; validation,cuiyan Xing,mingyi Wu and benhang Gong; formal analysis, cuiyan Xing; investigation, cuiyan Xing; resources, cuiyan Xing; data curation, cuiyan Xing; writing—original draft preparation, cuiyan Xing; writing—review and editing, cuiyan Xing and mingyi Wu . All authors have read and agreed to the published version of the manuscript. Funding This study did not receive any funding in any form. Availability of data and materials The datasets used and analyzed in the current study are available upon reasonable request from corresponding author. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests This manuscript has no potential conflict of interest to disclose. References Yellon, D.M. and D.J. Hausenloy, Myocardial reperfusion injury. N Engl J Med, 2007. 357 (11): p. 1121-35. 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Apoptosis, 2016. 21 (3): p. 252-68. Chen, G. and D.V. Goeddel, TNF-R1 signaling: a beautiful pathway. Science, 2002. 296 (5573): p. 1634-5. Jackman, R.W., et al., Microtubule-mediated NF-kappaB activation in the TNF-alpha signaling pathway. Exp Cell Res, 2009. 315 (19): p. 3242-9. Li, P., et al., The inflammatory cytokine TNF-alpha promotes the premature senescence of rat nucleus pulposus cells via the PI3K/Akt signaling pathway. Sci Rep, 2017. 7 : p. 42938. Li, J., et al., Dexmedetomidine Attenuates Myocardial Ischemia-Reperfusion Injury in Diabetes Mellitus by Inhibiting Endoplasmic Reticulum Stress. J Diabetes Res, 2019. 2019 : p. 7869318. Additional Declarations No competing interests reported. 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. 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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-5436669","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":386189262,"identity":"3b9b1028-6dc9-479c-8943-4e28c45d991d","order_by":0,"name":"Cuiyan Xing","email":"","orcid":"","institution":"Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Cuiyan","middleName":"","lastName":"Xing","suffix":""},{"id":386189263,"identity":"02138175-27c3-43db-9dd9-911a4e7e4e3d","order_by":1,"name":"Mingyi Wu","email":"","orcid":"","institution":"Shandong University Jinan","correspondingAuthor":false,"prefix":"","firstName":"Mingyi","middleName":"","lastName":"Wu","suffix":""},{"id":386189264,"identity":"89105849-986a-4e1f-8651-e36a2fca8908","order_by":2,"name":"Xiaoyang Zhou","email":"","orcid":"","institution":"Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyang","middleName":"","lastName":"Zhou","suffix":""},{"id":386189268,"identity":"e247d545-5843-4b59-a537-e04ab3c0e7f0","order_by":3,"name":"Benhang Gong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYDACCR6GAx8M/smxsbcfIFoL48EZBQeM+XjOJBCthfkwz4cDifMkHAyI0yEf3XvgAI/BnfQ2CYYEhh8V2whrMbxzLuGAhMGz3DbpxgOMPWduE6FlRo7BAQMD5tw2mQMJzIxtxGpJMGBOZ5MAkkRpkZcAajlgcDiBeC0GMmcMDjYYpBm2AQP5IFF+kZ/dY/z5zx8befn29oMPflQQY8sBJM4BHIrQbGkgStkoGAWjYBSMaAAAf31Bz2OgNBwAAAAASUVORK5CYII=","orcid":"","institution":"Shandong Provincial Hospital Affiliated to Shandong First Medical University","correspondingAuthor":true,"prefix":"","firstName":"Benhang","middleName":"","lastName":"Gong","suffix":""}],"badges":[],"createdAt":"2024-11-12 06:23:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5436669/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5436669/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70758919,"identity":"523d03e6-9f52-44ff-ab63-b2580e8efb66","added_by":"auto","created_at":"2024-12-06 10:54:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1410682,"visible":true,"origin":"","legend":"\u003cp\u003eDex and TPO promote proliferation and inhibit apoptosis of AC16 cells. (a) Effect of Dex and TPO on AC16 cell proliferation. (b) and (c) Effects of Dex and TPO on AC16 cell apoptosis. CNTL: control, Dex: dexmedetomidine, TPO: thrombopoietin.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5436669/v1/a494e8fda5a4e37adccb0f71.png"},{"id":70758922,"identity":"4f6ab8b7-7283-441b-9769-1b668d668f7f","added_by":"auto","created_at":"2024-12-06 10:54:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":653082,"visible":true,"origin":"","legend":"\u003cp\u003eDex and TPO reduce caspase-3 activities and autophagy in AC16 cells with H/R injury. (a) and (b) Effect of Dex and TPO on caspase-3 activity. (c) and (d) Effects of Dex and TPO on autophagy. CNTL: control, Dex: dexmedetomidine, TPO: thrombopoietin.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5436669/v1/ab0f501fdd230167593d1d12.png"},{"id":70759151,"identity":"e5b818e5-18aa-4f07-a82a-0cfef61f63bf","added_by":"auto","created_at":"2024-12-06 11:02:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":422992,"visible":true,"origin":"","legend":"\u003cp\u003eDex and TPO induce differential expression of genes in AC16 cells with H/R injury. (a) Number of differentially expressed genes in AC16 cells treated with vehicle (CTNL), Dex, or TPO. (b) Overlap of differentially expressed genes in different groups. (c) Volcano plot of differentially expressed genes in different groups. (d) GO functional analysis of differentially expressed genes in the TPO group. (e) GO functional analysis of differentially expressed genes in the Dex group. CNTL: control, Dex: dexmedetomidine, TPO: thrombopoietin, DE: differentially expressed.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5436669/v1/008d129c8b59672bdfec5495.png"},{"id":70758924,"identity":"d08fc7c8-146d-4e8f-b040-5a7b26013a05","added_by":"auto","created_at":"2024-12-06 10:54:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":425927,"visible":true,"origin":"","legend":"\u003cp\u003e(a) KEGG pathway enrichment analysis in Dex and TPO groups of AC16 cells. (b) Dex and TPO had no effect on splicing events in AC16 cells with H/R injury. (c) Effects of Dex and TPO on the counts of SNP, InDel, and total events. SNP: single-nucleotide polymorphisms, KEGG: Kyoto Encyclopedia of Genes and Genomes.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5436669/v1/7c96086213d10570ad570dd9.png"},{"id":70759152,"identity":"94919b52-a20c-4cb7-ad9a-f3b4fafcbac5","added_by":"auto","created_at":"2024-12-06 11:02:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1161292,"visible":true,"origin":"","legend":"\u003cp\u003eDex and TPO had no effect on the frequency distribution of SNP mutations (a) or InDel length (b) in AC16 cells with H/R injury. CNTL: control, Dex: dexmedetomidine, TPO: thrombopoietin.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5436669/v1/0fb2cfd0910a4a00439fc457.png"},{"id":81426018,"identity":"0a1d84b1-c1d8-491d-ab6d-a767f536e306","added_by":"auto","created_at":"2025-04-26 07:16:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5493595,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5436669/v1/cd34d180-0090-442e-b847-d87fed625aba.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bioinformatic Analysis of the Protective Effects of Dexmedetomidine and Thrombopoietin against Hypoxia/Reoxygenation-Induced Injury in AC16 Cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMyocardial ischemia-reperfusion injury (MIRI) is a complex pathophysiological syndrome involving many risk factors, including calcium overload, oxygen free radicals, metabolic disorders, apoptosis, and inflammation[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. MIRI causes organ damage and exacerbates tissue damage and is caused by blood flow restoration post-ischemia. MIRI is a serious clinical problem in patients undergoing coronary artery bypass grafting, percutaneous coronary intervention, cardiac transplantation, and thrombolysis therapy[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, effective treatment of MIRI in the clinic remains challenging.\u003c/p\u003e \u003cp\u003eDexmedetomidine (Dex) is a selective α2-adrenergic receptor agonist that has been extensively clinically applied as an anesthetic and a sedative. Dex is effective for alleviating ischemia-reperfusion injury, although its mechanism of action is unclear[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Dex pre-conditioning significantly improved the symptoms of patients post-surgery[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and exerted cardio-protective effects[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], indicating its potential for treating MIRI. Thrombopoietin (TPO) is a cytokine that stimulates the production of platelets and can alleviate thrombocytopenia[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Recent studies demonstrated that TPO can directly protect the brain and heart against ischemia-reperfusion injury[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].TPO can repair neurons and promote the formation of vascular endothelial cells[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the protective effects of TPO on cardiomyocytes and associated mechanisms are not completely clear, and its effects on myocardial hypoxia/reoxygenation (H/R) injury are also unknown.\u003c/p\u003e \u003cp\u003eThe present study evaluated the protective effects of Dex and TPO against myocardial H/R injury in an H/R injury model. The gene expression patterns of cardiomyocytes post H/R injury were examined by RNA-sequencing (RNA-seq) to identify Dex and TPO-associated genes and signaling pathways, and the mechanism through which Dex and TPO alleviate H/R injuries was investigated.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eCell culture and trial grouping\u003c/h2\u003e\n \u003cp\u003eThe human cardiomyocyte cell line AC16 was purchased from Shanghai Cell Bank at the Chinese Academy of Science (Shanghai, China) and cultured in Dulbecco\u0026apos;s modified Eagle\u0026rsquo;s medium (DMEM) supplemented with 1% (v/v) penicillin-streptomycin (Beyotime Biotech., Shanghai, China) and 10% fetal bovine serum (FBS, Gibco BRL, Grand Island, NY, USA), at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well, in 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C in a humidified incubator (Mode: MCO-15AC-SC, Sanyo, Tokyo, Japan) for 24 h, and then treated with Dex as the vehicle (Jiangsu Hengrui Medicine Co., Ltd., Jiangsu, China), or TPO (PeproTech, Rocky Hill, NJ, USA) for 30 min.\u003c/p\u003e\n \u003cp\u003eThe cells were divided into three groups: vehicle (CNTL), Dex, and TPO. The Dex group was divided into six sub-groups treated with increasing concentrations of Dex at 0.1, 0.3, 1.0, 3.0, 10.0, and 30 nM. The TPO group was similarly divided into six subgroups treated with 0.1, 0.3, 1.0, 3.0, 10.0, and 30 ng/mL of TPO. Three replicates were evaluated for each group.\u003c/p\u003e\n \u003cp\u003eFor H/R injury, AC16 cells were cultured in glucose-free DMEM supplemented with vehicle, Dex, or TPO for 30 min and subjected to hypoxic conditions under 95% nitrogen and 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C for 5 h.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eCell counting assay\u003c/h2\u003e\n \u003cp\u003eThe proliferative rates of AC16 cells were evaluated using Cell Counting Kit 8 (Cat. No. 96992, Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer\u0026rsquo;s instructions. Briefly, AC16 cells were cultured in 96-well plates (Corning-Costar, Corning, NY, USA) at a density of 6000 cells/well and treated with different concentrations of Dex or TPO. AC16 cells undergoing H/R were continuously cultured for 0, 12, and 24 h. CCK-8 solution was added to the 96-well plates and cultured for 4 h. The optical density was examined with a microplate reader (Mode: MK3, Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm. The inhibition rate (%) was calculated according to Eq. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"450\" height=\"65\"\u003e\u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eApoptosis assay\u003c/h2\u003e\n \u003cp\u003eThe rate of AC16 cell apoptosis during H/R injury was evaluated by flow cytometry, Hoechst33342 assay, and caspase-3 analysis. The Hoechst33342 assay was conducted to observe the morphological characteristics of the apoptotic AC16 cells according the manufacturer\u0026apos;s instructions (Hoechst33342 staining kit, Cat. No. C1022, Beyotime Biotech.). Briefly, AC16 cells were fixed in 4% formaldehyde (Sangon Biotech Co., Ltd., Shanghai, China) for 10 min at 4\u0026deg;C and then incubated with Hoechst33342 at a final concentration of 5 \u0026micro;g/mL in the dark for 10 min. The stained AC16 cells were observed using a laser-scanning confocal microscope (Bio-Tek, Winooski, VT, USA) at emission and excitation wavelengths of 460 and 350 nm, respectively.\u003c/p\u003e\n \u003cp\u003eThe Annexin V-PE/7-AAD Flow Cytometry Kit (Cat. No. 559763, BD Biosciences, Franklin Lakes, NJ, USA) was used to evaluate the rate of apoptosis according to the manufacturer\u0026apos;s instructions. AC16 cells were washed three times with D-Hanks buffer (Beyotime Biotech) at 4\u0026deg;C for 5 min, and the adjusted to 10\u003csup\u003e6\u003c/sup\u003e cells/mL with DMEM. Next, 7-AAD (5 \u0026micro;L) and Annexin V-PE (1 \u0026micro;L) were added to 100 \u0026micro;L of cell suspension per well and incubated at 24\u0026deg;C for 10 min in the dark. Binding buffer (450 \u0026micro;L) was added to each well prior to analysis using a FACSCalibur flow cytometer (BD Biosciences). The stained AC16 cells were detected at excitation and emission wavelengths of 488 and 578 nm, respectively, for Annexin V-PE and 546 and 647 nm for 7-AAD, respectively.\u003c/p\u003e\n \u003cp\u003eCaspase-3 activity was evaluated using the Caspase-3 Fluorometric Assay Kit (Cat. No. K105-25, BioVision, Inc. Mountain View, CA, USA) according to the manufacturer\u0026rsquo;s instructions. The cells were removed from the plates by digestion with trypsin (Cat. No. C0201, Beyotime Biotech) and collected by centrifugation at 600 \u0026times;\u003cem\u003eg\u003c/em\u003e for 5 min. The cells were lysed in an ice bath for 15 min, and cell debris was pelleted by centrifugation at 20,000 \u0026times;\u003cem\u003eg\u003c/em\u003e for 15 min at 4\u0026deg;C. The supernatants were collected to evaluate caspase-3 activity according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eMeasurement of light chain 3B (LC3B) expression\u003c/h2\u003e\n \u003cp\u003eAC16 cells were seeded onto cover slides at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/slide, cultured for 24 h, treated with vehicle, Dex, or TPO, washed with phosphate-buffered saline (PBS, Beyotime Biotech), pre-fixed with 4% paraformaldehyde (Beyotime Biotech) for 15 s, and fixed and permeabilized with ice-cold methanol (100% concentration, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) at \u0026minus;20\u0026deg;C for 10 min. The prepared slides were washed with PBS and air-dried at 24\u0026deg;C. The slides were incubated with rabbit anti-human LC3B (1:1000, Cat. No. ab168831, Abcam, Cambridge, UK) overnight at 4\u0026deg;C. The slides were continuously washed three times with PBS for 5 min and incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit antibody (1:800, Cat. No. A0562, Beyotime Biotech) at 37\u0026deg;C for 30 min. The stained slides were observed under a fluorescence microscope (Mode: H600L, Nikon, Tokyo, Japan). The number of LC3B-positive dots was evaluated and quantified using the FV10-ASW 3.0 Image software (Olympus, Tokyo, Japan).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eTotal RNA isolation\u003c/h2\u003e\n \u003cp\u003eAC16 cells were initially cultured in T25 flasks with Ready-to-Use Myocyte Growth Medium (PromoCell GmbH, Heidelberg, Germany) supplemented with 20% FBS (PromoCell GmbH) at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 h. The cell medium was replaced with DMEM containing 17% medium 199 (Cat. No. 11150059, Gibco BRL), 10% horse serum (Gibco BRL), 5% FBS (Gibco BRL), and 0.5% penicillin-streptomycin (Beyotime Biotech). After pretreatment with vehicle, Dex, or TPO for 30 min, AC16 cells were subjected to hypoxic conditions under 95% nitrogen and 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C for 5 h, and then reoxygenated in 5% CO\u003csub\u003e2\u003c/sub\u003e for 16 h. Total RNA was extracted using the TRIzol reagent (Beyotime Biotech) according to the manufacturer\u0026apos;s instructions. Total RNA was quantified using a spectrophotometer (Mode: UV754N, Shanghai Aucy Sci. Ins. Ltd., Shanghai, China). RNA integrity and purity were measured with an RNA 6000 Nano LabChip Kit (Cat. No. 5067\u0026thinsp;\u0026minus;\u0026thinsp;1511) and Bioanalyzer (Mode: Agilent 2100, Agilent Technologies, Santa Clara, CA, USA). The RNA integrity number was \u0026ge;\u0026thinsp;9 and 260/280 ratio was \u0026ge;\u0026thinsp;2.0 for all samples.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eRNA-sequencing\u003c/h2\u003e\n \u003cp\u003eRNA-sequencing was performed as previously described\u003csup\u003e11\u003c/sup\u003e with some modifications. The first- and second-strand cDNA were synthesized using 6-base random primers, dNTPs, RNase H, and DNA polymerase I. The cDNA was purified and washed using a QIAQuick PCR Purification Kit (Cat. No. 28104, Qiagen, Hilden, Germany) before end-repair, 3\u0026prime; adenylation, and ligation of a sequencing adapter using a TruSeq RNA Sample Preparation Kit (Cat. No. RS-122-2503, Illumina, San Diego, CA, USA). Purified cDNA was recovered by agarose gel electrophoresis, and PCR was performed to obtain the whole-transcriptome libraries. Finally, the samples were sequenced using the PE150 protocol.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eComputational analysis and gene functional annotation\u003c/h2\u003e\n \u003cp\u003eLow-quality sequence and base joints were removed from raw files obtained using the Illumina sequencing platform to obtain high-quality clean reads. Differential expression analysis was performed on the filtered high-quality reads using the Edge approach, which depends on a Poisson model to assess RNA-seq data variance. Functional analysis of the differential genes was performed using the Database for the Annotation, Visualization and integrated Discovery (DAVID functional annotation bioinformatic microarray analysis, version: 6.7). Detailed computational analysis was performed as previously reported[\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eFor differentially expressed genes, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was used to identify dysregulated functions in the H/R injury model[\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. For GO functional analysis, differential gene expression GO analysis and GO enrichment analysis[\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e] were conducted. GO terms were considered as enriched when their q value was \u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eAlternative splicing analysis\u003c/h2\u003e\n \u003cp\u003eAlternative splicing is a process that transforms mRNA precursors into multiple mRNA isoforms by differential splicing or different splice sites[\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. The OLego mapping method\u003csup\u003e15\u003c/sup\u003e and Quantas pipelines were applied to the RNA-seq data to identify potential alternative splicing events according to a previously published workflow[\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eGene variation analysis\u003c/h2\u003e\n \u003cp\u003eUsing Samtools[\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e], the sorted blast files with PCR repeats removed were compared to a reference sequence to identify treatment-specific gene variants, including single-nucleotide polymorphisms (SNPs) and insertions/deletions (InDels). The distribution and frequency of these events were determined based on a previously published study[\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eData are represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation and were analyzed using SPSS software 20.0 (SPSS, Inc., Chicago, IL, USA). Analysis of variance was performed to compare groups using Tukey\u0026rsquo;s post-hoc test to identify significantly dysregulated genes. Student\u0026apos;s \u003cem\u003et-\u003c/em\u003etest was used to analyze differences between two groups. A \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDex and TPO promote proliferation in H/R-treated AC16 cells\u003c/h2\u003e \u003cp\u003eAC16 cells were successfully cultured, and the effects of Dex or TPO were evaluated following H/R injury by using the CCK-8 assay. Compared with the vehicle group, the proliferation rate of AC16 cells treated with TPO increased significantly in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At the indicated concentration of TPO, the rate of proliferation decreased with increasing culture time ranging from 0 to 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The proliferation rate of AC16 cells treated with Dex similarly increased in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and decreased with increasing culture time (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The proliferation rates of H/R-treated AC16 cells in the TPO group were significantly higher than those in the Dex group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This suggests that TPO and Dex can protect AC16 cells against H/R injury. Based on the results of the CCK-8 assays, 10 ng/mL TPO and 10 nM Dex were used in subsequent experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDex and TPO inhibit apoptosis and caspase-3 activity in H/R-treated AC16 cells\u003c/h2\u003e \u003cp\u003eDex and TPO significantly improved the morphology of AC16 cells treated with H/R (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The percentage of necrotic cells, early apoptotic cells, and late apoptotic cells were evaluated by flow cytometry. The percentage of necrotic cells, early apoptotic cells, late apoptotic cells, and total apoptotic cells in the Dex and TPO groups were significantly decreased as compared to those in the CNTL group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, all \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The proportion of necrotic and apoptotic cells in the Dex group was significantly higher than that in the TPO group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eHoechst33342 staining in the Dex and TPO group decreased significantly compared to that in the CNTL group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The caspase-3 activity indicated by the concentration of pNA (p-nitroaniline) in the Dex and TPO groups was also significantly reduced as compared to that in the CNTL group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Therefore, TPO and Dex can ameliorate cell death caused by H/R injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDex and TPO reduce autophagy in H/R-treated AC16 cells\u003c/h2\u003e \u003cp\u003eAutophagy is a type of programmed cell death, the specific biomarker of which is LC3B[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Compared with the CNTL group, the numbers of LC3B-positive cells in the Dex and TPO groups were significantly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This result suggests that Dex and TPO can inhibit autophagy caused by H/R.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDex and TPO induce differential gene expression in AC16 cells\u003c/h2\u003e \u003cp\u003eCompared with the CNTL group, there were 31 upregulated genes and 134 downregulated genes in the Dex group and 19 upregulated genes and 118 downregulated genes in the TPO group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). However, there were no differentially expressed genes between the Dex and TPO groups. The treatment-specific shared and unique differentially expressed genes (DEGs) were identified. There were 120-shared DEGs between the Dex and TPO groups, 17 unique DEGs in the TPO group, and 45 unique DEGs in the Dex group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The volcano plots and gene Wayne map (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec) showed a similar distribution of gene expression levels and significance for the Dex and TPO groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eGO functional analysis\u003c/h2\u003e \u003cp\u003eThe results showed that there were 12 genes for cellular component (CC), 19 genes for biological process (BP), and 9 genes for molecular function (MF) in the TPO group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), and 13 genes for CC, 20 genes for BP, and 9 genes for MF in the Dex group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). The results illustrated similar GO terms, gene counts, and gene percentages between the TPO group and Dex group. Among the differentially expressed genes, the most differentially expressed genes were cell part genes in the CC category, cellular process genes in the BP category, and binding genes in the MF category (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eGO enrichment analysis\u003c/h2\u003e \u003cp\u003eGO enrichment analysis identified terms enriched for the differentially regulated proteins unique to the TPO group and to the Dex group. The enriched terms for the TPO group included negative regulation of cellular metabolic process, cell differentiation, and negative regulation of metabolic process. The enriched terms for the Dex group included regulation of multicellular organismal development, developmental process, and negative regulation of multicellular organismal process. This suggests that TPO treatment causes changes in cell differentiation and metabolic processes, whereas Dex treatment causes changes in cell growth and development.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eKEGG pathway enrichment analysis\u003c/h2\u003e \u003cp\u003eWe performed KEGG pathway enrichment analysis to identify biological pathways associated with TPO and Dex treatment. We identified five enriched KEGG pathways based on the unique dysregulated genes in the TPO group, including systemic lupus erythematosus, TGF-beta signaling pathway, alcoholism, and signaling pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Four KEGG pathways showed significant enrichment of differentially expressed genes in the Dex group, including MAPK signaling pathway, alcoholism, systemic lupus erythematosus, and TGF-beta signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Systemic lupus erythematosus, TGF-beta signaling pathway, and alcoholism were common KEGG pathways. The signaling pathways regulating pluripotency of stem cells and TNF signaling pathway were unique KEGG pathways in the TPO group. Notably, the Dex group uniquely involved the MAPK signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eEffects of Dex and TPO on alternative splicing of AC16 cells exposed to H/R\u003c/h2\u003e \u003cp\u003eThere were no differences in the alternative splicing events and alternative splicing counts between the CNTL, Dex, and TPO groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Therefore, Dex and TPO treatment did not result in alternative splicing in AC16 cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of Dex and TPO on SNP, InDel, and total mutations in AC16 cells exposed to H/R\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThere were no significant differences in the frequency of gene variants, including SNP, InDel, and total mutations, among the CNTL, Dex, and TPO groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Neither Dex nor TPO treatment affected the frequency distribution of SNP mutations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). There were no significant differences in the InDel length between the CNTL, Dex, and TPO groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Taken together, Dex and TPO did not cause changes in the frequency of gene mutations in AC16 cells exposed to H/R.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we demonstrated that Dex and TPO promoted the proliferation of AC16 cells undergoing H/R in a concentration-dependent manner, and protected the cells from H/R injury, which is consistent with previous reports on the cardio-protective effect of Dex[\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and TPO[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Dex and TPO treatment significantly suppressed apoptosis and autophagy in AC16 cells exposed to H/R. The mechanism of action of these drugs may be related to the inhibition of caspase-3 activities. Dex is a novel, highly selective α2-adrenergic receptor agonist\u003csup\u003e3\u003c/sup\u003e that can inhibit inflammation, reduce the incidence of encephalitis, decrease IL-1 and TNF-α levels, and suppress ischemia-reperfusion injury and cerebral injury[\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. TPO plays important roles in regulating the production of hemopoietic monocyte macrophages and platelets and modulating the early hematopoiesis of hematopoietic stem cells, which is mediated by the PI3K/Akt, JAK/STAT, and MAPK signaling pathways[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo further investigate the genes involved in the protective effect of Dex and TPO in H/R injury of AC16 cells, we performed bioinformatics analysis with RNA-seq.\u0026nbsp;There were no DEGs between the Dex and TPO groups. Compared with the CNTL group, multiple genes were differentially expressed (upregulated or downregulated), including 120 common DEGs between the Dex and TPO groups. These common genes, combined with the results of GO function analysis, suggested that Dex and TPO have similar mechanisms at the DNA level in protecting against H/R-induced injury in AC16 cells, from modulating CC, BP, and MF. However, the uniquely expressed genes in Dex- and TPO-treated AC16 cells suggested that they also have unique mechanisms, which was supported by the results of GO enrichment analysis. According to GO enrichment analysis of the treatment-specific differentially expressed genes, cells treated with Dex expressed genes enriched for the GO term \u0026lsquo;multicellular organismal development\u0026rsquo;, which is consistent with previously published studies[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. TPO regulated the transcription of human platelet glycoprotein 6, forkhead-related family of mammalian transcription factor 1, and GATA-family transcription factors by activating demethylation processes[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The modulation of gene transcription and nuclear or chromatin signaling by Dex and TPO requires further exploration.\u003c/p\u003e \u003cp\u003eTo further clarify the functional role of Dex and TPO in H/R-associated injury, we performed functional enrichment of differentially regulated genes that were unique to each treatment using KEGG pathway enrichment analysis. Systemic lupus erythematosus, TGF-beta signaling pathway, and alcoholism were enriched in both the Dex and TPO gene sets. The signaling pathway regulating the pluripotency of stem cells and TNF signaling were uniquely enriched in the TPO group, whereas the MAPK signaling pathway was uniquely enriched in the Dex group. Systemic lupus erythematosus is an autoimmune disease involved in heart failure and multiple non-cardiac disorders and seriously influences the quality of life of patients[\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. TGF-β (containing TGF-β1 and TGF-β2) is a cytokine with multiple effects in the processes of embryonic development, wound healing, cell proliferation, and differentiation[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Alcoholism-related signaling pathways are involved in cardiovascular diseases through inflammation and MAPK signaling[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].The signaling pathways regulating the pluripotency of stem cells and TNF signaling pathways were uniquely enriched in TPO-treated AC16 cells. These signaling pathways include signal transducers and activators of transcription 3, octamer-binding transcription factor 3/4, Kruppel-like factor 4, extracellular regulated protein kinase, fibroblast growth factor, Wnt, nuclear factor κB (NF-κB), and phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt), which have been implicated in cardiovascular diseases and stem cell regeneration[\u003cspan additionalcitationids=\"CR41 CR42 CR43 CR44\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The MAPK signaling pathway was uniquely enriched in Dex-treated AC16 cells after H/R injury. Previous studies indicated that Dex can reduce H/R-induced cardiomyocyte injury through the MAPK signaling pathway[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. These results suggest that Dex and TPO modulate H/R-induced injury through multiple common and specific signaling pathways. However, Dex or TPO did not significantly affect the rate of alternative splicing and gene mutations in AC16 cells undergoing H/R injury, suggesting that they do not cause damage at the DNA level. The phenomenon requires further study.\u003c/p\u003e \u003cp\u003eIn conclusion, Dex and TPO protected AC16 cells against the injury induced by H/R by promoting proliferation and inhibiting apoptosis, autophagy, and caspase-3 activities. Bioinformatics analysis indicated that the protective effect of Dex and TPO are related, with a complicated mechanism including upregulation and downregulation of expression of multiple genes. Further investigations are required to validate the results of the present study and establish whether the effects observed in this study occur in other cell lines and cardiac injury models. These results are promising and offer insight into a potential treatment strategy for cardiac injury induced by H/R. Furthermore, these findings reveal the advantages of using Dex in clinical anesthesia, particularly in cardiac surgery.\u003c/p\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\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization,cuiyan Xing and mingyi Wu; methodology,cuiyan Xing; software,xiaoyang Zhou; validation,cuiyan Xing,mingyi Wu and benhang Gong; formal analysis, cuiyan Xing; investigation, cuiyan Xing; resources, cuiyan Xing; data curation, cuiyan Xing; writing\u0026mdash;original draft preparation, cuiyan Xing; writing\u0026mdash;review and editing, cuiyan Xing and mingyi Wu . 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 study did not receive any funding in any form.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analyzed in the current study are available upon reasonable request from corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis manuscript has no potential conflict of interest to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYellon, D.M. and D.J. 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J Diabetes Res, 2019. \u003cstrong\u003e2019\u003c/strong\u003e: p. 7869318.\u003c/li\u003e\n\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":"dexmedetomidine, thrombopoietin, hypoxia/reoxygenation injury, cardiomyocytes, gene expression regulation","lastPublishedDoi":"10.21203/rs.3.rs-5436669/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5436669/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e To investigate the protective mechanisms of dexmedetomidine (Dex) and thrombopoietin (TPO) against hypoxia/reoxygenation (H/R)-induced myocardial injury.\u003cbr\u003e\n \u003cstrong\u003eMethods: \u003c/strong\u003eHuman cardiomyocyte AC16 cells were subjected to hypoxic conditions and treated with Dex and TPO. Cellular responses were assessed through proliferation, apoptosis, and autophagy assays. RNA sequencing and bioinformatic analyses were conducted to identify differentially expressed genes, followed by functional pathway enrichment analysis.\u003cbr\u003e\n \u003cstrong\u003eResults:\u003c/strong\u003e Dex and TPO significantly promoted cell proliferation, reduced apoptosis and autophagy, and inhibited caspase-3 activity and LC3B expression. Both agents induced changes in gene expression, with pathway enrichment analysis revealing involvement in MAPK, TGF-β, and TNF signaling pathways. While both treatments demonstrated overlapping effects, each exhibited unique gene regulation mechanisms.\u003cbr\u003e\n \u003cstrong\u003eConclusion:\u003c/strong\u003e Dex and TPO mitigate H/R-induced myocardial injury through complex gene regulatory mechanisms, offering potential intervention strategies for myocardial ischemia-reperfusion injury (MIRI).\u003c/p\u003e","manuscriptTitle":"Bioinformatic Analysis of the Protective Effects of Dexmedetomidine and Thrombopoietin against Hypoxia/Reoxygenation-Induced Injury in AC16 Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-06 10:54:16","doi":"10.21203/rs.3.rs-5436669/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":"53c4624a-868f-458e-95d2-8e0272c72d0f","owner":[],"postedDate":"December 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-26T07:08:32+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-06 10:54:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5436669","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5436669","identity":"rs-5436669","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}