{"id":26674,"date":"2024-01-03T21:37:13","date_gmt":"2024-01-03T19:37:13","guid":{"rendered":"https:\/\/hho-bulgaria.com\/h2-repairs-endothelial-progenitor-cells-injury\/"},"modified":"2024-01-29T21:22:26","modified_gmt":"2024-01-29T19:22:26","slug":"h2-repairs-endothelial-progenitor-cells-injury","status":"publish","type":"post","link":"https:\/\/hho-bulgaria.com\/en\/h2-repairs-endothelial-progenitor-cells-injury\/","title":{"rendered":"H2 Repairs Endothelial Progenitor Cells Injury"},"content":{"rendered":"<div class=\"jig-ncbiinpagenav\" data-jigconfig=\"smoothScroll: false, allHeadingLevels: ['h2'], headingExclude: ':hidden,.nomenu'\" id=\"ui-ncbiinpagenav-1\">\n<div class=\"fm-sec half_rhythm no_top_margin\">\n<div class=\"fm-flexbox\">\n<div class=\"fm-citation\">\n<div class=\"citation-default\">\n<div class=\"part1\"><span role=\"menubar\"><a href=\"#\" role=\"menuitem\" aria-expanded=\"false\" aria-haspopup=\"true\">Front Pharmacol.<\/a><\/span> 2022; 13: 894812. <\/div>\n<div class=\"part2\"><span class=\"fm-vol-iss-date\">Published online 2022 May 12. <\/span>  <span class=\"doi\"><span>doi:&nbsp;<\/span><a href=\"\/\/doi.org\/10.3389%2Ffphar.2022.894812\" target=\"_blank\" rel=\"noopener noreferrer\" ref=\"reftype=other&amp;article-id=9133378&amp;issue-id=400058&amp;journal-id=1524&amp;FROM=Article%7CFront%20Matter&amp;TO=Content%20Provider%7CCrosslink%7CDOI\">10.3389\/fphar.2022.894812<\/a><\/span><\/div>\n<\/div>\n<\/div>\n<div class=\"fm-ids\">\n<div class=\"fm-citation-pmcid\"><span class=\"fm-citation-ids-label\">PMCID: <\/span><span>PMC9133378<\/span><\/div>\n<div class=\"fm-citation-pmid\">PMID: <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/35645804\">35645804<\/a><\/div>\n<\/div>\n<\/div>\n<h1 class=\"content-title\">Hydrogen Repairs LPS-Induced Endothelial Progenitor Cells Injury <em>via<\/em> PI3K\/AKT\/eNOS Pathway<\/h1>\n<div class=\"half_rhythm\">\n<div class=\"contrib-group fm-author\"><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Mu%20Q%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm140208056121456\" co-class=\"co-affbox\">Qingjie Mu<\/a>,<sup><br \/>\n1<br \/>\n,<\/sup><sup><br \/>\n2<br \/>\n,<\/sup><sup><br \/>\n\u2020<br \/>\n<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Lv%20K%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm140208056105424\" co-class=\"co-affbox\">Kaixuan Lv<\/a>,<sup><br \/>\n3<br \/>\n,<\/sup><sup><br \/>\n\u2020<br \/>\n<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Yu%20J%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm140208059584736\" co-class=\"co-affbox\">Jielun Yu<\/a>,<sup><br \/>\n3<br \/>\n,<\/sup><sup><br \/>\n4<br \/>\n,<\/sup><sup><br \/>\n5<br \/>\n,<\/sup><sup><br \/>\n\u2020<br \/>\n<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Chu%20S%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm140208059578640\" co-class=\"co-affbox\">Shangmin Chu<\/a>,<sup><br \/>\n3<br \/>\n<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Zhang%20L%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm140208059576272\" co-class=\"co-affbox\">Lichun Zhang<\/a>,<sup><br \/>\n3<br \/>\n<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Kong%20L%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm140208059573904\" co-class=\"co-affbox\">Lingyu Kong<\/a>,<sup><br \/>\n6<br \/>\n<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Zhang%20L%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm140208059571168\" co-class=\"co-affbox\">Linlin Zhang<\/a>,<sup><br \/>\n3<br \/>\n<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Tian%20Y%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm140208059568800\" co-class=\"co-affbox\">Yan Tian<\/a>,<sup><br \/>\n7<br \/>\n<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Jia%20X%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm140208059566064\" co-class=\"co-affbox\">Xiaopeng Jia<\/a>,<sup><br \/>\n8<br \/>\n<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Liu%20B%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm140208059563696\" co-class=\"co-affbox\">Benhong Liu<\/a>,<sup><img decoding=\"async\" src=\"\/corehtml\/pmc\/pmcgifs\/corrauth.gif\" alt=\"corresponding author\"><\/sup><sup><br \/>\n9<br \/>\n,<\/sup><sup>*<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Wei%20Y%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm140208059560352\" co-class=\"co-affbox\">Youzhen Wei<\/a>,<sup><img decoding=\"async\" src=\"\/corehtml\/pmc\/pmcgifs\/corrauth.gif\" alt=\"corresponding author\"><\/sup><sup><br \/>\n10<br \/>\n,<\/sup><sup>*<\/sup> and  <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Yang%20N%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm140208059556640\" co-class=\"co-affbox\">Nana Yang<\/a><sup><img decoding=\"async\" src=\"\/corehtml\/pmc\/pmcgifs\/corrauth.gif\" alt=\"corresponding author\"><\/sup><sup><br \/>\n3<br \/>\n,<\/sup><sup><br \/>\n4<br \/>\n,<\/sup><sup><br \/>\n5<br \/>\n,<\/sup><sup>*<\/sup><\/div>\n<div style=\"display:none\" class=\"contrib-group aff-tip\">\n<div id=\"_co_idm140208056121456\">\n<h3 class=\"no_margin\">Qingjie Mu<\/h3>\n<p>\n<sup>1<\/sup><br \/>\nSchool of Clinical Medicine, Weifang Medical University, Weifang, China\n<\/p>\n<p>\n<sup>2<\/sup><br \/>\nUniversity of Health and Rehabilitation Sciences, Qingdao, China\n<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Mu%20Q%5BAuthor%5D\">Qingjie Mu<\/a><\/div>\n<\/div>\n<div id=\"_co_idm140208056105424\">\n<h3 class=\"no_margin\">Kaixuan Lv<\/h3>\n<p>\n<sup>3<\/sup><br \/>\nSchool of Bioscience and Technology, Weifang Medical University, Weifang, China\n<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Lv%20K%5BAuthor%5D\">Kaixuan Lv<\/a><\/div>\n<\/div>\n<div id=\"_co_idm140208059584736\">\n<h3 class=\"no_margin\">Jielun Yu<\/h3>\n<p>\n<sup>3<\/sup><br \/>\nSchool of Bioscience and Technology, Weifang Medical University, Weifang, China\n<\/p>\n<p>\n<sup>4<\/sup><br \/>\nMedical Laboratory Animal Center, Weifang Medical University, Weifang, China\n<\/p>\n<p>\n<sup>5<\/sup><br \/>\nWeifang Key Laboratory of Animal Model Research on Cardiovascular and Cerebrovascular Diseases, Weifang, China\n<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Yu%20J%5BAuthor%5D\">Jielun Yu<\/a><\/div>\n<\/div>\n<div id=\"_co_idm140208059578640\">\n<h3 class=\"no_margin\">Shangmin Chu<\/h3>\n<p>\n<sup>3<\/sup><br \/>\nSchool of Bioscience and Technology, Weifang Medical University, Weifang, China\n<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Chu%20S%5BAuthor%5D\">Shangmin Chu<\/a><\/div>\n<\/div>\n<div id=\"_co_idm140208059576272\">\n<h3 class=\"no_margin\">Lichun Zhang<\/h3>\n<p>\n<sup>3<\/sup><br \/>\nSchool of Bioscience and Technology, Weifang Medical University, Weifang, China\n<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Zhang%20L%5BAuthor%5D\">Lichun Zhang<\/a><\/div>\n<\/div>\n<div id=\"_co_idm140208059573904\">\n<h3 class=\"no_margin\">Lingyu Kong<\/h3>\n<p>\n<sup>6<\/sup><br \/>\nSchool of Rehabilitation Medicine, Weifang Medical University, Weifang, China\n<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Kong%20L%5BAuthor%5D\">Lingyu Kong<\/a><\/div>\n<\/div>\n<div id=\"_co_idm140208059571168\">\n<h3 class=\"no_margin\">Linlin Zhang<\/h3>\n<p>\n<sup>3<\/sup><br \/>\nSchool of Bioscience and Technology, Weifang Medical University, Weifang, China\n<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Zhang%20L%5BAuthor%5D\">Linlin Zhang<\/a><\/div>\n<\/div>\n<div id=\"_co_idm140208059568800\">\n<h3 class=\"no_margin\">Yan Tian<\/h3>\n<p>\n<sup>7<\/sup><br \/>\nResearch Center of Translational Medicine Shanghai East Hospital, Tongji University, Shanghai, China\n<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Tian%20Y%5BAuthor%5D\">Yan Tian<\/a><\/div>\n<\/div>\n<div id=\"_co_idm140208059566064\">\n<h3 class=\"no_margin\">Xiaopeng Jia<\/h3>\n<p>\n<sup>8<\/sup><br \/>\nShandong Qilu Stem Cell Engineering Co., Jinan, China\n<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Jia%20X%5BAuthor%5D\">Xiaopeng Jia<\/a><\/div>\n<\/div>\n<div id=\"_co_idm140208059563696\">\n<h3 class=\"no_margin\">Benhong Liu<\/h3>\n<p>\n<sup>9<\/sup><br \/>\nDepartment of Respiratory, Dongying People\u2019s Hospital, Dongying, China\n<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Liu%20B%5BAuthor%5D\">Benhong Liu<\/a><\/div>\n<\/div>\n<div id=\"_co_idm140208059560352\">\n<h3 class=\"no_margin\">Youzhen Wei<\/h3>\n<p>\n<sup>10<\/sup><br \/>\nResearch Center for Translational Medicine and Key Laboratory of Arrhythmias of the Ministry of Education of China, East Hospital, Tongji University School of Medicine, Shanghai, China\n<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Wei%20Y%5BAuthor%5D\">Youzhen Wei<\/a><\/div>\n<\/div>\n<div id=\"_co_idm140208059556640\">\n<h3 class=\"no_margin\">Nana Yang<\/h3>\n<p>\n<sup>3<\/sup><br \/>\nSchool of Bioscience and Technology, Weifang Medical University, Weifang, China\n<\/p>\n<p>\n<sup>4<\/sup><br \/>\nMedical Laboratory Animal Center, Weifang Medical University, Weifang, China\n<\/p>\n<p>\n<sup>5<\/sup><br \/>\nWeifang Key Laboratory of Animal Model Research on Cardiovascular and Cerebrovascular Diseases, Weifang, China\n<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Yang%20N%5BAuthor%5D\">Nana Yang<\/a><\/div>\n<\/div>\n<\/div>\n<\/div>\n<div class=\"half_rhythm\">\n<div class=\"togglers fm-copyright-license\"><a href=\"#\" class=\"pmctoggle\" rid=\"idm140208061449648_ai\">Author information<\/a> <a href=\"#\" class=\"pmctoggle\" rid=\"idm140208061449648_an\">Article notes<\/a> <a href=\"#\" class=\"pmctoggle\" rid=\"idm140208061449648_cpl\">Copyright and License information<\/a> <a href=\"\/pmc\/about\/disclaimer\/\" style=\"margin-left: 1em\">PMC Disclaimer<\/a><\/div>\n<div class=\"fm-authors-info hide half_rhythm\" id=\"idm140208061449648_ai\" style=\"display:none\">\n<div class=\"fm-affl\" id=\"aff1\">\n<sup>1<\/sup><br \/>\nSchool of Clinical Medicine, Weifang Medical University, Weifang, China\n<\/div>\n<div class=\"fm-affl\" id=\"aff2\">\n<sup>2<\/sup><br \/>\nUniversity of Health and Rehabilitation Sciences, Qingdao, China\n<\/div>\n<div class=\"fm-affl\" id=\"aff3\">\n<sup>3<\/sup><br \/>\nSchool of Bioscience and Technology, Weifang Medical University, Weifang, China\n<\/div>\n<div class=\"fm-affl\" id=\"aff4\">\n<sup>4<\/sup><br \/>\nMedical Laboratory Animal Center, Weifang Medical University, Weifang, China\n<\/div>\n<div class=\"fm-affl\" id=\"aff5\">\n<sup>5<\/sup><br \/>\nWeifang Key Laboratory of Animal Model Research on Cardiovascular and Cerebrovascular Diseases, Weifang, China\n<\/div>\n<div class=\"fm-affl\" id=\"aff6\">\n<sup>6<\/sup><br \/>\nSchool of Rehabilitation Medicine, Weifang Medical University, Weifang, China\n<\/div>\n<div class=\"fm-affl\" id=\"aff7\">\n<sup>7<\/sup><br \/>\nResearch Center of Translational Medicine Shanghai East Hospital, Tongji University, Shanghai, China\n<\/div>\n<div class=\"fm-affl\" id=\"aff8\">\n<sup>8<\/sup><br \/>\nShandong Qilu Stem Cell Engineering Co., Jinan, China\n<\/div>\n<div class=\"fm-affl\" id=\"aff9\">\n<sup>9<\/sup><br \/>\nDepartment of Respiratory, Dongying People\u2019s Hospital, Dongying, China\n<\/div>\n<div class=\"fm-affl\" id=\"aff10\">\n<sup>10<\/sup><br \/>\nResearch Center for Translational Medicine and Key Laboratory of Arrhythmias of the Ministry of Education of China, East Hospital, Tongji University School of Medicine, Shanghai, China\n<\/div>\n<div><sup><img decoding=\"async\" src=\"\/corehtml\/pmc\/pmcgifs\/corrauth.gif\" alt=\"corresponding author\"><\/sup>Corresponding author.<\/div>\n<div id=\"fn-a.l.b.p.a\">\n<strong>Edited by:<\/strong><br \/>\n<a href=\"https:\/\/loop.frontiersin.org\/people\/277275\/overview\" data-ga-action=\"click_feat_suppl\" ref=\"reftype=extlink&amp;article-id=9133378&amp;issue-id=400058&amp;journal-id=1524&amp;FROM=Article%7CFront%20Matter&amp;TO=External%7CLink%7CURI\" target=\"_blank\" rel=\"noopener\">Abdur Rauf<\/a>, University of Swabi, Pakistan<\/div>\n<div id=\"fn-a.l.b.p.b\">\n<strong>Reviewed by:<\/strong><br \/>\n<a href=\"https:\/\/loop.frontiersin.org\/people\/1729416\/overview\" data-ga-action=\"click_feat_suppl\" ref=\"reftype=extlink&amp;article-id=9133378&amp;issue-id=400058&amp;journal-id=1524&amp;FROM=Article%7CFront%20Matter&amp;TO=External%7CLink%7CURI\" target=\"_blank\" rel=\"noopener\">Xiaolu Zhang<\/a>, University of Toledo, United States<\/p>\n<p class=\"p p-last\">\n<a href=\"https:\/\/loop.frontiersin.org\/people\/317984\/overview\" data-ga-action=\"click_feat_suppl\" ref=\"reftype=extlink&amp;article-id=9133378&amp;issue-id=400058&amp;journal-id=1524&amp;FROM=Article%7CFront%20Matter&amp;TO=External%7CLink%7CURI\" target=\"_blank\" rel=\"noopener\">Pan Yu<\/a>, Nanjing General Hospital of Nanjing Military Command, China<\/p>\n<\/div>\n<div id=\"c001\">*Correspondence: Benhong Liu, <a href=\"mailto:dev@null\" data-email=\"moc.361@7970uiqakb\" class=\"oemail\">moc.361@7970uiqakb<\/a&gt;; Youzhen Wei, <a href=\"mailto:dev@null\" data-email=\"moc.361@nehzuoy-iew\" class=\"oemail\">moc.361@nehzuoy-iew<\/a&gt;; Nana Yang, <a href=\"mailto:dev@null\" data-email=\"moc.621@0891nebneb\" class=\"oemail\">moc.621@0891nebneb<\/a>\n<\/div>\n<div id=\"fn1\"><sup><br \/>\n\u2020<br \/>\n<\/sup>These authors have contributed equally to this work and share first authorship.<\/div>\n<div id=\"fn-a.l.b.p.e\">This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology<\/div>\n<\/div>\n<div class=\"fm-article-notes hide half_rhythm\" id=\"idm140208061449648_an\" style=\"display:none\">\n<div class=\"fm-pubdate half_rhythm\">Received 2022 Mar 12; Accepted 2022 Apr 21.<\/div>\n<\/div>\n<div class=\"permissions half_rhythm hide\" id=\"idm140208061449648_cpl\" style=\"display:none\">\n<div class=\"fm-copyright half_rhythm\"><a href=\"\/pmc\/about\/copyright\/\">Copyright<\/a>  \u00a9 2022 Mu, Lv, Yu, Chu, Zhang, Kong, Zhang, Tian, Jia, Liu, Wei and Yang.<\/div>\n<div class=\"license half_rhythm\">This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.<\/div>\n<\/div>\n<\/div>\n<div id=\"pmclinksbox\" class=\"links-box whole_rhythm hidden\" role=\"complementary\" aria-label=\"Related or updated information about this article.\"><\/div>\n<\/div>\n<div class=\"sec\"><\/div>\n<div id=\"ass-data\" class=\"tsec fm-sec whole_rhythm\" data-section=\"Featured_PMC_Datacitation\">\n<h2 class=\"nomenu\">Associated Data<\/h2>\n<dl data-count=\"1\" class=\"box-data-suppmats whole_rhythm no_bottom_margin\">\n<dt><a href=\"#\" rid=\"data-suppmats\" data-ga-action=\"click_feat_toggler\" data-ga-label=\"Supplementary Materials\" class=\"pmctoggle\">Supplementary Materials<\/a><\/dt>\n<dd id=\"data-suppmats\" style=\"display: none;\">\n<div class=\"half_rhythm\">\n<div><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/bin\/Image1.TIF\" data-ga-action=\"click_feat_suppl\">Image1.TIF<\/a><span style=\"color:gray\"> (3.0M)<\/span><\/div>\n<div class=\"small guid\">GUID:&nbsp;DF758B4D-2F52-4EAD-9102-B3DCEF75AC0A<\/div>\n<\/div>\n<\/dd>\n<\/dl>\n<dl data-length=\"120\" class=\"box-data-avail whole_rhythm no_bottom_margin\">\n<dt><a href=\"#\" rid=\"data-avl-stmnt\" data-ga-action=\"click_feat_toggler\" data-ga-label=\"Data Availability Statement\" class=\"pmctoggle\">Data Availability Statement<\/a><\/dt>\n<dd id=\"data-avl-stmnt\" style=\"display: none;\">\n<p class=\"p p-first-last\">The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.<\/p>\n<\/dd>\n<\/dl>\n<\/div>\n<div id=\"abstract-a.l.b.w\" lang=\"en\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><span role=\"menubar\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"menuitem\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/span><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"abstract-a.l.b.wtitle\">Abstract<\/h2>\n<p><!--article-meta--><\/p>\n<div>\n<p class=\"p p-first-last\">Endotoxins and other harmful substances may cause an increase in permeability in endothelial cells (ECs) monolayers, as well as ECs shrinkage and death to induce lung damage. Lipopolysaccharide (LPS) can impair endothelial progenitor cells (EPCs) functions, including proliferation, migration, and tube formation. EPCs can migrate to the damaged area, differentiate into ECs, and participate in vascular repair, which improves pulmonary capillary endothelial dysfunction and maintains the integrity of the endothelial barrier. Hydrogen (H<sub>2<\/sub>) contributes to the repairment of lung injury and the damage of ECs. We therefore speculate that H<sub>2<\/sub> protects the EPCs against LPS-induced damage, and it\u2019s mechanism will be explored. The bone marrow-derived EPCs from ICR Mice were treated with LPS to establish a damaged model. Then EPCs were incubated with H<sub>2<\/sub>, and treated with PI3K inhibitor LY294002 and endothelial nitric oxide synthase (eNOS) inhibitor L-NAME. MTT assay, transwell assay and tube formation assay were used to detect the proliferation, migration and angiogenesis of EPCs. The expression levels of target proteins were detected by Western blot. Results found that H<sub>2<\/sub> repaired EPCs proliferation, migration and tube formation functions damaged by LPS. LY294002 and L-NAME significantly inhibited the repaired effect of H<sub>2<\/sub> on LPS-induced dysfunctions of EPCs. H<sub>2<\/sub> also restored levels of phosphor-AKT (p-AKT), eNOS and phosphor-eNOS (p-eNOS) suppressed by LPS. LY294002 significantly inhibited the increase of p-AKT and eNOS and p-eNOS expression exposed by H<sub>2<\/sub>. L-NAME significantly inhibited the increase of eNOS and p-eNOS expression induced by H<sub>2<\/sub>. H<sub>2<\/sub> repairs the dysfunctions of EPCs induced by LPS, which is mediated by PI3K\/AKT\/eNOS signaling pathway.<\/p>\n<\/div>\n<div class=\"sec\"><strong class=\"kwd-title\">Keywords: <\/strong><span class=\"kwd-text\">endothelial progenitor cells, lung injury, hydrogen, PI3K\/AKT\/eNOS signaling pathway, lipopolysaccharide<\/span><\/div>\n<\/div>\n<div id=\"s1\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"s1title\">Introduction<\/h2>\n<p class=\"p p-first\">Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are a series of pulmonary pathological changes arising from a wide variety of lung injuries, which have high morbidity and mortality (<a href=\"#B3\" rid=\"B3\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Butt et al., 2016<\/a>), characterized by disruption of endothelial barrier integrity and diffuse lung damage. It can cause an imbalance between coagulation and inflammation to induce inflammation (<a href=\"#B14\" rid=\"B14\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Frantzeskaki et al., 2017<\/a>). Besides, macrophages, neutrophils inflammatory cells and their pro-inflammatory products can destroy pulmonary epithelial cells, increase pulmonary microvascular permeability, produce pulmonary edema, damage gas exchange, and lead to respiratory failure (<a href=\"#B3\" rid=\"B3\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Butt et al., 2016<\/a>). Therefore, how to maintain the integrity of the endothelial barrier through the regulation of the microenvironment in the inflammatory state is critical to the treatment.<\/p>\n<p>ECs dysfunction and inflammation contribute to the occurrence and development of lung injury. Thus, vascular endothelial repair is an integral part of lung injury repair (<a href=\"#B57\" rid=\"B57\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Zhao et al., 2020<\/a>). EPCs are a kind of progenitor cells that can differentiate into vascular ECs (<a href=\"#B1\" rid=\"B1\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Asahara et al., 1997<\/a>), which can migrate to the damaged area and differentiate into ECs to participate in angiogenesis or repair, and promote the improvement of endothelial functions (<a href=\"#B30\" rid=\"B30\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Li et al., 2017<\/a>). In addition, EPCs can repair the vascular injury and alleviate LPS-induced lung injury, reduce inflammation, and promote bacterial clearance of pneumonia (<a href=\"#B36\" rid=\"B36\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Mao et al., 2010<\/a>), which has a broad prospect in the treatment of lung injury.<\/p>\n<p class=\"p p-last\">Hydrogen (H<sub>2<\/sub>) is an important physiological regulatory factor that has protective effects of anti-oxidation, anti-inflammation, and anti-apoptosis on cells and organs (<a href=\"#B22\" rid=\"B22\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Huang et al., 2010b<\/a>). H<sub>2<\/sub> can reduce oxidative stress (<a href=\"#B44\" rid=\"B44\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Song et al., 2011<\/a>), promote the scavenging of free radicals, and inhibit vascular aging (<a href=\"#B23\" rid=\"B23\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Iketani et al., 2018<\/a>). A multicenter, open-label clinical trial showed that hydrogen\/oxygen mixed gas inhalation improved disease severity and dyspnea in patients with Coronavirus disease 2019 (COVID-19) (<a href=\"#B17\" rid=\"B17\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Guan et al., 2020<\/a>). In addition, H<sub>2<\/sub> has an excellent therapeutic effect on inflammation, ischemia-reperfusion injury, diabetes, cancer, atherosclerosis, and other diseases (<a href=\"#B27\" rid=\"B27\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Li et al., 2013<\/a&gt;; <a href=\"#B26\" rid=\"B26\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Lee et al., 2015<\/a&gt;; <a href=\"#B42\" rid=\"B42\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Shimada et al., 2016<\/a&gt;; <a href=\"#B31\" rid=\"B31\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Li et al., 2019<\/a>). It can reduce the levels of serum Low-Density Lipoprotein Cholesterol (LDL-C) and apolipoprotein- B (Apo-B), improve the high-density lipoprotein (HDL) functions damaged by dyslipidemia (<a href=\"#B43\" rid=\"B43\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Song et al., 2013<\/a>), reduce the formation of neointima after vein transplantation in rats (<a href=\"#B45\" rid=\"B45\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Sun et al., 2012<\/a>), and decrease hypertension, angiogenesis imbalance and oxidative stress caused by placental ischemia (<a href=\"#B46\" rid=\"B46\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Ushida et al., 2016<\/a>). It also can protect the pulmonary microvessels of mice from the endothelial function damage induced by septicemia, maintain the consistency of pulmonary endothelium (<a href=\"#B32\" rid=\"B32\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Li Y. et al., 2020<\/a>), and improve microvascular ECs viability in traumatic brain injury by inhibiting autophagy (<a href=\"#B49\" rid=\"B49\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Wang Y. et al., 2020<\/a>). Animal experimental studies have shown that H<sub>2<\/sub> inhalation can provide protection in animal models of lung injury caused by mechanical ventilation, sepsis, ischemia-reperfusion, LPS and hyperoxia, seawater infusion, etc. (<a href=\"#B38\" rid=\"B38\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Ohsawa et al., 2007<\/a&gt;; <a href=\"#B51\" rid=\"B51\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Xie et al., 2010<\/a&gt;; <a href=\"#B7\" rid=\"B7\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Chen et al., 2015<\/a&gt;; <a href=\"#B8\" rid=\"B8\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Diao et al., 2016<\/a&gt;; <a href=\"#B2\" rid=\"B2\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Audi et al., 2017<\/a>). Clinical studies show that the inhalation of H<sub>2<\/sub> by pregnant women can also inhibit the LPS-induced apoptosis and oxidative damage of fetal lung cells (<a href=\"#B18\" rid=\"B18\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Hattori et al., 2015<\/a>). Previous studies have found that EPCs can repair lung injury induced by LPS (<a href=\"#B53\" rid=\"B53\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Yang et al., 2019<\/a>), and H<sub>2<\/sub> has the same effect (<a href=\"#B18\" rid=\"B18\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Hattori et al., 2015<\/a>). We speculate that H<sub>2<\/sub> may have a protective effect on EPCs, and even repair LPS-induced lung injury by improving the functions of EPCs. In this study, the injury model of EPCs was established by LPS treatment, and the cell viability, migration, angiogenic ability, and related protein expression of EPCs were measured. The molecular mechanisms of H<sub>2<\/sub> on the functional damage and repair of EPCs induced by LPS were discussed.<\/p>\n<\/div>\n<div id=\"s2\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"s2title\">Materials and Methods<\/h2>\n<div id=\"s2-1\" class=\"sec sec-first\">\n<h3 id=\"s2-1title\">Animals<\/h3>\n<p class=\"p p-first-last\">ICR mice (4&nbsp;weeks old, males) were obtained from the Cavens Company (Jiang Su, China). All animal experiments were approved by the Animal Experimental Ethics Committee of Weifang Medical University (approval code: 2019SDL108).<\/p>\n<\/div>\n<div id=\"s2-2\" class=\"sec\">\n<h3 id=\"s2-2title\">Isolation and Culture of EPCs<\/h3>\n<p class=\"p p-first-last\">MNCs were isolated from the femurs of 4-week-old male ICR mice by density gradient centrifugation using Histopaque 1,083 (Sigma, St. Louis, MO, United States). The isolated MNC were seeded in a 6-well culture plate coated with fibronectin and cultured in EGM-2MV (Endothelial cell basal medium-2, plus FBS, VEGF, R-IGF-1, rhEGF, rhFGF-B, GA-1000, hydrocortisone and ascorbic acid) (Lonza, Basel, Switzerland). After 3&nbsp;days of culture at 37\u00b0C with 5% CO<sub>2<\/sub>, the culture medium was changed thoroughly with fresh culture medium, and non-adherent cells were removed, and the culture medium was changed every 2&nbsp;days.<\/p>\n<\/div>\n<div id=\"s2-3\" class=\"sec\">\n<h3 id=\"s2-3title\">Characterization of EPCs<\/h3>\n<p class=\"p p-first\">MNCs from mice bone marrow were cultured for 5&nbsp;days, incubated with 50&nbsp;ug\/ml Human Dil-Acetylated Low Density Lipoprotein (Dil-Ac-LDL, FuShen, Shanghai, China) at 37\u00b0C for 4&nbsp;h. Then cells were fixed in 4% paraformaldehyde (PFA), incubated with FITC-labeled Ulex europaeus agglutinin 1 (FITC-UEA-1, FuShen, Shanghai, China) for 1&nbsp;h. After setting the image acquisition parameters at each wavelength using background control, images were obtained under OLYMPUS, IX71 fluorescence microscope at 400x.<\/p>\n<p class=\"p p-last\">We further detected the expression of surface markers in cells at 10d and 21d. Cells were fixed in 4% PFA, treated with 0.1% Triton X-100 for 10&nbsp;min. After being blocked with 5% FBS for 1&nbsp;h at room temperature, the cells were incubated with primary antibodies against CD117 (C-Kit) (eBioscience, San Diego, CA, United States), SCA-1 (Abcam, Cambridge, MA, United States), VEGFR 2 (Abcam, Cambridge, MA, United States), CD31 (Abcam, Cambridge, MA, United States), eNOS (Cell Signaling Technologies, Danvers, MA, United States) overnight at 4\u00b0C. After being washed with PBS, EPCs were incubated with secondary antibodies conjugated with Cy3 (Goat anti-mouse cy3, 1:100, Proteintech Group, Chicago, IL, United States; Goat anti-Rat cy3, 1:100, Jackson Immunoresearch Laboratories, West Grove, PA, United States) or FITC (Goat anti-rabbit FITC, 1:100, Proteintech Group, Chicago, IL, United States) for 1&nbsp;h at 37\u00b0C. The immunofluorescence staining was evaluated under a fluorescence microscope (OLYMPUS, IX71, 400x).<\/p>\n<\/div>\n<div id=\"s2-4\" class=\"sec\">\n<h3 id=\"s2-4title\">EPCs Treatment<\/h3>\n<p class=\"p p-first-last\">Before the experiment, the cell culture medium was replaced by basic medium (M199 + 5% FBS). Then EPCs were treated with different concentrations (2.5&nbsp;\u03bcg\/ml, 5&nbsp;\u03bcg\/ml, 10&nbsp;\u03bcg\/ml, 20&nbsp;\u03bcg\/ml) of LPS (Solarbio, Beijing, China) at different time (24, 48, 72&nbsp;h) to establish a damaged model. The cell damaged model was treated with H<sub>2<\/sub> in different concentrations (20%, 40%, 60%) at different time (24, 48, 72&nbsp;h) to explore the suitable conditions of H<sub>2<\/sub>. The concentration of CO<sub>2<\/sub> in the H<sub>2<\/sub> incubator is 5%, the concentration of O<sub>2<\/sub> is 21%, and the concentration of H<sub>2<\/sub> is adjusted by N<sub>2<\/sub>. Finally, EPCs were treated with PI3K inhibitor LY294002 (Sigma-Aldrich, St Louis, MO, United States) (10&nbsp;\u03bcM, 20&nbsp;\u03bcM, 30&nbsp;\u03bcM) or eNOS inhibitor L-NAME (Beyotime, Shanghai, China) (100&nbsp;\u03bcM, 200&nbsp;\u03bcM) to find out the suitable concentration of inhibitors.<\/p>\n<\/div>\n<div id=\"s2-5\" class=\"sec\">\n<h3 id=\"s2-5title\">Cell Viability of EPCs<\/h3>\n<p class=\"p p-first-last\">EPCs were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Cells (100\u03bcl, 5\u00d710<sup>4<\/sup>&nbsp;cells\/mL) were seeded in 96-well plates and cultured for 24&nbsp;h until adhered to the wall. After different treatments, cells were incubated with MTT (20&nbsp;\u03bcl, 5&nbsp;mg\/ml) for 4&nbsp;h at 37\u00b0C with 5% CO<sub>2<\/sub>. 200 \u03bcl of dimethylsulfoxide (DMSO) was added to each well and shaken for 10&nbsp;min. The optical density (OD) values at 492&nbsp;nm were determined using a microplate spectrophotometer (Multiskan GO, Thermo, United States).<\/p>\n<\/div>\n<div id=\"s2-6\" class=\"sec\">\n<h3 id=\"s2-6title\">EPCs Migration Assay<\/h3>\n<p class=\"p p-first-last\">EPCs migration was measured by an 8&nbsp;\u03bcm pore 24-well Cell Migration Assay kit (BD Biosciences, San Jose, CA, United States). EPCs suspension (300&nbsp;\u03bcl, 5\u00d710<sup>4<\/sup>&nbsp;cells\/mL in M199 medium) was added to the upper chamber and cultured according to different groups, and 600&nbsp;\u03bcl EGM-2MV medium was added lower chamber. After different treatments, the chamber was taken out, scrubbed carefully with a cotton swab and rinsed with PBS. The lower cells were fixed and stained with 0.1% crystal violet. Carefully cut off the Polycarbonate film from the base of the upper chamber, seal the film and take a picture under a microscope.<\/p>\n<\/div>\n<div id=\"s2-7\" class=\"sec\">\n<h3 id=\"s2-7title\">Tube Formation <em>in Vitro<\/em><br \/>\n<\/h3>\n<p class=\"p p-first-last\">Matrigel (BD Biosciences, San Jose, CA, United States) matrix was dissolved overnight at 4\u00b0C. After being placed in a 37\u00b0C incubator for 30min, 250&nbsp;\u03bcl Matrigel was added to a 24-well plate. After treatments, 5\u00d710<sup>4<\/sup>&nbsp;cells were seeded in the Matrigel-coated plate. After 6&nbsp;h incubation, Calcein AM was added to staining for 30&nbsp;min, and the samples were observed and photographed under a fluorescence microscope.<\/p>\n<\/div>\n<div id=\"s2-8\" class=\"sec\">\n<h3 id=\"s2-8title\">Western Blot<\/h3>\n<p class=\"p p-first-last\">Total protein was extracted with Radio immunoprecipitation assay (RIPA, Beyotime, Shanghai, China) lysis buffer and quantified with a BCA assay kit (Solarbio, Beijing, China). In total, 25&nbsp;\u03bcg of protein was electrophoresed on a 10% denaturing polyacrylamide gel and transferred onto polyvinylidene difluoride (PVDF) membranes. After being blocked with 7% dried skimmed milk for 3&nbsp;h at room temperature, the membranes were incubated with primary antibodies against GAPDH (1: 20,000, Proteintech Group, Chicago, IL, United States), AKT (1: 1,000, Proteintech Group, Chicago, IL, United States), p-AKT (1: 2000, Proteintech Group, Chicago, IL, United States), eNOS (1: 1,000, Cell Signaling Technologies, Danvers, MA, United States), p-eNOS (1: 1,000, Cell Signaling Technologies, Danvers, MA, United States) overnight at 4&nbsp;\u00b0C under constant shaking. After washing with TBST buffer (Shandong Sparkjade Biotechnology Co., Ltd.) 4 times (each 5min), the membranes were incubated with the secondary antibodies conjugated to horseradish peroxidase (HRP) for 3&nbsp;h at room temperature under constant shaking. After washing with TBST buffer for three times (each 5&nbsp;min), the protein bands on the PVDF membrane were detected using the ECL chemiluminescence detection kit and chemiluminescence gel imaging system (FluorChem Q, ProteinSimple, CA, United States).<\/p>\n<\/div>\n<div id=\"s2-9\" class=\"sec sec-last\">\n<h3 id=\"s2-9title\">Statistical Analyses<\/h3>\n<p class=\"p p-first-last\">All data are presented as mean \u00b1 standard deviation (SD). The data were analyzed using SPSS software (version 26.0, SPSS Inc., Chicago, IL, United States). Differences between three groups or more were analyzed by one-way ANOVA. Values were considered significant at <em>p<\/em> &lt; 0.05.<\/p>\n<\/div>\n<\/div>\n<div id=\"s3\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"s3title\">Results<\/h2>\n<div id=\"s3-1\" class=\"sec sec-first\">\n<h3 id=\"s3-1title\">Isolation and Characterization of EPCs<\/h3>\n<p class=\"p p-first\">Bone marrow mononuclear cells (MNCs) isolated from mouse bone marrow showed cobblestone-like morphology (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F1\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"F1\" rid-ob=\"ob-F1\" co-legend-rid=\"lgnd_F1\" rel=\"noopener\"><span>Supplementary Figure 1<\/span><\/a>). These induced MNCs engulfed Dil-ac-LDL and FITC-UEA-1 (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F1\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"F1\" rid-ob=\"ob-F1\" co-legend-rid=\"lgnd_F1\" rel=\"noopener\"><span>Figure 1A<\/span><\/a>), the differentiation markers of EPCs. Then we examined cell-surface markers of EPCs. After 10&nbsp;days of culture, VEGFR-2 with C-kit were co-expressed in the isolated cells (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F1\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"F1\" rid-ob=\"ob-F1\" co-legend-rid=\"lgnd_F1\" rel=\"noopener\"><span>Figure 1B<\/span><\/a>), and VEGFR-2 with SCA-1 were also co-expressed. (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F1\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"F1\" rid-ob=\"ob-F1\" co-legend-rid=\"lgnd_F1\" rel=\"noopener\"><span>Figure 1C<\/span><\/a>). After 21&nbsp;days of culture, the cells expressed both eNOS and CD31. (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F1\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"F1\" rid-ob=\"ob-F1\" co-legend-rid=\"lgnd_F1\" rel=\"noopener\"><span>Figure 1D<\/span><\/a>). Therefore, these induced MNCs were characterized as EPCs and could be used in later experiments.<\/p>\n<p><!--fig ft0--><!--fig mode=article f1--><\/p>\n<div class=\"fig iconblock whole_rhythm\" id=\"F1\" co-legend-rid=\"lgnd_F1\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F1\/\" target=\"figure\" rid-figpopup=\"F1\" rid-ob=\"ob-F1\" rel=\"noopener\"><!--fig\/graphic|fig\/alternatives\/graphic mode=\"anchored\" m1--><\/a><\/p>\n<div class=\"figure\" data-largeobj=\"\" data-largeobj-link-rid=\"largeobj_idm140208060005088\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F1\/\" target=\"figure\" rid-figpopup=\"F1\" rid-ob=\"ob-F1\" rel=\"noopener\"><\/a><a class=\"inline_block ts_canvas\" href=\"\/core\/lw\/2.0\/html\/tileshop_pmc\/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&amp;p=PMC3&amp;id=9133378_fphar-13-894812-g001.jpg\" target=\"tileshopwindow\" rel=\"noopener\"><\/p>\n<div class=\"ts_bar small\" title=\"Click on image to zoom\"><\/div>\n<p><img decoding=\"async\" loading=\"lazy\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is fphar-13-894812-g001.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/fphar-13-894812-g001.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm140208060005088\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F1\/?report=objectonly\">Open in a separate window<\/a><\/div>\n<div class=\"icnblk_cntnt\" id=\"lgnd_F1\">\n<div><a class=\"figpopup\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F1\/\" target=\"figure\" rid-figpopup=\"F1\" rid-ob=\"ob-F1\" rel=\"noopener\">FIGURE 1<\/a><\/div>\n<p><!--caption a7--><\/p>\n<div class=\"caption\">\n<p>Isolation and characterization of mice bone marrow-derived EPCs. <strong>(A)<\/strong> DiI-ac-LDL (red) and FITC-UEA-1 (green) could be taken up by EPCs (blue) and merged images of red and green fluorescence. Scale bar = 20&nbsp;\u03bcm (400x). <strong>(B)<\/strong> Co-expression of VEGFR-2 (green) with C-kit (red) in cells at 10&nbsp;d. <strong>(C)<\/strong> Co-expression of VEGFR-2 (green) with SCA-1 (red) in cells at 10&nbsp;d. <strong>(D)<\/strong> Co-expression of eNOS (green) with CD31 (red) in cells at 21&nbsp;days. Scale bar = 20&nbsp;\u03bcm (400x).<\/p>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<div id=\"s3-2\" class=\"sec\">\n<h3 id=\"s3-2title\">LPS Impaired EPCs Functions<\/h3>\n<p class=\"p p-first\">MTT assay results showed that, compared with the control group, LPS reduced the viability of EPCs in a concentration-dependent and time-dependent manner. We used 20&nbsp;\u03bcg\/ml LPS to induce 72&nbsp;h for the follow-up experiments (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F2\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"F2\" rid-ob=\"ob-F2\" co-legend-rid=\"lgnd_F2\" rel=\"noopener\"><span>Figure 2<\/span><\/a>). In addition, we also found that LPS significantly reduced the ability of migration and tube formation of EPCs (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F3\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"F3\" rid-ob=\"ob-F3\" co-legend-rid=\"lgnd_F3\" rel=\"noopener\"><span>Figures 3B\u2013E<\/span><\/a>).<\/p>\n<p><!--fig ft0--><!--fig mode=article f1--><\/p>\n<div class=\"fig iconblock whole_rhythm\" id=\"F2\" co-legend-rid=\"lgnd_F2\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F2\/\" target=\"figure\" rid-figpopup=\"F2\" rid-ob=\"ob-F2\" rel=\"noopener\"><!--fig\/graphic|fig\/alternatives\/graphic mode=\"anchored\" m1--><\/p>\n<div class=\"figure\" data-largeobj=\"\" data-largeobj-link-rid=\"largeobj_idm140208059998160\"><img decoding=\"async\" loading=\"lazy\" class=\"fig-image\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is fphar-13-894812-g002.jpg\" title=\"An external file that holds a picture, illustration, etc.\nObject name is fphar-13-894812-g002.jpg\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/fphar-13-894812-g002.jpg\"><\/div>\n<p><\/a><\/p>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm140208059998160\" style=\"display: none;\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F2\/\" target=\"figure\" rid-figpopup=\"F2\" rid-ob=\"ob-F2\" rel=\"noopener\"><\/a><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F2\/?report=objectonly\">Open in a separate window<\/a><\/div>\n<div class=\"icnblk_cntnt\" id=\"lgnd_F2\">\n<div><a class=\"figpopup\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F2\/\" target=\"figure\" rid-figpopup=\"F2\" rid-ob=\"ob-F2\" rel=\"noopener\">FIGURE 2<\/a><\/div>\n<p><!--caption a7--><\/p>\n<div class=\"caption\">\n<p>EPCs viability was reduced after being induced by LPS. EPCs were treated with different concentrations (2.5&nbsp;\u03bcg\/ml, 5&nbsp;\u03bcg\/ml, 10&nbsp;\u03bcg\/ml, 20&nbsp;\u03bcg\/ml) LPS for different time (24, 48, 72&nbsp;h). Cell viability was measured by MTT assay. Data are presented as mean \u00b1 SD (<em>n<\/em> = 6); **<em>p<\/em> &lt; 0.01.<\/p>\n<\/div>\n<\/div>\n<\/div>\n<p><!--fig ft0--><!--fig mode=article f1--><\/p>\n<div class=\"fig iconblock whole_rhythm\" id=\"F3\" co-legend-rid=\"lgnd_F3\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F3\/\" target=\"figure\" rid-figpopup=\"F3\" rid-ob=\"ob-F3\" rel=\"noopener\"><!--fig\/graphic|fig\/alternatives\/graphic mode=\"anchored\" m1--><\/a><\/p>\n<div class=\"figure\" data-largeobj=\"\" data-largeobj-link-rid=\"largeobj_idm140208059992688\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F3\/\" target=\"figure\" rid-figpopup=\"F3\" rid-ob=\"ob-F3\" rel=\"noopener\"><\/a><a class=\"inline_block ts_canvas\" href=\"\/core\/lw\/2.0\/html\/tileshop_pmc\/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&amp;p=PMC3&amp;id=9133378_fphar-13-894812-g003.jpg\" target=\"tileshopwindow\" rel=\"noopener\"><\/p>\n<div class=\"ts_bar small\" title=\"Click on image to zoom\"><\/div>\n<p><img decoding=\"async\" loading=\"lazy\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is fphar-13-894812-g003.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/fphar-13-894812-g003.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm140208059992688\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F3\/?report=objectonly\">Open in a separate window<\/a><\/div>\n<div class=\"icnblk_cntnt\" id=\"lgnd_F3\">\n<div><a class=\"figpopup\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F3\/\" target=\"figure\" rid-figpopup=\"F3\" rid-ob=\"ob-F3\" rel=\"noopener\">FIGURE 3<\/a><\/div>\n<p><!--caption a7--><\/p>\n<div class=\"caption\">\n<p>H<sub>2<\/sub> alleviated EPCs dysfunctions induced by LPS. EPCs treated with LPS induced EPCs damage, and then EPCs were treated with different concentrations (20%, 40%, 60%) for different time (24, 48, 72&nbsp;h). The viability of EPCs was assessed by MTT assay <strong>(A)<\/strong>. EPCs migration was measured by transwell assay <strong>(B,C)<\/strong>. Tube formation was measured by a Tube formation assay <strong>(D,E)<\/strong>. Data are presented as mean \u00b1 SD (<em>n<\/em> = 6); **<em>p<\/em> &lt; 0.01.<\/p>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<div id=\"s3-3\" class=\"sec\">\n<h3 id=\"s3-3title\">H<sub>2<\/sub> Alleviated LPS-Induced EPCs Dysfunctions<\/h3>\n<p class=\"p p-first\">Compared with the control group, LPS reduced the viability of EPCs. H<sub>2<\/sub> significantly improved the LPS-induced reduction in EPCs viability in a concentration-dependent and time-dependent manner, and the effect of 60% concentration of H<sub>2<\/sub> on EPCs for 72&nbsp;h was the most significant (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F3\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"F3\" rid-ob=\"ob-F3\" co-legend-rid=\"lgnd_F3\" rel=\"noopener\"><span>Figure 3A<\/span><\/a>).<\/p>\n<p>Migration assay showed that LPS reduced EPCs migration ability, compared with the control group. H<sub>2<\/sub> reversed the LPS-induced changes. H<sub>2<\/sub> increased the migration of EPCs in a concentration-dependent and time-dependent manner, and 60% concentration of H<sub>2<\/sub> treatment for 72&nbsp;h has the most significant effect on the repairment of EPCs migration ability (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F3\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"F3\" rid-ob=\"ob-F3\" co-legend-rid=\"lgnd_F3\" rel=\"noopener\"><span>Figure 3<\/span><\/a> B and C).<\/p>\n<p class=\"p p-last\">The EPCs tube formation was detected with tube formation assay. Compared with the control group, LPS reduces the tube formation of EPCs, which could be reversed by H<sub>2<\/sub> treatment in a concentration-dependent manner (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F3\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"F3\" rid-ob=\"ob-F3\" co-legend-rid=\"lgnd_F3\" rel=\"noopener\"><span>Figure 3<\/span><\/a> D and E).<\/p>\n<\/div>\n<div id=\"s3-4\" class=\"sec\">\n<h3 id=\"s3-4title\">LY294002 and L-NAME Inhibited H<sub>2<\/sub>-Mediated Restoration of EPCs Functions<\/h3>\n<p class=\"p p-first\">The effects of specific inhibitors LY294002 (PI3K inhibitor) and L-NAME (eNOS inhibitor) on EPCs functions were measured to investigate the protective mechanism of H<sub>2<\/sub>. As shown in <a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F4\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"F4\" rid-ob=\"ob-F4\" co-legend-rid=\"lgnd_F4\" rel=\"noopener\"><span>Figure 4<\/span><\/a> A and B, LY294002 (10&nbsp;\u03bcM, 20&nbsp;\u03bcM, 30&nbsp;\u03bcM) and L-NAME (100&nbsp;\u03bcM, 200&nbsp;\u03bcM) inhibited the H<sub>2<\/sub>-mediated restoration of EPCs viability impaired by LPS. 20&nbsp;\u03bcM LY294002 and 200&nbsp;\u03bcM&nbsp;L-NAME were employed in the subsequent experiments. <a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F4\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"F4\" rid-ob=\"ob-F4\" co-legend-rid=\"lgnd_F4\" rel=\"noopener\"><span>Figure 4<\/span><\/a> C-E showed that LY294002 and L-NAME significantly inhibited the H<sub>2<\/sub>-mediated restoration of EPCs migration and tube formation ability damaged by LPS.<\/p>\n<p><!--fig ft0--><!--fig mode=article f1--><\/p>\n<div class=\"fig iconblock whole_rhythm\" id=\"F4\" co-legend-rid=\"lgnd_F4\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F4\/\" target=\"figure\" rid-figpopup=\"F4\" rid-ob=\"ob-F4\" rel=\"noopener\"><!--fig\/graphic|fig\/alternatives\/graphic mode=\"anchored\" m1--><\/a><\/p>\n<div class=\"figure\" data-largeobj=\"\" data-largeobj-link-rid=\"largeobj_idm140208059972480\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F4\/\" target=\"figure\" rid-figpopup=\"F4\" rid-ob=\"ob-F4\" rel=\"noopener\"><\/a><a class=\"inline_block ts_canvas\" href=\"\/core\/lw\/2.0\/html\/tileshop_pmc\/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&amp;p=PMC3&amp;id=9133378_fphar-13-894812-g004.jpg\" target=\"tileshopwindow\" rel=\"noopener\"><\/p>\n<div class=\"ts_bar small\" title=\"Click on image to zoom\"><\/div>\n<p><img decoding=\"async\" loading=\"lazy\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is fphar-13-894812-g004.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/fphar-13-894812-g004.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm140208059972480\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F4\/?report=objectonly\">Open in a separate window<\/a><\/div>\n<div class=\"icnblk_cntnt\" id=\"lgnd_F4\">\n<div><a class=\"figpopup\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F4\/\" target=\"figure\" rid-figpopup=\"F4\" rid-ob=\"ob-F4\" rel=\"noopener\">FIGURE 4<\/a><\/div>\n<p><!--caption a7--><\/p>\n<div class=\"caption\">\n<p>LY294002 and L-NAME inhibited the H<sub>2<\/sub>-induced repair of EPCs dysfunctions damaged by LPS. EPCs were treated with the PI3K inhibitor LY294002 (10&nbsp;\u03bcM, 20&nbsp;\u03bcM, 30&nbsp;\u03bcM) or the eNOS inhibitor L-NAME (100&nbsp;\u03bcM, 200&nbsp;\u03bcM), incubated with LPS (20&nbsp;\u03bcg\/ml) and H<sub>2<\/sub> (60%) for 72&nbsp;h. The viability of EPCs was assessed by MTT assay <strong>(A,B)<\/strong>, <em>n<\/em> = 6). EPCs migration was measured by transwell assay <strong>(C,D)<\/strong><br \/>\n<em>n<\/em> = 15. Tube formation was measured by Tube formation assay <strong>(E,F)<\/strong><br \/>\n<em>n<\/em> = 15. Data are presented as mean \u00b1 SD; *<em>p<\/em> &lt; 0.05 and **<em>p<\/em> &lt; 0.01.,<\/p>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<div id=\"s3-5\" class=\"sec sec-last\">\n<h3 id=\"s3-5title\">H<sub>2<\/sub> Restored the PI3K\/AKT\/eNOS Pathway Inhibited by LPS<\/h3>\n<p class=\"p p-first\">We further investigated the protein levels of AKT, p-AKT, eNOS and p-eNOS to clarify the relationship between H<sub>2<\/sub> and PI3K\/AKT\/eNOS signaling pathway. As shown in <a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F5\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"F5\" rid-ob=\"ob-F5\" co-legend-rid=\"lgnd_F5\" rel=\"noopener\"><span>Figure 5<\/span><\/a>, LPS (20&nbsp;\u03bcg\/ml) decreased the protein levels of p-AKT, eNOS, p-eNOS in EPCs. H<sub>2<\/sub> restored these protein levels in a time-dependent manner, and H<sub>2<\/sub> treatment for 2&nbsp;h was employed in the subsequent experiments. As shown in <a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F6\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"F6\" rid-ob=\"ob-F6\" co-legend-rid=\"lgnd_F6\" rel=\"noopener\"><span>Figure 6<\/span><\/a>, LY294002 significantly inhibited the increased levels of p-AKT, eNOS, p-eNOS induced by 60% H<sub>2<\/sub>. <a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F7\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"F7\" rid-ob=\"ob-F7\" co-legend-rid=\"lgnd_F7\" rel=\"noopener\"><span>Figure 7<\/span><\/a> showed that L-NAME significantly inhibited the increased levels of eNOS and p-eNOS induced by 60% H<sub>2<\/sub>, however, there was no effect of L-NAME on the expression levels of p-AKT.<\/p>\n<p><!--fig ft0--><!--fig mode=article f1--><\/p>\n<div class=\"fig iconblock whole_rhythm\" id=\"F5\" co-legend-rid=\"lgnd_F5\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F5\/\" target=\"figure\" rid-figpopup=\"F5\" rid-ob=\"ob-F5\" rel=\"noopener\"><!--fig\/graphic|fig\/alternatives\/graphic mode=\"anchored\" m1--><\/a><\/p>\n<div class=\"figure\" data-largeobj=\"\" data-largeobj-link-rid=\"largeobj_idm140208059960400\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F5\/\" target=\"figure\" rid-figpopup=\"F5\" rid-ob=\"ob-F5\" rel=\"noopener\"><\/a><a class=\"inline_block ts_canvas\" href=\"\/core\/lw\/2.0\/html\/tileshop_pmc\/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&amp;p=PMC3&amp;id=9133378_fphar-13-894812-g005.jpg\" target=\"tileshopwindow\" rel=\"noopener\"><\/p>\n<div class=\"ts_bar small\" title=\"Click on image to zoom\"><\/div>\n<p><img decoding=\"async\" loading=\"lazy\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is fphar-13-894812-g005.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/fphar-13-894812-g005.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm140208059960400\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F5\/?report=objectonly\">Open in a separate window<\/a><\/div>\n<div class=\"icnblk_cntnt\" id=\"lgnd_F5\">\n<div><a class=\"figpopup\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F5\/\" target=\"figure\" rid-figpopup=\"F5\" rid-ob=\"ob-F5\" rel=\"noopener\">FIGURE 5<\/a><\/div>\n<p><!--caption a7--><\/p>\n<div class=\"caption\">\n<p>\n<strong>(A)<\/strong> Representative western blot images. H<sub>2<\/sub> up-regulated the levels of p-AKT <strong>(B)<\/strong>, eNOS <strong>(C)<\/strong>, and p-eNOS <strong>(D)<\/strong> in EPCs at different time points. Cells were treated with LPS (20&nbsp;\u03bcg\/ml) and H<sub>2<\/sub> (60%) for 0, 1, 1.5 and 2&nbsp;h, respectively. Western blotting was used to detect the expression levels of target protein. Data are presented as mean \u00b1 SD; **<em>p<\/em> &lt; 0.01.<\/p>\n<\/div>\n<\/div>\n<\/div>\n<p><!--fig ft0--><!--fig mode=article f1--><\/p>\n<div class=\"fig iconblock whole_rhythm\" id=\"F6\" co-legend-rid=\"lgnd_F6\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F6\/\" target=\"figure\" rid-figpopup=\"F6\" rid-ob=\"ob-F6\" rel=\"noopener\"><!--fig\/graphic|fig\/alternatives\/graphic mode=\"anchored\" m1--><\/a><\/p>\n<div class=\"figure\" data-largeobj=\"\" data-largeobj-link-rid=\"largeobj_idm140208059954752\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F6\/\" target=\"figure\" rid-figpopup=\"F6\" rid-ob=\"ob-F6\" rel=\"noopener\"><\/a><a class=\"inline_block ts_canvas\" href=\"\/core\/lw\/2.0\/html\/tileshop_pmc\/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&amp;p=PMC3&amp;id=9133378_fphar-13-894812-g006.jpg\" target=\"tileshopwindow\" rel=\"noopener\"><\/p>\n<div class=\"ts_bar small\" title=\"Click on image to zoom\"><\/div>\n<p><img decoding=\"async\" loading=\"lazy\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is fphar-13-894812-g006.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/fphar-13-894812-g006.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm140208059954752\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F6\/?report=objectonly\">Open in a separate window<\/a><\/div>\n<div class=\"icnblk_cntnt\" id=\"lgnd_F6\">\n<div><a class=\"figpopup\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F6\/\" target=\"figure\" rid-figpopup=\"F6\" rid-ob=\"ob-F6\" rel=\"noopener\">FIGURE 6<\/a><\/div>\n<p><!--caption a7--><\/p>\n<div class=\"caption\">\n<p>\n<strong>(A)<\/strong> Representative western blot images. LY294002 inhibited the increase of p-AKT <strong>(B)<\/strong>, eNOS <strong>(C)<\/strong> and p-eNOS <strong>(D)<\/strong> expression induced by H<sub>2<\/sub> (60%). Cells were treated with LPS (20&nbsp;\u03bcg\/ml), H<sub>2<\/sub> (60%) and LY294002 (20&nbsp;\u03bcM) for 2&nbsp;h, respectively. Western blotting was used to detect the expression levels of target protein. Data are presented as mean \u00b1 SD; **<em>p<\/em> &lt; 0.01.<\/p>\n<\/div>\n<\/div>\n<\/div>\n<p><!--fig ft0--><!--fig mode=article f1--><\/p>\n<div class=\"fig iconblock whole_rhythm\" id=\"F7\" co-legend-rid=\"lgnd_F7\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F7\/\" target=\"figure\" rid-figpopup=\"F7\" rid-ob=\"ob-F7\" rel=\"noopener\"><!--fig\/graphic|fig\/alternatives\/graphic mode=\"anchored\" m1--><\/a><\/p>\n<div class=\"figure\" data-largeobj=\"\" data-largeobj-link-rid=\"largeobj_idm140208059949104\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F7\/\" target=\"figure\" rid-figpopup=\"F7\" rid-ob=\"ob-F7\" rel=\"noopener\"><\/a><a class=\"inline_block ts_canvas\" href=\"\/core\/lw\/2.0\/html\/tileshop_pmc\/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&amp;p=PMC3&amp;id=9133378_fphar-13-894812-g007.jpg\" target=\"tileshopwindow\" rel=\"noopener\"><\/p>\n<div class=\"ts_bar small\" title=\"Click on image to zoom\"><\/div>\n<p><img decoding=\"async\" loading=\"lazy\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is fphar-13-894812-g007.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/fphar-13-894812-g007.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm140208059949104\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F7\/?report=objectonly\">Open in a separate window<\/a><\/div>\n<div class=\"icnblk_cntnt\" id=\"lgnd_F7\">\n<div><a class=\"figpopup\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F7\/\" target=\"figure\" rid-figpopup=\"F7\" rid-ob=\"ob-F7\" rel=\"noopener\">FIGURE 7<\/a><\/div>\n<p><!--caption a7--><\/p>\n<div class=\"caption\">\n<p>\n<strong>(A)<\/strong> Representative western blot images. L-NAME inhibited the increase of eNOS <strong>(C)<\/strong> and p-eNOS <strong>(D)<\/strong> expression induced by H<sub>2<\/sub> (60%), and did not affect p-AKT levels <strong>(B)<\/strong>. Cells were treated with LPS (20&nbsp;\u03bcg\/ml), H<sub>2<\/sub> (60%) and L-NAME (200&nbsp;\u03bcM) for 2&nbsp;h, respectively. Western blotting was used to detect the expression levels of target protein. Data are presented as mean \u00b1 SD; **<em>p<\/em> &lt; 0.01.<\/p>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<div id=\"s4\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"s4title\">Discussion<\/h2>\n<p class=\"p p-first\">EPCs were first initially discovered in 1997 by Asahara et al. EPCs are defined as a cell population capable of differentiating into mature ECs and have vasculogenic potential, which contributes to vasculogenic, wound healing (<a href=\"#B1\" rid=\"B1\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Asahara et al., 1997<\/a>) and repair of ischemic tissue damage (<a href=\"#B12\" rid=\"B12\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Fan et al., 2014<\/a>). As a potential therapeutic agent, EPCs have attracted attention for a variety of diseases including cerebral ischemia (<a href=\"#B59\" rid=\"B59\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Zhou et al., 2021<\/a>), diabetes (<a href=\"#B47\" rid=\"B47\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Wang K. et al., 2020<\/a>), ALI etc. (<a href=\"#B1\" rid=\"B1\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Asahara et al., 1997<\/a>). However, due to differences in the isolation, amplification and identification of EPCs, as well as controversy over therapeutic function, the further development and clinical application of EPCs were limited. In this study, we successfully isolated the mouse bone marrow-derived EPCs for subsequent research of the repair mechanism of H<sub>2<\/sub> on EPCs dysfunctions induced by LPS. This study is the first report of the effect of H<sub>2<\/sub> on EPCs.<\/p>\n<p>H<sub>2<\/sub> is an odorless, colorless, tasteless, and insoluble multifunctional medical gas. It can cross the cellular membranes and has the functions of anti-oxidation, anti-inflammatory and anti-apoptosis (<a href=\"#B20\" rid=\"B20\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Hong et al., 2010<\/a>). In 1975, Dole et al. found that high-pressure hydrogen gas has an antioxidant effect and can inhibit the growth of tumors in mice (<a href=\"#B9\" rid=\"B9\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Dole et al., 1975<\/a>). However, the study was not taken seriously because of the limitations of the experiment and the difficulty of reproducibility. In recent years, research on H<sub>2<\/sub> in the medical field has gradually widened. Clinical studies have found that H<sub>2<\/sub> plays a therapeutic role in diseases which related to the respiratory system (<a href=\"#B17\" rid=\"B17\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Guan et al., 2020<\/a>), nervous system, cardiovascular (<a href=\"#B4\" rid=\"B4\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Camara et al., 2019<\/a>), digestive system (<a href=\"#B10\" rid=\"B10\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Eryilmaz et al., 2020<\/a>), reproductive system, urinary system (<a href=\"#B16\" rid=\"B16\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Gokalp et al., 2017<\/a>), and metabolic exercise (<a href=\"#B39\" rid=\"B39\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Qiu et al., 2020<\/a>). It is recommended to inhale a mixture of H<sub>2<\/sub> and oxygen (O<sub>2<\/sub>) (33.3% O<sub>2<\/sub> and 66.6% H<sub>2<\/sub>) in the \u201cClinical Guidance for COVID-19 Pneumonia Diagnosis and Treatment (Trial Version 7)\u201d issued by the China National Health Commission. The recommendation recognized the importance of H<sub>2<\/sub> in contemporary medical gas research.<\/p>\n<p>In this study, LPS (20&nbsp;\u03bcg\/ml) significantly reduced the proliferation, migration, and tube formation of EPCs. This is consistent with Yu et al. research, which found that LPS (10&nbsp;\u03bcg\/ml) impaired the viability, migration, adhesion abilities, and tube formation of late EPCs (<a href=\"#B54\" rid=\"B54\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Yu et al., 2017<\/a>). Compared with the control group, the proliferation and adhesion activities of bone marrow-derived EPCs were impaired in LPS (100&nbsp;ng\/ml) induced group in dose and time dependence (<a href=\"#B29\" rid=\"B29\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Li et al., 2014<\/a>). Our previous study also demonstrated that LPS (30&nbsp;\u03bcg\/ml) inhibited the proliferation, migration, and tube formation of EPCs (<a href=\"#B53\" rid=\"B53\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Yang et al., 2019<\/a>). Furthermore, western blot results showed that LPS reduced the expression levels of p-AKT, eNOS, and p-eNOS in EPCs. LPS had no effect on AKT expression. Our previous research proved that LPS (30&nbsp;\u03bcg\/ml) decreases the levels of p-AKT, eNOS, and p-eNOS in EPCs (<a href=\"#B53\" rid=\"B53\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Yang et al., 2019<\/a>). Yang et al. found that LPS (10&nbsp;\u03bcg\/ml) reduced the expression of p-eNOS in human pulmonary microvascular endothelial cells (HPMECs) (<a href=\"#B52\" rid=\"B52\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Yang et al., 2018<\/a>). Liu et al. found that p-AKT was markedly suppressed in H9c2 cells after treatment with LPS (1&nbsp;\u03bcg\/ml) for 24&nbsp;h (<a href=\"#B35\" rid=\"B35\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Liu et al., 2021<\/a>). The effect of LPS on AKT expression in out study was consistent with the study of Li et al. and Zhan et al. Li et al. showed that there was no significant change in AKT expression of LPS-induced (20&nbsp;\u03bcg\/ml) mouse microvascular ECs (<a href=\"#B28\" rid=\"B28\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Li H. et al., 2020<\/a>). Zhan et al. reported that the AKT level of HPMECs did not change markedly under LPS (1&nbsp;mg\/L) induction (<a href=\"#B56\" rid=\"B56\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Zhan et al., 2020<\/a>). In addition, Wang et al. found that p-AKT protein expression was significantly increased in LPS-induced (100&nbsp;ng\/ml) rat microvascular ECs (<a href=\"#B48\" rid=\"B48\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Wang et al., 2013<\/a>). Fan et al. found that LPS (100&nbsp;ng\/ml) stimulation significantly increased the phosphorylation of both AKT and eNOS in HPMECs (<a href=\"#B13\" rid=\"B13\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Fan et al., 2020<\/a>). Taken together, we speculate that the inconsistent study results may be due to the differences in LPS concentration. In addition to toxic effects, LPS also has extensive biological activity. Low doses of LPS have immune-activating effects (<a href=\"#B37\" rid=\"B37\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Morris and Li, 2012<\/a>) (<a href=\"#B34\" rid=\"B34\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Lin et al., 2009<\/a>). Therefore, there are differences in the functional gene expression and cell functions under different concentrations LPS treatment. However, the relevant mechanism still needs further confirmation.<\/p>\n<p>The effectiveness of molecular hydrogen has been proven in the prevention and treatment of many diseases. H<sub>2<\/sub> had a protective effect on the rat model of ALI (<a href=\"#B24\" rid=\"B24\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Jiang et al., 2013<\/a&gt;; <a href=\"#B2\" rid=\"B2\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Audi et al., 2017<\/a>) by significantly improving lung endothelial permeability, reducing cell apoptosis and histopathological changes (<a href=\"#B8\" rid=\"B8\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Diao et al., 2016<\/a>), and preventing LPS-induced pulmonary ECs dysfunction (<a href=\"#B32\" rid=\"B32\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Li Y. et al., 2020<\/a>). Fu et al. demonstrated that hydrogen-rich saline has a protective effect on LPS-induced ALI by regulating cell apoptosis and inhibiting endothelial dysfunction (<a href=\"#B15\" rid=\"B15\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Fu et al., 2020<\/a>). Inhalation was the most direct way to administer molecular hydrogen (<a href=\"#B21\" rid=\"B21\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Huang et al., 2010a<\/a&gt;; <a href=\"#B25\" rid=\"B25\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Kawamura et al., 2010<\/a>). At the same time, molecular hydrogen could be dissolved in physiological saline to make hydrogen-rich water for intravenous injection, or it could be taken orally (<a href=\"#B6\" rid=\"B6\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Cardinal et al., 2010<\/a&gt;; <a href=\"#B19\" rid=\"B19\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">He et al., 2013<\/a>). A hydrogen incubator was used in this study. It could simulate the <em>in vivo<\/em> environment after inhaling hydrogen, which was more conducive to our research. H<sub>2<\/sub> has a protective effect on the ECs barrier, mechanism of which has not been fully elucidated, and the effect of H<sub>2<\/sub> on EPCs has not been reported. Our study indicated that H<sub>2<\/sub> attenuated the dysfunctions of EPCs induced by LPS, improved EPCs proliferation, migration, tube formation, and restored the expression levels of p-AKT, eNOS, p-eNOS. Recent studies revealed that H<sub>2<\/sub> could inhibit the expression of inflammatory factors, reduce sepsis-induced endothelial damage and inflammation, improve endothelial dysfunction (<a href=\"#B6\" rid=\"B6\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Cardinal et al., 2010<\/a&gt;; <a href=\"#B19\" rid=\"B19\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">He et al., 2013<\/a>).<\/p>\n<p>Previous studies showed that the PI3K\/AKT\/eNOS pathway is involved in the changes of the LPS-induced ECs barrier function (<a href=\"#B58\" rid=\"B58\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Zheng et al., 2018<\/a>), but whether it is involved in the effect of H<sub>2<\/sub> on EPCs repairment remains unclear. The results of the present study discovered the unique molecular basis for H<sub>2<\/sub> to inhibit LPS-induced EPCs dysfunctions. After inhibiting the PI3K\/AKT\/eNOS signaling pathway by LY294002 and L-NAME, the H<sub>2<\/sub>-mediated restoration of EPCs functions was partially prevented. The western blot results demonstrated that H<sub>2<\/sub> up-regulated p-AKT, eNOS and p-eNOS levels were inhibited by LPS. LY294002 significantly inhibited the increase of p-AKT, eNOS and p-eNOS induced by 60% H<sub>2<\/sub>. L-NAME significantly inhibited the increase of eNOS and p-eNOS induced by 60% H<sub>2<\/sub>, and had no effect on p-AKT levels. PI3K\/AKT\/eNOS activation plays a crucial role in our study. PI3K\/AKT signaling pathway mediates a variety of pathophysiological processes and involves multiple important cellular activities, such as cell proliferation, apoptosis and autophagy (<a href=\"#B40\" rid=\"B40\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Ravikumar et al., 2010<\/a&gt;; <a href=\"#B33\" rid=\"B33\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Liby and Sporn, 2012<\/a&gt;; <a href=\"#B41\" rid=\"B41\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Saiprasad et al., 2014<\/a>). Additionally, numerous studies show that the PI3K\/AKT signaling pathway plays a vital role in the process of EPCs proliferation, migration, homing and tube formation (<a href=\"#B11\" rid=\"B11\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Everaert et al., 2010<\/a&gt;; <a href=\"#B50\" rid=\"B50\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Wang et al., 2011<\/a>). Our previous results demonstrate that Rev-D4F mediates restoration of EPCs functions by PI3K\/AKT\/eNOS signaling pathway (<a href=\"#B53\" rid=\"B53\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Yang et al., 2019<\/a>). The PI3K\/AKT\/eNOS pathway was involved in restoring the dysfunctions of EPCs in diabetic mice (<a href=\"#B5\" rid=\"B5\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Cao et al., 2017<\/a>). Yu et al. suggested that the proliferation, migration and survival of EPCs impaired by LDL cholesterol via the PI3K\/AKT signaling pathway (<a href=\"#B55\" rid=\"B55\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Yu et al., 2010<\/a>).<\/p>\n<p class=\"p\">In summary, we concluded that the PI3K\/AKT\/eNOS signaling pathway was contributed to H<sub>2<\/sub> repairment of EPCs dysfunctions induced by LPS (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F8\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"F8\" rid-ob=\"ob-F8\" co-legend-rid=\"lgnd_F8\" rel=\"noopener\"><span>Figure 8<\/span><\/a>).<\/p>\n<p><!--fig ft0--><!--fig mode=article f1--><\/p>\n<div class=\"fig iconblock whole_rhythm\" id=\"F8\" co-legend-rid=\"lgnd_F8\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F8\/\" target=\"figure\" rid-figpopup=\"F8\" rid-ob=\"ob-F8\" rel=\"noopener\"><!--fig\/graphic|fig\/alternatives\/graphic mode=\"anchored\" m1--><\/p>\n<div class=\"figure\" data-largeobj=\"\" data-largeobj-link-rid=\"largeobj_idm140208098755056\"><img decoding=\"async\" loading=\"lazy\" class=\"fig-image\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is fphar-13-894812-g008.jpg\" title=\"An external file that holds a picture, illustration, etc.\nObject name is fphar-13-894812-g008.jpg\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/fphar-13-894812-g008.jpg\"><\/div>\n<p><\/a><\/p>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm140208098755056\" style=\"display: none;\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F8\/\" target=\"figure\" rid-figpopup=\"F8\" rid-ob=\"ob-F8\" rel=\"noopener\"><\/a><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F8\/?report=objectonly\">Open in a separate window<\/a><\/div>\n<div class=\"icnblk_cntnt\" id=\"lgnd_F8\">\n<div><a class=\"figpopup\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/figure\/F8\/\" target=\"figure\" rid-figpopup=\"F8\" rid-ob=\"ob-F8\" rel=\"noopener\">FIGURE 8<\/a><\/div>\n<p><!--caption a7--><\/p>\n<div class=\"caption\">\n<p>H<sub>2<\/sub>-mediated restoration of EPCs dysfunctions is mediated by the PI3K\/AKT\/eNOS pathway.<\/p>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<div id=\"s5\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"s5title\">Conclusion<\/h2>\n<p class=\"p p-first-last\">Our results showed that H<sub>2<\/sub> reversed the LPS-induced EPCs dysfunctions. Moreover, H<sub>2<\/sub> restored the LPS-attenuated levels of p-AKT, eNOS and p-eNOS. Therefore, this study proves that H<sub>2<\/sub>-mediated restoration of EPCs dysfunctions is mediated by the PI3K\/AKT\/eNOS pathway.<\/p>\n<\/div>\n<div id=\"s6\" class=\"tsec bk-sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"s6title\">Data Availability Statement<\/h2>\n<p><!--\/article\/back\/sec\/--><\/p>\n<p class=\"p p-first-last\">The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.<\/p>\n<\/div>\n<div id=\"s7\" class=\"tsec bk-sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"s7title\">Ethics Statement<\/h2>\n<p><!--\/article\/back\/sec\/--><\/p>\n<p class=\"p p-first-last\">The animal study was reviewed and approved by All animal experiments were approved by the Animal Experimental Ethics Committee of Weifang Medical University (approval code: 2019SDL108).<\/p>\n<\/div>\n<div id=\"s8\" class=\"tsec bk-sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"s8title\">Author Contributions<\/h2>\n<p><!--\/article\/back\/sec\/--><\/p>\n<p class=\"p p-first-last\">YW, NY, and BL, Conceptualization. QM and KL, Data curation. JY, Formal analysis. NY, Funding acquisition. QM, KL, JY, SC, LcZ, LK, LlZ, XJ, and YT, Investigation. QM, YW, KL, JY, and SC, Methodology. YW, NY, and BL, Project administration. LK, Software. QM, YW, KL, LcZ, LlZ, and SC, Validation. KL and JY, Writing\u2013original draft. NY, Writing\u2013review and editing.<\/p>\n<\/div>\n<div id=\"s9\" class=\"tsec bk-sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"s9title\">Funding<\/h2>\n<p><!--\/article\/back\/sec\/--><\/p>\n<p class=\"p p-first-last\">This research was funded by the National Natural Science Foundation of China (No. 81600360), the Province Natural Science Foundation of Shandong (No. ZR2020KH008 and No. ZR2012HL18), the Province Science and Technology Development Foundation of Shandong (2014GSF118105), the Province Higher University Science and Technology Development Project of Shandong (No. J14LK03) the Yuandu Scholar Project, the Introduction Plan of Young Creative Talents in Colleges and Universities of Shandong Province (No. 205), Shanghai Asclepius Meditec Co., Ltd.<\/p>\n<\/div>\n<div id=\"s10\" class=\"tsec bk-sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"s10title\">Conflict of Interest<\/h2>\n<p><!--\/article\/back\/sec\/--><\/p>\n<p class=\"p p-first\">XJ was employed by the Shandong Qilu Stem Cell Engineering Co.<\/p>\n<p class=\"p p-last\">The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.<\/p>\n<\/div>\n<div id=\"s11\" class=\"tsec bk-sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"s11title\">Publisher\u2019s Note<\/h2>\n<p><!--\/article\/back\/sec\/--><\/p>\n<p class=\"p p-first-last\">All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.<\/p>\n<\/div>\n<div id=\"s12\" class=\"tsec bk-sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"s12title\">Supplementary Material<\/h2>\n<p><!--\/article\/back\/sec\/--><\/p>\n<p class=\"p p-first\">The Supplementary Material for this article can be found online at: <a href=\"https:\/\/www.frontiersin.org\/articles\/10.3389\/fphar.2022.894812\/full#supplementary-material\" data-ga-action=\"click_feat_suppl\" ref=\"reftype=extlink&amp;article-id=9133378&amp;issue-id=400058&amp;journal-id=1524&amp;FROM=Article%7CBody&amp;TO=External%7CLink%7CURI\" target=\"_blank\" rel=\"noopener\">https:\/\/www.frontiersin.org\/articles\/10.3389\/fphar.2022.894812\/full#supplementary-material<\/a>\n<\/p>\n<div class=\"sec suppmat\" id=\"SM1\">\n<div class=\"sup-box half_rhythm\" id=\"media-a.n.g.c.a\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC9133378\/bin\/Image1.TIF\" data-ga-action=\"click_feat_suppl\">Click here for additional data file.<\/a><sup>(3.0M, TIF)<\/sup><\/div>\n<\/div>\n<\/div>\n<div id=\"ref-list-a.n.h\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"ref-list-a.n.htitle\">References<\/h2>\n<div class=\"ref-list-sec sec\" id=\"reference-list\">\n<ul class=\"back-ref-list\" style=\"list-style-type:decimal;\">\n<li id=\"B1\"><span class=\"mixed-citation\"><br \/>\nAsahara T., Murohara T., Sullivan A., Silver M., van der Zee R., Li T., et al. 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href=\"#abstract-a.l.b.wtitle\">Abstract<\/a><\/li>\n<li><a href=\"#s1title\">Introduction<\/a><\/li>\n<li><a href=\"#s2title\">Materials and Methods<\/a><\/li>\n<li><a href=\"#s3title\">Results<\/a><\/li>\n<li><a href=\"#s4title\">Discussion<\/a><\/li>\n<li><a href=\"#s5title\">Conclusion<\/a><\/li>\n<li><a href=\"#s6title\">Data Availability Statement<\/a><\/li>\n<li><a href=\"#s7title\">Ethics Statement<\/a><\/li>\n<li><a href=\"#s8title\">Author Contributions<\/a><\/li>\n<li><a href=\"#s9title\">Funding<\/a><\/li>\n<li><a href=\"#s10title\">Conflict of Interest<\/a><\/li>\n<li><a href=\"#s11title\">Publisher\u2019s Note<\/a><\/li>\n<li><a href=\"#s12title\">Supplementary Material<\/a><\/li>\n<li><a href=\"#ref-list-a.n.htitle\">References<\/a><\/li>\n<\/ul>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"<p>Hydrogen Repairs LPS-Induced Endothelial Progenitor Cells Injury via PI3K\/AKT\/eNOS Pathway<\/p>\n","protected":false},"author":1,"featured_media":17899,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[130],"tags":[],"disease":[843],"body-organ":[1027],"applications":[686],"test_subjects":[1520],"report-topic":[1304],"class_list":["post-26674","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-hydrogen-health","disease-sepsis-2","body-organ-lung-2","applications-ventilation-2","test_subjects-cell-culture-2","report-topic-acute-respiratory-distress-syndrome-ards-2"],"acf":[],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v28.1 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>H2 Repairs Endothelial Progenitor Cells Injury<\/title>\n<meta name=\"description\" content=\"Hydrogen Repairs LPS-Induced Endothelial Progenitor Cells Injury via PI3K\/AKT\/eNOS Pathway\" \/>\n<meta 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