{"id":26785,"date":"2024-01-03T21:38:21","date_gmt":"2024-01-03T19:38:21","guid":{"rendered":"https:\/\/hho-bulgaria.com\/h2-enhances-macrophage-polarization-in-necrotizing-enterocolitis\/"},"modified":"2024-01-29T21:14:38","modified_gmt":"2024-01-29T19:14:38","slug":"h2-enhances-macrophage-polarization-in-necrotizing-enterocolitis","status":"publish","type":"post","link":"https:\/\/hho-bulgaria.com\/en\/h2-enhances-macrophage-polarization-in-necrotizing-enterocolitis\/","title":{"rendered":"H2 Enhances Macrophage Polarization in Necrotizing Enterocolitis"},"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 Pediatr.<\/a><\/span> 2021; 9: 710382. <\/div>\n<div class=\"part2\"><span class=\"fm-vol-iss-date\">Published online 2021 Nov 17. <\/span>  <span class=\"doi\"><span>doi:&nbsp;<\/span><a href=\"\/\/doi.org\/10.3389%2Ffped.2021.710382\" target=\"_blank\" rel=\"noopener noreferrer\" ref=\"reftype=other&amp;article-id=8635714&amp;issue-id=374910&amp;journal-id=2282&amp;FROM=Article%7CFront%20Matter&amp;TO=Content%20Provider%7CCrosslink%7CDOI\">10.3389\/fped.2021.710382<\/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>PMC8635714<\/span><\/div>\n<div class=\"fm-citation-pmid\">PMID: <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/34869093\">34869093<\/a><\/div>\n<\/div>\n<\/div>\n<h1 class=\"content-title\">Hydrogen Promotes the M1 Macrophage Conversion During the Polarization of Macrophages in Necrotizing Enterocolitis<\/h1>\n<div class=\"half_rhythm\">\n<div class=\"contrib-group fm-author\"><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Yu%20S%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139877725268720\" co-class=\"co-affbox\">Shenghua Yu<\/a>,<sup><br \/>\n1<br \/>\n<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Lv%20Z%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139877725266592\" co-class=\"co-affbox\">ZhiBao Lv<\/a>,<sup><br \/>\n1<br \/>\n,<\/sup><sup><br \/>\n*<br \/>\n<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Gao%20Z%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139877764103296\" co-class=\"co-affbox\">Zhimei Gao<\/a>,<sup><br \/>\n2<br \/>\n<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Shi%20J%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139877764100928\" co-class=\"co-affbox\">Jingyi Shi<\/a>,<sup><br \/>\n3<br \/>\n<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Sheng%20Q%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139877764098560\" co-class=\"co-affbox\">Qingfeng Sheng<\/a>,<sup><br \/>\n1<br \/>\n<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Zheng%20L%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139877764095824\" co-class=\"co-affbox\">Lulu Zheng<\/a>,<sup><br \/>\n1<br \/>\n<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Zhou%20J%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139877764093456\" co-class=\"co-affbox\">Junmei Zhou<\/a>,<sup><br \/>\n2<br \/>\n<\/sup> and  <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Wang%20X%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139877764091088\" co-class=\"co-affbox\">Xueli Wang<\/a><sup><br \/>\n4<br \/>\n<\/sup><\/div>\n<div style=\"display:none\" class=\"contrib-group aff-tip\">\n<div id=\"_co_idm139877725268720\">\n<h3 class=\"no_margin\">Shenghua Yu<\/h3>\n<p><sup>1<\/sup>Department of Pediatric Surgery, Shanghai Children&#8217;s Hospital, Shanghai, China<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Yu%20S%5BAuthor%5D\">Shenghua Yu<\/a><\/div>\n<\/div>\n<div id=\"_co_idm139877725266592\">\n<h3 class=\"no_margin\">ZhiBao Lv<\/h3>\n<p><sup>1<\/sup>Department of Pediatric Surgery, Shanghai Children&#8217;s Hospital, Shanghai, China<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Lv%20Z%5BAuthor%5D\">ZhiBao Lv<\/a><\/div>\n<\/div>\n<div id=\"_co_idm139877764103296\">\n<h3 class=\"no_margin\">Zhimei Gao<\/h3>\n<p><sup>2<\/sup>Department of Center Laboratory, Shanghai Children&#8217;s Hospital, Shanghai, China<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Gao%20Z%5BAuthor%5D\">Zhimei Gao<\/a><\/div>\n<\/div>\n<div id=\"_co_idm139877764100928\">\n<h3 class=\"no_margin\">Jingyi Shi<\/h3>\n<p><sup>3<\/sup>Department of Critical Care Medicine, Shanghai Children&#8217;s Hospital, Shanghai, China<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Shi%20J%5BAuthor%5D\">Jingyi Shi<\/a><\/div>\n<\/div>\n<div id=\"_co_idm139877764098560\">\n<h3 class=\"no_margin\">Qingfeng Sheng<\/h3>\n<p><sup>1<\/sup>Department of Pediatric Surgery, Shanghai Children&#8217;s Hospital, Shanghai, China<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Sheng%20Q%5BAuthor%5D\">Qingfeng Sheng<\/a><\/div>\n<\/div>\n<div id=\"_co_idm139877764095824\">\n<h3 class=\"no_margin\">Lulu Zheng<\/h3>\n<p><sup>1<\/sup>Department of Pediatric Surgery, Shanghai Children&#8217;s Hospital, Shanghai, China<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Zheng%20L%5BAuthor%5D\">Lulu Zheng<\/a><\/div>\n<\/div>\n<div id=\"_co_idm139877764093456\">\n<h3 class=\"no_margin\">Junmei Zhou<\/h3>\n<p><sup>2<\/sup>Department of Center Laboratory, Shanghai Children&#8217;s Hospital, Shanghai, China<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Zhou%20J%5BAuthor%5D\">Junmei Zhou<\/a><\/div>\n<\/div>\n<div id=\"_co_idm139877764091088\">\n<h3 class=\"no_margin\">Xueli Wang<\/h3>\n<p><sup>4<\/sup>Department of Pathology, Shanghai Children&#8217;s Hospital, Shanghai, China<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Wang%20X%5BAuthor%5D\">Xueli Wang<\/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=\"idm139877765108656_ai\">Author information<\/a> <a href=\"#\" class=\"pmctoggle\" rid=\"idm139877765108656_an\">Article notes<\/a> <a href=\"#\" class=\"pmctoggle\" rid=\"idm139877765108656_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=\"idm139877765108656_ai\" style=\"display:none\">\n<div class=\"fm-affl\" id=\"aff1\"><sup>1<\/sup>Department of Pediatric Surgery, Shanghai Children&#8217;s Hospital, Shanghai, China<\/div>\n<div class=\"fm-affl\" id=\"aff2\"><sup>2<\/sup>Department of Center Laboratory, Shanghai Children&#8217;s Hospital, Shanghai, China<\/div>\n<div class=\"fm-affl\" id=\"aff3\"><sup>3<\/sup>Department of Critical Care Medicine, Shanghai Children&#8217;s Hospital, Shanghai, China<\/div>\n<div class=\"fm-affl\" id=\"aff4\"><sup>4<\/sup>Department of Pathology, Shanghai Children&#8217;s Hospital, Shanghai, China<\/div>\n<div id=\"fn-a.j.b.i.a\">Edited by: Giovanni Vento, Catholic University of the Sacred Heart, Italy<\/div>\n<div id=\"fn-a.j.b.i.b\">Reviewed by: Merih Cetinkaya, University of Health Sciences, Turkey; Dr. Aftab Shaukat, Huazhong Agricultural University, China<\/div>\n<div id=\"c001\">*Correspondence: ZhiBao Lv <a href=\"mailto:dev@null\" data-email=\"moc.361@uyloabihz\" class=\"oemail\">moc.361@uyloabihz<\/a><\/div>\n<div id=\"fn001\">This article was submitted to Neonatology, a section of the journal Frontiers in Pediatrics<\/div>\n<\/div>\n<div class=\"fm-article-notes hide half_rhythm\" id=\"idm139877765108656_an\" style=\"display:none\">\n<div class=\"fm-pubdate half_rhythm\">Received 2021 May 16; Accepted 2021 Sep 16.<\/div>\n<\/div>\n<div class=\"permissions half_rhythm hide\" id=\"idm139877765108656_cpl\" style=\"display:none\">\n<div class=\"fm-copyright half_rhythm\"><a href=\"\/pmc\/about\/copyright\/\">Copyright<\/a>  \u00a9 2021 Yu, Lv, Gao, Shi, Sheng, Zheng, Zhou and Wang.<\/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-length=\"190\" 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 datasets presented in this study can be found in online repositories. The names of the repository\/repositories and accession number(s) can be found in the article\/supplementary material.<\/p>\n<\/dd>\n<\/dl>\n<\/div>\n<div id=\"abstract-a.j.b.p\" 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.j.b.ptitle\">Abstract<\/h2>\n<p><!--article-meta--><\/p>\n<div>\n<p class=\"p p-first\"><strong>Background:<\/strong> Hydrogen is protective against intestinal injury in necrotizing enterocolitis (NEC), mainly through to alleviate inflammation response. The M1 macrophages can promote inflammation. We hypothesized that hydrogen would promote the M1 macrophages conversion during the polarization and reduce the inflammatory factors in NEC.<\/p>\n<p><strong>Methods:<\/strong> We used M1 and M2 macrophages induced from RAW264.7 cells and bone marrow-derived macrophages, models of NEC and macrophages derived from spleens, abdominal lymph nodes and lamina propria in model mice. Cytokines, CD16\/32 and CD206 were measured by quantitative PCR, flow cytometry. Nuclear factor-\u03baB (NF-\u03baB) p65 were determined by western blot. Histology staining were used to assess the severity of NEC.<\/p>\n<p><strong>Results:<\/strong> Macrophages were successfully polarized to M1 or M2 by assessing the expression of inflammatory factors. Pro-inflammatory factors and CD16\/32 in M1 macrophages were decreased, and the expression of CD16\/32 in lamina propria were inhibited after treatment with hydrogen, but the changes has no effects in other tissues. Hydrogen inhibited the NF-\u03baB p65 in M1 macrophages nucleus and distal ileum of NEC. HE staining showed hydrogen could attenuate the severity of NEC.<\/p>\n<p class=\"p p-last\"><strong>Conclusion:<\/strong> Hydrogen could attenuate the severity of NEC through promoting M1 macrophages conversion by inhibited the expression of NF-\u03baB p65 in the nucleus.<\/p>\n<\/div>\n<div class=\"sec\"><strong class=\"kwd-title\">Keywords: <\/strong><span class=\"kwd-text\">necrotizing enterocolitis, macrophage polarization, hydrogen molecule, NF-\u03baB, mice<\/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\">Necrotizing enterocolitis (NEC) is a serious gastrointestinal disease, mainly affecting premature neonates, especially in extremely low-birth-weight infants. Despite over six decades of clinical and basic research, the pathogenesis of NEC remains unclear (<a href=\"#B1\" rid=\"B1\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">1<\/a>). The overall mortality rate associated with NEC is between 20 and 30% (<a href=\"#B2\" rid=\"B2\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">2<\/a>, <a href=\"#B3\" rid=\"B3\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">3<\/a>). Premature infants have immature intestinal and deficient immune function. Some scholars expressed that insufficient immune function is involved in the pathogenesis of NEC, and macrophages play a vital role in the development of NEC as well (<a href=\"#B4\" rid=\"B4\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">4<\/a>\u2013<a href=\"#B7\" rid=\"B7\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">7<\/a>).<\/p>\n<p>Macrophages play an important role in the development, progression, and resolution of inflammation disease (<a href=\"#B8\" rid=\"B8\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">8<\/a>). Two major subtypes of polarized macrophages are called classical macrophages (M1) and alternative macrophages (M2). Activated M1 macrophages produce pro-inflammatory cytokines, such as interleukin 6 (IL-6), IL-1\u03b2, nitric oxide, and tumor necrosis factor-\u03b1 (TNF-\u03b1), and then contribute to inflammatory reaction. In contrast, M2 macrophages produce anti-inflammatory factors, including IL-10, tumor necrosis factor-\u03b2 (TNF-\u03b2), Arg-1, and protein YM-1, which mainly promote tissue remodeling and anti-inflammatory response (<a href=\"#B8\" rid=\"B8\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">8<\/a>\u2013<a href=\"#B11\" rid=\"B11\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">11<\/a>). Previous studies have shown that macrophages are plastic cells and M1 or M2 macrophages can alter their functional profiles in response to the micro-environmental changes (<a href=\"#B12\" rid=\"B12\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">12<\/a>). Wei et al. investigated the role of macrophages in murine model of NEC and showed that M1 macrophages can promote NEC by increasing intestinal epithelial apoptosis (<a href=\"#B13\" rid=\"B13\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">13<\/a>). Moreover, polarization of M2 macrophages has been reported to stimulate the ability of mesenchymal stem cells to ameliorate acute kidney injury in metabolic endotoxemia and protect the lungs against acute lung injury (<a href=\"#B14\" rid=\"B14\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">14<\/a>).<\/p>\n<p>Molecular hydrogen as a medical gas to measure the local blood flow firstly reported in 1964, and then hydrogen was used as a biomarker for the early diagnosis of NEC (<a href=\"#B15\" rid=\"B15\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">15<\/a>, <a href=\"#B16\" rid=\"B16\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">16<\/a>). Ohsawa et al. demonstrated that hydrogen can protect against oxidative damage by selectively reducing cytotoxic oxygen radicals, and our previous study also revealed that hydrogen-rich saline protects NEC from oxidative stress and inflammation (<a href=\"#B17\" rid=\"B17\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">17<\/a>, <a href=\"#B18\" rid=\"B18\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">18<\/a>). It is well-known that NF-\u03baB plays a key role in the immune system and polarization of macrophages (<a href=\"#B19\" rid=\"B19\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">19<\/a>\u2013<a href=\"#B21\" rid=\"B21\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">21<\/a>). Xu et al. showed that inhalation of hydrogen gas protects liver against ischemia\/reperfusion injury and also attenuates renal injury in severe acute pancreatitis and ameliorates bowel injury in a model of NEC by the NF-\u03baB signaling pathway (<a href=\"#B22\" rid=\"B22\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">22<\/a>\u2013<a href=\"#B24\" rid=\"B24\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">24<\/a>).<\/p>\n<p class=\"p p-last\">The aim of this study was to examine that the regulation of hydrogen on inflammatory factors in macrophages and mice model, and the role of NF-\u03baB in M1 macrophages conversion, the then effect of hydrogen on macrophages in newborn mice of NEC.<\/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\">Methods<\/h2>\n<div id=\"sec-a.k.b.b\" class=\"sec sec-first\">\n<h3 id=\"sec-a.k.b.btitle\">RAW264.7 Macrophage Cell Culture<\/h3>\n<p class=\"p p-first-last\">RAW264.7 macrophages were purchased from the Cell Bank of the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Cells were grown in Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) containing 10% fetal bovine serum (FBS; Gibco) and cultured at 37\u00b0C with 5% CO<sub>2<\/sub> in a humidified incubator.<\/p>\n<\/div>\n<div id=\"sec-a.k.b.c\" class=\"sec\">\n<h3 id=\"sec-a.k.b.ctitle\">Born Marrow-Derived Macrophages<\/h3>\n<p class=\"p p-first-last\">Bone marrows were harvested from femurs and tibia of C57B6\/J mice (aged 8\u201312 weeks) and cultured in the presence of recombinant mouse M-CSF (20 ng\/ml; R&amp;D Systems) in RPMI 1640 medium for 7 days as described previously (<a href=\"#B21\" rid=\"B21\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">21<\/a>, <a href=\"#B25\" rid=\"B25\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">25<\/a>). BMDMs were washed and cultured in different mediums to polarize M1 or M2 macrophages.<\/p>\n<\/div>\n<div id=\"sec-a.k.b.d\" class=\"sec\">\n<h3 id=\"sec-a.k.b.dtitle\">Animal Experiments<\/h3>\n<p class=\"p p-first-last\">The following experimental protocols were performed according to the Ethical Guidelines for the Use of Animals in Research, and this study was approved by the Animal Care Committee of the Children&#8217;s Hospital of Shanghai. In addition, 8-day-old C57BL\/6J mice were used to establish the model of NEC as previously described (<a href=\"#B26\" rid=\"B26\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">26<\/a>\u2013<a href=\"#B28\" rid=\"B28\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">28<\/a>). The mice were purchased from Shanghai Super B&amp;K Laboratory Animal Corp. (Shanghai, China), and randomized into the following groups: Group 1, CON (<em>n<\/em> = 18); Group 2, NEC (<em>n<\/em> = 19); Group 3, CON + H<sub>2<\/sub> (<em>n<\/em> = 18); and Group 4, NEC + H<sub>2<\/sub> (<em>n<\/em> = 20). All the mice were hand-fed trans-orally every 4\u20135 h with artificial formula using a 24-G catheter. Groups 2 and 4 had asphyxia for 10 min exposure to 95% nitrogen, followed by daily cold exposure at 4\u00b0C for 10 min twice before feeding. Groups 3 and 4 were daily treated with hydrogen-rich water (20 ml\/kg\/day, 0.5 mmol\/L) twice before stress. Groups 1 and 2 were fed with cleaned water as control. We fed model mice water in the last 12 h to remove the resident in the intestine of mice.<\/p>\n<\/div>\n<div id=\"sec-a.k.b.e\" class=\"sec\">\n<h3 id=\"sec-a.k.b.etitle\">Preparation and Stimulation of Macrophage Conditioned Medium<\/h3>\n<p class=\"p p-first-last\">RAW264.7 macrophages and BMDM were polarized to M1 and M2 macrophages as described previously (<a href=\"#B25\" rid=\"B25\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">25<\/a>, <a href=\"#B29\" rid=\"B29\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">29<\/a>). The following additives were added to the culture medium: (1) CON: no additional additive to maintain M0 macrophages; (2) M1: 20 ng\/ml LPS and 50 ng\/ml interferon gamma (IFN-\u03b3) to generate M1 macrophages; (3) M2: 20 ng\/ml IL-4 to generate M2 macrophages; (4) CON + H<sub>2<\/sub>: 0.6 mmol\/L molecular hydrogen in normal culture medium; (5) M1 + H<sub>2<\/sub>: 0.6 mmol\/L molecular hydrogen + 20 ng\/ml LPS and 50 ng\/ml IFN-\u03b3; (6) M2 + H<sub>2<\/sub>: 0.6 mmol\/L molecular hydrogen + 20 ng\/ml IL-4.<\/p>\n<\/div>\n<div id=\"sec-a.k.b.f\" class=\"sec\">\n<h3 id=\"sec-a.k.b.ftitle\">Hydrogen Treatment<\/h3>\n<p class=\"p p-first-last\">The preparation of hydrogen-rich water was performed as described previously (<a href=\"#B18\" rid=\"B18\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">18<\/a>). Hydrogen was dissolved in water or DMEM medium for 4 h under high pressure (0.4 MPa) up to a supersaturation. The saturated hydrogen water or DMEM medium was sterilized by gamma radiation and stored under atmospheric pressure at 4\u00b0C. It was freshly produced every day and detected by a portable determination instrument ENH-1000 to ensure a stable concentration of 0.6 mmol\/L (<a href=\"#B18\" rid=\"B18\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">18<\/a>, <a href=\"#B30\" rid=\"B30\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">30<\/a>).<\/p>\n<\/div>\n<div id=\"sec-a.k.b.g\" class=\"sec\">\n<h3 id=\"sec-a.k.b.gtitle\">Histology Staining and NEC Scoring<\/h3>\n<p class=\"p p-first-last\">Distal ileum samples were first fixed in 4% formalin and embedded in paraffin, and then stained with hematoxylin and eosin (H&amp;E). The histological slides were blindly evaluated by two independent observers using a published NEC scoring system. Grade 0, normal; grade 1, focal mild injury confined to villous tips; grade 2, partial loss of villi; grade 3, necrosis extending to submucosa; grade 4, transmural necrosis. NEC was here considered as grade 2 or above.<\/p>\n<\/div>\n<div id=\"sec-a.k.b.h\" class=\"sec\">\n<h3 id=\"sec-a.k.b.htitle\">Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR)<\/h3>\n<p class=\"p p-first\">Total RNA was extracted from macrophages using TRIzol reagent (Catalog No. 15596-026; Life Technologies, USA) according to the manufacturer&#8217;s protocol. Aliquots of 1 mg of total RNA were reversely transcribed using Super-Script Reverse Transcriptase (Invitrogen, USA) and Oligo-dT (<a href=\"#B18\" rid=\"B18\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">18<\/a>)-primers (Invitrogen, USA). The RT-qPCR was carried out by using SYBR Green Master Mix Kit in ABI Prism 7000 Sequence Detection System (Applied Biosystems, USA) according to the manufacturer&#8217;s instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as an internal standard. The primers used are shown in <a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/table\/T1\/\" target=\"table\" class=\"fig-table-link figpopup\" rid-figpopup=\"T1\" rid-ob=\"ob-T1\" co-legend-rid=\"\" rel=\"noopener\"><span>Table 1<\/span><\/a>. The expression levels of the messenger RNAs (mRNAs) were reported as fold changes vs. control.<\/p>\n<p><!--table ft1--><!--table-wrap mode=\"anchored\" t5--><\/p>\n<div class=\"table-wrap anchored whole_rhythm\" id=\"T1\">\n<h3>Table 1<\/h3>\n<p><!--caption a7--><\/p>\n<div class=\"caption\">\n<p>List of primers.<\/p>\n<\/div>\n<div class=\"xtable\">\n<table frame=\"hsides\" rules=\"groups\" class=\"rendered small default_table\">\n<thead>\n<tr>\n<th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<strong>Gene<\/strong>\n<\/th>\n<th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<strong>Primer sequence (5&#8217;\u22123&#8242;)<\/strong>\n<\/th>\n<th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">\n<strong>Product length<\/strong>\n<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-1\u03b2<\/td>\n<td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(F) GCAACTGTTCCTGAACTCAACT<\/td>\n<td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">89 bp<\/td>\n<\/tr>\n<tr>\n<td rowspan=\"1\" colspan=\"1\"><\/td>\n<td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(R) ATCTTTTGGGGTCCGTCAACT<\/td>\n<td rowspan=\"1\" colspan=\"1\"><\/td>\n<\/tr>\n<tr>\n<td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">iNOS<\/td>\n<td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(F) CAGCTGGGCTGTACAAACCTT<\/td>\n<td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">310 bp<\/td>\n<\/tr>\n<tr>\n<td rowspan=\"1\" colspan=\"1\"><\/td>\n<td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(R) CATTGGAAGTGAAGCGTTTCG<\/td>\n<td rowspan=\"1\" colspan=\"1\"><\/td>\n<\/tr>\n<tr>\n<td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TNF-\u03b1<\/td>\n<td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(F) CAGGAGGGAGAACAGAAA<\/td>\n<td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">68 bp<\/td>\n<\/tr>\n<tr>\n<td rowspan=\"1\" colspan=\"1\"><\/td>\n<td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(R) CCTGGTTGGCTGCTTGCTT<\/td>\n<td rowspan=\"1\" colspan=\"1\"><\/td>\n<\/tr>\n<tr>\n<td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Arg-1<\/td>\n<td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(F) AGACAGCAGAGGAGGTGAAGAG<\/td>\n<td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">63 bp<\/td>\n<\/tr>\n<tr>\n<td rowspan=\"1\" colspan=\"1\"><\/td>\n<td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(R) CGAAGCAAGCCAAGGTTAAAGC<\/td>\n<td rowspan=\"1\" colspan=\"1\"><\/td>\n<\/tr>\n<tr>\n<td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">YM-1<\/td>\n<td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(F) GGATGGCTACACTGGAGAAA<\/td>\n<td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">178 bp<\/td>\n<\/tr>\n<tr>\n<td rowspan=\"1\" colspan=\"1\"><\/td>\n<td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(R) AGAAGGGTCACTCAGGATAA<\/td>\n<td rowspan=\"1\" colspan=\"1\"><\/td>\n<\/tr>\n<tr>\n<td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-10<\/td>\n<td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(F) GCCGGGAAGACAATAACTGC<\/td>\n<td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">223 bp<\/td>\n<\/tr>\n<tr>\n<td rowspan=\"1\" colspan=\"1\"><\/td>\n<td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(R) GCCTGGGGCATCACTTCTAC<\/td>\n<td rowspan=\"1\" colspan=\"1\"><\/td>\n<\/tr>\n<tr>\n<td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GAPDH<\/td>\n<td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(F) TGATGGGTGTGAACCACGAG<\/td>\n<td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">126 bp<\/td>\n<\/tr>\n<tr>\n<td rowspan=\"1\" colspan=\"1\"><\/td>\n<td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(R) GCCCTTCCACAATGCCAAAG<\/td>\n<td rowspan=\"1\" colspan=\"1\"><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139877758565936\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/table\/T1\/?report=objectonly\">Open in a separate window<\/a><\/div>\n<\/div>\n<\/div>\n<div id=\"sec-a.k.b.i\" class=\"sec\">\n<h3 id=\"sec-a.k.b.ititle\">Flow Cytometry for Analysis of Macrophage Subtypes<\/h3>\n<p class=\"p p-first\">M1 macrophages were identified with monoclonal antibodies specific for BB515-CD11b (BD, 564454) and APC-Cy7-CD16\/32 (BD, 560541), and M2 macrophages were detected with antibodies specific for BB515-CD11b and Alexa Flour@647-CD206 (BD Biosciences, San Jose, CA, USA) antibodies, then CD16\/32 or CD206 represent M1 or M2 macrophages, respectively. For immunophenotypic analysis, macrophages were gently detached with cold phosphate-buffered saline (PBS), pipetted into single cells, and then suspended at 1 \u00d7 10<sup>6<\/sup> cells\/ml. Cell suspensions were washed with PBS and then followed by incubation with the antibody mixtures for 30 min on ice boxes in the dark. Cells were then washed with buffer solution twice. Data were immediately acquired by using BD LSR II using FlowJo software.<\/p>\n<p class=\"p p-last\">To isolate the cells from the lamina propria of newborn mice with the method previously described (<a href=\"#B21\" rid=\"B21\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">21<\/a>, <a href=\"#B31\" rid=\"B31\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">31<\/a>), 2-cm distal ileum samples were cleaned from mesentery, finely minced with scissors, and then incubated in DMEM containing 10% FBS and 10 mM dithioerythritol (D8255; Sigma-Aldrich, USA), which was preheated to 37\u00b0C. Tissue was incubated for 20 min with gentle agitation. Supernatants containing the enterocyte were discarded. Lamina propria leukocytes were then isolated from the remaining tissue digestion at 37\u00b0C for 40 min in RPMI, 10% FBS, 100 U ml<sup>\u22121<\/sup> collagenase (C0130; Sigma-Aldrich, USA), and 15 \u03bcg\/ml DNase (D4527; Sigma-Aldrich, USA) with gentle agitation. After that, cells were washed twice in cold PBS with 1% BSA and filtered through sequential 70-\u03bcm and 40-\u03bcm cell strainers.<\/p>\n<\/div>\n<div id=\"sec-a.k.b.j\" class=\"sec\">\n<h3 id=\"sec-a.k.b.jtitle\">Western Blot Analysis<\/h3>\n<p class=\"p p-first-last\">Distal ileum tissues were weighted and cut into small pieces according to the manufacturer&#8217;s instructions. Macrophages were washed with PBS and collected for extracting proteins. Cytoplasmic and nuclear proteins were extracted using the EpiQuickTM Nuclear Extraction Kit I (OP-0002; EpiGentek, Farmingdale, NYUSA) (<a href=\"#B32\" rid=\"B32\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">32<\/a>). The nucleoprotein concentration was quantified with bicinchoninic acid (BCA) Assay Kit (Thermo Fisher Scientific, USA) and stored at \u221280\u00b0C. The protein samples were denatured at 100\u00b0C for 5 min, separated on 10% acrylamide gels, and then electrotransferred to polyvinylidene fluoride (PVDF) membranes. Afterwards, the membranes were blocked in TBST containing 5% fat free milk at room temperature for 2 h. The blots were incubated overnight at 4\u00b0C with primary antibodies: rabbit anti-mice P65 (ab32536; Abcam, UK) and Lamin B1 (12987-1-AP; Proteintech, USA) at 1:1,000 and 1:2,000, respectively. The membranes were washed three times with TBST and incubated for 1 h at room temperature with goat anti-rabbit IgG and goat anti-mouse IgG (1:5,000; Proteintech, USA). After washing three times for 10 min with TBST, the membranes were visualized and imaged using a chemiluminescent substrate (Clinx, Chemiscope 3500, China). Lamin B1 protein was used as an endogenous control as well.<\/p>\n<\/div>\n<div id=\"sec-a.k.b.k\" class=\"sec sec-last\">\n<h3 id=\"sec-a.k.b.ktitle\">Statistical Analysis<\/h3>\n<p class=\"p p-first-last\">Statistical analysis was performed using SPSS 22.0 software (IBM, USA), and the data were presented as mean \u00b1 standard deviation (SD) of at least three independent experiments. The incidence of NEC was compared with chi-squared test. A <em>p<\/em>-value &lt; 0.05 was considered statistically significant.<\/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=\"sec-a.k.c.b\" class=\"sec sec-first\">\n<h3 id=\"sec-a.k.c.btitle\">Effect of Hydrogen on Inflammation Cytokines in M1 or M2 Macrophages<\/h3>\n<p class=\"p p-first\">RAW264.7 macrophages induced by LPS (20 ng\/ml) and IFN-\u03b3 (50 ng\/ml) at 0, 3, 6, 12, 24, and 48 h were polarized to M1 macrophages, and the axons were small and elongated (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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>). RAW264.7 macrophages induced by IL-4 (20 ng\/ml) were polarized to M2 macrophages, which contained stunted axons (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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>). Raw264.7 macrophages and BMDM were cultured with LPS and IFN-\u03b3, and the mRNA levels of inducible nitric oxide synthase (iNOS) (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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>), TNF-\u03b1 (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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>), and IL-1\u03b2 (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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 1E<\/span><\/a>) released by M1 macrophages were significantly upregulated (<em>p<\/em> &lt; 0.001). The mRNA levels of IL-10 (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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 1F<\/span><\/a>), YM-1 (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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 1G<\/span><\/a>), and Arg-1 (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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 1H<\/span><\/a>) produced by M2 macrophages induced by IL-4 were markedly increased as well (<em>p<\/em> &lt; 0.001). These results show that M1 and M2 macrophages were successfully polarized. RAW264.7 macrophages were pretreated with or without hydrogen-rich water and then polarized to M1 or M2 macrophages in different mediums. The mRNA levels of TNF-\u03b1 (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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 1J<\/span><\/a>) and IL-1\u03b2 (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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 1K<\/span><\/a>) were decreased in the M1+H<sub>2<\/sub> group compared to the M1 group (<em>p<\/em> &lt; 0.05). The mRNA levels of IL-10 (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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 1L<\/span><\/a>) were increased in the M2+H<sub>2<\/sub> group compared to the M2 group (<em>p<\/em> &lt; 0.05). However, there is no effects of hydrogen on iNOS (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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 1I<\/span><\/a>), YM-1 (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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 1M<\/span><\/a>), Arg-1 (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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 1N<\/span><\/a>) (<sup>*<\/sup>, # P &lt; 0.05, <sup>**<\/sup> P &lt; 0.01, <sup>***<\/sup> P &lt; 0.001).<\/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\/PMC8635714\/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_idm139877758475072\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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=8635714_fped-09-710382-g0001.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 fped-09-710382-g0001.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/fped-09-710382-g0001.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139877758475072\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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\/PMC8635714\/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>Effects of macrophages on stimulations of macrophages (M1, 20 ng\/ml LPS and 50 ng\/ml IFN-\u03b3; M2, 20 ng\/ml IL-4). <strong>(A)<\/strong> M1 macrophages (scale bars = 50 \u03bcm); <strong>(B)<\/strong> M2 macrophages (scale bar = 50 \u03bcm). The changes of inflammation factors in RAW264.7 and BMDM in different mediums and different times, and mRNA levels of iNOS <strong>(C)<\/strong>, TNF-\u03b1 <strong>(D)<\/strong>, IL-1\u03b2 <strong>(E)<\/strong>, IL-10 <strong>(F)<\/strong>, Arg-1 <strong>(H)<\/strong>, and protein YM-1 <strong>(G)<\/strong>. The mRNA levels of in RAW264.7 macrophages with and without hydrogen, iNOS <strong>(I)<\/strong>, TNF-\u03b1 <strong>(J)<\/strong>, IL-1\u03b2 <strong>(K)<\/strong>, IL-10 <strong>(L)<\/strong>, YM-1 <strong>(M)<\/strong>, Arg-1 <strong>(N)<\/strong>, <sup>*<\/sup> M1 or M2 vs. M0, <sup>#<\/sup> M1+H<sub>2<\/sub> or M2+H<sub>2<\/sub> vs. M0 (<sup>*<\/sup>,<sup>#<\/sup><br \/>\n<em>p<\/em> &lt; 0.05, <sup>**<\/sup><br \/>\n<em>p<\/em> &lt; 0.01, <sup>***<\/sup><br \/>\n<em>p<\/em> &lt; 0.001).<\/p>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<div id=\"sec-a.k.c.c\" class=\"sec\">\n<h3 id=\"sec-a.k.c.ctitle\">Expressions of CD16\/32 in M1 and CD206 in M2 Macrophages<\/h3>\n<p class=\"p p-first\">In this study, we further investigated the effects of hydrogen on the markers of different macrophages. After treatment with hydrogen for 6 h and then polarizing to M1 or M2 macrophages, respectively, the expression of CD16\/32 in M1 macrophages was significantly lower than that of the control cells (<em>t<\/em> = 24.846, <em>p<\/em> &lt; 0.05) (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/figure\/F2\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"F2\" rid-ob=\"ob-F2\" co-legend-rid=\"lgnd_F2\" rel=\"noopener\"><span>Figures 2A,C<\/span><\/a>). However, hydrogen has no significant influence on the expression of CD206 in M2 macrophages (<em>t<\/em> = 1.284, <em>p<\/em> &gt; 0.05) (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/figure\/F2\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"F2\" rid-ob=\"ob-F2\" co-legend-rid=\"lgnd_F2\" rel=\"noopener\"><span>Figures 2B,D<\/span><\/a>). The result shows that the expression of CD206 in M2 macrophages was significantly higher than RAW264.7 macrophages (<em>t<\/em> = 3.451, <em>p<\/em> &lt; 0.05), and after stimulation with LPS and IFN-\u03b3, the expression of CD16\/32 in M1 macrophages was similar to RAW264.7 macrophages (<em>t<\/em> = 0.976, <em>p<\/em> &gt; 0.05) (<sup>*<\/sup>, # P &lt; 0.05, <sup>**<\/sup> P &lt; 0.01, <sup>***<\/sup> P &lt; 0.001).<\/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\/PMC8635714\/figure\/F2\/\" target=\"figure\" rid-figpopup=\"F2\" rid-ob=\"ob-F2\" rel=\"noopener\"><!--fig\/graphic|fig\/alternatives\/graphic mode=\"anchored\" m1--><\/a><\/p>\n<div class=\"figure\" data-largeobj=\"\" data-largeobj-link-rid=\"largeobj_idm139877758458288\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/figure\/F2\/\" target=\"figure\" rid-figpopup=\"F2\" rid-ob=\"ob-F2\" 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=8635714_fped-09-710382-g0002.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 fped-09-710382-g0002.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/fped-09-710382-g0002.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139877758458288\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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\/PMC8635714\/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>Flow cytometry analysis of macrophages and their markers. Macrophages cultured in normal medium <strong>(A)<\/strong> and in hydrogen medium <strong>(B)<\/strong> with different additives, and quantification of CD16\/32 <strong>(C)<\/strong> and CD206 <strong>(D)<\/strong> represented M1 and M2 macrophages from <strong>(A)<\/strong> and <strong>(B)<\/strong>, respectively (<sup>*<\/sup><em>p<\/em> &lt; 0.05, <sup>**<\/sup><em>p<\/em> &lt; 0.01, <sup>***<\/sup><em>p<\/em> &lt; 0.001).<\/p>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<div id=\"sec-a.k.c.d\" class=\"sec\">\n<h3 id=\"sec-a.k.c.dtitle\">The Incidence of NEC in Mice Model and Histological Evaluation<\/h3>\n<p class=\"p p-first\">The incidence of NEC was 0% in the control group, 52.63% in the NEC group, and 45% in the NEC+ H<sub>2<\/sub> group. <a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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 3A\u2013C,E,F<\/span><\/a> shows the score of HE staining ranging from 0-4 respectively. <a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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 4G<\/span><\/a> shows the histological NEC scores of ileum in each group. <a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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 3D,H<\/span><\/a> shows the general body of NEC and NEC+ H<sub>2<\/sub> mice, respectively. The mean mucosal injury score in different groups was 0.50 \u00b1 0.51 in the control group, 2.26 \u00b1 0.99 in the NEC group, 0.61 \u00b1 0.50 in the control + H<sub>2<\/sub> group, and 1.60 \u00b1 0.68 in the NEC+ H<sub>2<\/sub> group (CON vs. NEC: <em>t<\/em> = 6.77, <em>p<\/em> &lt; 0.001; NEC vs. NEC + H<sub>2<\/sub>, <em>t<\/em> = 2.45, <em>p<\/em> &lt; 0.05; and CON vs. CON + H<sub>2<\/sub>, <em>t<\/em> = 0.66, <em>p<\/em> &gt; 0.05).<\/p>\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\/PMC8635714\/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_idm139877758442496\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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=8635714_fped-09-710382-g0003.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 fped-09-710382-g0003.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/fped-09-710382-g0003.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139877758442496\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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\/PMC8635714\/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>The pathological changes of the different groups. In brief, grade 0-2 <strong>(A\u2013C)<\/strong>, grade 3 <strong>(E)<\/strong>, grade 4 <strong>(F)<\/strong>, <strong>(D,H)<\/strong> shows the general body of NEC and NEC+ H<sub>2<\/sub> mice, respectively. Histological NEC scores of ileum in each group <strong>(G)<\/strong>. (<sup>*<\/sup>P &lt; 0.05, <sup>**<\/sup>P &lt; 0.01, <sup>***<\/sup>P &lt; 0.001).<\/p>\n<\/div>\n<\/div>\n<\/div>\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\/PMC8635714\/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_idm139877758436096\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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=8635714_fped-09-710382-g0004.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 fped-09-710382-g0004.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/fped-09-710382-g0004.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139877758436096\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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\/PMC8635714\/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>The percentage of macrophages in different tissues. H<sub>2<\/sub>: fed with hydrogen; H<sub>2<\/sub>O: fed with water as control. <strong>(A,C,E)<\/strong> Expression of CD16\/32 in spleens, abdominal lymph nodes, and lamina propria, respectively. <strong>(B,D)<\/strong> Tof HE staining ranginghe expression of CD206 in spleens, abdominal lymph nodes respectively <strong>(B,D)<\/strong>. <strong>(F)<\/strong> Quantification of CD16\/32 or CD206 macrophages in different tissues obtained from <strong>(A\u2013E)<\/strong> (<sup>*<\/sup>P &lt; 0.05, <sup>**<\/sup>P &lt; 0.01, <sup>***<\/sup>P &lt; 0.001).<\/p>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<div id=\"sec-a.k.c.e\" class=\"sec\">\n<h3 id=\"sec-a.k.c.etitle\">Analysis of the Percentages of M1 and M2 Macrophages in Different Tissues of Mice<\/h3>\n<p class=\"p p-first-last\">Numerous macrophages with few neutrophils were found in resected tissue samples of neonates affected by NEC. Thus, we attempted to investigate the percentages of macrophages in different tissues of mice model of NEC. Our results showed that they have no significant effect on the percentage of M1 macrophages or M2 macrophages in spleens (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/figure\/F4\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"F4\" rid-ob=\"ob-F4\" co-legend-rid=\"lgnd_F4\" rel=\"noopener\"><span>Figures 4A,B,F<\/span><\/a>) (NEC vs. CON: CD16\/32: <em>t<\/em> = 1.27, <em>p<\/em> &gt; 0.05; CD206: <em>t<\/em> = 0.71, <em>p<\/em> &gt; 0.05) or in abdominal lymph nodes (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/figure\/F4\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"F4\" rid-ob=\"ob-F4\" co-legend-rid=\"lgnd_F4\" rel=\"noopener\"><span>Figures 4C,D,F<\/span><\/a>) (NEC vs. CON: CD16\/32: <em>t<\/em> = 2.06, <em>p<\/em> &gt; 0.05; CD206: <em>t<\/em> = 2.364, <em>p<\/em> &gt; 0.05). However, in lamina propria mucosae of distal ileum, the percentage of M1 macrophages in the NEC group was remarkably higher than that in the control group and the percentage of M1 macrophages was significantly decreased after treatment with hydrogen (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/figure\/F4\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"F4\" rid-ob=\"ob-F4\" co-legend-rid=\"lgnd_F4\" rel=\"noopener\"><span>Figures 4E,F<\/span><\/a>) (NEC vs. CON, <em>t<\/em> = 9.51, <em>p<\/em> &lt; 0.001; NEC vs. NEC + H<sub>2<\/sub>, <em>t<\/em> = 12.51, <em>p<\/em> &lt; 0.000) (<sup>*<\/sup> P &lt; 0.05, <sup>**<\/sup> P &lt; 0.01, <sup>***<\/sup> P &lt; 0.001).<\/p>\n<\/div>\n<div id=\"sec-a.k.c.f\" class=\"sec sec-last\">\n<h3 id=\"sec-a.k.c.ftitle\">Expression of NF-\u03baB p65 in Distal Ileum and M1 Macrophages<\/h3>\n<p class=\"p p-first\">To understand the effects of hydrogen on polarization of macrophages in M1 macrophages and the mice model of NEC, the level of NF-\u03baB p65 in the nucleus of M1 macrophages (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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 5A<\/span><\/a>) or distal ileum of mice (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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 5B<\/span><\/a>) was studied by Western blot analysis. The NF-\u03baB p65 was decreased in M1 macrophage cell nucleus after treatment with hydrogen compared to M1 macrophages (<em>t<\/em> = 16.65, <em>p<\/em> &lt; 0.05) and significantly increased in M1 macrophages after being induced by LPS and IFN-\u03b3. As the same time, we found that the NF-\u03baB p65 was significantly elevated in the NEC group compared with the CON group (1.70 \u00b1 0.09 vs. 1.02 \u00b1 0.02, <em>p<\/em> &lt; 0.01), and decreased in the NEC + H<sub>2<\/sub> group compared to the NEC group (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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 5C<\/span><\/a>) (1.70 \u00b1 0.09 vs. 1.39 \u00b1 0.04, <em>p<\/em> &lt; 0.05).<\/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\/PMC8635714\/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_idm139877723490304\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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=8635714_fped-09-710382-g0005.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 fped-09-710382-g0005.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/fped-09-710382-g0005.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139877723490304\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC8635714\/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\/PMC8635714\/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>Expression of NF-\u03baB p65 signaling pathway in RAW264.7 and distal ileum of mice. <strong>(A)<\/strong> Western blotting was undertaken for the expression of NF- \u03baB p65 in the cytoplasm and nucleus of macrophages cell line, <strong>(B)<\/strong> the expression of NF- \u03baB p65 in distal ileum of NEC mice model. <strong>(C)<\/strong> Densitometric analysis of the bands of NF-\u03baB p65 in different groups (<sup>*<\/sup>P &lt; 0.05, <sup>**<\/sup>P &lt; 0.01, <sup>***<\/sup>P &lt; 0.001).<\/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\">Macrophages are such important components of the innate immune system, and the functional diversity of the macrophages can be related to their ability on responding to the different micro-environments. With the different stimulated micro-environments, two main phenotypes of macrophages are suggested, M1 macrophages (called classically activated macrophages) and M2 macrophages (called alternatively activated macrophages). M1 macrophages are mainly stimulated by LPS and IFN-\u03b3, and have a key role in host defense against infection. However, M2 macrophages could be stimulated by IL-4 or other factors; they mainly contribute to anti-inflammatory response, and associate with tissue remodeling (<a href=\"#B9\" rid=\"B9\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">9<\/a>\u2013<a href=\"#B11\" rid=\"B11\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">11<\/a>, <a href=\"#B33\" rid=\"B33\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">33<\/a>). We try to polarize bone marrow-derived macrophages with LPS and IFN-gamma or IL-4 to M1 or M2 in our research; however, with the culture of the time, the less macrophages residue. Afterwards, we only detect the inflammatory cytokines produced by M1 or M2 with rt-PCR, and then we used RAW264.7 cells polarized to M1 or M2 macrophages. The results show that hydrogen has no effect on CD16\/32 in RAW264.7 cells; however, the percentage of CD16\/32 in M1 macrophages was decreased in the hydrogen group. We supposed that CD16\/32 were expressed in most RAW264.7 macrophages, and hydrogen may decrease the expression of CD16\/32 in RAW264.7 macrophages. After treatment with hydrogen in RAW264.7 macrophages, the inflammatory cytokines produced by M1 were significantly reduced, and the NF-\u03baB p65 was decreased in M1 macrophage cell nuclei. There may be a connection with the results we found, and that needs to be confirmed further.<\/p>\n<p>NEC is a common gastrointestinal disease, mainly affecting prematurity, and extremely low-birth-weight infants; however, the pathogenesis of NEC is still not fully clear. Hence, in the present study, the NEC model was established to explore the mechanism and therapeutic effects (<a href=\"#B2\" rid=\"B2\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">2<\/a>, <a href=\"#B34\" rid=\"B34\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">34<\/a>, <a href=\"#B35\" rid=\"B35\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">35<\/a>). There are many experimental models of NEC available, and researchers may choose different models depending on the purpose of the study. However, we have no idea as to which of the models is truly NEC. In our study, hypoxia, cold stimulation, and artificial formula feeding were used to establish the NEC model, without LPS induction. It was done to exclude the effect of LPS-induced inflammatory stimulation on the outcome. A positive control with LPS may be more appropriate in study design. The older the model animal, the more mature the intestinal development, and the lower incidence of NEC. However, the incidence of NEC in different age groups has not been reported. Pierro et al. found that osmolality of enteral formula had no significant correlation with the incidence of NEC. Therefore, the NEC model was established by hypoxia and cold stimulation and formula feeding (<a href=\"#B36\" rid=\"B36\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">36<\/a>, <a href=\"#B37\" rid=\"B37\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">37<\/a>). The incidence of NEC was 52.63% higher than the control group (<em>p<\/em> &lt; 0.001). By comparing the HE staining score of terminal ileum, it was revealed that the NEC score was decreased from 2.26 \u00b1 0.99 to 1.60 \u00b1 0.68 after treatment with hydrogen. There were no remarkable changes in incidence of NEC between the NEC group and the NEC+ H<sub>2<\/sub> group, which may be related to the mice used for the established model, which are too mature even though they are not beyond 14\u201321 days of age (<a href=\"#B35\" rid=\"B35\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">35<\/a>). We analyzed the weight of mice during the experiment, in which the results showed that the decrease of body weight in the NEC group was higher than that in the CON group, while there was no significant difference among other groups during the experiment. We supposed hydrogen may not increase the body weight of NEC mice.<\/p>\n<p>We also investigated the percentages of M1 and M2 macrophages in abdominal lymph nodes and spleen in the NEC model; however, the results disclosed that the changes observed among different groups were not statistically significant. Previous studies reported that macrophages in the lamina propria of the intestine plays a key role in the development of an immune response (<a href=\"#B38\" rid=\"B38\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">38<\/a>\u2013<a href=\"#B40\" rid=\"B40\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">40<\/a>). Therefore, we assessed the percentage of M1 macrophages in lamina propria. The results showed that expression of CD16\/32 M1 macrophages was higher in the NEC group than in the CON group. Moreover, after intervention with hydrogen, the expression of CD16\/32 in M1 macrophages was remarkably decreased.<\/p>\n<p>Hydrogen has been shown to have selectively reduced ROS and anti-inflammation by Ohsawa et al. (<a href=\"#B18\" rid=\"B18\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">18<\/a>). Hydrogen is a new therapeutic medical gas with anti-oxidant, anti-inflammatory, and anti-apoptotic effects on cells and organs, but not applied extensively in clinical practice. Instead of hydrogen molecule, hydrogen-rich saline or water, which is safer and easier to apply, may be more suitable for clinical applications. However, there are many issues that need to be resolved (molecular mechanism, the optimal dose and period, long-term effects, etc.) before the practical clinical application. Our team provides a new method for the role of hydrogen in NEC (<a href=\"#B17\" rid=\"B17\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">17<\/a>), thus giving hope to solve the previous problems.<\/p>\n<p class=\"p p-last\">There are several limitations in the present study. Firstly, we used mice fed with clean water as a control group, other than bed with their mother together. Secondly, we did not analyze the correlation between the NF-\u03baB signaling pathway and the inflammatory cytokines. We will further analyze the molecular mechanisms of hydrogen regarding polarization of macrophages in NEC.<\/p>\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\">Conclusions<\/h2>\n<p class=\"p p-first-last\">In conclusion, the achieved results suggest that M1 macrophages play an important role in the pathogenesis of NEC. Hydrogen protects against intestinal injury through reducing the inflammation factors produced by M1 macrophage <em>via<\/em> preventing M1 macrophage conversion. Hydrogen preventing M1 macrophage conversion may be related to the decrease of NF-\u03baB p65 in the nucleus.<\/p>\n<\/div>\n<div id=\"s6\" 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=\"s6title\">Data Availability Statement<\/h2>\n<p class=\"p p-first-last\">The datasets presented in this study can be found in online repositories. The names of the repository\/repositories and accession number(s) can be found in the article\/supplementary material.<\/p>\n<\/div>\n<div id=\"s7\" 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=\"s7title\">Ethics Statement<\/h2>\n<p class=\"p p-first-last\">The animal study was reviewed and approved by and the experimental protocols were performed according to the Ethical Guidelines for the Use of Animals in Research, and this study was approved by the Animal Care Committee of the Children&#8217;s Hospital of Shanghai.<\/p>\n<\/div>\n<div id=\"s8\" 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=\"s8title\">Author Contributions<\/h2>\n<p class=\"p p-first-last\">SY and JS contributed to the project conception and design. SY and ZG collected the data and prepared the article. XW and JZ observed the histological slides. QS and ZL interpreted the data and critically revised and approved the final article. All authors contributed to the article and approved the submitted version.<\/p>\n<\/div>\n<div id=\"s9\" 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=\"s9title\">Funding<\/h2>\n<p class=\"p p-first-last\">National Natural Science Foundation of China (Award Number: 81370743), Recipient: Zhibao LV. 2. Interdisciplinary Funding of Medicine and Engineering obtained from Shanghai Jiao Tong University (Award Number: YG2015ZD13), Recipient: Zhibao LV. 3. Shanghai Natural Science Foundation (Award Number: 17ZR1423100), Recipient: Zhimei Gao.<\/p>\n<\/div>\n<div id=\"conf1\" 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=\"conf1title\">Conflict of Interest<\/h2>\n<p class=\"p p-first-last\">The 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=\"s10\" 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=\"s10title\">Publisher&#8217;s Note<\/h2>\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=\"ref-list-a.l.a\" 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.l.atitle\">References<\/h2>\n<div class=\"ref-list-sec sec\" id=\"reference-list\">\n<div class=\"ref-cit-blk half_rhythm\" id=\"B1\">1. <span class=\"mixed-citation\">Lim JC, Golden JM, Ford HR. <span class=\"ref-title\">Pathogenesis of neonatal necrotizing enterocolitis<\/span>. <span class=\"ref-journal\">Pediatr Surg Int.<\/span> (2015) <span class=\"ref-vol\">31<\/span>:509\u201318. 10.1007\/s00383-015-3697-9 [<a 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href=\"https:\/\/scholar.google.com\/scholar_lookup?journal=J+Surg+Res.&amp;title=Role+of+neutrophils+and+macrophages+in+the+pathogenesis+of+necrotizing+enterocolitis+caused+by+Cronobacter+sakazakii&amp;author=CN+Emami&amp;author=R+Mittal&amp;author=L+Wang&amp;author=HR+Ford&amp;author=NV+Prasadarao&amp;volume=172&amp;publication_year=2012&amp;pages=18-28&amp;pmid=21601887&amp;doi=10.1016\/j.jss.2011.04.019&amp;\" target=\"_blank\" rel=\"noopener noreferrer\" ref=\"reftype=other&amp;article-id=8635714&amp;issue-id=374910&amp;journal-id=2282&amp;FROM=Article%7CCitationRef&amp;TO=Content%20Provider%7CLink%7CGoogle%20Scholar\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Google Scholar<\/a>]<\/span><\/span><\/div>\n<\/div>\n<\/div>\n<div style=\"display: none; width: 200px; top: -100px; left: -100px;\" aria-live=\"assertive\" aria-hidden=\"true\" class=\"ui-helper-reset ui-ncbipopper-wrapper ui-ncbilinksmenu\">\n<ul id=\"ui-ncbiinpagenav-2\">\n<li><a href=\"#abstract-a.j.b.ptitle\">Abstract<\/a><\/li>\n<li><a href=\"#s1title\">Introduction<\/a><\/li>\n<li><a href=\"#s2title\">Methods<\/a><\/li>\n<li><a href=\"#s3title\">Results<\/a><\/li>\n<li><a href=\"#s4title\">Discussion<\/a><\/li>\n<li><a href=\"#s5title\">Conclusions<\/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=\"#conf1title\">Conflict of Interest<\/a><\/li>\n<li><a href=\"#s10title\">Publisher&#8217;s Note<\/a><\/li>\n<li><a href=\"#ref-list-a.l.atitle\">References<\/a><\/li>\n<\/ul>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"<p>Hydrogen Promotes the M1 Macrophage Conversion During the Polarization of Macrophages in Necrotizing Enterocolitis<\/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":[898],"body-organ":[1025],"applications":[680],"test_subjects":[1518],"report-topic":[1348],"class_list":["post-26785","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-hydrogen-health","disease-intestinal-injury-2","body-organ-intestine-2","applications-ingestion-2","test_subjects-mouse-2","report-topic-necrotizing-enterocolitis-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 Enhances Macrophage Polarization in Necrotizing Enterocolitis<\/title>\n<meta name=\"description\" content=\"Hydrogen Promotes the M1 Macrophage Conversion During the Polarization of Macrophages in Necrotizing 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