{"id":26882,"date":"2024-01-03T21:39:37","date_gmt":"2024-01-03T19:39:37","guid":{"rendered":"https:\/\/hho-bulgaria.com\/h2-rich-saline-reduces-neuron-apoptosis-after-cerebral-ischemia\/"},"modified":"2024-01-29T21:18:08","modified_gmt":"2024-01-29T19:18:08","slug":"h2-rich-saline-reduces-neuron-apoptosis-after-cerebral-ischemia","status":"publish","type":"post","link":"https:\/\/hho-bulgaria.com\/en\/h2-rich-saline-reduces-neuron-apoptosis-after-cerebral-ischemia\/","title":{"rendered":"H2-rich saline reduces neuron apoptosis after cerebral ischemia"},"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\">Iran J Basic Med Sci.<\/a><\/span> 2020 Apr; 23(4): 494\u2013499. <\/div>\n<div class=\"part2\">  <span class=\"doi\"><span>doi:&nbsp;<\/span><a href=\"\/\/doi.org\/10.22038%2Fijbms.2020.41751.9857\" target=\"_blank\" rel=\"noopener noreferrer\" ref=\"reftype=other&amp;article-id=7239416&amp;issue-id=358089&amp;journal-id=1931&amp;FROM=Article%7CFront%20Matter&amp;TO=Content%20Provider%7CCrosslink%7CDOI\">10.22038\/ijbms.2020.41751.9857<\/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>PMC7239416<\/span><\/div>\n<div class=\"fm-citation-pmid\">PMID: <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/32489564\">32489564<\/a><\/div>\n<\/div>\n<\/div>\n<h1 class=\"content-title\">Hydrogen-rich saline ameliorates hippocampal neuron apoptosis through up-regulating the expression of cystathionine \u03b2-synthase (CBS) after cerebral ischemia- reperfusion in rats<\/h1>\n<div class=\"half_rhythm\">\n<div class=\"contrib-group fm-author\"><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Cong%20Hm%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139678172636784\" co-class=\"co-affbox\">Hai-ming Cong<\/a>,<sup>1<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Gao%20Qp%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139678179239952\" co-class=\"co-affbox\">Qiu-ping Gao<\/a>,<sup>2<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Song%20Gq%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139678180683456\" co-class=\"co-affbox\">Guo-qiang Song<\/a>,<sup>3<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Ye%20Yx%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139678172857920\" co-class=\"co-affbox\">Ying-xin Ye<\/a>,<sup>4<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Li%20Xl%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139678172856160\" co-class=\"co-affbox\">Xiao-li Li<\/a>,<sup>5<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Zhang%20Ls%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139678172854176\" co-class=\"co-affbox\">Lian-shuang Zhang<\/a>,<sup>6<\/sup> and  <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Wang%20Xf%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139678175914832\" co-class=\"co-affbox\">Xi-feng Wang<\/a><sup>5,<\/sup><sup>*<\/sup><\/div>\n<div style=\"display:none\" class=\"contrib-group aff-tip\">\n<div id=\"_co_idm139678172636784\">\n<h3 class=\"no_margin\">Hai-ming Cong<\/h3>\n<p>\n<sup>1<\/sup>Department of Rehabilitation Medicine, Weihai Central Hospital, Wendeng 264400, China<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Cong%20Hm%5BAuthor%5D\">Hai-ming Cong<\/a><\/div>\n<\/div>\n<div id=\"_co_idm139678179239952\">\n<h3 class=\"no_margin\">Qiu-ping Gao<\/h3>\n<p>\n<sup>2<\/sup>Department of Emergency, Ji Nan Zhang Qiu District Hospital of Traditional Chinese Medicine, Zhang Qiu 250200, China<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Gao%20Qp%5BAuthor%5D\">Qiu-ping Gao<\/a><\/div>\n<\/div>\n<div id=\"_co_idm139678180683456\">\n<h3 class=\"no_margin\">Guo-qiang Song<\/h3>\n<p>\n<sup>3<\/sup>Department of Neurosurgery, Ji Nan Zhang Qiu District Hospital of Traditional Chinese Medicine, Zhang Qiu 250200, China<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Song%20Gq%5BAuthor%5D\">Guo-qiang Song<\/a><\/div>\n<\/div>\n<div id=\"_co_idm139678172857920\">\n<h3 class=\"no_margin\">Ying-xin Ye<\/h3>\n<p>\n<sup>4<\/sup>Department of Neurology, Ji Nan Zhang Qiu District Hospital of TraditionalChinese Medicine, Zhang Qiu 250200, China<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Ye%20Yx%5BAuthor%5D\">Ying-xin Ye<\/a><\/div>\n<\/div>\n<div id=\"_co_idm139678172856160\">\n<h3 class=\"no_margin\">Xiao-li Li<\/h3>\n<p>\n<sup>5<\/sup>Department of Critical Care Medicine, Yu Huang Ding Hospital, Qingdao University, Yantai, P. R. China<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Li%20Xl%5BAuthor%5D\">Xiao-li Li<\/a><\/div>\n<\/div>\n<div id=\"_co_idm139678172854176\">\n<h3 class=\"no_margin\">Lian-shuang Zhang<\/h3>\n<p>\n<sup>6<\/sup>Department of Histology and Embryology, Binzhou Medical University, Yantai, P. R. China<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Zhang%20Ls%5BAuthor%5D\">Lian-shuang Zhang<\/a><\/div>\n<\/div>\n<div id=\"_co_idm139678175914832\">\n<h3 class=\"no_margin\">Xi-feng Wang<\/h3>\n<p>\n<sup>5<\/sup>Department of Critical Care Medicine, Yu Huang Ding Hospital, Qingdao University, Yantai, P. R. China<\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Wang%20Xf%5BAuthor%5D\">Xi-feng 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=\"idm139678181347344_ai\">Author information<\/a> <a href=\"#\" class=\"pmctoggle\" rid=\"idm139678181347344_an\">Article notes<\/a> <a href=\"#\" class=\"pmctoggle\" rid=\"idm139678181347344_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=\"idm139678181347344_ai\" style=\"display:none\">\n<div class=\"fm-affl\" id=\"aff1\">\n<sup>1<\/sup>Department of Rehabilitation Medicine, Weihai Central Hospital, Wendeng 264400, China<\/div>\n<div class=\"fm-affl\" id=\"aff2\">\n<sup>2<\/sup>Department of Emergency, Ji Nan Zhang Qiu District Hospital of Traditional Chinese Medicine, Zhang Qiu 250200, China<\/div>\n<div class=\"fm-affl\" id=\"aff3\">\n<sup>3<\/sup>Department of Neurosurgery, Ji Nan Zhang Qiu District Hospital of Traditional Chinese Medicine, Zhang Qiu 250200, China<\/div>\n<div class=\"fm-affl\" id=\"aff4\">\n<sup>4<\/sup>Department of Neurology, Ji Nan Zhang Qiu District Hospital of TraditionalChinese Medicine, Zhang Qiu 250200, China<\/div>\n<div class=\"fm-affl\" id=\"aff5\">\n<sup>5<\/sup>Department of Critical Care Medicine, Yu Huang Ding Hospital, Qingdao University, Yantai, P. R. China<\/div>\n<div class=\"fm-affl\" id=\"aff6\">\n<sup>6<\/sup>Department of Histology and Embryology, Binzhou Medical University, Yantai, P. R. China<\/div>\n<div id=\"cor1\"><sup>*<\/sup>Corresponding author: Xi-feng Wang. Department of Critical Care Medicine, Yu Huang Ding Hospital, Qingdao University, Yantai, P. R. China.264000. Tel; 86-535-6691999; Email: wxf197400@126.com<\/div>\n<\/div>\n<div class=\"fm-article-notes hide half_rhythm\" id=\"idm139678181347344_an\" style=\"display:none\">\n<div class=\"fm-pubdate half_rhythm\">Received 2019 Jul 10; Accepted 2019 Nov 9.<\/div>\n<\/div>\n<div class=\"permissions half_rhythm hide\" id=\"idm139678181347344_cpl\" style=\"display:none\">\n<div class=\"fm-copyright half_rhythm\"><a href=\"\/pmc\/about\/copyright\/\">PMC Copyright notice<\/a> <\/div>\n<div class=\"license half_rhythm\">This is an Open Access article distributed under the terms of the Creative Commons Attribution License, (<a href=\"http:\/\/creativecommons.org\/licenses\/by\/3.0\/\" data-ga-action=\"click_feat_suppl\" ref=\"reftype=extlink&amp;article-id=7239416&amp;issue-id=358089&amp;journal-id=1931&amp;FROM=Article%7CFront%20Matter&amp;TO=External%7CLink%7CURI\" target=\"_blank\" rel=\"noopener\">http:\/\/creativecommons.org\/licenses\/by\/3.0\/<\/a>) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.<\/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=\"abstract-a.i.b.s\" 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.i.b.stitle\">Abstract<\/h2>\n<p><!--article-meta--><\/p>\n<div>\n<div id=\"sec-a.i.b.s.a\" class=\"sec sec-first\">\n<h3 id=\"sec-a.i.b.s.atitle\">Objective(s):<\/h3>\n<p class=\"p p-first-last\">This study aimed to evaluate the potential role of hydrogen in rats after cerebral ischemic\/reperfusion (I\/R) injury. <\/p>\n<\/div>\n<div id=\"sec-a.i.b.s.b\" class=\"sec\">\n<h3 id=\"sec-a.i.b.s.btitle\">Materials and Methods:<\/h3>\n<p class=\"p p-first-last\">The experimental samples were composed of sham group, model group of rats that received middle cerebral artery occlusion (MCAO) for 2 hr followed by reperfusion for 24 hr, and the hydrogen saline group treated by hydro\u00acgen-rich saline (1 ml\/kg) after MCAO. Hydrogen sulfide (H2S), S100-\u03b2protein (S100-\u03b2), and neuron-specific enolase (NSE) levels were measured; the levels of malondialdehyde (MDA), reactive oxygen species (ROS), and superoxide dismutase (SOD) were detected; the histologic structure and apoptotic cells of hippocampus were observed; the expressions of cystathionine \u03b2-synthase (CBS), nuclear factor erythroid 2-related factor 2 (Nrf2), and hemeoxygenase-1 (HO-1) were measured. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Fisher\u2019s least significant difference (LSD) test.<\/p>\n<\/div>\n<div id=\"sec-a.i.b.s.c\" class=\"sec\">\n<h3 id=\"sec-a.i.b.s.ctitle\">Results:<\/h3>\n<p class=\"p p-first-last\">Our results showed that hydrogen up-regulated H2S levels via promoting the expression of CBS in the hippocampus, and its treatment alleviated oxidative stress via activating the expression of Nrf2 and HO-1, and then cell apoptosis reduced, furthermore, brain function improved by down-regulating the levels of S100-\u03b2and NSE. <\/p>\n<\/div>\n<div id=\"sec-a.i.b.s.d\" class=\"sec sec-last\">\n<h3 id=\"sec-a.i.b.s.dtitle\">Conclusion:<\/h3>\n<p class=\"p p-first-last\">This study showed that hydrogen-rich saline ameliorates cell injury through up-regulating the expression of CBS in the hippocampus after cerebral ischemia reperfusion (I\/R) in rats, this provides new experimental evidence for the treatment of stroke with hydrogen saline.<\/p>\n<\/div>\n<\/div>\n<div class=\"sec\"><strong class=\"kwd-title\">Key Words: <\/strong><span class=\"kwd-text\">Cerebral ischemia \u2013 reperfusion, Cystathionine \u03b2-synthase, Hippocampus, Hydrogen, Rats<\/span><\/div>\n<\/div>\n<div id=\"sec-a.j.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=\"sec-a.j.atitle\">Introduction<\/h2>\n<p class=\"p p-first\">It is known that ischemic stroke characterized by the sudden loss of blood circulation is a major type of stroke (<a href=\"#B1\" rid=\"B1\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">1<\/a>) and has become the second leading cause of death globally (<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>). Moreover, its high recurrence rate has economic burdens for the society. Studies have confirmed that cerebral ischemia has a complicated pathology closely related to oxidative stress (<a href=\"#B4\" rid=\"B4\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">4<\/a>&#8211;<a href=\"#B7\" rid=\"B7\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">7<\/a>). Accumulating evidence demonstrated that the main cause of neuron damage is not ischemia itself but the overproduction of reactive oxygen species (ROS) which attacks cells, resulting in ischemia\/reperfusion (I\/R) injury and neuronal cell death (<a href=\"#B8\" rid=\"B8\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">8<\/a>&#8211;<a href=\"#B10\" rid=\"B10\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">10<\/a>). Thus, a better understanding of the molecular and cellular mechanisms underlying oxidative stress injury after I\/R may provide novel treatment for ischemic stroke. It has generated considerable interest in developing antioxidant therapies to combat ischemia-induced damage due to the close relationship between cerebral ischemia and oxidative stress (<a href=\"#B11\" rid=\"B11\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">11<\/a>). <\/p>\n<p>Hydrogen sulfide (H<sub>2<\/sub>S), known as a toxic gas in nature (<a href=\"#B12\" rid=\"B12\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">12<\/a>) with an odorous smell, is synthesized from L-cysteine by enzymes such as cystathionine \u03b3-lyase (CSE), cystathionine \u03b2-synthase (CBS), and mercaptopyruvate sulfurtransferase (3MST) (<a href=\"#B13\" rid=\"B13\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">13<\/a>). CBS is predominantly responsible for the production of H<sub>2<\/sub>S in the central nervous system (<a href=\"#B14\" rid=\"B14\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">14<\/a>, <a href=\"#B15\" rid=\"B15\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">15<\/a>), recent studies have shown that small amounts of H<sub>2<\/sub>S are produced in the brain (<a href=\"#B14\" rid=\"B14\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">14<\/a>), and it has been proven to be an endogenous factor that regulates cellular function (<a href=\"#B16\" rid=\"B16\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">16<\/a>). Previous research has investigated that H<sub>2<\/sub>S has a protective effect against cerebral injury in rodent models (17). So, up-regulating the expression of CBS to promote H<sub>2<\/sub>S synthesis may be a therapeutic strategy for stroke.<\/p>\n<p class=\"p p-last\">Hydrogen is a gas that can be used for diving (<a href=\"#B18\" rid=\"B18\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">18<\/a>). However, studies have confirmed its anti-oxidative capabilities in animal models since Ohsawa <em>et al<\/em>. reported that hydrogen inhalation could protect the brain against cerebral I\/R injury (<a href=\"#B19\" rid=\"B19\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">19<\/a>). In our previous researches, we also found that hydrogen has a neuroprotective effect in ischemia reperfusion rats (<a href=\"#B20\" rid=\"B20\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">20<\/a>, <a href=\"#B21\" rid=\"B21\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">21<\/a>). The novelty of this study is investigating whether hydrogen protected the brain against I\/R injury through up-regulating the expression of CBS and changing the levels of neurological function indices such as neuron specific enolase (NSE) and S100-\u03b2protein (S100-\u03b2) in the hippocampus and its related mechanism.<\/p>\n<\/div>\n<div id=\"sec-a.j.b\" 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=\"sec-a.j.btitle\">Materials and Methods<\/h2>\n<p class=\"p p-first\">\n<strong><em>Preparation of hydrogen-rich saline<\/em><\/strong>\n<\/p>\n<p>The hydrogen-rich saline was purchased from Second Military Medical University (Shanghai, China) and stored under atmospheric pressure at 4 <sup>\u00b0<\/sup>C in an aluminum bag. Hydrogen-rich saline was freshly prepared within one week to ensure a constant concentration no less than 0.6 mmol\/l (<a href=\"#B22\" rid=\"B22\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">22<\/a>).<\/p>\n<p>\n<strong><em>Experiment design<\/em><\/strong>\n<\/p>\n<p>In this study, 36 adult male Sprague-Dawley Rats (weighing 280\u2013320 g) were obtained from the Experimental Animal Center of Shandong University (Jinan, China). The experiment protocols were approved by the Ethics Committee of Bin Zhou Medical University and performed in accordance with the guidelines of National Institutes of Health Guide for Laboratory Animals.<\/p>\n<p>After one week acclimation, the rats were divided into three groups randomly, 12 rats in each group. The animals were anesthetized with 3.5% chloral hydrate solution (1 ml\/100 g, IP). Middle cerebral artery occlusion (MCAO) was induced as our previous research described (<a href=\"#B21\" rid=\"B21\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">21<\/a>). In brief, the left common carotid artery (CCA) and the external carotid artery (ECA) were exposed, and then, a 3-0 surgical monofilament nylon suture was inserted from the external carotid artery into the internal carotid artery (ICA) carefully and was advanced in a forward manner to occlude the origin of the left middle cerebral artery (MCA) until a light resistance was felt (18\u201320 mm from the CCA bifurcation). After 2 hr of MCAO, the nylon suture was withdrawn, followed by 46 hr of reperfusion. Twelve rats were used as sham group (suture only after exposure of carotid artery), 12 rats were used as I\/R group after MCAO, and the I\/R + hydrogen group comprised 12 MCAO rats treated with hydrogen-rich saline (1 ml\/kg) after the beginning of reperfusion. After 24 hr, all rats were euthanized with chloral hydrate (7%, 5 ml\/kg). The blood and brain tissues from each animal were collected for analysis. <\/p>\n<p>\n<strong><em>Measurement of S100-\u03b2and neuron-specific enolase (NSE) levels<\/em><\/strong>\n<\/p>\n<p>The blood samples were allowed to clot and then centrifuged at 3,000 rpm for 10&nbsp;min, sera were separated and used to determine S100-\u03b2 protein and NSE by Enzyme-Linked Immunosorbent Assay (ELISA) (Yuchen, Shanghai) according to the manufacturer\u2019s protocol.<\/p>\n<p>\n<strong><em>Measurement of H<\/em><\/strong><br \/>\n<sub>2<\/sub><br \/>\n<strong><em>S levels<\/em><\/strong>\n<\/p>\n<p>The biosynthesis of H<sub>2<\/sub>S in the brain was measured as described previously (<a href=\"#B24\" rid=\"B24\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">24<\/a>). The optical absorbance was measured at 655 nm with a microplate reader (iMark; Bio-Rad). The H<sub>2<\/sub>S concentration of each sample was calculated.<\/p>\n<p>\n<strong><em>Measurement of ROS, malondialdehyde (MDA) and superoxide dismutase (SOD)<\/em><\/strong>\n<\/p>\n<p>ROS and SOD are the indicators of oxidative stress. The homogenates of brain tissue were centrifuged at 3,000 rpm for 20 min at 4 <sup>\u00b0<\/sup>C. The supernatant was separated and the activity of ROS and SOD were determined using a detection kit (Jiancheng, China) as manual protocol. Optical density was determined using a spectrometer, both at 550 nm.<\/p>\n<p>The concentration of MDA as a marker of lipid peroxidation, was measured using a detection kit (Jiancheng, China) following the manufacturer\u2019s protocol. Optical density was determined by a spectrometer at 532 nm.<\/p>\n<p>\n<strong><em>Histopathological examination<\/em><\/strong>\n<\/p>\n<p>Isolated ischemic cerebral tissues were fixed with 10% methanol, embedded with paraffin, tissues were sectioned at a thickness of 5 \u03bcm, stained with hematoxylin and eosin (HE), and observed under a light microscope (Olym-pusX71-F22PH, Japan) at 400 magnification. The total and injured neurons were counted in 12 different fields of microscope per sample, six samples in each group were counted.<\/p>\n<p>\n<strong><em>Immunohistochemical staining<\/em><\/strong>\n<\/p>\n<p>Immunohistochemical staining method refers to our previous studies (<a href=\"#B21\" rid=\"B21\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">21<\/a>).The ischemic cerebral tissues were fixed in 10% formalin, embedded in paraffin, and cut into 5-\u03bcm thick sections, stained with a CBS antibody (diluted 1:500, Cell Signaling Technology (CST), USA), followed by a second IgG antibody. Immunostaining was performed with diaminobenzidine (DAB).The DAB staining density was assessed with a microscopic image analysis system (GX51, Olympus, Japan).<\/p>\n<p>\n<strong><em>TdT-mediated dUTP nick end labeling (TUNEL)<\/em><\/strong>\n<\/p>\n<p>The ischemic cerebral tissue was fixed in 10% formalin, embedded in paraffin, and sectioned at a thickness of 5 \u03bcm; TUNEL staining was performed with an <em>in situ<\/em> cell death detection kit (Jiancheng, China) according to the manufacturer\u2019s protocol. <\/p>\n<p>First, the sections were rinsed with PBS and were treated with 1% Triton X-100 for 3 min. Terminal deoxynucleotidyl transferase (TdT) was used to catalyze the addition of biotinconjugated dUTP to the 3\u2032-OH ends of the DNA fragments subsequently. Streptavidin-HRP solution was added and reacted at 37 \u00b0C for 30 min. Finally, the slides were placed in DAB for 3 min and stained with Hematoxylin Harris. These analyses were performed at 100\u00d7 magnification under a light microscope in 12 different fields using computer-aided software (Olympus X71-F22PH, Japan). The apoptosis cells were quantified using computer assisted image analysis (Leica LAS Image Analysis V4.0).<\/p>\n<p>\n<strong><em>Western blotting<\/em><\/strong>\n<\/p>\n<p>The protein was extracted using an extraction kit (<a href=\"http:\/\/www.baidu.com\/link?url=iQDaGdbLxoBRgUXcwIMKPQ3WuxFbrV1C8RrwCMNsKIxVITSb8eHz66OvFhcIiJgz\" data-ga-action=\"click_feat_suppl\" ref=\"reftype=extlink&amp;article-id=7239416&amp;issue-id=358089&amp;journal-id=1931&amp;FROM=Article%7CBody&amp;TO=External%7CLink%7CURI\" target=\"_blank\" rel=\"noopener\">Beyotime Biotechnology<\/a>, china). Equal amounts of lysate proteins (20 \u03bcg) were loaded onto SDS-polyacrylamide gels and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocking with 5% nonfat milk in TBS and Tween 20 (TBST) for 1 hr, the PVDF membrane was incubated with \u03b2-actin (1:2000, CST, USA), Nrf-2 (1:800, CST, USA), HO-1(1:800, CST, USA) overnight at 4 <sup>\u00b0<\/sup>C. Then, membranes were washed three times with TBST and incubated with IgG secondary antibody (1:5000, Beyotime Biotechnology, China) for 1 hr at room temperature. After washing with TBST, the antibody-bound proteins were detected with the ECL chemiluminescence reagent (Beyotime Biotechnology, china). Protein levels were calculated relative to that of \u03b2-actin. The images were analyzed using the software package (Bio-Rad Laboratories Inc, Hercules, CA, USA).<\/p>\n<p>\n<strong><em>Statistical analysis<\/em><\/strong>\n<\/p>\n<p class=\"p p-last\">When data were normally distributed, they were analyzed using SPSS 21.0 and are expressed as mean \u00b1standard error (SEM). Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Fisher\u2019s least significant difference (LSD) test, and a value of <em>P<\/em>&lt;0.05 was considered statistically significant.<\/p>\n<\/div>\n<div id=\"sec-a.j.c\" 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=\"sec-a.j.ctitle\">Results<\/h2>\n<p class=\"p p-first\">\n<strong><em>Effect of hydrogen on H<\/em><\/strong><br \/>\n<sub>2<\/sub><br \/>\n<strong><em>S levels<\/em><\/strong>\n<\/p>\n<p>The levels of H<sub>2<\/sub>S in the brain tissue decreased in the I\/R group compared with the sham group (<em>P<\/em>&lt;0.05), but hydrogen increased the endogenous H<sub>2<\/sub>S levels in the brain compared to the I\/R group (<em>P<\/em>&lt;0.05) (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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 1<\/span><\/a>). <\/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\/PMC7239416\/figure\/F1\/\" target=\"figure\" rid-figpopup=\"F1\" rid-ob=\"ob-F1\" rel=\"noopener\"><!--fig\/graphic|fig\/alternatives\/graphic mode=\"anchored\" m1--><\/p>\n<div class=\"figure\" data-largeobj=\"\" data-largeobj-link-rid=\"largeobj_idm139678176124368\"><img decoding=\"async\" loading=\"lazy\" class=\"fig-image\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is IJBMS-23-494-g001.jpg\" title=\"An external file that holds a picture, illustration, etc.\nObject name is IJBMS-23-494-g001.jpg\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/IJBMS-23-494-g001.jpg\"><\/div>\n<p><\/a><\/p>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139678176124368\" style=\"display: none;\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/figure\/F1\/\" target=\"figure\" rid-figpopup=\"F1\" rid-ob=\"ob-F1\" rel=\"noopener\"><\/a><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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\/PMC7239416\/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 hydrogen on hydrogen sulfide levels in middle cerebral artery occlusion rats (mean\u00b1SEM). * indicates significant difference compared with sham group (<em>P<\/em>&lt;0.05), # indicates significant difference compared with I\/R group (<em>P<\/em>&lt;0.05). I\/R: ischemia\/reperfusion<\/p>\n<\/div>\n<\/div>\n<\/div>\n<p>\n<strong><em>Effect of hydrogen on S100-<\/em><\/strong>\u03b2<strong><em>and NSE levels<\/em><\/strong><\/p>\n<p>The levels of S100-\u03b2 and NSE have similar trends, these increased in the I\/R group compared with those of the sham group (<em>P<\/em>&lt;0.05), but hydrogen treatment decreased their levels in the brain compared to the I\/R group (<em>P<\/em>&lt;0.05) (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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>). It indicated that hydrogen played a protective role against ischemic injury.<\/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\/PMC7239416\/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_idm139678174575248\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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=7239416_IJBMS-23-494-g002.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 IJBMS-23-494-g002.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/IJBMS-23-494-g002.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139678174575248\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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\/PMC7239416\/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>Effects of hydrogen on S100-\u03b2protein and neuron-specific enolase levels in middle cerebral artery occlusion rats (mean\u00b1SEM). S100-\u03b2 protein level in brain (A), neuron-specific enolase level in brain (B). * indicates significant difference compared with sham group (<em>P<\/em>&lt;0.05), # indicates significant difference compared with I\/R group (<em>P<\/em>&lt;0.05). I\/R: ischemia\/reperfusion<\/p>\n<\/div>\n<\/div>\n<\/div>\n<p>\n<strong><em>Changes of histopathological structure<\/em><\/strong>\n<\/p>\n<p>The neurons in the CA1 area of the hippocampus were observed. Normal neurons had round nuclei, but the nuclei of necrotic neurons were irregular, shrinkage, and with deep staining. <a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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> shows obvious morphological changes in the I\/R group in which the body and nuclei of neurons were reduced with shrinkage, and percentage of injured cells detected in the I\/R group was significantly higher than that in the sham group (<em>P<\/em>&lt;0.05), but it decreased after hydrogen treatment (<em>P<\/em>&lt;0.05) (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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 3B<\/span><\/a>). <\/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\/PMC7239416\/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_idm139678171874704\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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=7239416_IJBMS-23-494-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 IJBMS-23-494-g003.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/IJBMS-23-494-g003.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139678171874704\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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\/PMC7239416\/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>Histopathological observation of the hippocampus in middle cerebral artery occlusion rats. Sham group (a), I\/R group (b), I\/R+hydrogen group (c). Black arrows denote neurons in the hippocampus. HE \u00d7400 (A). Percentage of injury cells in the three groups (B). Scale bar: 30 \u03bcm. * indicates significant difference compared with sham group (<em>P<\/em>&lt;0.05), # indicates significant difference compared with I\/R group (<em>P<\/em>&lt;0.05). I\/R: ischemia\/reperfusion<\/p>\n<\/div>\n<\/div>\n<\/div>\n<p>\n<strong><em>Effect of hydrogen on ROS, MDA, and SOD levels<\/em><\/strong>\n<\/p>\n<p>As shown in <a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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 4 B and C<\/span><\/a>, ROS and MDA levels were much higher in the I\/R group than those in the sham group (<em>P<\/em>&lt;0.05). Treatments with hydrogen decreased ROS and MDA levels compared with the I\/R group (<em>P<\/em>&lt;0.05). On the contrary, the levels of SOD in the brain tissue decreased in the I\/R group compared with the sham group (<em>P<\/em>&lt;0.05), but hydrogen increased the endogenous H<sub>2<\/sub>S levels in the brain compared with the I\/R group (<em>P<\/em>&lt;0.05) (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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 4A<\/span><\/a>). <\/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\/PMC7239416\/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_idm139678170997904\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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=7239416_IJBMS-23-494-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 IJBMS-23-494-g004.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/IJBMS-23-494-g004.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139678170997904\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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\/PMC7239416\/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>Effects of hydrogen on reactive oxygen species, malondialdehyde and superoxide dismutase levels in middle cerebral artery occlusion rats (mean\u00b1SEM). SOD level in the brain (A), ROS level in the brain (B), MDA level in the brain (C). * indicates significant difference compared with sham group (<em>P<\/em>&lt;0.05), # indicates significant difference compared with I\/R group (<em>P<\/em>&lt;0.05). I\/R: ischemia\/reperfusion; SOD: superoxide dismutase ; ROS: reactive oxygen species<\/p>\n<\/div>\n<\/div>\n<\/div>\n<p>\n<strong><em>Changes of apoptosis<\/em><\/strong>\n<\/p>\n<p>The TUNEL staining is shown in <a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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>, the gray value of positive TUNEL cells found in the I\/R group was significantly higher than that of the sham group (<em>P<\/em>&lt;0.05), but it decreased after hydrogen treatment (<em>P<\/em>&lt;0.05) (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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>).<\/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\/PMC7239416\/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_idm139678172677776\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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=7239416_IJBMS-23-494-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 IJBMS-23-494-g005.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/IJBMS-23-494-g005.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139678172677776\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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\/PMC7239416\/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>Effects of hydrogen on expressions of TdT-mediated dUTP nick end labeling positive cells in middle cerebral artery occlusion rats. Sham group (a), I\/R group (b), I\/R+hydrogen group (c). Black arrows denote TUNEL positive neurons in the hippocampus (A). Gray values of the TUNEL positive cells in the three groups (B). Scale bar: 30 \u03bcm. * indicates significant difference compared with sham group (<em>P<\/em>&lt;0.05), # indicates significant difference compared with I\/R group (<em>P<\/em>&lt;0.05). I\/R: ischemia\/reperfusion<\/p>\n<\/div>\n<\/div>\n<\/div>\n<p>\n<strong><em>Changes of CBS expression <\/em><\/strong>\n<\/p>\n<p>The expression of CBS showed brown staining in cells (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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 A<\/span><\/a>). Its mean density in I\/R group increased compared with the sham group (<em>P<\/em>&lt;0.05). Moreover, it increased significantly higher in the hydrogen treatment group compared with I\/R group (<em>P<\/em>&lt;0.05) (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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 B<\/span><\/a>). It indicated that hydrogen up-regulated the CBS expression in ischemic hippocampus.<\/p>\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\/PMC7239416\/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_idm139678178644144\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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=7239416_IJBMS-23-494-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 IJBMS-23-494-g006.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/IJBMS-23-494-g006.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139678178644144\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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\/PMC7239416\/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>Effects of hydrogen on the expression of cystathionine \u03b2-synthase in middle cerebral artery occlusion rats. Sham group (a), I\/R group (b), I\/R+hydrogen group (c). Black arrows denote cystathionine \u03b2-synthase positive neurons in the hippocampus (A). Gray values of cystathionine \u03b2-synthase positive cells in the three groups (B). Scale bar: 30 \u03bcm. * indicates significant difference compared with sham group (<em>P<\/em>&lt;0.05), # indicates significant difference compared with I\/R group (<em>P<\/em>&lt;0.05). I\/R: ischemia\/reperfusion<\/p>\n<\/div>\n<\/div>\n<\/div>\n<p>\n<strong><em>Changes of Nrf-2 and HO-1 expressions<\/em><\/strong>\n<\/p>\n<p class=\"p\">The expression levels of Nrf-2 and HO-1 increased in the I\/R group compared with the sham group (<em>P<\/em>&lt;0.05), and the hydrogen treatment further up-regulated the protein levels of those compared to the I\/R group (<em>P<\/em>&lt;0.05) (<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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>). It indicated that endogenous expression of Nrf-2 and HO-1 were activated after I\/R, and hydrogen could further up-regulate the expressions of those.<\/p>\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\/PMC7239416\/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_idm139678175447744\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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=7239416_IJBMS-23-494-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 IJBMS-23-494-g007.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/IJBMS-23-494-g007.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139678175447744\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7239416\/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\/PMC7239416\/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>Effects of hydrogen on protein expression in middle cerebral artery occlusion rats. Expressions of Nrf-2 and HO-1in each group (A). Relative expressions of Nrf-2 (B) and HO-1 (C) in each group were measured. Data are reported as means\u00b1SE. * indicates significant difference compared with sham group (<em>P<\/em>&lt;0.05), # indicates significant difference compared with I\/R group (<em>P<\/em>&lt;0.05). I\/R: ischemia\/reperfusion; Nrt2: nuclear factor erythroid 2-related factor 2; HO-1: hemeoxygenase-1<\/p>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<div id=\"sec-a.j.d\" 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=\"sec-a.j.dtitle\">Discussion<\/h2>\n<p class=\"p p-first\">In the present study, we investigated the protective role of hydrogen in rats after I\/R injury through up-regulation of H<sub>2<\/sub>S levels, it was confirmed by H<sub>2<\/sub>S assessment and CBS expression in the hippocampus. Meanwhile, the treatment of hydrogen reduced oxidative stress and down-regulated the percentage of apoptotic neurons in ischemic hippocampus, and then injuries of neurons were improved. These results indicated the protective effect of hydrogen on injured neurons. These are consistent with our previous research showing that hydrogen protected the neurons against I\/R injury (<a href=\"#B20\" rid=\"B20\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">20<\/a>, <a href=\"#B21\" rid=\"B21\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">21<\/a>). Furthermore, the novelty of this study is that hydrogen may achieve its protective effect by up-regulating the expression of CBS and H<sub>2<\/sub>S levels in the hippocampus. <\/p>\n<p class=\"p p-last\">H<sub>2<\/sub>S is the third gas signal molecule (<a href=\"#B24\" rid=\"B24\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">24<\/a>), has been recognized to play crucial physiological functions in the central nervous system (<a href=\"#B25\" rid=\"B25\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">25<\/a>, <a href=\"#B26\" rid=\"B26\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">26<\/a>). The results were in accordance with previous reports that the expression of CBS and concentration of H<sub>2<\/sub>S increased in the hippocampus of rats after brain ischemia (<a href=\"#B27\" rid=\"B27\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">27<\/a>), it indicated that hydrogen up-regulated the H<sub>2<\/sub>S levels in ischemic brain, and H<sub>2<\/sub>S could reduce cerebral I\/R injury in the animal model (<a href=\"#B28\" rid=\"B28\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">28<\/a>&#8211;<a href=\"#B31\" rid=\"B31\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">31<\/a>). To further elucidate the mechanism of hydrogen in alleviating MCAO-induced cerebral ischemic injury, the present study investigated the effects on antioxidants. Oxidative stress is a core pathological component closely related to reperfusion injury accompanied with excessive ROS production (<a href=\"#B32\" rid=\"B32\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">32<\/a>, <a href=\"#B33\" rid=\"B33\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">33<\/a>). Our results demonstrated that induction of I\/R leads to elevated levels of ROS and MDA and a decrease of SOD. Increasing studies have shown that treatment with H<sub>2<\/sub>S could inhibit apoptosis via blocking an ROS activated Ca<sup>2+ <\/sup>signaling pathway in hypoxia-induced hippocampal neurons (<a href=\"#B30\" rid=\"B30\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">30<\/a>) and improved ischemic damage and apoptosis in cerebral ischemia through its antioxidant effects (<a href=\"#B34\" rid=\"B34\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">34<\/a>&#8211;<a href=\"#B36\" rid=\"B36\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">36<\/a>). As expected, we found that treatment with hydrogen reduced ROS and MDA levels in the ischemic brain of I\/R rats, and increased the SOD activity as well as the expression of CBS, nuclear factor erythroid 2-related factor 2 (Nrf2), and hemeoxygenase-1 (HO-1). All these implied that treatment with hydrogen saline significantly suppressed oxidative stress in ischemic brain. Nrf2 is an endogenous cytoprotective factor that activates the transcription of antioxidant stress genes, including HO-1 against oxidative stress (<a href=\"#B37\" rid=\"B37\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">37<\/a>, <a href=\"#B38\" rid=\"B38\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">38<\/a>). The activation of Nrf2\/HO-1 antioxidant pathway has been shown to play an important neuroprotective role after ischemia reperfusion-induced brain injury (<a href=\"#B39\" rid=\"B39\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">39<\/a>&#8211;<a href=\"#B41\" rid=\"B41\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">41<\/a>). The results showed that hydrogen significantly regulated Nrf2\/HO-1 levels in the ischemic model.<\/p>\n<\/div>\n<div id=\"sec-a.j.e\" 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=\"sec-a.j.etitle\">Conclusion<\/h2>\n<p class=\"p p-first-last\">The present study demonstrated that hydrogen could protect neurons in the hippocampus against ischemic injury through up-regulation of the CBS expression and activating the Nrf2\/HO-1 antioxidant pathway. This provides a new experimental basis for clinical application of hydrogen in the treatment of cerebral ischemic injury. But the specific mechanism of how hydrogen up-regulated CBS expression remains to be further explored, this is the subsequent target for us and other researchers.<\/p>\n<\/div>\n<div id=\"ack-a.k.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=\"ack-a.k.atitle\">Acknowledgment<\/h2>\n<div class=\"sec\">\n<p>This study was supported by Nature Science Foundation from Shandong Province (No. ZR2016HL24)<\/p>\n<\/div>\n<\/div>\n<div id=\"sec-a.k.b\" 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=\"sec-a.k.btitle\">Conflicts of Interest<\/h2>\n<p><!--\/article\/back\/sec\/--><\/p>\n<p class=\"p p-first-last\"> There are no conflicts of interest in this article.<\/p>\n<\/div>\n<div id=\"ref-list-a.k.c\" 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.k.ctitle\">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=\"element-citation\">Benjamin EJ, Blaha MJ, Chiuve SE, Cushman M, Das SR, Deo R, et al. 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ref=\"reftype=pubmed&amp;article-id=7239416&amp;issue-id=358089&amp;journal-id=1931&amp;FROM=Article%7CCitationRef&amp;TO=Entrez%7CPubMed%7CRecord\">PubMed<\/a>] <span class=\"nowrap\">[<a href=\"https:\/\/scholar.google.com\/scholar_lookup?journal=+PLoS+One&amp;title=the+Nrf2\/HO-1+activation+of+antioxidant+pathway+contributes+to+the+protective+of+Lycium+effects+barbarum+polysaccharides+in+the+rodent+retina+after+ischemia-reperfusion-induced+damage&amp;author=M+He&amp;author=H+Pan&amp;author=RC+Chang&amp;author=KF+So&amp;author=NC+Brecha&amp;volume=9&amp;publication_year=2014&amp;pages=e84800&amp;pmid=24400114&amp;\" target=\"_blank\" rel=\"noopener noreferrer\" ref=\"reftype=other&amp;article-id=7239416&amp;issue-id=358089&amp;journal-id=1931&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: 202px; 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.i.b.stitle\">Abstract<\/a><\/li>\n<li><a href=\"#sec-a.j.atitle\">Introduction<\/a><\/li>\n<li><a href=\"#sec-a.j.btitle\">Materials and Methods<\/a><\/li>\n<li><a href=\"#sec-a.j.ctitle\">Results<\/a><\/li>\n<li><a href=\"#sec-a.j.dtitle\">Discussion<\/a><\/li>\n<li><a href=\"#sec-a.j.etitle\">Conclusion<\/a><\/li>\n<li><a href=\"#ack-a.k.atitle\">Acknowledgment<\/a><\/li>\n<li><a href=\"#sec-a.k.btitle\">Conflicts of Interest<\/a><\/li>\n<li><a href=\"#ref-list-a.k.ctitle\">References<\/a><\/li>\n<\/ul>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"<p>Hydrogen-rich saline ameliorates hippocampal neuron apoptosis through up-regulating the expression of cystathionine \u03b2-synthase (CBS) after cerebral ischemia- reperfusion in rats<\/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":[881],"body-organ":[1019],"applications":[682],"test_subjects":[1517],"report-topic":[1297],"class_list":["post-26882","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-hydrogen-health","disease-stroke-2","body-organ-brain-2","applications-injection-2","test_subjects-rat-2","report-topic-ischemia-reperfusion-i-r-injury-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-rich saline reduces neuron apoptosis after cerebral ischemia<\/title>\n<meta name=\"description\" content=\"Hydrogen-rich saline ameliorates hippocampal neuron apoptosis through up-regulating the expression of cystathionine 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