title: Hydrogen-Rich Saline Attenuated Subarachnoid Hemorrhage-Induced Early Brain Injury in Rats by Suppressing Inflammatory Response: Possible Involvement of NF-κB Pathway and NLRP3 Inflammasome.
abstract: Early brain injury (EBI), highlighted with inflammation and apoptosis, occurring within 72 h after subarachnoid hemorrhage (SAH), is associated with the prognosis of SAH. Recent studies have revealed that hydrogen-rich saline (HS) exerted multiple neuroprotective properties in many neurological diseases including SAH, involved to anti-oxidative and anti-apoptotic effect. We have previously reported that HS could attenuate neuronal apoptosis as well as vasospasm. However, the underlying mechanism of HS on inflammation in SAH-induced EBI remains unclear. In this study, we explored the influence of HS on nuclear factor-κB (NF-κB) pathway and nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome at early stage after SAH, by injecting HS intraperitoneally to SAH rats. One hundred and twenty-nine SD rats were randomly divided into four groups: sham group, SAH group, SAH+vehicle group, and SAH+HS group. SAH model was conducted using endovascular perforation method; all rats were sacrificed at 24 h after SAH. Protein level of pIκBα, cytosolic and nuclear p65, NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, interleukin-1β (IL-1β), and cleaved caspase-3 were measured by western blot. mRNA level of IL-1β, interleukin-6 (IL-6), tumor necrosis factor-c (TNF-α) were evaluated by RT-PCR. Cellular injury and death was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Nissl staining, respectively. Our results showed that pIκBα, nuclear p65, NLRP3, ASC, caspase-1, IL-1β, cleaved caspase-3 proteins, as well as the mRNA of IL-1β, IL-6, and TNF-ɑ increased at 24 h after SAH, while cytosolic p65 decreased. TUNEL and Nissl staining presented severe cellular injury at 24 h post-SAH. However, after HS administration, the changes mentioned above were reversed. In conclusion, HS may inhibit inflammation in EBI and improve neurobehavioral outcome after SAH, partially via inactivation of NF-κB pathway and NLRP3 inflammasome. Graphical Abstract Schematic representation of the mechanism of HS-mediated anti-inflammatory effect in EBI after SAH. The NF-κB inflammatory pathway and NLRP3 inflammasome are involved in the anti-neuroinflammatory effect of HS post-SAH. SAH-induced oxidative stress enhances the activation of NF-κB, thus promoting the translocation of p65 subunit into nucleus and increasing the mRNA level of its downstream proinflammatory cytokines (IL-1β, IN-6, TNF-α) and NLRP3. Elevated expression of NLRP3 mRNA increases the assembly of NLRP3 inflammasome. In addition, oxidative stress after SAH stimulates the activation of NLRP3 inflammasome, therefore, promoting caspase-1 activation and the cleavage of pro-IL-1β into mature IL-1β. Finally, activation of NF-κB pathway and NLRP3 inflammasome contribute to the inflammation response and cellular injury in EBI after SAH. HS treatment reversed the detrimental effect mentioned above via inactivation of NF-κB pathway and NLRP3 inflammasome. NF-κB nuclear factor-κB, IκB inhibitor of NF-κB, IKK Iκ kinase, NLRP3 nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3, ASC apoptosis-associated speck-like protein containing a caspase recruitment domain.
authors.affiliation: Department of Neurosurgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310009, China.
authors.forename: Anwen
authors.initials: A
authors.lastname: Shao
authors.affiliation: Department of Neurosurgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310009, China.
authors.forename: Haijian
authors.initials: H
authors.lastname: Wu
authors.affiliation: Department of Neurosurgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310009, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Hong
authors.affiliation: Department of Thoracic Oncology, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310009, China.
authors.forename: Sheng
authors.initials: S
authors.lastname: Tu
authors.affiliation: Department of Diving Medicine, The Second Military Medical University, Shanghai, 200433, China.
authors.forename: Xuejun
authors.initials: X
authors.lastname: Sun
authors.affiliation: Department of Neurosurgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310009, China.
authors.forename: Qun
authors.initials: Q
authors.lastname: Wu
authors.affiliation: Department of Thoracic Oncology, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310009, China. doczq.2008@gmail.com.
authors.forename: Qiong
authors.initials: Q
authors.lastname: Zhao
authors.affiliation: Department of Neurosurgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310009, China. zjm153vip@sina.com.|Brain Research Institute, Zhejiang University, Hangzhou, 310009, China. zjm153vip@sina.com.
authors.forename: Jianmin
authors.initials: J
authors.lastname: Zhang
authors.affiliation: State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310009, China.|Department of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310009, China.
authors.forename: Jifang
authors.initials: J
authors.lastname: Sheng
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D001921:Brain / Q000473:pathology; D001929:Brain Edema / Q000150:complications / Q000473:pathology; D001930:Brain Injuries / Q000188:drug therapy* / Q000201:enzymology / Q000209:etiology / Q000473:pathology; D002540:Cerebral Cortex / Q000473:pathology; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D006859:Hydrogen / Q000494:pharmacology / Q000627:therapeutic use*; D058847:Inflammasomes / Q000378:metabolism*; D007249:Inflammation / Q000150:complications / Q000378:metabolism / Q000473:pathology*; D018836:Inflammation Mediators / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism; D015227:Lipid Peroxidation / Q000187:drug effects; D008297:Male; D008315:Malondialdehyde / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D012965:Sodium Chloride / Q000494:pharmacology / Q000627:therapeutic use*; D013345:Subarachnoid Hemorrhage / Q000150:complications* / Q000188:drug therapy
chemical_list: D000975:Antioxidants; D016207:Cytokines; D058847:Inflammasomes; D018836:Inflammation Mediators; D053583:Interleukin-1beta; D016328:NF-kappa B; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D012965:Sodium Chloride; D008315:Malondialdehyde; D006859:Hydrogen
keywords: Early brain injury; Hydrogen; Inflammation; NLRP3 inflammasome; Nuclear factor κB; Subarachnoid hemorrhage