| Part I Maresin 1 protects against early cerebral ischemia/reperfusion injury by attenuating the pro-inflammatory responseObjective:To explore the effect of different doses of MaRl on early inflammatory response of mice cerebral ischemia/reperfusion(I/R).Methods:The cerebral ischemia/reperfusion model was introduced by middle cerebral artery occlusion (MCAO) using an intraluminal silicon coated suture, and reperfusion was simulated by removing the suture after one hour of occlusion. Totally, sixty male C57 mice weighing 22-27g were randomly divided into five groups (n=12):(1) Sham group:Mice received anesthesia and cervical vascular separation operation without suture occlusion, one hour later 2.5ul normal saline(NS) was intracerebroventricular(i.c.v) injected.(2) MCAO group:Mice received MCAO surgery, after reperfusion 2.5ul of NS was i.c.v. injected.(3) MCAO+0.1ng MaRl group:Mice received MCAO surgery, after reperfusion O.lng of MaRl was i.c.v. injected.(4) MCAO+lng MaRl group:Mice received MCAO surgery, after reperfusion 1ng of MaRl was i.c.v. injected.(5) MCAO+10ng MaRl group: Mice received MCAO surgery, after reperfusion 10ng of MaRl was i.c.v. injected. After 24h,48h or 72h,brain tissue was collected, TTC staining, Hematoxylin and Eeosin(H&E) staining, Fluoro-JadeB (FJ) staining, transferase-mediated deoxyuridine triphosphate-biotin nick end labeling(TUNEL) staining were performed to estimate cerebral infarction and injury, neurological deficit score was used to evaluate neurological function, Immunohistochemical staining was performed to determine the infiltration of neutrophil and activation of glia. Myeloperoxidase (MPO) of brain tussue was detected by spectrophotometer. Cytokines in brain tissue were analyzed by enzyme-linked immunosorbent assay (ELISA) Kits, the expression of synaptic associated protein was measured by Western Blotting.Results:compared with Sham group, after reperfusion the mice in MCAO group showed infarction and tissue damage, the neurocytes exhibited vacuolar degeneration accompanied by apoptosis or necrosis, and inflammatory cells infiltrated into brain. After 48h and 72h of reperfusion the mice showed neurologic deficits (P<0.001). Furthermore, the expression of synaptic associated protein was significantly decreased (P<0.05), while cytokines(P<0.01) and activated glia were sharply increased. With an optimal dose of 1 ng, MaR1 significantly reduced the infarct volume and neurological defects, preserved brain tissue and neurons, reduced apoptosis and cytokines (P<0.01) also suppressed glial activation and MPO activity. In addition, 0.1ng of MaRl had slight protection while 10ng of MaR1 had no difference with MCAO group. Data were expressed as the mean ± the standard error of the mean (S.E.M.). Group comparisons were analyzed using one-way ANOVA followed by the Student-Newman-Keuls (SNK) test. Statistical analyses were performed using GraphPad Prism 5.0. Significant differences were defined as P<0.05.Conclusion:MaR1 significantly alleviated MCAO induced brain injury, probably through attenuating early pro-inflammatory reaction.Part II the mechanism of MaRl on inflammatory reaction of mice cerebral ischemia/reperfusion injuryObjective:To investigate the possible mechanisms of MaRl in the early stage of cerebral ischemia/reperfusion.Methods:The MCAO model was established by intraluminal suture method. Experiments were divided into two parts. Part A, a total of 30 healthy male C57 mice were divided into 5 groups(n=6):(1) sham group::Mice received anesthesia and cervical vascular separation operation without suture occlusion, one hour later 2.5ul normal saline(NS) was intracerebroventricular(i.c.v) injected.(2) MCAO group:Mice received MCAO surgery, after reperfusion 2.5ul of NS was i.c.v. injected.(3) MCAO+O.lng MaRl group:Mice received MCAO surgery, after reperfusion 0.1ng of MaR1 was i.c.v. injected.(4) MCAO+1ng MaRl group:Mice received MCAO surgery, after reperfusion 1ng of MaRl was i.c.v. injected.(5) MCAO+lOng MaRl group:Mice received MCAO surgery, after reperfusion 10ng of MaR1 was i.c.v. injected. All the mice were euthanized fter 24h of reperfusion, the localization of NF-kB P65 was detected by immunofluorescence, the expression of acetylated or phosphorylated P65 were analyzed by Western Blotting. Part B,48 of male C57 mice were randomly divided into 4 groups:(1) MCAO group:1%DMSO vehicle solution was intraperitoneal injected every two days before MCAO for three times, and then mice received MCAO surgery. (2) MCAO+1ng MaRl group:1%DMSO vehicle solution was intraperitoneal injected every two days before MCAO for three times, and then mice received MCAO surgery and 1ng MaRl i.c.v injection.(3) MCAO+lng MaRl+EX527 group, 5mg/kg EX527 was intraperitoneal injected every two days before MCAO for three times, and then mice received MCAO surgery and 1ng MaRl i.c.v injection.(4) MCAO+EX527 group,5mg/kg EX527 was intraperitoneal injected every two days before MCAO for three times, and then mice received MCAO surgery. After reperfusion, the infarction volume and tissue damage were detected by TTC and H&E staining, cytokines of TNF-a and IL-1β were determined by ELISA kits, the expression of SIRT1, acetylated(AC)-NF-κB, Bcl2 and Bax were analyzed by Western Blotting. Statistical analyses were the same as part I.Results:Part A, the increased expression of AC-NF-κB, phosphorylated P65, nuclear located P65 and Bax that induced by MCAO were significantly suppressed by Ing MaRl, on the other hand, the decreased expression of SIRT1 and Bcl2 were improved by Ing MaRl. Part B, specifically, MCAO+MaRl+EX527 showed more expression of acetylated P65 and Bax, as well as less SIRT1 and Bcl2 compared with MCAO+MaR1.Furthermore, EX527 administration partially reversed the protective effect of MaRl on infarction and neurologic dificits.Conclusion:The anti-inflammatory effect of MaRl on cerebral I/R was partially SIRT1-NF-kB dependent.PartⅢThe effect of MaRl on blood brain barrier of mice cerebral ischemia/reperfusion injuryObjective:To investigate the protective effect of MaRl on blood brain barrier of mice cerebral ischemia/reperfusion injury.Methods:Mice received MCAO operation (n=15), (1) sham group:Mice received anesthesia and cervical vascular separation operation without suture occlusion, one hour later 2.5ul NS was i.c.v injected.(2) MCAO group:Mice received MCAO surgery, after reperfusion 2.5ul of NS was i.c.v. injected.(3) MCAO+MaRl group:Mice received MCAO surgery, after reperfusion Ing or O.lng or lOng of MaRl was i.c.v. injected. After 24h of reperfusion brain water content was calculated, blood brain barrier permeability was evaluated by Intravenous injected Evans blue, activity of MMP9 was analyzed by gelatin zymography, while activity of MMP3 was calculated by fluorometric immune-capture, the expression of zo-1,claudin5,MMP9 were determined by western blotting or immunofluorescence.Results:Cerebral I/R injury significantly increased water content (p<0.001) and Evans Blue leakage (p<0.001) of ischemic hemisphere, correlating with BBB damage, also the activity of MMP9 and MMP3 were elevated. Meanwhile, the component of BBB such as zo-1 and claudin5 were sharply decreased, furthermore, the trend in immunofluorescence and western blotting were uniform. However, Ing MaRl significantly alleviated brain edema and BBB damage, suppressed MMPs’activity and expression. Additionally, MaRl administration could up-regulate the expression of zo-1 and claudin5.Conclusion:After cerebral I/R, MaR1 preserved component protein of BBB, suppressed MMPs’activity, resulting in decreased BBB permeability and brain edema, thus protecting brain from I/R injury. |
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