The Study Of Hypertonic Saline Attenuates Proinflammatory Mediators In Cerebral Ischemia Through Notch Signaling

Ischemic stroke is a common neurological disease in the clinic. It has shown that neuroinflammatory mediators can exert deleterious effects and exacerbate the progression of cerebral ischemia injury. These inflammatory mediators, are widely thought to be derived from the microglia, activated microglia can release amounts inflammation mediators of such as tumor necrosis factor-a (TNF-a), reactive oxygen species (ROS), interleukin-6 (IL-6), which aggravating brain damage.NF-κB is widely thought to be an important regulator of neuroinflammatory and is crucial in cerebral ischemia. Recently some studies have considered that Notch is the upstream of NF-κB. It has been reported that inhibition of Notch with pharmacological attenuates the nuclear distribution of NF-κB, thus reducing transcriptional activity. On the other hand, Notch signaling can up-regulate the expression of the active NF-κB signaling components, thus enhancing NF-κB activation.Notch signaling plays an important role in regulating the activation of immune cells when responses to external stimuli and infection. When blocking Notch signaling pathway, the apoptosis of neuronal and the number of activated microglia were significantly been reduced; the release of inflammatory mediators IL-6, IL-1β, iNOS, M-CSF and the infarct size were also been significantly reduced; the neurological function were been improved.Hypertonic saline, which has been shown to effectively reduce intracranial pressure and hemispheric edema, is widely utilized in acute brain injuries including ischemic stroke, traumatic brain injury (TBI) and intracranial hemorrhage (ICH) Hypertonic saline (HS) and been a guidance for the treatment of brain edema. The osmotic property of HS has been well studied in brain injury, its extraosmotic effect is following more and more attention. Many reports have indicated that HS has neuroprotective properties and could reduce mortality after cerebral ischemia. Hypertonic saline was reported to prevent the activation of macrophages though reducing the inflammatory response.Previous study in our group demonstrated that 10%HS could reduce brain water inthe content of cerebral infarction; reduce infarct size and decrease the release of neuroinflammatory mediators such as IL-1β and TNF-α in activated microglia. On the basis of literature and our previous study, We established model of ischemic stroke in rats with middle cerebral artery occlusion; Meanwhile, oxygen glucose deprivation culturing BV-2 microglia cells in vitro to imitate the cerebral ischemia/reperfusion model in vivo, the changes of Notch-1, NICD, RBP-JK, Hes-1, iNOS, IL-1β and ROS were detected after ishemia/hypoxia; the effect of HS on the release of inflammatory mediators and Notch signaling pathway were detected; In addition, the effect the Notch signaling on the release of inflammatory mediators in BV-2 cells was observed when Notch signaling is blocked with the specific inhibitor named DAPT.Chapterl The effect of HS on proinflammatory mediators in microglia in peri-ischemic cortexObjective Establish the model of ischemic stroke in rats with middle cerebral artery occlusion, to explore whether hypertonic saline would effect on proinflammatory mediators TNF-α and IL-1β in microglia in peri-ischemic cortex in experimentally induced cerebral ischemia.Methods SPF-adult male Sprague-Dawley (SD) rats weighing 220-250g were divided into sham group, cerebral ischemia group, normal saline group (NS group), 10% hypertonic saline group (HS group), except the rats in sham group, other rats were used to establish the model of cerebral ischemia by using middle cerebral artery occlusion (MCAO). At 2h following MCAO, rats in the normal saline group and 10% HS group were respectively treated with a continuous intravenous injection of normal saline (0.3 ml/h) and 10% HS (0.3 ml/h) by tail vein until the end of the experiment. It was divided into two subgroups as 12h group and 24h group according to the treatment time. Following treatment for 12h or 24h, the cerebral hemisphere of ischemic side was extracted, the co-location expression of the iNOS, IL-1β, with microglia cells (marked by Lectin) were detected by double immunofluorescence method; Follonwing treatment for 12h or 24h, the protein expression of iNOS in peri-ischemic cortex were detected by Western Blot. All data was analyzed by one-way analysis of variance (ANOVA), The intergroup comparisons were analyzed by the least-significant-difference (LSD) tests. Differences were considered statistically significant if P＜0.05.Results Immunofluorescence showed that the microglia cells in the sham group were ramified; the expressions of iNOS, IL-1β were almost not observed in the microglia in sham group; Following cerebral ischemia, the microglia were been activated, the morphology of microglia been round or amoeba, and the expression of iNOS, IL-1β were significantly increased in cerebral ischemia group and normal saline group. The expression of iNOS, IL-1β were significantly decreased after treatment with 10% hypertonic saline for 24h. Western Blot showed that the protein expression of iNOS was significantly increased in ischemia group and NS group compared to sham group following cerebral ischemia 12h or 24h (P＜0.05); the protein expression of iNOS in NS group has no significantly changed compared to ischemia group when treatment with NS for 12h or 24h (P>0.05); but it was significantly reduced in 10% HS group compared to ischemia group and NS group when treatment with 10%HS for 12h or 24h(P<0.05).Conclusion Our results show that HS markedly suppresses the activation of microglia; and suppresses the activated microglia release of inflammation mediators iNOS、IL-1β peri-ischemia tissue in experimental induced cerebral ischemia rats.Chapter2 The effect of HS on Notch signaling in peri-ischemic cortexObjective Establish the model of ischemic stroke in rats with middle cerebral artery occlusion, to explore the change of Notch signaling and whether hypertonic saline would effect on Notch signaling in microglia in experimentally induced cerebral ischemia.Methods SPF-adult male Sprague-Dawley (SD) rats weighing 220-250g were divided into sham group, cerebral ischemia group, normal saline group (NS group), 10%hypertonic saline group (HS group), except the rats in sham group, other rats were used to establish the model of cerebral ischemia by using middle cerebral artery occlusion (MCAO). At 2h following MCAO, rats in the normal saline group and 10% HS group were respectively treated with a continuous intravenous injection of normal saline (0.3 ml/h) and 10% HS (0.3 ml/h) by tail vein until the end of the experiment. It was divided into two subgroups as 12h group and 24h group according to the treatment time. Following treatment for 12h or 24h, the cerebral hemisphere of ischemic side was extracted, the co-location expression of the members of Notch signaling including of Notch-1, NICD, RBP-JK, Hes-1, with microglia cells (marked by Lectin)were detected by double immunofluorescence method; The protein expression of the members of Notch signaling including of Notch-1, NICD, RBP-JK, Hes-1 in peri-ischemic cortex were detected by Western Blot. All data was analyzed by one-way analysis of variance (ANOVA), The intergroup comparisons were analyzed by the least-significant-difference (LSD) tests. Differences were considered statistically significant if P＜0.05.Results Immunofluorescence showed that the microglia cells in the sham group were ramified; the expression of Notch-1,NICD, RBP-JK, Hes-1 were almost not observed in the microglia in sham group; Following cerebral ischemia, the microglia were acticated, the morphology of microglia been round or amoeba, and the expression of Notch-1, NICD, RBP-JK, Hes-1 in microglia were significantly increased in cerebral ischemia group and normal saline group. The expression of Notch-1, NICD, RBP-JK, Hes-1 were significantly decreased in microgliaafter treatment with 10% hypertonic saline for 24h. Western Blot showed that the protein expression of Notch-1, NICD, RBP-JK, Hes-1 were significantly increased in ischemia group and NS group compared to sham group following ischemic for 12h or 24h (P<0.05); the protein expression of Notch-1, NICD, RBP-JK, Hes-1 in NS group have no significantly changed compared to ischemia group when with the treatment of NS for 12h or 24h(P>0.05); but it was significantly reduced in 10% HS group compared to ischemia group and NS group when with the treatment of 10%HS for 12h or 24h(P<0.05).Conclusion Our results show that the Notch signaling in microglia can been activated around the ischemia tissue area; the expression of Notch-1, NICD, RBP-JK and Hes-1 were significantly increased following cerebral ischemic. HS markedly suppresses the activation of Notch signaling and the expression of Notch-1, NICD, RBP-JK and Hes-1 in microglia around the ischemia tissue area in experimental induced cerebral ischemia rats.Chapter3 The mechanism of HS effect on BV-2 microglia releasing proinflammatory mediatorsObjective To explore the effect of 80mM HS on BV-2 microglia releasing proinflammatory mediators after hypoxia and whether HS would effect on it through Notch signaling.Methods Oxygen glucose deprivation culturing BV-2 microglia cells model was used to imitate the cerebral ischemia/reperfusion model in vivo, the cells were divided into control group, oxygen glucose deprivation group (OGD group), oxygen glucose deprivation + DAPT (DAPT group) and oxygen glucose deprivation + 80mM hypertonic saline (HS group). In addition to the control group continued to normal culture, cells from different groups were incubated in an air-tight hypoxia chamber with 3% O2,5% CO2,92%N2 for 4h. After OGD, the cells were incubated in a normoxia incubator for 1h for re-oxygenation. Immnunofluorescence double labeling and western blotting were performed to the changes of Notch signaling including Notch-1, NICD, RBP-JK, Hes-1 and inflammation mediators Phos-NF-κB, iNOS and IL-1β after hypoxia; the effect of HS on Notch signaling pathway including Notch-1, NICD, RBP-JK, Hes-1 and the release of inflammatory mediators Phos-NF-KB, iNOS, IL-ip and were detected on activated BV-2 cells; In addition, the effect the Notch signaling on the release of inflammatory mediators in BV-2 cells was observed when Notch signaling is blocked with the specific inhibitor named DAPT. DCFH-DA method was used to detect the expression of ROS in BV-2 cells after hypoxia and the effect of HS on microglia relaeasing of ROS; in addition, the change of ROS in BV-2 cells when Notch signaling was blocked with DAPT. All data was analyzed by one-way analysis of variance (ANOVA), the intergroup comparisons were analyzed by the least-significant-difference (LSD) tests. Differences were considered statistically significant if P<0.05.Results The expression of Phos-NF-κB, iNOS, IL-1β, ROS were significantly increased when BV-2 microglia cells subjected to OGD for 4h compared to control group(*P<0.05); however, the expression level of Phos-NF-κB, iNOS, IL-1β,ROS were significantly depressed with the treatment of 80 mM or 10μM DAPT (#P<0.05). Phos-NF-κB expression was markedly enhanced and translocated to the nucleus in OGD BV-2 microglia cells at 4h; the Phos-NF-KB expression in the nucleus was markedly reduced when with the trearment of 80 mM or 10μM DAPT. The expression of members of Notch signaling pathway including Notch-1, NICD, RBP-JK, and Hes-1 were significantly increased in BV-2 cells following OGD treatment for 4h (*P<0.05). The expression level of Notch-1, NICD, RBP-JK, and Hes-1 were significantly suppressed with the treatment of 80mM HS or 10μM DAPT in OGD-activated BV-2 cells (#P<0.05).Conclusion BV-2 microglia can be activated when subjected to hypoxia and release a large number of inflammatory mediators Phos-NF-κB, iNOS, IL-1pβ, ROS; At the same time, the Notch signaling pathway in BV-2 microglia cells was also been activated, the expression of Notch-1, NICD, RBP-JK, and Hes-1 were significantly increased. HS can suppress the activation of BV-2 microglia and the releasing of inflammatory mediators and the activation of Notch signaling. Pharmacological inhibition of Notch signaling attenuates the inflammatory mediators Phos-NF-κB, iNOS, IL-1β, ROS released by activated BV-2 microglia. It is suggested that HS may suppress the activation of Notch signaling and thus suppress of Phos-κB translocated to the nucleus and thus suppress the releasing of inflammatory mediators by activated BV-2 microglia following ischemia/hypoxic.