Neuroprotective Effects And Mechanisms Of Sodium Butyrate On Cerebral Ischemia Reperfusion Injury In Mice

The cerebral ischemia injury can result in neuronal death and/or functional impairment, which leads to further the damage and dysfunction after recovery of blood. Cerebral ischemia/reperfusion(I/R) injury often causes irreversible brain damage and neuronal injury and death, which is involved in many complex pathogenesis such as endogenous substances release, oxidative stress, amino acid toxicity, inflammation and apoptosis. Therefore, the prevention and therapy of cerebral ischemia/reperfusion injury has been focus in neurological areas. Sodium butyrate(NaB) is a sort of short chain fatty acids, which has been previously shown to maintain gastrointestinal homeostasis through antioxidant, anti-inflammatory and anti-apoptotic. Meanwhile, recent studies have indicated that NaB could provide anti-inflammatory, antioxidant, anti-apoptotic and neuroprotective effects in neurodegenerative disorders and also improve spatial learning and memory ability. Although the neuroprotective effect of NaB has been well studied, its neuroprotective effects on the cerebral I/R injury have not yet been clear. Na B(5, 10 mg/kg) was administered intragastrically 3 h after the onset of reperfusion in bilateral common carotid artery occlusion(BCCAO) mice. After 24 h of reperfusion, the neurological deficit, pathological morphology of brain, ultramarine structure, oxidative stress, inflammation response and neurons apoptosis of mice were estimated, and its possible mechanism of NaB against cerebral I/R inury would be further explored. Additionally, we treated mice with NaB for 4 weeks continuously after cerebral I/R injury, and investigated the cognitive behaviors, pathological changes of brain tissue and the brain derived neurophie faetor(BDNF) protein expression in the recovery phase of cerebral I/R injury in mice. The research is divided into three parts, and the contents of each part are summarized as follows:PartⅠ Effects of sodium butyrate on acute cerebral ischemia reperfusion injury in miceObjective: To observe the protective effects of NaB on acute cerebral ischemia reperfusion injury in mice.Methods: The bilateral common carotid artery occlusion(BCCAO) method was applied to adult male ICR mice by clamping bilateral common carotid arteries for 20 minutes. After a global cerebral ischemia model 3 h after the onset of reperfusion, NaB was intragastrically administered 5 mg/kg NaB or 10mg/kg. After 24 h of reperfusion, locomotors activity in open field test were estimated. Morphological examination was performed by electron microscopy and hematoxylin-eosin(HE) staining.Results: The acute cerebral ischemia reperfusion injury led to the changes of locomotors activity, as well as morphology and ultrastructure in brain tissue. Compared with Model group, NaB treatment significantly ameliorated changes of locomotors activity(10 NaB group, P < 0.01), histopathology and ultrastructure in cerebral I/R injury mice.PartⅡ Mechanisms of sodium butyrate on acute cerebral ischemia reperfusion injury in miceObjective: To investigate the protective mechanisms of NaB on acute cerebral ischemia reperfusion injury in mice.Methods: Male ICR mice were randomly divided into 4 groups: Sham group(sham operation group), Model group(cerebral ischemia reperfusion injury), 5 NaB group(5 mg/kg NaB treatment group) and 10 NaB group(10 mg/kg NaB treatment group). We used a cerebral ischemic injury mouse model induced by BCCAO. 3 h after the onset of reperfusion, NaB was intragastrically administered 5 mg/kg or 10mg/kg. The levels of oxidative stress(SOD activity and MDA content) and inflammatory cytokines(IL-1β, TNF-α and IL-8) in the brain tissues were assessed. Apoptotic neurons were measured by TUNEL; apoptosis-related protein Bcl-2, Bax and the phosphorylation Akt(p-Akt) were assayed by western blot and immunohistochemistry.Results: After 24 h of reperfusion, the content of MDA wasincreased in the model group compared with the sham group(P < 0.01). The activity of SOD in the hippocampus of the model group was diminished remarkably compared with the sham group(P < 0.01). Levels of IL-1β(P < 0.01), IL-8(P < 0.05), and TNF-α(P < 0.01) were markedly increased in the model group in relative to the sham group. By TUNELstaining, the neurons in the hippocampus of mice with model showed obvious apoptosis. The ratio of Bcl-2/Bax(antiapoptotic/proapoptotic) was significantly reduced in the model group compared with the sham group(P < 0.01). The protein level of p-Akt was significantly descended in the model group in comparison to the sham group(P < 0.05). However, the SOD activity in the 10 mg/kg NaB treatment group was significantly enhanced(P < 0.05), and the content of MDA was evidently deceased in comparison to the model group(P < 0.05). 10mg/kg NaB treatment group significantly decreased the levels of these cytokines in relative to the model group(P <0.05), especially the level of IL-1β(P < 0.01). The 10mg/kg NaB treatment markedly increased the levels of Bcl-2 and decreased the levels of Bax in mice after cerebral I/R injury. The ratio of Bcl-2/Bax in 10mg/kg NaB treatment group was remarkably increased compared with the model group. The treatment groups significantly increased the protein level of p-Akt compared with the model groupPart Ⅲ The effects of sodium butyrate on convalescent cerebral ischemia reperfusion injuryObjective: To investigate the protective effects of NaB on the cerebral ischemia reperfusion injury in convalescent mice.Methods: Male ICR mice were randomly divided into 3 groups: Sham group(sham operation group), Model group(cerebral I/R injury), NaB group(10mg/kg NaB group). After a ischemic cerebral injury mouse model induced by BCCAO was established, NaB was intragastrically administered for 4 weeks. As for the behavioral testm mice were assessed by the Morris Water Maze test. Morphological examination was performed by Nissl staining in the hippocampus. BDNF protein were assayed by western blot and immunohistochemistry.Results: In the Morris water maze test, the escape latency in the model group was significantly longer than in the sham group on days 3, 4, and 5(Day 3, P < 0.05;Day 4 and Day 5, P < 0.01). There was a significant reduction of the time spent in the target quadrant in the model group compared to the sham group(P < 0.05). By Nissl staining, the model mice exhibited many morphological changes in the hippocampal region,including a cell body diminution, karyorrhexis and cloudiness or disappearance of the nucleolus. By Western blot and immunohistochemistry, the protein level of BDNF in the model group was significantly decreased compared with the sham group. After treatment with NaB, the escape latency was decreased significantly in mice(P < 0.05). There was obvious increase of the time spent in the target quadrant in NaB-treated group, compared to model group(P < 0.05). NaB treatment attenuated the morphological changes of the hippocampal neuron of model mice. The protein level of BDNF in the NaB-treated group was significantly increased compared with the model group.Conclusion:1. NaB can attenuate the locomotor activity and pathological damage with acute cerebral ischemia reperfusion injury in mice2. NaB exerted neuroprotective effects on cerebral I/R injury by anti-oxidant, anti-inflammatory, and anti-apoptotic properties and PI3K/Akt pathway may be involved in the anti-apoptotic effect.3. Na B can attenuate the behavioral dysfuction and pathological damage, upregulate BDNF protein level with convalescent cerebral I/R injury in mice.