| Objective: Ischemia,with high mortality and serious disability,is the most common type of cerebral vascular disease. Thrombolytic treatment in early stage has proved to be one of the effective therapies in acute cerebral infarction. The theory of ischemia-reperfusion injury contains several reactive mechanisms, such as inflammation and apoptosis and excitotoxic amino acids injury and so on. Oxidative stress from reactive oxygen species (ROS) enhances inflammatory responses during tissue injury, possibly through activation of redox-sensitive chemokines and transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2).Recent studies reviewed that Kelch-like ECH -associated protein1 (Keap1)-nuclear factor erythroid 2-related factor 2(Nrf2)/ antioxidant response element (ARE) signal pathway was one of important mechanism of cytoprotective and antioxidant genes that attenuate tissue injury. Nrf2 coordinates expression of genes required for free radical scavenging, upregulation GSH and SOD1 level, detoxification of xenobiotics, and maintenance of redox potential.Our experiment successfully establishing the animal model of middle cerebral artery occlusion/ reperfusion( MCAO/R), observed the expression of Nrf2 and HO-1 at different time point, investigated the neuroprotective effects of OMT and upregulation Keap1-Nrf2/ARE signaling pathway .Methods:⑴Male, healthy Sprague-Dawley rats were used and randomly assigned to three groups: Sham group, middle cerebral artery occlusion/ reperfusion group (MCAO/R group), middle cerebral artery occlusion/ reperfusion group OMT group (OMT group). Each group was divided into 6 subgroups randomly: 3 h, 6 h,12 h, 24 h, 48 h and 72 h after operation. MCAO/R model was induced by using intraluminal filament technique in rats. OMT (120 mg/kg) was administrated by intraperitoneal injection after cerebral ischemia and once daily on the following days. For MCAO/R group and Sham group, equal volume saline was administered in the same manner. Neurological behavior was evaluated at 3 h, 6 h,12 h, 24 h, 48 h and 72 h after operation then rats were sacrificed. Brain water content was measured by wet-dry method;Nrf2 and HO-1 expression were measured by immunohistochemistry.⑵Evaluating the expression of Nrf2 and HO-1. Male, healthy Sprague-Dawley rats evaluated the dynamic expression were used and randomly assigned to seven groups:①MCAO/R 3 h group;②MCAO/R 6 h group;③MCAO/R 12 h group;④MCAO/R 24 h group;⑤MCAO/R 48 h group;⑥MCAO/R 72 h group;⑦Sham group. Male, healthy Sprague-Dawley rats detected OMT's neuroprotection were used and randomly assigned to three groups:①M CAO/R group;②O MT group; ③S ham group,at time point 48 h after operation. Using Western blot detected the expression of Nrf2 and HO-1.⑶Male, healthy Sprague-Dawley rats determined infarct volume were used and randomly assigned to three groups:①M CAO/R group;②OMT group;③Sham group, infarct volume was analyzed with 2, 3, 5- triphenyltetrazolium chloride (TTC) staining at 72 h time point after operation.Results:1 Rats in MCAO/R group and OMT group performed a right palsy. Neurological deficit score in OMT group was decreased compared with MCAO/R group (P<0.05).2 No infarction was observed in Sham group. Compared with MCAO/R group, the infarct volume was significantly reduced in OMT group (P<0.01).3 The water content in MCAO/R group and OMT group was increased at 6 h, with the peak at 48 h and decreased at 72 h, compared with the Sham group, the difference was statistically significant (P<0.05).Compared with MCAO/R group, the water content was significantly reduced in OMT group(P<0.05).4 The number of Nrf2 nuclear positive cells in MCAO/R group and OMT group was increased after ischemia at 6 h, reached the peak at 48 h and decreased at 72 h, compared with the Sham group, the difference was statistically significant (P<0.01). The number of nuclear positive cells was more in OMT group than MCAO/R group. Western blot indicated ,the expression of Nrf2 total protein in MCAO/R group at any time points was not statistically significant compared with Sham group (P>0.05);compared with MCAO/R group and Sham group, the expression of Nrf2 total protein in OMT group at 48 h time point increased significantly(P<0.05).5 The number of NO-1 positive cells in MCAO/R group and OMT group was increased after ischemia at 6 h, reached the peak at 48 h and decreased at 72 h, compared with the Sham group, the difference was statistically significant (P<0.01). The number of positive cells was more in OMT group than MCAO/R group. Western blot indicated ,the expression of HO-1 total protein in MCAO/R group at any time points was statistically significant compared with Sham group (P<0.01);compared with MCAO/R group and Sham group, the expression of HO-1 total protein in OMT group at 48 h time point increased significantly(P<0.01).Conclusion: Nrf2 and HO-1 were induced at the early stage after MCAO/R. OMT protected the brain from damage caused by MCAO/R, decreased the brain water content, infarct size and neurological deficit scores, and upregulated the expression of Nrf2 and HO-1. OMT could protect the brain against ischemic injury, and may be through upregulation Keap1-Nrf2/ARE pathway. |
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