The Role Of Nodal/Smads Signaling Pathway Involved Cerebral Ischemic Injury

Cerebral ischemia is the main cause of disability and the third cause of death worldwide following cardiovascular diseases and cancer,and its incidence rises with the increase of age.The pathological process of cerebral ischemic injury is very complicated,and the current research is not clear.Because it involves a variety of mechanisms,such as energy metabolism disorders,free radical damage,inflammation,excitatory amino acid toxicity,intracellular calcium overload,cytotoxic effects of NO,and abnormal opening of the blood-brain barrier.Thus,comprehensive elucidation of the molecular mechanisms underlying ischemic brain damage and the search for key signaling pathways and protein molecules are important for guiding the clinical treatment of cerebral ischemia.In recent years,studies showedthat Nodal is carcinogenic in many cancers.However,few studies have investigated Nodal expression in ischemic brain injury.Meanwhile,the mechanism mediated by Nodal/Smads pathway in ischemic brain injury remains to be further elucidated.In this study,the role of Nodal and downstream Smads pathways in cerebral ischemic injury were detected through in vivo and in vitro experiments by constructing a model of cerebral ischemic injury,which can be divided into the following three parts:1.Establishment of rat model of cerebral ischemic injury and the expression of Nodal/SmadsObjective:To establish middle cerebral artery occlusion?MCAO?model in rats to simulate the pathological process of cerebral ischemia in vivo.Methods:The Longa suture method was used to establish the rat model of MCAO model with ischemia at different time points.Neurological score was used to detect the degree of nerve injury,and TTC staining was used to detect brain tissue injury.Pathological sections and Tunel were used to observe the extent of brain tissue damage and apoptosis.The expression of Nodal/Smads pathway related genes and proteins in brain tissues was detected by Realtime-PCR and WB.Immunohistochemistry was used to detect the expression of Nodal and Smads.Results:Neurological score showed that the nerve injury was more obvious with the prolongation of ischemia time.Compared with the sham group,TTC staining showed that the percentage of cerebral infarct volume was increased with the extension of ischemic time.Compared with those results of sham group,0.5h,1.5h and 3h groups markedly aggravate pathological damage,promoted the neuronal apoptosis,increased the expression of Nodal and p-Smad2 in protein and mRNA.Conclusion:The Nodal/Smad2 pathway was activated during the successful establishment of rat cerebral ischemia model.2.The mechanism of Nodal/Smad2 signaling involved in OGD model.Objective:Established Oxygen-Glucose Deprivation?OGD?model to primary cortical neurons cells and simulated the ischemic injury in vitro.,to investigate the mechanism of of Nodal/Smad2 pathway.Methods:Glucose free DMEM and NaS₂O₄ were introduced to cells toestablish OGD model in vitro.Primary cortical neurons were cultured in conditions of oxygen deprivation for 0?h,12?h,and 24?h and glucose-free culture,then reoxygenated for24?h in a complete medium.Cell viability was measured using CCK8.Flow cytometry was used to detect cell apoptosis and ROS,and Realtime-PCR was used to detect Nodal/Smad2.Results:The results of CCK8 assay showed that the cell survival rate decreased gradually with the prolongation of hypoxia time,and the decrease was more obvious after 24h of OGD.Annexin V-FITC and PI staining results showed that apoptosis rates increased with prolonged ischemia.The results of ROS flow cytometry showed that with the extension of hypoxia time,a large number of ROS were produced due to the cell hypoxia injury after 24h of hypoxia.The expression of Nodal and Smad2 in OGD injury model were significantly increased by Realtime-PCR,indicating that Nodal/Smad2 was activated in the anoxic injury of neurons.Conclusion:The Nodal/Smad2 pathway was activated in the OGD/R model.3.The mechanism of Nodal/Smad2 pathway in cerebral ischemic injury.Objective:To investigate the effect of Nodal gene on ischemic injury.Methods:The Nodal-specific small interfering RNAs?siRNA?,as well as the corresponding non-targeting scrambled control siRNA were designed and chemically synthesized.Primer Cultured Cortex Neurons were transfected with target-specific siRNA or control siRNA using Lipofectamine 2000 according to the manufacturer's protocol.After 48 h of transfection,the cells were subjected to a 24h OGD challenge followed by reoxygenation for 24 h.The cell samples were collected for the apoptosis and ROS assay by flow cytometry.The expressions of apoptosis-related proteins bcl-2and Caspase3,as well as the expressions of oxidative stress related proteins SOD1and NOX4 were detected by Western blot.Results:After the silencing of Nodal gene,the apoptosis results showed that the apoptosis of Nodal-RNAi-OGD24h cells was significantly increased compared with OGD24h cells,indicating that the injury caused by the decrease of Nodal expression was increased after cerebral ischemia.Western blot analysis of apoptosis-related proteins also showed that caspase-3 expression was significantly increased compared with the negative control group.Bcl-2 protein expression was significantly lower than that of the invisible control group.The experimental results further indicated that reducing the expression of Ndoal would increase the production of pro-apoptotic proteins and inhibit the production of anti-apoptotic proteins,further suggesting that Nodal may play a protective role.The ROS test results showed that the cell Nodal gene silencing increased the intracellular ROS content,indicating that the neuron cell injury increased,resulting in a large number of ROS production.Relevant protein detection also showed that NOX4 protein significantly increased the production of SOD1 protein after the silencing of Nodal gene,further indicating that Nodal may play a protective role.Conclusion:The expression of Nodal gene is negatively correlated with apoptosis and oxidative stress.Low expression of Nodal through Nodal interference can increase the apoptosis and oxidative stress of neurons after OGD,suggesting that Nodal plays a neuronal protective role through downregulation of the expression levels of Smad2 in the ischemic injury.