Apoptosis Of Astrocytes Induced By Activation Of Wnt/β-catenin Signaling Pathway After Spinal Cord Injury In Rats

ObjectiveTo investigate the changes ofβ-catenin,SIRT1 and apoptosis and investigate the role ofβ-catenin and SIRT1 signaling in the regulation of apoptosis after spinal cord injury in rats,we established SD rat SCI model and glial cell stress model.MethodsAnimal experiments:90 adult male Sprague-Dawley rats weighing210g±10g（provided by Animal Center of Jinzhou Medical University）were randomly divided into five groups:sham operation group（laminectomies only）and after injury groups,which invited to 3,7,14 and 28 d groups,18 rats in each group.All rats were given a median incision in the rat’s T₉-T₁₀0 vertebral body after intraperitoneal anesthesia with 10%chloral hydrate.The incision length was about 3cm.Moderate SCI models were made at T₁₀0 spinal cord segments using modified Allen’s impactor and were harvested at 3,7,14 and 28 days after operation.Frozen sections were prepared for HE staining,Nissl staining and immunofluorescence staining（n=6）,BBB score（n=6）and Western blot（n=6）were used to detect the protein expression ofβ-catenin,SIRT1 and caspese-3in spinal cord semi-quantitatively.Cell Assay:Primary astrocytes were cultured for two weeks and were randomly divided into four groups:control group,treatment group,treatment control group and treatment group.Western blot and immunofluorescence were used to detect the protein expression ofb-catenin,SIRT1,foxo4 and caspese-3 semi-quantitatively.Results1.BBB score test:After spinal cord injury in rats,the score on the first day was 0,indicating that the rats completely lost their motor function;as the time went by,the score score was getting higher and higher,indicating that the motor function was recovering.2.Histopathology of spinal cord injury area:hemorrhage and necrosis in the damaged area,empty cyst cavity,partial neuron nucleus pyknosis,nuclear fragmentation,cell vacuolation,inflammatory cell invasion,inflammation prolonged over time.3.Western blot analysis showed that the expression ofβ-catenin protein increased firstly and then decreased after spinal cord injury in rats,with the peak value at the 7th day（P<0.05）;apoptotic markers（Caspese-3）increased after injury and then decreased,with the peak value at the 7th day（P<0.05）;protein SIRT1 and downstream pathway protein foxo4 increased with time after injury,and increased significantly compared with the control group（P<0.05）.In the cell experiment-treated group,proteinβ-catenin and apoptotic marker（Caspese-3）were significantly higher than those in the treated group（P<0.05）,while protein SIRT1 and foxo4 expression were decreased（P<0.05）.4.Immunofluorescence assay showed that the number of neurons positively expressed by proteinβ-catenin after rat spinal cord injury increased first and then decreased;the number of neurons positively expressed by protein SIRT1 gradually increased（P<0.05）.The number of astrocytes positively expressed byβ-catenin in the treatment group was higher than that in the treated group;the astrocytes positively expressed by the protein SIRT1 in the treated group.The number of astrocytes positively expressed by SIRT1 was less than the number of histones treated（P<0.05）.ConclusionExperiments show that Wnt/β-catenin signal is related to the pathogenesis of spinal cord injury.Wnt/β-catenin signal is activated by LiCl to induce apoptosis of astrocytes and down-regulateSIRT1 signaling pathway after spinal cord injury in rats.