Guixing Zhang1, Mingyue Ge1, Ziwei Han2, Sheng Wang3, Jiangwen Yin1, Li Peng1, Feng Xu1, Qingtong Zhang1, Zhigang Dai1, Liping Xie1, Yan Li1, Junqiang Si2, Ketao Ma2. 1. Department of Anesthesiology, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi, 832002, China. 2. Department of Physiology, School of Medicine, Shihezi University and the Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Shihezi, 832002, China. 3. Department of Anesthesiology, First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230001, China; Department of Anesthesiology, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi, 832002, China. Electronic address: iamsheng2006@163.com.
Abstract
AIM: The Wnt/β-catenin signaling pathway plays an important role in ischemia-reperfusion(I/R) injury, and the transforming growth factor(TGF)-β/Smad signaling pathway participates in the neuroprotection effect induced by isoflurane(ISO) postconditioning. In this study, we aimed to explore the role of the Wnt/|[beta]|-catenin β-catenin signaling pathway in the neuroprotection effect induced by ISO postconditioning, and investigate the interaction of Wnt/β-catenin and TGF-β/Smad signaling pathway in this neuroprotection effect. METHODS: Cerebral I/R injury was established in Sprague-Dawley rats by using the middle cerebral artery occlusion (MCAO) model for 90 min followed by 24 h reperfusion. Postconditioning by inhalation of ISO was performed for 60 min after ischemia at the onset of reperfusion. Neurological deficit scoring, 2,3,5-triphenyl tetrazolium chloride staining and Nissl staining were adopted to evaluate brain injury. Apoptosis of the hippocampus and cortex neurons was detected by TUNEL staining. The expression levels of Wnt3a, GSK-3β, β-catenin, Cyclin D1, VEGF, Caspase 3, TGF-β1, Smad3 and p-Smad3 were determined by immunofluorescence (IF) staining, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. Various targeted inhibitors were administered via intraperitoneal injection or lateral ventricle injection. RESULTS: In the cortex region, the neurological deficit score, infarct volumes and neuron apoptosis increased, and the expression level of the Wnt3a, GSK-3β, β-catenin, VEGF and Cyclin D1 decreased in the MCAO group compared with the Sham group. In the MCAO + ISO group, the neurological deficit score, infarct volumes and neuron apoptosis reduced significantly, the expression levels of Wnt3a, β-catenin, VEGF and Cyclin D1 increased, while the expression level of GSK-3β and Caspase 3 decreased relative to MCAO group. When Wnt inhibitor(DKK-1) was given in advance followed by ISO postconditioning, the neurological deficit score, infarct volumes, neuron apoptosis and the expression level of GSK-3β and Caspase 3 increased. qRT-PCR and IF showed similar changes in the protein levels of all groups. However, the expression level of β-catenin in nuclear and cytoplasm both decreased significantly after pre-injection with the TGF-β1 inhibitor(LY2157299) and Smad3 inhibitor(SIS3), whereas the expression levels of TGF-β1, Smad3 and p-Smad3 were almost unchanged. The expression levels of all the related proteins and morphological changes in the hippocampus region were consistent with that of the cortex. CONCLUSION: ISO postconditioning can reduce cerebral I/R injury by activating the Wnt/β-catenin signaling pathway and may be related to the TGF-β/Smad3 signaling pathway.
AIM: The Wnt/β-catenin signaling pathway plays an important role in ischemia-reperfusion(I/R) injury, and the transforming growth factor(TGF)-β/Smad signaling pathway participates in the neuroprotection effect induced by isoflurane(ISO) postconditioning. In this study, we aimed to explore the role of the Wnt/|[beta]|-catenin β-catenin signaling pathway in the neuroprotection effect induced by ISO postconditioning, and investigate the interaction of Wnt/β-catenin and TGF-β/Smad signaling pathway in this neuroprotection effect. METHODS: Cerebral I/R injury was established in Sprague-Dawley rats by using the middle cerebral artery occlusion (MCAO) model for 90 min followed by 24 h reperfusion. Postconditioning by inhalation of ISO was performed for 60 min after ischemia at the onset of reperfusion. Neurological deficit scoring, 2,3,5-triphenyl tetrazolium chloride staining and Nissl staining were adopted to evaluate brain injury. Apoptosis of the hippocampus and cortex neurons was detected by TUNEL staining. The expression levels of Wnt3a, GSK-3β, β-catenin, Cyclin D1, VEGF, Caspase 3, TGF-β1, Smad3 and p-Smad3 were determined by immunofluorescence (IF) staining, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. Various targeted inhibitors were administered via intraperitoneal injection or lateral ventricle injection. RESULTS: In the cortex region, the neurological deficit score, infarct volumes and neuron apoptosis increased, and the expression level of the Wnt3a, GSK-3β, β-catenin, VEGF and Cyclin D1 decreased in the MCAO group compared with the Sham group. In the MCAO + ISO group, the neurological deficit score, infarct volumes and neuron apoptosis reduced significantly, the expression levels of Wnt3a, β-catenin, VEGF and Cyclin D1 increased, while the expression level of GSK-3β and Caspase 3 decreased relative to MCAO group. When Wnt inhibitor(DKK-1) was given in advance followed by ISO postconditioning, the neurological deficit score, infarct volumes, neuron apoptosis and the expression level of GSK-3β and Caspase 3 increased. qRT-PCR and IF showed similar changes in the protein levels of all groups. However, the expression level of β-catenin in nuclear and cytoplasm both decreased significantly after pre-injection with the TGF-β1 inhibitor(LY2157299) and Smad3 inhibitor(SIS3), whereas the expression levels of TGF-β1, Smad3 and p-Smad3 were almost unchanged. The expression levels of all the related proteins and morphological changes in the hippocampus region were consistent with that of the cortex. CONCLUSION:ISO postconditioning can reduce cerebral I/R injury by activating the Wnt/β-catenin signaling pathway and may be related to the TGF-β/Smad3 signaling pathway.