Wnt/β-catenin signaling pathway contributes to isoflurane postconditioning against cerebral ischemia-reperfusion injury and is possibly related to the transforming growth factorβ1/Smad3 signaling pathway.

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. 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. 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. 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.

Zhang G ，Ge M ，Han Z ，Wang S ，Yin J ，Peng L ，Xu F ，Zhang Q ，Dai Z ，Xie L ，Li Y ，Si J ，Ma K ... -  《-》

Transforming growth-beta 1 contributes to isoflurane postconditioning against cerebral ischemia-reperfusion injury by regulating the c-Jun N-terminal kinase signaling pathway.

Cerebral ischemia-reperfusion (I/R) injury is a devastating complication in the perioperative period. Transforming growth factor beta (TGF-β) is a key protein that can participate in the repair and control process responses after I/R injury. Isoflurane is widely used in neurosurgery. Previous studies have shown that isoflurane preconditioning plays an important role in neuroprotection. However, the effects of isoflurane postconditioning on cerebral I/R injury have not yet been elucidated. In the present study, we evaluated the protective effect of isoflurane postconditioning against cerebral I/R injury and investigated the role of the TGF-β signaling pathway and the downstream c-Jun N-terminal kinase (JNK) signaling pathway in neuroprotective mechanism. In particular, the JNK signaling pathway emerges as a possible target for brain repair after stroke. Cerebral I/R injury was produced in SD rat by using the middle cerebral artery occlusion model for 90 min, followed by 24h reperfusion. Postconditioning by inhalation of isoflurane was performed at different concentrations (1.5%, 3.0%, and 4.5%) for 1h after ischemia at the starting time point of reperfusion. The protective effect was tested by neurological deficit scoring with 2,3,5-triphenyl tetrazolium chloride and propidium iodide (PI) staining. Apoptosis of CA1 cells in the hippocampus was detected by TUNEL method. Expression levels of TGF-β1, Smad 2/3, p-Smad2/3, JNK, and p-JNK were determined by immunostaining and Western blot. Postconditioning by isoflurane at 1.5% and 3.0% concentrations significantly decreased the neurobehavioral deficit scores and infarct volume compared with the I/R group, but no significant difference in neurobehavioral deficit score was detected between the I/R and 4.5% isoflurane postconditioning groups. Additionally, 1.5% isoflurane postconditioning decreased the numbers of PI-positive cells at 24h after reperfusion compared with the I/R group. TGF-β1 and p-Smad2/3 protein gradually increased after I/R injury, with the highest values observed in the 1.5% and 3% isoflurane postconditioning groups. For Smad2/3 protein expression, no differences existed among all groups. After inducing the TGF-β/SMAD3 signaling pathway specific blocker (LY2157299), the neurological deficit scores increased, infarct volumes enlarged, apoptosis increased, and PI-positive CA1 cells in the hippocampus also increased. The expression levels of TGF-β1 and p-Smad2/3 proteins were downregulated. During the pre-injection of LY2157299, the expression levels of TGF-β1 and p-Smad2/3 decreased significantly, but compared with the sham group, the expression level of p-JNK significantly increased. When the injection of LY2157299 was abolished, the expression of p-JNK significantly decreased. The expression levels of p-JNK and TGF-β1 significantly decreased when LY2157299 and SP600125 were injected simultaneously. However, the protective effect mediated by SP600125 completely disappeared, and the role of LY2157299 became dominant. Compared with the sham group, the expression of TGF-β1 was almost unchanged by the injection of SP600125 alone, but the expression of p-JNK significantly decreased. Up to 1.5% isoflurane can upregulate the expression of TGF-β1 and downregulate that of p-JNK, which significantly mitigated I/R injury, leading to cerebral injury. However, this protective effect was abrogated when the TGF-β1 signaling pathway was blocked by LY2157299. Overall, the present results provided valid evidence to demonstrate that TGF-β1 contributes to isoflurane postconditioning against cerebral I/R injury by inhibiting the JNK signaling pathway.

Wang S ，Yin J ，Ge M ，Dai Z ，Li Y ，Si J ，Ma K ，Li L ，Yao S ... -  《-》

TGF-β2/Smad3 Signaling Pathway Activation Through Enhancing VEGF and CD34 Ameliorates Cerebral Ischemia/Reperfusion Injury After Isoflurane Post-conditioning in Rats.

Peng L ，Yin J ，Wang S ，Ge M ，Han Z ，Wang Y ，Zhang M ，Xie L ，Li Y ... -  《-》

Isoflurane post-conditioning down-regulates expression of aquaporin 4 in rats with cerebral ischemia/reperfusion injury and is possibly related to bone morphogenetic protein 4/Smad1/5/8 signaling pathway.

Aquaporins (AQPs) are water-channels that play important roles in brain water homeostasis and cerebral edema induced by brain injury. This study aimed to investigate the relationship between AQP4, bone morphogenetic protein 4 (BMP4)/Smad1/5/8 signaling pathway and isoflurane post-conditiong, which has effects on brain edema in rats with cerebral ischemia/reperfusion (I/R) injury. Cerebral I/R injury was induced in rats by using the middle cerebral artery occlusion (MCAO) model for 90min, followed by 24h of reperfusion. Isoflurane post-conditioning (ISO) group received 90min ischemia and underwent 1.5% isoflurane post-conditioning for 60min after initiating reperfusion. Neurobehavior, brain water content, thionine staining and 2, 3, 5-triphenyl tetrazolium chloride staining were evaluated to measure levels of brain edema and damage. Expressions of AQP4, BMP4, Smad1/5/8 and phosphorylated Smad1/5/8 were detected by using Western blot, quantitative real-time polymerase chain reaction (qRT-PCR), and immunofluorescence (IF) staining. Compared with the Sham group, neurological behavior score, brain infarct volume and water content of MCAO model rats increased with reperfusion injury. However, in the ISO group, cell edema and damage of brain was significantly ameliorated (P<0.01). qRT-PCR showed less AQP4 mRNA expression in the hippocampal tissue of the ISO group than in the I/R group (P<0.01). Western blot and immunofluorescence results showed similar changes in protein levels of both groups. Related protein expressions showed expressions of BMP4 and Smad1/5/8 increased in the ISO group (P<0.01), whereas total Smad1/5/8 expression didn't change in all groups. When BMP4 inhibitor (LDN193189) was injected, expression levels of AQP4 increased and neuronal density decreased (P<0.05). By contrast, expression levels of BMP4 did not change significantly after pre-injection of AQP4 inhibitor (TGN020) (P>0.05), but neuronal density increased (P<0.05). Isoflurane post-conditioning may inhibit occurrence of brain edema and reduce cerebral I/R injury through down-regulating expression of AQP4, This process may be related to the activation of BMP4/Smad1/5/8 signaling pathway.

Yuan M ，Ge M ，Yin J ，Dai Z ，Xie L ，Li Y ，Liu X ，Peng L ，Zhang G ，Si J ，Ma K ，Wang S ... -  《-》

TGF-β3/Smad3 Contributes to Isoflurane Postconditioning Against Cerebral Ischemia-Reperfusion Injury by Upregulating MEF2C.

Yang Y ，Chen L ，Si J ，Ma K ，Yin J ，Li Y ，Yang C ，Wang S ... -  《-》