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

Abstract

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.