TIGAR Contributes To Ischemic Tolerance Induced By Cerebral Preconditioning Through Scavenging Of Reactive Oxygen Species And Inhibition Of Apoptosis

Aim: Previous study showed that TIGAR(TP53-induced glycolysis and apoptosis regulator) protected ischemic brain injury via enhancing pentose phosphate pathway(PPP) flux and preserving mitochondria function. This study was aimed to study the role of TIGAR in cerebral preconditioning.Methods: The ischemic preconditioning(IPC) and isoflurane preconditioning(ISO)models were established in primary cultured cortical neurons and in mice. The cortical neurons were transfected with LV-shTIGAR or LV-sh-scramble(LV-negative control,LV-NC). Cell Counting Kit-8 and LDH assay were used to determine cell viability. The isoflurane preconditioning(ISO) and middle cerebral artery occlusion(MCAO) models were established in ICR. The neuronal NADPH and GSH were measured 3 h after reperfusion with the EnzyChrom NADP+/NADPH assay kit and GSH Kit. The ROS level of cortical neurons were measured 3 h after reperfusion with the DHE staining.Immunoblotting was used to examine the protein expression of TIGAR, Bcl-2, Bax and Caspase-3.Results: Both IPC and ISO increased TIGAR expression in cortical neurons.Preconditioning might upregaulte TIGAR through SP1 transcription factor. Lentivirus mediated knockdown of TIGAR significantly abolished the ischemic tolerance induced by IPC and ISO. ISO also increased TIGAR in mice cortex and hippocampus and alleviated subsequent brain ischemia-reperfusion injury, while the ischemic tolerance induced by ISO was eliminated with TIGAR knockdown in mouse brain. ISO increased the production of NADPH and glutathione(GSH), and scavenged reactive oxygen species(ROS), while TIGAR knockdown decreased GSH and NADPH production and increased the level of ROS. Supplementation of ROS scavenger N-acetyl-L-cysteineand PPP product NADPH effectively rescue the neuronal injury caused by TIGAR deficiency. Notably, TIGAR knockdown inhibited ISO-induced anti-apoptotic effects in cortical neurons. These results suggest that TIGAR participates in the cerebral preconditioning through reduction of ROS and subsequent cell apoptosis.Conclusion: Our results showed that TIGAR participated in the cerebral preconditioning through PPP pathway mediated clearance of ROS and inhibition of neuronal apoptosis. The combination of TIGAR and cerebral preconditioning may be a new therapeutic target for stroke prevention and treatment.