The Protective Effect Of TIAGR On Ischemia/Reperfusion Induced Brain Injury Through Increased NADPH

Aim: To observe the effects of cerebral ischemia on TIAGR expression; the influenceof TIGAR on ischemic neuronal damage and preliminary mechanism of the protectiveeffect of TIGAR on ischemia/reperfusion induced brain injury.Methods: Transient middle cerebral artery occlusion (tMCAO) was produced byusing an intraluminal filament technique in mice; in vitro the OGD model of primarycortical neurons were established by deprivation of oxygen and glucose and againreoxygenation. In animal model, cell type of TIGAR expression was detected with Westernblot analysis and immunofluorescence analysis. The changes in the expression of TIGAR,p53, NOX4in in cerebral cortex were assessed with RT-PCR and Western blot analysis.The effect of p53inhibitors (Pifithrin-α, PFT-α) on expression of TIGAR protein in micebrain ischemic cortex was detected with Western blot. DHE probe detects the brainischemic cortex reactive oxygen species (ROS). Lentivirus-overexpression or knockdownof TIGAR was lateral ventricle injected in mouse brain, infection rate and proteinexpression of TIGAR were detected with immunofluorescence and Western blot. Theneuroprotective effect of TIGAR was analyzed by assessing brain infarction volume;scoring neurological deficits and determining the brain water content24h after reperfusion.Lentivirus-overexpression or knockdown of TIGAR was injected in primary corticalneuron, infection rate and protein expressions of TIGAR were detected withimmunofluorescence and Western blot. The protection of TIGAR on OGD/R inducedneuronal death was detected with CCK-8, LDH, TUNEL. The effect of NADPH onOGD/R induced intracellular ROS level and mitochondrial membrane potential wasdetected by flow cytometry though DCFH2-DA and probe JC-1marker. The changes in theexpression of apoptosis protein caspase-3caused OGD/R was detected by Western blot.NADPH was Lateral ventricleadministered immediately before MCAO. The effects ofNADPH, NOX inhibitor apocynin and VAS2870were analyzed by assessing braininfarction volume, scoring neurological deficits and determining the brain water content24 h after reperfusion.Results: TIGAR highly expressed in the neurons (P0.01). ischemia-reperfusioninduced increases in the expression of TIGAR,p53, NOX4. PFT-αdid not restrain TIGARexpression caused by ischemia-reperfusion. ROS appeared two peaks in0.5h and12hafter cerebral ischemia-reperfusion. Lentivirus-TIGAR could efficiently infect brain tissue,can improve or silence TIGAR expression (P<0.05, P<0.01). Overexpression of TIGARcould significantly reduce infarction volume, reduce the brain water content, and improvebehavioral deficits (P <0.05, P <0.01). In primary cortical neurons lentivirus TIGAReffectively increased or reduced TIGAR expression. CCK-8, LDH, TUNEL showed thatoverexpression of TIAGR could significantly reduce neuron death induced by OGD/R (P <0.01). Interference TIGAR aggravated the neurons induced by OGD/R injury. NADPHsignificantly suppressed cell death induced by interference TIGAR. Flow cytometry resultsshowed that overexpression of TIGAR dramatically reduced intracellular ROS levelinduced by OGD/R enhancement and inhibition of mitochondrial membrane potentialdecreased. Interference TIGAR is increased intracellular ROS level and reduce themitochondrial membrane potential. NADPH blocked the effect of interference TIGAR.Western blot showed that overexpression of TIGAR reduced the caspase-3activationinduced by OGD/R; interference TIGAR enhanced the caspase-3activation induced byOGD/R. Combined NADPH and NOX inhibitor more significantly reduced infarctionvolume, alleviated cerebral edema, and improved neurobehavioral deficits than the role ofsingle application of NADPH.Conclusion: TIGAR has a protective effect on mouse ischemia-reperfusion injury, itsmechanism may be related to enhance endogenous antioxidant NADPH inhibiting ROS,inhibiting neuronal cell apoptosis. Exogenous NADPH protects neurons againstischemia-reperfusion injury...