TIGAR Plays A Role In Regulation Of Autophagy In Cerebral Ischemic Stroke

Aim: To investigate the role of TIGAR in the regulation of autophagy and the corresponding molecular mechanisms in cerebral ischemia and reperfusion.Methods: The animal and cellular models of ischemia/reperfusion in vitro and in vivo were established by transient middle cerebral artery occlusion and reperfusion(t MCAO / R)and cultured neurons by Oxygen and Glucose Deprivation / Reoxygenation(OGD / R)model in vitro. After ischemia / reperfusion in vivo and OGD / R in vitro at different time points, the expression of TIGAR and autophagy-related protein LC3, Beclin1 and P62 and other proteins in mouse cortical and primary neurons were analyzed by Western blot. Then after 3h reperfusion which was the highest point of TIGAR, the expression of TIGAR,autophagy-related protein LC3, Beclin1 and m TOR-S6 K pathway protein in transgenic mice tested by Western blot, thereby detecting the impact of TIGAR on autophagy.Cultured primary neurons were infected with lentivirus TIGAR( LV- sh_TIGAR) and the expression of TIGAR and autophagy-related protein were detected with Western blot assay.meanwhile we use fluorescence microscopy for detecting lentivirus transfection efficiency.Primary neurons(8 days) wereincubated with Neurobasal medium containing 10 μmol / L NADPH for 4 h. Then we detected the expression levels of LC3 and TIGAR, thereby detecting the impact of TIGAR on autophagy after giving antioxidants NADPH.Results: In this study, we found that in C57 mice subjected to t MCAO / R, LC3 and Beclin1 protein expression were found significantly increased, the level of P62 was reduced and at 0.5 h and 6 h reached the peak(P <0.001, P <0.05, P <0.001). In TG-TIGAR transgenic mice subjected to t MCAO/R, the expression of LC3 and Beclin1 were also significantly increased, the level of P62 was lower and peaked at 12 h(P <0.05).Primary cortical neurons after OGD / R, TIGAR levels were significantly improved,peaked at 3 h(P <0.001). Western blot showed that knocked out TIGAR with lentivirus and reperfusion 6 h and 12 h, autophagy was activated( P < 0.05).Wild-type mice(WT), TIGAR transgenic(TG) and knockout(KO) mice were subjected to t MCAO for 2 h and reperfusion for 3 h. Then cerebral cortical tissues were detected for the changes of autophagy-related proteins. Compared with WT mice, under normal conditions the TIGAR expression in TG-TIGAR mouse was significantly increased,LC3-II level was significantly reduced( P <0.05), p-m TOR protein expression, and p-S6 K significantly increased( P <0.05), m TOR and S6 K protein levels did not change. After 3and 12 h reperfusion, compared with WT mice, TIGAR level in TG-TIGAR mice was significantly upregulated( P <0.001), the increase of LC3-II level was diminished(P<0.05). In contrast, Western Blot results showed that compared with WT mice, under normal conditions TIGAR expression was significantly reduced in KO-TIGAR mice(P<0.001). Knockdown of TIGAR significantly increased the level of LC3-II in normal conditions(P < 0.05), TIGAR, p-m TOR and p-S6 K protein expression was significantly reduced(P < 0.001, P < 0.001, P < 0.05). After reperfusion 3 and 12 h, TIGAR was slightly enhanced in KO-TIGAR mice compared with WT mice. The LC3-II protein levels were more robustly increased. These results indicate that TIGAR plays a role in autophagy regulation in ischemia.TIGAR overexpression significantly reduced the magnitude of the activation of autophagy after 3 and 12 h of reperfusion, indicating ischemia reperfusion TIGAR inhibit excessive activation of autophagy, and TIGAR expression was correlated with p-m TOR and p-S6 K levels.Primary neurons were supplemented with antioxidants nicotinamide adenine dinucleotide phosphate NADPH(10 μ M) before, during and after OGD. The LC3 and TIGAR expression levels were decreased(P < 0.001, P < 0.05), which may be indicated that TIGAR can partially inhibit excessive activation of autophagy by supplementation of exogenous NADPH.Conclusions:In the present investigation, we confirmed that autophagy was activated in wild type and TG-TIGAR mice. Knocking down of TIGAR with LV- sh_TIGAR enhanced autophagy activation after 6 h and 12 h reoxygenation. In TG-TIGAR mice,overexpression of TIGAR significantly diminished the magnitude of autophagy activation after 3 and 12 h of reperfusion. In contrast, knockdown of TIGAR remarkably enhanced autophagy activation. TIGAR expression was correlated with the levels of p-m TOR and p-S6 K. Overactivation of autophagy after knockout of TIGAR was partially suppressedwith supplementation of reduced nicotinamide adenine dinuclectid phosphate(NADPH).These studies suggest that TIGAR plays a negative role in ischemic/reperfusion-induced autophagy activation through generation of NADPH.