Fig. 5

SIRT3 expression is regulated by the classical AMPK/PGC-1α signaling pathway and inhibition of AMPK attenuates the NMDA-induced neuroprotective effect. A, B Altered protein expression of NMDAR2B, T-AMPK, p-AMPK, PGC-1α and SIRT3 in neurons treated with NMDA was detected by Western blotting at different time points. Relative expression was normalized to the level of the control (n = 3/group). C, D Western blotting was used to detect the expression of T-AMPK, p-AMPK, and PGC-1α after sham or I/R treatment with or without RIPC. Relative expression was normalized to the level of the sham group (n = 5/group). E, F In response to NMDA, altered protein expression levels of p-AMPK, PGC-1α and SIRT3 in neurons when blocking AMPK using AMPK shRNA and compound C (an AMPK inhibitor) were detected using Western blotting. Relative expression was normalized to the level of control (n = 3/group). G Flow cytometry was used to detect the effect of compound C pretreatment on apoptosis of NMDA-OGD/R treated neurons (n = 3/group). H LDH was detected to assess neuronal damage after pretreatment with compound C in NMDA-OGD/R treated neurons (n = 3/group). I The relative activity of MnSOD, CAT, and GSH content was measured after pretreatment with compound C in NMDA-OGD/R treated neurons (n = 3/group). J, K The production of ROS was measured by flow cytometry after pretreatment with compound C in NMDA-OGD/R treated neurons (n = 3/group). Statistical analysis: mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001