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Fig. 6 | Cell & Bioscience

Fig. 6

From: Remote ischemic preconditioning protects against spinal cord ischemia–reperfusion injury in mice by activating NMDAR/AMPK/PGC-1α/SIRT3 signaling

Fig. 6

Toxic concentrations of glutamate inhibits AMPK activation through PP4 activation. A Flow cytometry was used to detect the apoptosis of neurons stimulated with different concentrations of glutamate. Apoptotic neurons were defined as Annexin V-positive cells (n = 3/group). B LDH was detected to assess neuronal damage after stimulation with different concentrations of glutamate (n = 3/group). C, D Altered protein expression levels of NMDAR2B, T-AMPK, p-AMPK, PGC-1α and SIRT3 in neurons treated with different concentrations of glutamate were detected by Western blotting at different time points (n = 3/group). E Flow cytometry was used to detect intracellular Ca2+ concentration in neurons stimulated with different concentrations of glutamate. Relative intensity was normalized to the level of control (n = 3/group). F, G Western blotting was used to detect the expression of T-AMPK, p-AMPK, PGC-1α and SIRT3 after treatment with intracellular calcium chelator BAPTA in 100Glu-treated neurons. Relative expression was normalized to the level of control (n = 3/group). H, I The expression of protein phosphatase-4 was detected using western blotting in neurons after treatment with 100Glu at different time points. Relative expression was normalized to the level of control. * p < 0.05, **p < 0.01, compared to the former timepoint (n = 3/group). J Western blotting was used to detect the expression of T-AMPK, p-AMPK, PGC-1α and SIRT3 after treatment with the PP4 inhibitor cantharidin in 100Glu-treated neurons. Relative expression was normalized to the level of control (n = 3/group). Statistical analysis: mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001

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