6-O-Galloylpaeoniflorin Attenuates Cerebral Ischemia Reperfusion-Induced Neuroinflammation and Oxidative Stress via PI3K/Akt/Nrf2 Activation
Nrf2 siRNA and Ly294002 attenuated the antioxidative and cell protective activities of GPF. PC12 cells pretreated with Ly294002 (50 μM) for 1 h or transfected with Nrf2 siRNA (50 nM) or scramble RNA was administrated with GPF (100 μM) for 1 h and then subjected to OGD as mentioned above. PC12 cells only treated with GPF and OGD were set as the control group. The ROS level (green fluorescence, in (a)) and MDA level (right panel of (c)) were remarkably increased after Nrf2 siRNA or Ly294002 treatment, compared with the control cells. The MMP (b) and SOD activity (right panel of (c)) were downregulated by Nrf2 siRNA or Ly294002 administration. The mRNA expression (detected by qRT-PCR; upper panel of (d)) in the PC12 cells as well as the protein levels (detected by ELISA; lower panel of (d)) of TNF-α and IL-1β in the cell culture medium was remarkably upregulated by Nrf2 siRNA or Ly294002 treatment, compared with the control group. The results of the CCK-8 assay and LDH activity detection revealed that Nrf2 siRNA or Ly294002 weakened the protective effects of GPF against OGD (e). Finally, Nrf2 siRNA or Ly294002 administration upregulated the ratio of TUNEL-positive cells and the generation of caspase-3 (f). The data of each group were from three independent assays. versus the control group.
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