Research Article

Aescin Protects Neuron from Ischemia-Reperfusion Injury via Regulating the PRAS40/mTOR Signaling Pathway

Figure 5

PRAS40 knockdown and rapamycin treatment undermined the protective effects of aescin against I/R injury. (a) Vectors containing PRAS40 shRNA were transferred to neurons 96 hours before OGD; the cultures were maintained without OGD or with 2 hours of OGD, followed by 24 hours of simulated reperfusion with aescin treatment; rapamycin was used at the beginning of simulated reperfusion after OGD. LDH release was measured and compared with the groups without PRAS40 shRNA or rapamycin treatment, and all results of LDH release after OGD were normalized to the values of the sham group. PRAS40 knockdown (PRAS40 KD) and rapamycin treatment reversed the decrease of LDH release induced by aescin treatments which indicated that they undermine the protective effects of the aescin treatments in primary cultured neurons after OGD and reperfusion. (b) Representative images showed the protein bands of phosphorylated PRAS40, mTOR, and S6K expression with or without aescin treatment after OGD and reperfusion. Bands of proteins of the group without OGD were displayed. β-Actin was used as a reference of equal protein loading. (c) The bar graphs represented the relative optical densities of phosphorylated and total PRAS40, mTOR, and S6K from the protein bands shown above. (d) Representative protein bands showed phosphorylated and total mTOR, S6K, and 4E-BP1 with or without rapamycin treatment followed by OGD and reperfusion measured by western blot; β-actin was used as a control to ensure equal protein loading. (e) The bar graphs represented the relative expression of phosphorylated mTOR, S6K, and 4E-BP1 from the protein bands shown above. All results are given as . , respectively, vs. sham (no OGD) or Con (OGD); # vs. aescin; , respectively, vs. PRAS40 KD or rapamycin. per group. Sham group: no OGD samples; Con group: OGD/R samples without treatment; aescin group: OGD/R samples treated with aescin; KD group: OGD/R samples treated with aescin and PRAS40 shRNA; Rapa group: OGD/R samples treated with aescin and rapamycin.
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