p53 Inhibition Protects against Neuronal Ischemia/Reperfusion Injury by the p53/PRAS40/mTOR Pathway

Figure 1

p53 exacerbated cerebral I/R injury by inhibiting the mTOR pathway both in vitro and in vivo. (a) In primary cultured rat neurons after OGD/R injury (6 h OGD followed by 6 h reperfusion), the LDH level increased in the p53-overexpressing group (p53 OE+OGD) compared with the control groups (control+OGD and GFP+OGD). In the p53 knockdown group (p53 KD+OGD), LDH release decreased compared with that in the control groups (control+OGD and scramble shRNA+OGD). vs. the control+OGD group; ^# vs. the GFP+OGD or scramble shRNA+OGD group. (b) Forty-eight hours after dMCAO in SD rats, the infarction size, normalized to the contralateral cortex, greatly increased in the p53-overexpressing group (p53 OE); however, administration of p53 shRNA (p53 shRNA group) reduced the total infarction size. vs. the GFP group; ^# vs. the scramble shRNA group. (c, d) In the p53-overexpressing group, the expression levels of p-mTOR and p-S6K decreased compared to those in the control groups (control+OGD and GFP+OGD). In the p53 knockdown group, the levels of p-mTOR and p-S6K obviously increased compared to those in the control groups (control+OGD and scramble shRNA+OGD). vs. the control+OGD group; ^# vs. the GFP+OGD or scramble shRNA+OGD group. –21/group in at least 3 independent experiments. (e) Confirmation of p53 gene KO in mice. PCR for genotyping p53 KO mice. Lane M: DNA size marker; lanes 1 to 11: C57 heterozygote (+/−); lane 12: homozygote (-/-); lane 13: homozygote (+/+). (f) The LDH release ratio after 6 hrs OGD followed by 6 hrs reperfusion was much higher in primary cultured WT mice neurons than that in p53^+/- or p53^-/- mice. vs. WT+No OGD; ^# vs. WT+OGD. (g) In primary cultured p53^-/- mice neurons, when p53 was overexpressed (OE), the LDH level greatly increased compared to control group (p53^-/- + GFP). vs. WT+No OGD; ^# vs. p53^-/- + GFP. (h) Inhibition of p53 reduced the LDH level via activating the mTOR pathway. The LDH level can be reduced by administrating phosphatidic acid (an agonist of mTOR pathway, P.A.) when p53 was overexpressed. Similarly, when p53 shRNA was administrated, rapamycin (an antagonist of mTOR pathway, Rapa) could increase the LDH release ratio. Moreover, in primary cultured p53^-/- mice neurons, the LDH release was more severe when treated with Rapa compared with PBS. vs. WT+p53 OE+PBS; ^# vs. WT+p53 shRNA+PBS; ^& vs. p53^-/-+PBS. -21/group in at least 3 independent experiments.

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