mTOR activates hypoxia-inducible factor-1α and inhibits neuronal apoptosis in the developing rat brain during the early phase after hypoxia-ischemia

Abstract

The mammalian target of rapamycin (mTOR) exerts neuroprotective effects under hypoxic or ischemic conditions. To explore whether mTOR participates in neuroprotective signaling through regulation of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and neuronal apoptosis in developing rat brain with hypoxia-ischemia (HI), we operated on postnatal day 10 rats by ligating the common carotid artery followed by exposure to systemic hypoxia. Brains were collected at various intervals to detect the expression of mTOR, phosphorylated mTOR (p-mTOR), HIF-1α, VEGF and cleaved caspase 3 (CC3), using immunohistochemistry and Western blot analysis. We also used terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) to detect neuronal apoptosis. The p-mTOR protein expression increased at 2h after HI, peaked at 8h, lasted 24h, and then dropped to the basal level. Also, the expression of HIF-1α and VEGF was significantly enhanced and peaked at 8h after HI. Up-regulated expression of CC3 was observed at 2h, peaked at 24h, and lasted 72h after HI. Increased neuronal apoptosis is associated with reduced HIF-1α and VEGF expression. Furthermore, pretreatment with rapamycin, a mTOR specific inhibitor, significantly inhibited HIF-1α and VEGF protein after HI. The expression of CC3 and the number of TUNEL-positive cells were up-regulated at 8h and down-regulated at 24h after HI in the rapamycin-treated group. Our findings suggest that mTOR may participate in the regulation of HIF-1α, VEGF and neuronal apoptosis, serving neuroprotective functions after HI in developing rat brain.

Fig. 1

Immunohistochemistry of mTOR, p-mTOR, HIF-1α, VEGF and CC3 expression in P10 rats brain after HI. The total mTOR-positive cells were not obviously changed compared with the sham control. The p-mTOR, HIF-1α, VEGF and CC3-positive cells were weakly detected in sham controls. By contrast, p-mTOR, HIF-1α and VEGF-positive cells were significantly increased and peaked at 8 h in cortex after HI. CC3-positive cells were significantly increased and peaked at 24 h, lasted 72 h after HI. Arrows show the positive staining cells (n=4). 400× (HI, hypoxia-ischemia).

Fig. 2

mTOR, p-mTOR, HIF-1α, VEGF and CC3 proteins expression in P10 rat brain after HI detected by Western blot analysis. Equal amount of protein samples (100 μg) was loaded, and β-actin served as loading control. Values are expressed in relative optical density and represented as mean ± SD (n=4). Total mTOR protein was not obviously changed at different time points compared with sham controls (A). However, p-mTOR protein was induced in ischemia hemisphere, peaked at 8 h, and maintained a high level at 24 h, and then declined to baseline at 72 h (A). HIF-1α and VEGF proteins were obviously induced and peaked at 8 h, then reduced at 24 h after HI (C). CC3 protein was induced at 2 h, peaked at 24 h and maintained a high level at 72 h after HI (C). mTOR, p-mTOR, HIF-1α, VEGF and CC3 expression in HI groups and sham controls were quantified. Data were obtained by densitometry and normalized by β-actin. *p < 0.05, **p < 0.01 compared with the sham control (B and D) (HI, hypoxia-ischemia).

Fig. 3

mTOR involvement in the regulation of HIF-1α, VEGF, and CC3 after HI in P10 rat brains. Western blot was used to analyse mTOR, p-mTOR, HIF-1α, VEGF, and CC3 expression in rapamycin- and DMSO-treated rats (A and C). In rapamycin-treated rats, p-mTOR was significantly reduced but the total mTOR remained unchanged compared with DMSO-treated rats (A). HIF-1α and VEGF protein expression were significantly inhibited by rapamycin. CC3 protein expression was increased at 8 h but reduced at 24 h after HI in rapamycin-treated rats (C). β-actin served as a loading control. The values are expressed in relative optical density and represented as mean ± SD (n=4). *p < 0.05, **p < 0.01 DMSO-treated group compared with the rapamycin-treated group at the same time points (B and D) (HI, hypoxia-ischemia; DM, DMSO; RA, rapamycin).

Fig. 4

The effect of mTOR on apoptosis after HI in P10 rat brains. TUNEL-positive cells were hardly detected in the sham control (data not shown), but significantly induced at 8 and 24 h after HI in the DMSO-treated group. The number of TUNEL positive cells increased at 8 h but decreased at 24 h after HI in rapamycin-treated rats (n=4). Arrows show the positive staining cells. 400× (HI, hypoxia-ischemia; DM, DMSO; RA, rapamycin).