Curcumin targets vascular endothelial growth factor via activating the PI3K/Akt signaling pathway and improves brain hypoxic-ischemic injury in neonatal rats

Abstract

This study aimed to evaluate the effect of curcumin on brain hypoxicischemic (HI) damage in neonatal rats and whether the phosphoinositide 3-kinase (PI3K)/Akt/vascular endothelial growth factor (VEGF) signaling pathway is involved. Brain HI damage models were established in neonatal rats, which received the following treatments: curcumin by intraperitoneal injection before injury, insulin-like growth factor 1 (IGF-1) by subcutaneous injection after injury, and VEGF by intracerebroventricular injection after injury. This was followed by neurological evaluation, hemodynamic measurements, histopathological assessment, TUNEL assay, flow cytometry, and western blotting to assess the expression of p-PI3K, PI3K, p-Akt, Akt, and VEGF. Compared with rats that underwent sham operation, rats with brain HI damage showed remarkably increased neurological deficits, reduced right blood flow volume, elevated blood viscosity and haematocrit, and aggravated cell damage and apoptosis; these injuries were significantly improved by curcumin pretreatment. Meanwhile, brain HI damage induced the overexpression of p-PI3K, p-Akt, and VEGF, while curcumin pretreatment inhibited the expression of these proteins. In addition, IGF-1 treatment rescued the curcumin-induced down-regulated expression of p- PI3K, p-Akt, and VEGF, and VEGF overexpression counteracted the inhibitory effect of curcumin on brain HI damage. Overall, pretreatment with curcumin protected against brain HI damage by targeting VEGF via the PI3K/Akt signaling pathway in neonatal rats.

Keywords: Brain; Curcumin; Hypoxic-ischemic; PI3K/Akt signaling pathway; Vascular endothelial growth factor.

Fig. 1. Curcumin improves brain HI damage in neonatal rats (n = 12 per group).

(A) HI damage in brain tissues of the sham, HI, and HI + CUR groups by TUNEL staining. (B) Neurological deficits evaluated in the sham, HI, and HI + CUR groups by Longa’s score at 24 h and 72 h after treatment. (C) Histopathological changes of brain tissues in the sham, HI, and HI + CUR groups by H&E staining (×400). (D) Cell apoptosis in brain tissues of the sham, HI, and HI + CUR groups by TUNEL staining (×400). (E) Early and late apoptotic in brain tissues of the sham, HI, and HI + CUR groups by flow cytometry. HI, hypoxic-ischemic injury; CUR, curcumin. **p < 0.01, ***p < 0.001 vs. sham group; ^##p < 0.01 vs. HI group.

Fig. 2. Curcumin promotes the activation of PI3K/Akt signaling pathway and VEGF expression in neonatal rats with brain HI damage (n = 12 per group).

(A) Band intensities from western blots were calculated. (B) The protein expression of p-PI3K, PI3K, p-Akt, Akt, and VEGF in the sham, HI, and HI + CUR groups by western blotting. HI, hypoxic-ischemic injury; CUR, curcumin; VEGF, vascular endothelial growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. **p < 0.01 vs. sham group; ^##p < 0.01 vs. HI group.

Fig. 3. Inhibitory effect of curcumin on VEGF expression disappears via activating PI3K/Akt signaling pathway in neonatal rats with brain HI damage (n = 12 per group).

(A) Band intensities from western blots were calculated. (B) The protein expression of p-PI3K, PI3K, p-Akt, Akt, and VEGF in the sham, HI, HI + CUR and HI + CUR + IGF-1 groups by western blotting. HI, hypoxic-ischemic injury; CUR, curcumin; IGF-1, insulin-like growth factor 1; VEGF, vascular endothelial growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. **p < 0.01 vs. sham group; ^##p < 0.01 vs. HI group.

Fig. 4. The inhibitory effect of curcumin on brain HI damage is counteracted by VEGF overexpression in neonatal rats (n = 12 per group).

(A) The VEGF expression in the sham, HI, HI + CUR, and HI + CUR + ov-VEGF groups by western blotting. (B) HI damage in brain tissues of the sham, HI, HI + CUR, and HI + CUR + ov-VEGF groups by TUNEL staining. (C) Neurological deficits evaluated in the sham, HI, HI + CUR, and HI + CUR + ov-VEGF groups by Longa’s score at 24 h and 72 h after treatment. (D) Histopathological changes of brain tissues in the sham, HI, HI + CUR, and HI + CUR + ov-VEGF groups by H&E staining (×400). (E) Cell apoptosis in the sham, HI, HI + CUR, and HI + CUR + ov-VEGF groups by TUNEL staining (×400). (F) Early and late apoptotic in brain tissues of the sham, HI, HI + CUR, and HI + CUR + ov-VEGF groups by flow cytometry. HI, hypoxic-ischemic injury; CUR, curcumin; VEGF, vascular endothelial growth factor. **p < 0.01 vs. sham group; ^##p < 0.01 vs. HI group.