Echinocystic acid alleviated hypoxic-ischemic brain damage in neonatal mice by activating the PI3K/Akt/Nrf2 signaling pathway

Abstract

Neonatal hypoxic-ischemic encephalopathy (HIE) is considered a major cause of death and long-term neurological injury in newborns. Studies have demonstrated that oxidative stress and apoptosis play a major role in the progression of neonatal HIE. Echinocystic acid (EA), a natural plant extract, shows great antioxidant and antiapoptotic activities in various diseases. However, it has not yet been reported whether EA exerts a neuroprotective effect against neonatal HIE. Therefore, this study was undertaken to explore the neuroprotective effects and potential mechanisms of EA in neonatal HIE using in vivo and in vitro experiments. In the in vivo study, a hypoxic-ischemic brain damage (HIBD) model was established in neonatal mice, and EA was administered immediately after HIBD. Cerebral infarction, brain atrophy and long-term neurobehavioral deficits were measured. Hematoxylin and eosin (H&E), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and dihydroethidium (DHE) staining were performed, and the contents of malondialdehyde (MDA) and glutathione (GSH) were detected. In the in vitro study, an oxygen-glucose deprivation/reperfusion (OGD/R) model was employed in primary cortical neurons, and EA was introduced during OGD/R. Cell death and cellular ROS levels were determined. To illustrate the mechanism, the PI3K inhibitor LY294002 and Nrf2 inhibitor ML385 were used. The protein expression levels of p-PI3K, PI3K, p-Akt, Akt, Nrf2, NQO1, and HO-1 were measured by western blotting. The results showed that EA treatment significantly reduced cerebral infarction, attenuated neuronal injury, and improved brain atrophy and long-term neurobehavioral deficits in neonatal mice subjected to HIBD. Meanwhile, EA effectively increased the survival rate in neurons exposed to OGD/R and inhibited oxidative stress and apoptosis in both in vivo and in vitro studies. Moreover, EA activated the PI3K/Akt/Nrf2 pathway in neonatal mice following HIBD and in neurons after OGD/R. In conclusion, these results suggested that EA alleviated HIBD by ameliorating oxidative stress and apoptosis via activation of the PI3K/Akt/Nrf2 signaling pathway.

Keywords: HIBD; OGD/R; PI3K/Akt/Nrf2 pathway; apoptosis; echinocystic acid; neonatal; oxidative stress.

FIGURE 1

Effects of EA on cerebral infarction following HIBD. (A) The chemical structure of EA. (B) Schedule of the surgery, treatment protocols and experimental tests in vivo study. (C) Representative images of TTC-stained coronal brain sections from the sham group, HI group and groups treated with different doses of EA. (D) Quantitative analysis of the brain infarct volume. Data are presented as the means ± SEM, n = 10. ^# p < 0.05 vs. the HI group.

FIGURE 2

Effects of EA on neuronal damage and apoptosis following HIBD. (A) Representative photomicrographs of H&E staining in the ipsilateral parietal cortex and the hippocampal CA1 region (scale bar: 20 μm), n = 1. (B) Representative photomicrographs of TUNEL staining in the ipsilateral parietal cortex and the CA1 area of hippocampus (scale bar: 20 μm). (C,D) The percentage of apoptotic cells in the ipsilateral parietal cortex and the hippocampal CA1 area, n = 3. (E) Levels of the Bcl-2, Bax, cleaved-caspase-3 and β-actin. (F) Quantification of Bcl-2, Bax and cleaved-caspase-3 expressions. The red arrowheads indicate the damaged neurons. Data are presented as the means ± SEM, n = 3. *p < 0.05, ***p < 0.001, ****p < 0.0001 vs. the sham group; ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 vs. the HI group.

FIGURE 3

Effects of EA on oxidative stress following HIBD. (A) Representative pictures of DHE staining in the ipsilateral parietal cortex (scale bar: 20 μm). (B) Semiquantitative analysis of the DHE fluorescence intensity in the ipsilateral parietal cortex, n = 3. (C) Representative pictures of DHE staining in the CA1 area of hippocampus (scale bar: 20 μm). (D) Semiquantitative analysis of the DHE fluorescence intensity in the hippocampal CA1 region, n = 3. (E,F) Levels of MDA and GSH, n = 6. Data are presented as the means ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001 vs. the sham group; ^# p < 0.05, ^### p < 0.001, ^#### p < 0.0001 vs. the HI group.

FIGURE 4

Effects of EA on long-term neurobehavioral deficits and brain atrophy following HIBD. (A–C) Neurological outcomes were measured by the grip strength test, the corner test, and the Y maze test (n = 9 in the sham group, n = 8 in the HI group, n = 10 in the HI + EA group). (D) Representative images of the whole brains at 21 days after HIBD (scale bar: 1 mm). (E) Statistics of the ratio of brain tissue loss (n = 9 in the sham group, n = 8 in the HI group, n = 10 in the HI + EA group). Data are presented as the means ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001 vs. the sham group; ^# p < 0.05, ^## p < 0.01, ^#### p < 0.0001 vs. the HI group.

FIGURE 5

Effects of EA on the PI3K/Akt/Nrf2 signaling pathway following HIBD. (A) Representative western blot bands of the p-PI3K, PI3K, p-Akt, Akt, Nrf2, NQO1, HO-1 and β-actin. (B–F) Western blot analysis of p-PI3K, PI3K, p-Akt, Akt, Nrf2, NQO1, and HO-1, respectively. Data are presented as the means ± SEM, n = 6. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. the HI group; ^&&& p < 0.001, ^&&&& p < 0.0001 vs. the HI + EA group.

FIGURE 6

Effects of EA on OGD/R-induced neuronal injury. (A) Diagram of the experimental design for the in vitro study. (B) Viability of primary cortical neurons after treatment with 0, 10, 15, 20, 25 μM EA, n = 8. (C) Viability of primary cortical neurons subjected to OGD/R with or without EA (5, 10, 15, 20 μM) treatment, n = 8. (D) Representative fluorescence images of PI staining (scale bar: 20 μm). (E) Quantitative analysis of PI-positive cells, n = 4. Data are presented as the means ± SEM. ****p < 0.0001 vs. the control group; ^# p < 0.05, ^## p < 0.01, ^#### p < 0.0001 vs. the OGD/R group.

FIGURE 7

Effects of EA on OGD/R-induced ROS production. (A) Representative fluorescence images of ROS (scale bar: 20 μm). (B) ROS level analysis. Data are presented as the means ± SEM, n = 4. ****p < 0.0001 vs. the control group; ^#### p < 0.0001 vs. the OGD/R group.

FIGURE 8

Effects of EA on the PI3K/Akt/Nrf2 signaling pathway following OGD/R. (A) Representative western blot bands of p-PI3K, PI3K, p-Akt, Akt, Nrf2, NQO1, HO-1 and β-actin. (B–F) Western blot analysis of p-PI3K, PI3K, p-Akt, Akt, Nrf2, NQO1, and HO-1, respectively. Data are presented as the means ± SEM, n = 3. ^# p < 0.05, ^### p < 0.001 vs. the OGD/R group; ^& p < 0.05, ^&& p < 0.01 vs. the OGD/R + EA group.