Puerarin inhibited oxidative stress and alleviated cerebral ischemia-reperfusion injury through PI3K/Akt/Nrf2 signaling pathway

Abstract

Introduction: Puerarin (PUE) is a natural compound isolated from Puerariae Lobatae Radix, which has a neuroprotective effect on IS. We explored the therapeutic effect and underlying mechanism of PUE on cerebral I/R injury by inhibiting oxidative stress related to the PI3K/Akt/Nrf2 pathway in vitro and in vivo. Methods: The middle cerebral artery occlusion and reperfusion (MCAO/R) rats and oxygen-glucose deprivation and reperfusion (OGD/R) were selected as the models, respectively. The therapeutic effect of PUE was observed using triphenyl tetrazolium and hematoxylin-eosin staining. Tunel-NeuN staining and Nissl staining to quantify hippocampal apoptosis. The reactive oxygen species (ROS) level was detected by flow cytometry and immunofluorescence. Biochemical method to detect oxidative stress levels. The protein expression related to PI3K/Akt/Nrf2 pathway was detected by using Western blotting. Finally, co-immunoprecipitation was used to study the molecular interaction between Keap1 and Nrf2. Results: In vivo and vitro studies showed that PUE improved neurological deficits in rats, as well as decreased oxidative stress. Immunofluorescence and flow cytometry indicated that the release of ROS can be inhibited by PUE. In addition, the Western blotting results showed that PUE promoted the phosphorylation of PI3K and Akt, and enabled Nrf2 to enter the nucleus, which further activated the expression of downstream antioxidant enzymes such as HO-1. The combination of PUE with PI3K inhibitor LY294002 reversed these results. Finally, co-immunoprecipitation results showed that PUE promoted Nrf2-Keap1 complex dissociation. Discussion: Taken together, PUE can activate Nrf2 via PI3K/Akt and promote downstream antioxidant enzyme expression, which could further ameliorate oxidative stress, against I/R-induced Neuron injury.

Keywords: PI3K/Akt/Nrf2 pathway; cerebral ischemia reperfusion injury (CIRI); hippocampal neurons; oxidative stress; puerarin.

FIGURE 1

PUE reduced neurological scores and alleviated neuronal damage in MCAO/R rat brains. (A) PUE reduced the neurological score assessed by the Zea Longa test after cerebral I/R (n = 10). (B) Representative images of TTC staining. (C) Quantitative analysis of cerebral infarction volume (n = 3). (D) The morphological alterations of cells in the hippocampal CA1 region were measured by HE staining (×400). (E) The morphological alterations of cells in the hippocampal CA1 region were examined by Nissl staining (×400). (a) Sham group; (b) model group; (c) PUE (25 mg/kg) group; (d) PUE (50 mg/kg) group; (e) PUE (100 mg/kg) group; (f) edaravone. The scale bar was 20 µm. All data were expressed as mean ± SD. ^## p < 0.01 vs. sham group, *p < 0.05 or **p < 0.01 vs. model group.

FIGURE 2

PUE alleviates the neuro apoptosis of cerebral ischemic tissue. (A) Tunel-NeuN staining: green, Tunel-positive staining; blue, DAPI core dye; red, neuron-specific nucleus protein (NeuN) (400×). (B) Quantitative analysis of the proportion of Tunel-positive neurons in each group. (C) Bax, Caspase-3 and Bcl-2 protein expression in rat hippocampal neurons; Quantification results of protein expression. All data are expressed as mean ± SD. ^## p < 0.01 vs. sham group, *p < 0.05 or **p < 0.01 vs. model group.

FIGURE 3

PUE regulated the oxidative stress level in the MCAO/R model. (A–F) The levels of ROS, SOD, MDA, GSH, GSH-px, and CAT in the brain tissues were measured by the corresponding kits. All data are expressed as mean ± SD (n = 6). ^## p < 0.01 vs. sham group, *p < 0.05 or **p < 0.01 vs. model group, ^△ p < 0.05 or ^△△ p < 0.01 vs. PUE group.

FIGURE 4

PUE attenuates hippocampal neuron injury after MCAO/R via PI3K/Akt/Nrf2. (A) The expression and location of Nrf2 were determined by immunohistochemistry assay (×400). (B) Representative protein imprinting image of PI3K, Akt, P-PI3K, P-Akt, Keap1, Nucleus Nrf2, Cytoplasm Nrf2 and HO-1 in hippocampal neurons of the ischemic side of rat brain tissue. (C) Quantitative analysis of p-PI3K/PI3K protein expression. (D) Quantitative analysis of p-Akt/Akt protein expression. (E) Quantitative analysis of Keap1 protein expression. (F) Quantitative analysis of Nucleus Nrf2 protein expression. (G) Quantitative analysis of Cytoplasm Nrf2 protein expression. (H) Quantitative analysis of HO-1 protein expression. All data are expressed as mean ± SD. ^## p < 0.01 vs. sham group, *p < 0.05 or **p < 0.01 vs. model group, ^△ p < 0.05 or ^△△ p < 0.01 vs. PUE group.

FIGURE 5

PUE promotes the viability of hippocampal neurons induced by oxygen-glucose deprivation/reperfusion (OGD/R). (A) Hippocampal neurons were treated with PUE at various concentrations (50–750 nM) and the cell viability was measured by the CCK-8 assay. (B) Cell viability was measured by the CCK-8 assay after OGD/R and normal conditions. (C) Effect of PUE on the release of LDH from hippocampal neurons damaged by OGD/R. (D) The percentage of HT22 cell apoptosis was analyzed by staining cells with Hoechst 33258 (blue represents viable cells, × 200). (E,F) Apoptotic cells rate were detected by flow cytometry. (a) Control group; (b) OGD/R group; (c) PUE (150 nM) + OGD/R; (d) PUE (200 nM)+OGD/R; (e) PUE (250 nM)+OGD/R; (f) edaravone. All data are expressed as means ± SD (n = 6). ^## p < 0.01 vs. control group, *p < 0.05 or **p < 0.01 vs. OGD/R group.

FIGURE 6

The effect of PUE on oxygen-glucose deprivation/reperfusion (OGD/R)-induced ROS release of hippocampal neurons. (A) The reactive oxygen species (ROS) level induced by OGD/R was detected by fluorescence microscopy (×200). The mean of fluorescence intensity is shown in (B). (C) ROS generation was determined with DCFH-DA staining and analyzed by Flow Jo. The mean of fluorescence intensity is shown in (D). (a) Control group; (b) OGD/R group; (c) PUE (150 nM) + OGD/R; (d) PUE (200 nM) + OGD/R; (e) PUE (250 nM) + OGD/R; (f) edaravone. All data are expressed as means ± SD (n = 3). *p < 0.05 or **p < 0.01 vs. OGD/R group.

FIGURE 7

PUE changed the level of enzymatic activity related to oxidative stress in HT22 cells. (A-E) The levels of SOD, MDA, CAT, GSH, and GSH-px were measured by the corresponding kits. All data are expressed as mean ± SD (n = 6). ^## p < 0.01 vs. control group, *p < 0.05 or **p < 0.01 vs. OGD/R group, ^△p < 0.05 or ^△△ p < 0.01 vs. PUE group.

FIGURE 8

PUE affects the expression of proteins related to PI3K/AKT/Nrf2. (A) The expression of NRF2 (green labeled) and nuclear (blue labeled) in HT22 cells was measured by confocal laser scanning microscope. Bar = 25 μm. (B) Co-immunoprecipitation evaluation of Nrf2-Keap1 interaction in HT22 cells. (C) Representative protein imprinting image of PI3K, Akt, P-PI3K, P-Akt, Keap1, Nucleus Nrf2, Cytoplasm Nrf2 and HO-1 in hippocampal neurons of the ischemic side of rat brain tissue. (D) Quantitative analysis of p-PI3K/PI3K protein expression. (E) Quantitative analysis of p-Akt/Akt protein expression. (F) Quantitative analysis of Keap1 protein expression. (G) Quantitative analysis of Nucleus Nrf2 protein expression. (H) Quantitative analysis of Cytoplasm Nrf2 protein expression. (I) Quantitative analysis of HO-1 protein expression. All data are expressed as mean ± SD. ^## p < 0.01 vs. control group, *p < 0.05 or **p < 0.01 vs. OGD/R group, ^△ p < 0.05 or ^△△ p < 0.01 vs. PUE group.

FIGURE 9

PUE promotes the upregulation of HO-1 expression by activating Nrf2, and HO-1 inhibitor ZnPP reverses the protective effect of PUE. (A) Protein expression of Nrf2 after administration of ML385. (B) Protein expression of HO-1 after administration of ML385. (C) Cell viability after ZnPP administration. (D) Protein expression of HO-1 after administration of ZnPP. All data are expressed as mean ± SD. ^## p < 0.01 vs. control group, *p < 0.05 or **p < 0.01 vs. OGD/R group, ^△ p < 0.05 or ^△△ p < 0.01 vs. PUE group.

FIGURE 10

Puerarin can reduce oxidative stress by activating PI3K/Akt/Nrf2 pathway to achieve neuroprotective effect on ischemic stroke.