Antiepileptic Effects of Protein-Rich Extract from Bombyx batryticatus on Mice and Its Protective Effects against H2O2-Induced Oxidative Damage in PC12 Cells via Regulating PI3K/Akt Signaling Pathways

Abstract

Bombyx batryticatus is a known traditional Chinese medicine (TCM) utilized to treat convulsions, epilepsy, cough, asthma, headaches, and purpura in China for thousands of years. This study is aimed at investigating the antiepileptic effects of protein-rich extracts from Bombyx batryticatus (BBPs) on seizure in mice and exploring the protective effects of BBPs against H₂O₂-induced oxidative stress in PC12 cells and their underlying mechanisms. Maximal electroshock-induced seizure (MES) and pentylenetetrazole- (PTZ-) induced seizure in mice and the histological analysis were carried out to evaluate the antiepileptic effects of BBPs. The cell viability of PC12 cells stimulated by H₂O₂ was determined by MTT assay. The apoptosis and ROS levels of H₂O₂-stimulated PC12 cells were determined by flow cytometry analysis. Furthermore, the levels of malondialdehyde (MDA), superoxide dismutase (SOD), lactate dehydrogenase (LDH), and glutathione (GSH) in PC12 cells were assayed by ELISA and expressions of caspase-3, caspase-9, Bax, Bcl-2, PI3K, Akt, and p-Akt were evaluated by Western blotting and quantitative real-time polymerase chain reaction (RT-qPCR) assays. The results revealed that BBPs exerted significant antiepileptic effects on mice. In addition, BBPs increased the cell viability of H₂O₂-stimulated PC12 cells and reduced apoptotic cells and ROS levels in H₂O₂-stimulated PC12 cells. By BBPs treatments, the levels of MDA and LDH were reduced and the levels of SOD and GSH-Px were increased in H₂O₂-stimulated PC12 cells. Moreover, BBPs upregulated the expressions of PI3K, Akt, p-Akt, and Bcl-2, whereas they downregulated the expressions of caspase-9, caspase-3, and Bax in H₂O₂-stimulated PC12 cells. These findings suggested that BBPs possessed potential antiepileptic effects on MES and PTZ-induced seizure in mice and protective effects on H₂O₂-induced oxidative stress in PC12 cells by exerting antioxidative and antiapoptotic effects via PI3K/Akt signaling pathways.

Figure 1

SDS-PAGE analysis of BBPs. (a) SDS-PAGE patterns of BBPs. (b) Electrophoresis lane of the marker. (c) Electrophoresis lane of BBPs. M: standard marker; BBPs: protein extracts in Bombyx batryticatus. SDS-PAGE patterns were analyzed using Image Lab software 6.0.1.

Figure 2

Histological analysis. (a) Hematoxylin and eosin stain (40x). (b) Toluidine blue stain (40x). (c) Number of black neurons in the hippocampal CA1 and CA3 areas stained by toluidine blue. BBPs: protein extracts in Bombyx batryticatus. The values represent mean ± SD (n = 6). ^#P < 0.05 and ^##P < 0.01 vs. the normal group; ^∗P < 0.05 and ^∗∗P < 0.01 vs. the model group.

Figure 3

Protective effects of BBPs on the cell viability of H₂O₂-stimulated PC12 cells. (a) Effects of BBPs on cell viability of PC12 cell. PC12 cells were treated with BBPs at concentrations ranging from 0 to 800 μg/mL for 24 h. (b) Effects of BBPs on cell viability of H₂O₂-induced PC12 cells. PC12 cells were treated with BBPs at concentrations ranging from 0 to 800 μg/mL for 24 h, subsequently subjected to H₂O₂ at the concentration of 300 μmol/L for 4 h. The cell viability was determined by MTT assay. BBPs: protein extracts in Bombyx batryticatus. The values represent mean ± SD (n = 6). ^##P < 0.01 vs. the normal group; ^∗∗P < 0.01 vs. the model group.

Figure 4

Effects of BBPs on H₂O₂-stimulated apoptosis of PC12 cells. (a) Normal group. (b) Model group. (c) 200 μg/mL of BBP group. (d) 400 μg/mL of BBP group. (e) 800 μg/mL of BBP group. (f) Apoptosis rate of different groups. PC12 cells were treated with BBPs at concentrations of 200, 400, and 800 μg/mL for 24 h, subsequently subjected to H₂O₂ at the concentration of 300 μmol/L for 4 h. Cell apoptosis was detected by the flow cytometry assay. BBPs: protein extracts in Bombyx batryticatus. The values represent mean ± SD (n = 3). ^##P < 0.01 vs. the normal group; ^∗∗P < 0.01 vs. the model group.

Figure 5

Effects of BBPs on ROS levels of H₂O₂-stimulated PC12 cells. (a) Normal group. (b) Model group. (c) 200 μg/mL of BBP group. (d) 400 μg/mL of BBP group. (e) 800 μg/mL of BBP group. (f) Fluorescence intensity of different groups. PC12 cells were treated with BBPs at concentrations of 200, 400, and 800 μg/mL for 24 h, subsequently subjected to H₂O₂ at the concentration of 300 μmol/L for 4 h. The intracellular ROS level by the flow cytometry (FCM) assay. BBPs: protein extracts in Bombyx batryticatus. The values represent mean ± SD (n = 3). ^##P < 0.01 vs. the normal group; ^∗P < 0.05 and ^∗∗P < 0.01 vs. the model group.

Figure 6

Effects of BBPs on MDA, SOD, LDH, and GSH-Px in H₂O₂-stimulated PC12 cells. The levels of MDA, SOD, LDH, and GSH-Px were determined by the corresponding ELISA kits. PC12 cells were treated with BBPs at concentrations of 200, 400, and 800 μg/mL for 24 h, subsequently subjected to H₂O₂ at the concentration of 300 μmol/L for 4 h. BBPs: protein extracts in Bombyx batryticatus. The values represent mean ± SD (n = 6). ^##P < 0.01 vs. the normal group; ^∗P < 0.05 and ^∗∗P < 0.01 vs. the model group.

Figure 7

Effects of BBPs on mRNA expressions of caspase-3, caspase-9, Bax, Bcl-2, PI3K, Akt, and p-Akt in H₂O₂-stimulated PC12 cells. PC12 cells were treated with BBPs at concentrations of 200, 400, and 800 μg/mL for 24 h, subsequently subjected to H₂O₂ at the concentration of 300 μmol/L for 4 h. Caspase-3, caspase-9, Bax, Bcl-2, PI3K, Akt, and p-Akt mRNAs were detected by RT-qPCR, whereas β-actin was detected as the control. BBPs: protein extracts in Bombyx batryticatus. The values represent mean ± SD (n = 3). ^##P < 0.01 vs. the normal group; ^∗P < 0.05 and ^∗∗P < 0.01 vs. the model group.

Figure 8

Effects of BBPs on protein expressions of caspase-3, caspase-9, Bax, Bcl-2, PI3K, Akt, and p-Akt in H₂O₂-stimulated PC12 cells. PC12 cells were treated with BBPs at concentrations of 200, 400, and 800 μg/mL for 24 h, subsequently subjected to H₂O₂ at the concentration of 300 μmol/L for 4 h. Caspase-3, caspase-9, Bax, Bcl-2, PI3K, Akt, and p-Akt proteins were detected by Western blotting, whereas β-actin was detected as the control. BBPs: protein extracts in Bombyx batryticatus. The values represent mean ± SD (n = 3). ^##P < 0.01 vs. the normal group; ^∗P < 0.05 and ^∗∗P < 0.01 vs. the model group.