Effects of Gryllus bimaculatus and Oxya chinensis sinuosa extracts on brain damage via blood-brain barrier control and apoptosis in mice with pentylenetetrazol-induced epilepsy

Abstract

The demand for environmentally friendly foods with high nutritional value and low carbon emissions is increasing with the aging of the global population and the crisis of food resources. Edible insects are becoming increasingly well-known as such foods. This study evaluated the effects and mechanisms of Gryllus bimaculatus (Cricket) (Gb) and Oxya chinensis sinuosa (Grasshopper) (Ocs) extracts on epilepsy. A pentylenetetrazol (PTZ)-induced seizure mouse model was used for the study, and Gb and Ocs extracts were administered for 29 days on alternate days at concentrations of 8 g/kg and 16 g/kg. The integrity of the blood-brain barrier (BBB) and brain edema was measured using the perfusion of Evans blue dye and brain water content. Gb and Ocs extracts prevented BBB permeabilization and cerebral edema through increasing the expression of tight junction-associated proteins in the endothelial cells and reducing water content in PTZ-treated mice. Additionally, Gb and Ocs extracts protected neurons from oxidative stress and apoptosis in different brain areas. These protective effects were demonstrated through the restoration of the expression of neuronal nuclear protein and postsynaptic density protein-95, thus increasing the levels of glutathione and superoxide dismutase, decreasing lipid peroxidation, and recovering apoptosis-associated proteins, such as Bax, cleaved PARP, and cleaved caspase-3, in epileptic mice. In addition, Gb and Ocs extracts rescued PTZ-induced hyperexcitable neurons to control mice level, as supported by the restored expression of gamma-aminobutyric acid (GABA) transporter 1, the metabotropic glutamate receptors-GRM2/3, and BDNF. This study suggested that Gb and Ocs extracts are novel medicinal candidates that can help ameliorate epilepsy by improving BBB health and preventing oxidative stress-mediated apoptosis.

Fig 1. Schematic diagram of the experimental design of the pentylenetetrazol (PTZ)-induced epilepsy model, drug treatment, and biochemical analysis in mice.

PTZ was administered intraperitoneally (i.p.) to the mice on alternating days (15 injections over 29 days). VPA (i.p.), Gryllus bimaculatus (Gb), and Oxya chinensis sinuosa (Ocs) extracts were administered orally 30 min before initiating the PTZ treatment.

Fig 2. Determination of subconvulsant PTZ kindling dose for seizure induction and effects of Gb and Ocs extracts on PTZ kindling-induced seizure severity in mice.

(A) Statistical results showing a PTZ dose-dependent increase in the seizure score. (B) A statistical analysis of seizure severity in the PTZ-induced epilepsy mouse model after administrating Gb and Ocs extracts. N = 4. Mean ± standard deviation (SD). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, vs. control group; ^#P ≤ 0.05 vs. PTZ group.

Fig 3. Effects of Gb and Ocs extracts on blood-brain barrier integrity and brain edema in a PTZ-induced epilepsy mouse model.

(A) Representative photographs of Evans blue extravasation in the whole brain and individual hemispheres of various groups at day 31 (D31). (B-C) A quantitative analysis of Evans blue leakage from the PFC (B) and HC (C). (D) A quantitative analysis of brain edema based on brain water content measurements. (E-F) Immunoblots for ZO-1, claudin-5, occludin, and MMP-2 in the PFC I and HC (F) tissue lysates at D31 using different combination treatments. Bars denote a densitometric analysis of the immunoblots. Mean ± SD. *P ≤ 0.05 and **P ≤ 0.01 vs. control group; ^#P ≤ 0.05, ^##P ≤ 0.01, and ^###P ≤ 0.001 vs. PTZ group.

Fig 4. Effects of Gb and Ocs extracts on enzymes associated with the regulation of acetylcholine activity and neuronal damage-induced histopathological changes in the mouse model of PTZ-induced epilepsy.

(A-D) Immunoblots for acetylcholinesterase (AchE), choline acetyltransferase (ChAT), postsynaptic density protein-95, and neuronal nuclear protein from the PFC and HC tissue lysates at D31. Bars denote a densitometric analysis of the immunoblots. (E-I) Representative sections of Nissl staining of different brain regions in each group at D31. Coronal brain sections (20 μm thick) passing through the prefrontal cortex (E) and HC (F), with the CA1 (G), CA3 (H), and dentate gyrus (I) stained with toluidine blue. Mean (n = 3) ± SD. *P ≤ 0.05 and **P ≤ 0.01 vs. control group; not significant, ns, ^#P ≤ 0.05 and ^##P ≤ 0.01 vs. PTZ group.

Fig 5. Effects of Gb and Ocs extracts on excitatory and inhibitory neurotransmitter receptor-associated epileptogenesis in the PTZ-induced epilepsy mouse model.

(A, B) Immunoblots for GRM2/3, GAT1, and BDNF levels in the PFC (A) and HC (B) tissue lysates at D31 treated with different combinations of drugs. Bars indicate a densitometric analysis of the immunoblots. α-Tubulin was used as a loading control. Mean (n = 3) ± SD. **P ≤ 0.01 and ***P≤0.001 vs. control group; ns, ^#P ≤ 0.05, ^##P ≤ 0.01, and ^###P≤0.001 vs. the PTZ group.

Fig 6. Effects of Gb and Ocs extracts on antioxidant enzyme activity in the brain parenchyma of the PTZ-induced epilepsy mouse model.

(A-B) The quantitative measurement of antioxidant enzymes from whole-brain tissue lysates at D31 in each group treated with various combinations of drugs. (A) Reduced glutathione (GSH) and (B) superoxide dismutase (SOD) levels. Mean (n = 3) ± SD. **P ≤ 0.01 and ***P ≤ 0.001 vs. control group; ^#P ≤ 0.05, ^##P ≤ 0.01, and ^###P ≤ 0.001 vs. the PTZ group.

Fig 7. Effects of Gb and Ocs extracts on apoptosis in the brain tissues of the PTZ-induced epilepsy mouse model.

(A, B) Immunoblots of the PFC (A) and HC (B) tissue lysates at D31 treated with different combinations of drugs. Bars indicate the densitometric analysis of the immunoblots. α-Tubulin was used as a loading control. Mean (n = 3) ± SD. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 vs. control group; ns, ^#P ≤ 0.05, ^##P ≤ 0.01, and ^###P ≤ 0.001 vs. PTZ group.

Fig 8. Schematic illustration of the possible role of Gb and Ocs extracts in the PTZ-induced epilepsy mouse model.

PTZ stimulates ROS formation via the reduction of GSH and SOD, but increases lipid peroxidation. Increased ROS may promote oxidative stress, thus actively triggering multiple signaling pathways, including the activation of MMP2 and the inhibition of apoptosis and tight junction proteins, such as claudin-5, occludin, and ZO-1, partly contributing to BBB damage. Furthermore, PTZ-induced oxidative stress accelerates neuronal hyperexcitability via the abnormal regulation of ChAT, AChE, GRM2/3, GAT1, and BDNF. This leads to neuronal damage and cell death. Abbreviations: PTZ, pentylenetetrazole; Gb, Gryllus bimaculatus; Ocs, Oxya chinensis sinuosa; ROS, reactive oxygen species; MDA, malondialdehyde; GSH, glutathione; SOD, superoxide dismutase; MMP-2, matrix metalloproteinase-2; BBB, brain blood barrier; ChAT, choline acetyltransferase; AChE, acetylcholinesterase; GRM2/3, metabotropic glutamate receptor 2/3; GAT1, gamma-aminobutyric acid (GABA) transporter 1; BDNF, brain-derived neurotrophic factor; Neu-N, neuronal nuclear protein; PSD-95, postsynaptic density protein-95.