Geraniol Ameliorates Pentylenetetrazol-Induced Epilepsy, Neuroinflammation, and Oxidative Stress via Modulating the GABAergic Tract: In vitro and in vivo studies

Abstract

Introduction: Geraniol (Ger), a monoterpene, is a common constituent of several essential oils. This study explored the anticonvulsant effect of Ger in-vitro using nerve growth factor (NGF) prompted PC12 cell injured by Glutamate (Glu) and in-vivo using Pentylenetetrazole (PTZ)-induced kindling through the GABAergic pathway.

Materials: To assess the effect of Ger on NGF prompted PC12 cells injured by Glu, Ger at concentrations of 25, 50, 100, 200 and 400 μg/mL was used. GABA, 5-HT, IL-1β, IL-4, and TNF-α levels and the gene expressions of GABAA-Rα1, NMDAR1, GAD 65, GAD 67, GAT 1 and GAT 3 were measured in NGF-induced PC12 cells treated with Ger (100, and 200 μg/mL). Mice were randomly separated into five groups. Normal and PTZ groups in which mice were injected with saline or PTZ, respectively. PTZ + Ger 100, PTZ + Ger 200 and PTZ + SV groups in which mice orally administered Ger or sodium valproate (SV), respectively, then injected with PTZ.

Results: Ger up to 400 μg/mL did not display any toxicity or injury in PC12 cells. Ger (100 to 200 μg/mL) reduced the injury induced by Glu, increased the gene expression of GABAA-Rα1, GAD65 and GAD67 and decreased GAT 1, GAT 3 and NMDAR1 expression in NGF-induced PC12 cells damaged by Glu. Ger (100 to 200 μg/mL) increased GABA and reduced TNF-α, IL-4 and IL-1β levels in NGF-induced PC12 cells injured by Glu. As for the in-vivo results, Ger increased GABA, GAD, GAT 1 and 3 and lowered GABA T. Ger mitigated MDA, NO, IL-1β, IL-6, TNF-α and IFN-γ, GFAP, caspase-3, and -9 levels and Bax gene expression and escalated GSH, SOD, catalase, BDNF and Bcl2 gene expression.

Conclusion: Ger reduced the oxidative stress status, neuroinflammation and apoptosis and activated GABAergic neurotransmission, which might clarify its anticonvulsant. Ger protects animals against PTZ prompted kindling as established by the enhancement in short term as well as long-term memory. Ger mitigated the injury induced by Glu in NGF prompted PC12 cell.

Keywords: GABAergic; apoptosis; geraniol; neuroinflammation; oxidative stress; pentylenetetrazole.

Figure 1

Experimental design for chronic administration of PTZ experiment.

Figure 2

The outcome of Ger at different concentrations on the (A) normal cells viability (B) NGF-induced PC12 cells injured by Glu (20 mmol/L) viability. Effects of Ger (100 and 200 μg/mL) on the gene expressions of (C) GABAA-Rα1, (D) NMDAR1, (E) GAD 65, (F) GAD 67, (G) GAT 1 and (H) GAT 3 in NGF prompted PC12 cells damage by Glu. PC12 cells (induced by 50 ng/mL NGF for 48 h) were treated with Ger (100 or 200 μg/mL) for 24 h, then Glu (20 mmol/L) for another 24 h. Normal cells are NGF-induced PC12 cells without Ger and Glu. ^#P≤0.0219 vs normal cells ^###P≤ 0.001 vs normal cells; **P≤ 0.0013 vs model cells; ***P≤ 0.001 vs model cells.

Figure 3

Outcomes of Ger on (A) GABA, (B) 5-HT, (C) IL-1β, (D) IL-4 and (E) TNF-α in model cells. PC12 cells (induced by 50 ng/mL NGF for 48 h) were treated with Ger (100 or 200 μg/mL) for 24 h, then Glu (20 mmol/L) for another 24 h. ^##P≤0.01 vs normal cells; ^###P≤ 0.001 vs normal cells; ***P≤ 0.001 vs model cells. Normal cells are NGF prompted cells without Ger and Glu; Model cells are NGF prompted cells damaged by Glu (20 mmol/L).

Figure 4

The outcomes of Ger (100 and 200 mg/kg) and SV (300 mg/kg) administration in PTZ (35 mg/kg, 14 ip injections, every other day) kindled mice on different behavioral assessments including (A) Racine seizures scale, (B) latency to enter the dark room, (C) % of spontaneous alternations, (D) number of arms entries, (E) exploration time of the familiar object, (F) exploration time of the novel object (G) discrimination index, (H) rearing, (I) crossing, (J) grooming (K) center time and (L) time on the rotating bar. Data are expressed as mean ± SD (n=6); ^###P<0.001 compared to the normal group, *P<0.002 compared to the PTZ group, P<0.001 compared to the PTZ group.

Figure 5

The outcomes of Ger (100 and 200 mg/kg) and SV (300 mg/kg) administration in PTZ (35 mg/kg, 14 ip injections, every other day) kindled mice on GABAergic pathway specifically (A and B) GABA, (C and D) GAD and (E and F) GABA T levels in the hippocampus and cortex respectively. Data are expressed as mean ± SD (n=6); ^###compared to normal group (P<0.001), ***Compared to PTZ group (P<0.001) using One-Way ANOVA tailed by Tukey’s post hoc test.

Figure 6

The outcomes of Ger (100 and 200 mg/kg) and SV (300 mg/kg) administration in PTZ (35 mg/kg, 14 ip injections, every other day) kindled mice on GABAergic pathway specifically on the gene and protein expression (A and B) GAT 1, and (C and D) GAT 3 in the hippocampus. Data are expressed as mean ± SD (n=6); ^###compared to normal group (P<0.001), ***Compared to PTZ group (P<0.001) using One-Way ANOVA tailed by Tukey’s post hoc test.

Figure 7

The outcomes of Ger (100 and 200 mg/kg) and SV (300 mg/kg) administration in PTZ (35 mg/kg, 14 ip injections, every other day) kindled mice on lipid peroxidation and oxidative/nitrosative stress specifically on (A and B) MDA, (C and D) NO, (E and F) GSH, (G and H) SOD and (I and J) Catalase in the hippocampus and cortex respectively. Data are expressed as mean ± SD (n=6); ^###Compared to normal group (P<0.001), *Compared to PTZ group (P<0.0002), ***Compared to PTZ group (P<0.001) using One-Way ANOVA tailed by Tukey’s post hoc test.

Figure 8

The outcomes of Ger (100 and 200 mg/kg) and SV (300 mg/kg) administration in PTZ (35 mg/kg, 14 ip injections, every other day) kindled mice on inflammatory markers, specifically (A and B) IL- 1β, (C and D) IL-6, (E and F) IFN-γ and (G and H)TNF-α levels in the hippocampus and cortex respectively. Data are expressed as mean ± SD (n=6); ^###Compared to normal group. (P<0.001), ***Compared to PTZ group (P<0.001) using One-Way ANOVA tailed by Tukey’s post hoc test.

Figure 9

The outcomes of Ger (100 and 200 mg/kg) and SV (300 mg/kg) administration in PTZ (35 mg/kg, 14 ip injections, every other day) kindled mice on (A) GFAP and (B) BDNF levels and on apoptotic markers specifically (C) caspase 3, (D) caspase 9, the gene expression of (E) BAX, and (F) Bcl2 levels in the hippocampus. Data are expressed as mean ± SD (n=6); ^###Compared to normal group (P<0.001), ***Compared to PTZ group (P<0.001) using One-Way ANOVA tailed by Tukey’s post hoc test.

Figure 10

The action of Ger on the histopathological alteration of hippocampus stained with H&E stain showing the GL includes 4–6 cell layers (long arrow), PTZ showing apparent reduction in the number of GL cell layers with swelling cells (long arrow) and dilated blood vessel (arrow head), Ger (100 and 200 mg/kg) and SV (300 mg/kg) administration with PTZ revealed amendment in the hippocampus structure, with a noticeable increase in the number of GL cell layers and normally sized blood vessels, and histopathological alteration scoring. ^###Compared to normal group (P<0.001), ***Compared to PTZ group (P<0.001).