Trimetazidine Modulates Mitochondrial Redox Status and Disrupted Glutamate Homeostasis in a Rat Model of Epilepsy

Abstract

Mitochondrial oxidative status exerts an important role in modulating glia-neuron interplay during epileptogenesis. Trimetazidine (TMZ), a well-known anti-ischemic drug, has shown promising potential against a wide range of neurodegenerative disorders including epilepsy. Nevertheless, the exact mechanistic rationale behind its anti-seizure potential has not been fully elucidated yet. Herein, the impact of TMZ against mitochondrial oxidative damage as well as glutamate homeostasis disruption in the hippocampus has been investigated in rats with lithium/pilocarpine (Li/PIL) seizures. Animals received 3 mEq/kg i.p. LiCl₃ followed by PIL (single i.p.; 150 mg/kg) 20 h later for induction of seizures with or without TMZ pretreatment (25 mg/kg; i.p.) for five consecutive days. Seizure score and seizure latency were observed. Mitochondrial redox status as well as ATP and uncoupling protein 2 was recorded. Moreover, glutamate homeostasis was unveiled. The present findings demonstrate the TMZ-attenuated Li/PIL seizure score and latency. It improved mitochondrial redox status, preserved energy production mechanisms, and decreased reactive astrocytes evidenced as decreased glial fibrillary acidic protein immune-stained areas in hippocampal tissue. In addition, it modulated phosphorylated extracellular signal-regulated kinases (p-ERK1/2) and p-AMP-activated protein kinase (p-AMPK) signaling pathways to reflect a verified anti-apoptotic effect. Consequently, it upregulated mRNA expression of astroglial glutamate transporters and reduced the elevated glutamate level. The current study demonstrates that TMZ exhibits robust anti-seizure and neuroprotective potentials. These effects are associated with its ability to modulate mitochondrial redox status, boost p-ERK1/2 and p-AMPK signaling pathways, and restore glutamate homeostasis in hippocampus.

Keywords: ERK1/2; astrogliosis; glutamate transporters; mitochondrial oxidative stress; trimetazidine.

FIGURE 1

Effect of TMZ on Li/PIL-induced seizures. (A) Racine score, data are expressed as box plots of the median of 15 animals. Statistical analysis was done using the Kruskal–Wallis test followed by Dunn's test. (B) Seizure latency, data are expressed as mean of 15 animals ± SD. Two-way ANOVA followed by the Tukey–Kramer post-hoc test was used for statistical analyses. *Significantly different from the normal control group (Saline) at p < 0.05; ****Significantly different from the normal control group (Saline) at p < 0.0001; ^@@Significantly different from the Li/PIL control group at p < 0.01; ^@@@@Significantly different from the Li/PIL control group at p < 0.0001.

FIGURE 2

Effect of TMZ on glial glutamate transporters and hippocampal glutamate level in rats with Li/PIL-induced seizures. (A) hippocampal mRNA expression of GLT-1 as indicated by qRT-PCR analysis, (B) hippocampal mRNA expression of GLAST as indicated by qRT-PCR analysis, (C) hippocampal glutamate level. Values are expressed as mean of six animals ± SD. Two-way ANOVA followed by the Tukey–Kramer post-hoc test was used for statistical analyses of data. **Significantly different from the normal control group (Saline) at p < 0.01; ***Significantly different from the normal control group (Saline) at p < 0.001; ****Significantly different from the normal control group (Saline) at p < 0.0001; ^@@@@Significantly different from the Li/PIL control group at p < 0.0001.

FIGURE 3

Effect of TMZ on GFAP expression in the hippocampi of rats with Li/PIL-induced seizures as indicated by immunohistochemistry (×400 original magnification). Normal control rats (A), Normal + TMZ rats (B), Li/PIL control rats (C), and TMZ + Li/PIL rats (D). Area % of GFAP (E). Values are expressed as mean of three animals ± SD. Two-way ANOVA followed by the Tukey–Kramer post-hoc test was used for statistical analyses of data. **Significantly different from the normal control group (Saline) at p < 0.01; ****Significantly different from the normal control group (Saline) at p < 0.0001; ^@@@@Significantly different from the Li/PIL control group at p < 0.0001.

FIGURE 4

Effect of TMZ on the protein expression of p-44/42 Erk1/2 (Thr202/Tyr204) and p-AMPK (Thy172) in the hippocampi of rats with Li/PIL-induced seizures. (A) The expression of p-44/42 Erk1/2 (Thr202/Tyr204) as indicated by Western blot analysis; (B) the expression of p-AMPK (Thy172) as indicated by Western blot analysis. Values are expressed as mean of three animals ± SD. Two-way ANOVA followed by the Tukey–Kramer post-hoc test was used for statistical analyses of data. *Significantly different from the normal control group (Saline) at p < 0.05; **Significantly different from the normal control group (Saline) at p < 0.01; ***Significantly different from the normal control group (Saline) at p < 0.001; ****Significantly different from the normal control group (Saline) at p < 0.0001; ^@Significantly different from the Li/PIL control group at p < 0.05; ^@@@@Significantly different from the Li/PIL control group at p < 0.0001.

FIGURE 5

Effect of TMZ on mitochondrial oxidative stress, mitochondrial uncoupling protein 2 (UCP2), and ATP production in the hippocampi of rats with Li/PIL-induced seizures. (A) TAC, (B) reduced GSH contents, (C) MDA levels, (D) UCP2, (E) ATP. Values are expressed as mean of six animals ± SD. Two-way ANOVA followed by the Tukey–Kramer post-hoc test was used for statistical analyses of data. *Significantly different from the normal control group (Saline) at p < 0.05; **Significantly different from the normal control group (Saline) at p < 0.01; ****Significantly different from the normal control group at p < 0.0001; ^@@@Significantly different from the Li/PIL control group at p < 0.001; ^@@@@Significantly different from the Li/PIL control group at p < 0.0001.

FIGURE 6

Effect of TMZ on the Cyt c content and caspase-3 activity in rats with Li/PIL-induced seizures. (A) Cyt c content, (B) caspase-3 activity. Values are expressed as mean of six animals ± SD. Two-way ANOVA followed by the Tukey–Kramer post-hoc test was used for statistical analyses of data. *Significantly different from the normal control group (Saline) at p < 0.05; ****Significantly different from the normal control group (Saline) at p < 0.0001; ^@@@@Significantly different from the Li/PIL control group at p < 0.0001.

FIGURE 7

Effect of TMZ on histopathological alterations in the hippocampi of rats with Li/PIL-induced seizures (×400 original magnification). Normal control rats and normal rats treated with TMZ showed normal histology of CA1, CA3, and hilar regions (A–C) and (D–F), respectively. Li/PIL control showed a significant loss of pyramidal cells layer (dotted arrows), many apoptotic figures, edema (star), and sloughing of capillary endothelium in CA1 and CA3 regions (arrows); in addition to many degenerated neurons (arrow), pyknotic nuclei, and glial cells infiltration (dotted arrow) (G,H), and edematous fluid effusion and congested blood capillaries in the hilar region (I). TMZ + Li/PIL rats demonstrated partial protection with less edema fluid and congested capillaries (star), more organizing effect on pyramidal cells in CA1 and CA3 regions with mixed arrangement of fewer damaged (arrow) and intact (dotted arrow) neurons (J,K), and lower number of degenerated and apoptotic neurons in the hilar region (arrow) (L). (M) Total histology lesion score; data are expressed as box plots of the median of three animals. Statistical analysis was done using the Kruskal–Wallis test followed by Dunn's test. *Significantly different from the normal control group (Saline) at p < 0.05; ****Significantly different from the normal control group (Saline) at p < 0.0001; ^@@@@Significantly different from the Li/PIL control group at p < 0.0001.

FIGURE 8

Effect of TMZ on hippocampal neuronal loss of rats with Li/PIL-induced seizures. Photomicrographs demonstrating toluidine blue–stained hippocampal tissue sections of CA1 and CA3 sub regions and DC hilar cells in normal control rats (A,E,I), TMZ control rats (B,F,J), Li/PIL control rats (C,G,K), and TMZ + Li/PIL rats (D,H,L). Intact neurons count in CA1 (M), CA3 (N), and DG hilar cells (O). Values are expressed as mean of three animals ± SD. Two-way ANOVA followed by the Tukey–Kramer post-hoc test was used for statistical analyses of data. *Significantly different from the normal control group (Saline) at p < 0.05; **Significantly different from the normal control group (Saline) at p < 0.01; ****Significantly different from the normal control group (Saline) at p < 0.0001; ^@@Significantly different from the Li/PIL control group at p < 0.01; ^@@@@Significantly different from the Li/PIL control group at p < 0.0001.