Zingerone Targets Status Epilepticus by Blocking Hippocampal Neurodegeneration via Regulation of Redox Imbalance, Inflammation and Apoptosis

Abstract

Epilepsy is an intricate neurological disease where the neurons are severely affected, leading to the mortality of millions worldwide. Status epilepticus (SE), induced by lithium chloride (LiCl) and pilocarpine, is the most accepted model for epilepsy. The current work aims to unravel the mechanisms underlying the anti-epileptic efficacy of zingerone (an active ingredient of ginger), which has beneficial pharmacological activities on seizure-induced behavioral, histological, neurochemical, and molecular patterns in mice. Zingerone restored cognitive function by diminishing seizure activity, escape latency, and subsequent hippocampal damage manifested in histology. Seizures are associated with local inflammation, redox imbalance, and neural loss, confirmed by the present study of SE, and was attenuated by zingerone treatment. Nuclear factor-kappa B and its downstream signaling molecules (TNF-α, IL-1β, IL-6, NO, MPO) were activated in the LiCl-and-pilocarpine-induced group leading to inflammatory signaling, which was substantially ameliorated by zingerone treatment. The intrinsic apoptotic process was triggered subsequent to SE, as demonstrated by augmentation of cleaved caspase-3, downregulation of Bcl-2. However, zingerone treatment downregulated caspase-3 and upregulated Bcl-2, increasing cell survival and decreasing hippocampal neural death, deciphering involvement of apoptosis in SE. Therefore, zingerone plays an essential role in neuroprotection, probably by precluding oxidative stress, inflammation, and obstructing the mitochondrial pathway of apoptosis.

Keywords: Bcl-2; NF-κB; apoptosis; caspase-3; cognition; histology; inflammation; redox status; status epilepticus; zingerone.

Figure 1

Effect of zingerone on the cognitive function of LiCl-and-pilocarpine-induced SE in mice by Morris water maze. (A) Effect on escape latency (sec) on pilocarpine-induced status epilepticus. Results are representative of mean ± SE of fourteen mice per group. In group-II, the escape latency was increased significantly (*** p < 0.001) as compared to control group (group I). Treatment with sodium valproate and zingerone (25 and 50 mg/kg b.w.) significantly attenuated escape latency level in group III (^### p < 0.001), group IV (^## p < 0.01), and group V (^### p < 0.001) as compared to group II. Group I: Normal saline (10 mL/kg b.w.), Group II: LiCl (3 mEq/kg b.w.) + pilocarpine (30 mg/kg b.w.), Group III: Sodium Valproate (300 mg/kg b.w.) + LiCl (3 mEq/kg b.w.) + pilocarpine (30 mg/kg b.w.), Group IV: zingerone (25 mg/kg b.w.) + LiCl (3 mEq/kg b.w.) + pilocarpine (30 mg/kg b.w.), Group V: zingerone (50 mg/kg b.w.) + LiCl (3 mEq/kg b.w.) + pilocarpine (30 mg/kg b.w.) (B) Effect of zingerone on % time in target quadrant (seconds) on LiCl-and-pilocarpine-induced SE. Results are representative of mean ± SE of eight mice per group. The results that we got are significantly different from pilocarpine group as the main comparison is with pilocarpine group only (^## p < 0.01 and ^### p < 0.001). Group I: Normal saline (10 mL/kg b.w.), Group II: LiCl (3 mEq/kg b.w.) + pilocarpine (30 mg/kg b.w.), Group III: Sodium Valproate (300 mg/kg b.w.) + LiCl (3 mEq/kg b.w.) + pilocarpine (30 mg/kg b.w.), Group IV: zingerone (25 mg/kg b.w.) + LiCl (3 mEq/kg b.w.) + pilocarpine (30 mg/kg b.w.), Group V: zingerone (50 mg/kg b.w.) + LiCl (3 mEq/kg b.w.) + pilocarpine (30 mg/kg b.w.).

Figure 2

Panel of graphs represents effects of zingerone on AChE (a), MPO (b), ROS (c), and NO (d) levels on LiCl-and-pilocarpine-induced SE. Values are significantly different in LiCl and pilocarpine group (*** p < 0.001) as compared to control group. Results that we got are significantly different from LiCl and pilocarpine group as the main comparison is with LiCl and pilocarpine group only (^# p < 0.05, ^## p < 0.01 and ^### p < 0.001). Group I: Normal saline (10 mL/kg b.w.), Group II: LiCl (3 mEq/kg b.w.) + pilocarpine (30 mg/kg b.w.), Group III: Sodium Valproate (300 mg/kg b.w.) + LiCl (3 mEq/kg b.w.) + pilocarpine (30 mg/kg b.w.), Group IV: zingerone (25 mg/kg b.w.) + LiCl (3 mEq/kg b.w.) + pilocarpine (30 mg/kg b.w.), Group V: zingerone (50 mg/kg b.w.) + LiCl (3 mEq/kg b.w.) + pilocarpine (30 mg/kg b.w.)

Figure 3

Zingerone effects on histomorphological features in LiCl-and-pilocarpine-induced SE. There is normal histology of group I (A,B) with intact and appropriately sized nuclei and neural cells. In group II (C,D) there is distortion of neurons along with neural condensation and necrosis, pyknotic nuclei in pilocarpine-administered rats as compared to negative control mice. Group V (E,F) treatment of zingerone (50 mg/kg) recovered the damage which was evident by retaining of normal histology and decrease in neural distortion and death. Values are expressed as mean ± SEM (n = 6). Photomicrographs of hippocampus depicting Hematoxylin and eosin staining analyses. Below photomicrographs is the panel which shows quantitative evaluation of neural loss. Significant difference was indicated by *** p < 0.001 when compared with group I and (^### p < 0.001) when compared with group II. Zingerone treatment significantly protected neural loss in group V (^### p < 0.001) when compared with group II (G). Group I: Normal saline (10 mL/kg b.w.), Group II: LiCl (3 mEq/kg b.w.) + pilocarpine (30 mg/kg b.w.), Group V: zingerone (50 mg/kg b.w.) + LiCl (3 mEq/kg b.w.) + pilocarpine (30 mg/kg b.w.).

Figure 4

Effect of zingerone on choline acetyl transferase (ChAT) in LiCl-and-pilocarpine-induced SE. Photomicrographs of hippocampus depicting immunohistochemical analyses. Below photomicrographs is the panel which shows quantitative evaluation of ChAT. Values are expressed as mean ± SEM (n = 6). Significant differences were indicated by ^### p < 0.001 when compared with group II. Brain sections showing immunohistochemical results demonstrate specific immune positive staining of ChAT with brown color. The CA1 section of hippocampus in LiCl and pilocarpine-administered group-II (C,D) has decreased immunopositive staining of ChAT as quantified by brown color in comparison to negative control (A,B). But treatment of zingerone (50 mg/kg b.w.) in group V (E,F) increased ChAT immune-staining compared to group II (### p < 0.001) (G). Group I: Normal saline (10 mL/kg b.w.), Group II: LiCl (3 mEq/kg b.w.) + pilocarpine (30 mg/kg b.w.), Group V: zingerone (50 mg/kg b.w.) + LiCl (3 mEq/kg b.w.) + pilocarpine (30 mg/kg b.w.); *** p < 0.001.

Figure 5

Zingerone upregulates Bcl-2 expression in LiCl-and-pilocarpine-induced SE. Photomicrographs of hippocampus depicting immunohistochemical analyses indicating specific immune positive staining of Bcl-2 with brown color. Below photomicrographs is the panel which show quantitative evaluation of Bcl-2. Values are expressed as mean ± SEM (n = 6). Significant differences were indicated by *** p < 0.001 when compared with group I and (### p < 0.001) when compared with group II. The CA1 section of hippocampus in LiCl and pilocarpine administered group-II (C,D) has decreased immuno-positive staining of Bcl-2 as specified by brown color in comparison to negative control (group I) (A,B). However, treatment with zingerone (50 mg/kg b.w.) in group V (E,F) enhanced immune positive staining of Bcl-2 in comparison to positive control (group II) (### p < 0.001) (G). Group I: Normal saline (10 mL/kg b.w.), Group II: LiCl (3 mEq/kg b.w.) + pilocarpine (30 mg/kg b.w.), Group V: zingerone (50 mg/kg b.w.) + LiCl (3 mEq/kg b.w.) + pilocarpine (30 mg/kg b.w.).

Figure 6

Zingerone downregulates caspase-3 expression in LiCl-and-pilocarpine-induced SE. Photomicrographs of hippocampus depicting immunohistochemical analyses. Below photomicrographs is the panel which shows quantitative evaluation of activated caspase-3 indicating specific immune positive staining of activated caspase-3 with brown color. Values are expressed as mean ± SEM (n = 6). Significant differences were indicated by *** p < 0.001 when compared with group I and (^### p < 0.001) when compared with group II. The CA1 section of hippocampus in LiCl and pilocarpine administered group-II (C,D) has enhanced caspase-3 immuno-positive staining as stipulated by brown color in comparison to negative control group (group I) (A,B). But zingerone treatment (50 mg/kg b.w.) in group V (E,F) decreased activated caspase-3 in comparison to positive control (group I) (^### p < 0.001) (G). Group I: Normal saline (10 mL/kg b.w.), Group II: LiCl (3 mEq/kg b.w.) + pilocarpine (30 mg/kg b.w.), Group V: zingerone (50 mg/kg b.w.) + LiCl (3 mEq/kg b.w.) + pilocarpine (30 mg/kg b.w.).

Figure 7

A schematic of status epilepticus induced by pilocarpine treatment protocol.