Vanillic acid (VA) exhibited antioxidant and neuroprotective properties in some
neurodegenerative disorders. So, the current study examined the neuroprotective
potential of VA as an antiepileptic agent in pentylenetetrazole (PTZ)-induced
epileptic rats and the prospective role of Insulin like growth factor-1 (IGF-1)
and nuclear factor-2 erythroid-related factor-2 (Nrf2)/heme oxygenase-1 (HO-1)
pathway in this respect. Thirty male albino rats were equally subdivided into 3
groups; (1) normal control (NC) group, (2) PTZ-group: received PTZ (50 mg/Kg,
i.p. every other day) for 14 days, and (3) PTZ + VA group: received PTZ and VA (50
mg/Kg daily for 2 weeks). The seizure score and latency were evaluated after PTZ
injection. Also, the markers of oxidative stress (malondialdehyde (MDA),
catalase, and reduced glutathione (GSH)), histopathological examination, the
expression of glial fibrillary acidic protein (GFAP) (a marker of astrocytes)
IGF-1, Nrf2, and HO-1 were assessed in the brain tissues by the end of the
experiment. PTZ caused significant decrease in seizure latency and significant
increase in seizure score by the end of the experiment (p
Epilepsy is a serious neurological disorder that impacts about 1%–2% of the world’s population [1]. Epilepsy is accompanied with cognitive deficits which negatively influence life’s quality [2] and about one third of patients with epilepsy show resistance to the established lines of treatment [3]. So, looking into the possible mechanisms of the process of epileptogenesis is mandatory to develop new agents that regulate epileptic seizures during epilepsy. During the period of epileptogenesis, molecular events bring about various changes in the brain structure including loss of neurons, sprouting of mossy fibers, reorganization of synapses, astrocytosis, neurogenesis, etc. [4]. The process of epileptogenesis can be replicated through kindling. Kindling is a process through which we can ignite prolonged seizures with gradually increased duration and degree of behavioral disorder. Kindling is achieved by repetitive sub-threshold applications of any convulsing agent. Chemical kindling via pentylenetetrazole (PTZ) is one way to induce animal models for temporal lobe epilepsy, which is the most common symptomatic refractory form of epilepsy [5]. Oxidative stress is one of the major mechanisms which initiates epilepsy and leads to its progression next to a primary brain insult [6]. Oxidative stress in neurons during epilepsy results from excessive production of reactive oxygen radicals (ROS), apoptotic, inflammatory, immune changes and dysfunction of blood-brain barrier [7, 8]. So, the use of antioxidants as ascorbic acid [9], flavonoids [10], vitamin E [11], L-carnitine [12] etc., protected against epilepsy in animal models.
Nuclear factor-2 erythroid-related factor-2 (Nrf2) is a transcription factor that promotes antioxidant enzymes production such as heme oxygenase-1 (HO-1), superoxide dismutase (SOD) glutathione peroxidase (GPx), thus renders cells oxidative stress resistant [13]. Nrf2 is the principal transcription factor activated succeeding oxidative stress in epilepsy representing an endogenous adaptive mechanism that protect against the neuronal oxidant insult [14]. In case of temporal lope epilepsy, Nrf2/antioxidant response element (ARE)-dependent HO-1/NAD(P)H dehydrogenase (quinone)-1 (NQO-1) production was found to ameliorate oxidative injury induced by glutamate. Nrf2/HO-1 pathway activation can be considered a potential therapeutic avenue in intractable epilepsy [13, 15]. Moreover, IGF-1 refines the central nervous system development, aids neurons to differentiate, mature and connect properly. It urges axons to grow and neurons to survive [16]. Insulin like growth factor-1 (IGF-1)/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway has been recently featured as a protective pathway against oxidative stress in astrocytes. It was also implicated in ameliorating neurodegenerative changes in Alzheimer’s [17]. Recently, it has been found that IGF-1 induce a transcriptional program for mitochondrial biogenesis through induction of Nrf2 expression [18]. So, we suggest that there would be a cross talk between IGF-1 and Nrf2/HO-1 pathways.
Plant phenolics including vanillic acid (VA), the major constituent of vanilla
bean and pod extracts [19], are commonly used as a food flavour. It has been
reported that they have neuroprotective properties and can be used in treatment
of several disorders [20]. Also, vanillic acid demonstrated antioxidative,
antihypertensive and anti-inflammatory properties [21, 22, 23]. Recently, VA was
reported to activate the Nrf2/HO-1 pathway in neurodegenerative disorders as
Alzheimer’s disease. Also, VA exerted its antioxidant effect through the protein
kinase B (Akt)/glycogen synthase kinase 3
Thirty male sprague-dawely rats weighs 170–190 g were accommodated in standard cages at Medical Experimental Research, Mansoura, Egypt. Animals fed on standard diet and water adlibitum. Animal care was done according to The Care and Use of Laboratory Animals (1996, published by National Academy Press, 2101 Constitution Ave. NW, Washington, D.C. 20055, USA). The IRB-committee approved all experimental procedures (code #R.20.11.1098.r).
Rats were randomly (sealed envelopes) partitioned into 3 equal groups (10 rats in each) as the following: (a) Normal group: normal animals received 0.2 mL saline via intraperitoneal injection (i.p.) for 2 weeks, (b) PTZ group: rats were i.p injected with 50 mg/kg PTZ (purchased from Sigma Aldrich, USA) in 0.2 mL every other day for 2 weeks [25] and (c) VA + PTZ group: the same as PTZ group except that rats were pretreated with VA (purchased from Sigma Aldrich, USA) daily 50 mg/kg VA by oral gavage 30 min prior to PTZ for 2 weeks [22].
Subsequent to each PTZ injection, rats were placed in transparent Plexiglas cages to record their 30 min convulsive behavior by a video camera after PTZ administration for 2 weeks or 7 records or trials (every other day). Latency to seizure onset (sec) and seizure score were recorded. Scoring of seizure severity was done based on Racine’s scale (0 = normal, non-epileptic activity, 1 = movements of mouth and face, hyperactivity, grooming, sniffing, scratching, wet dog shakes, 2 = head nodding, staring, tremor, 3 = forelimb clonus, forelimb extension, 4 = rearing, salivating, tonic clonic activity and 5 = falling, status epilepticus) [26].
At the end of the study, a high dose of Na
Hippocampal regions of the brain were homogenized in 1–2 mL cold phosphate
buffered saline (50 mM in EDTA (1 mM) at pH 7.5. Then, centrifugation done at
4000 rpm at 4
Brain tissues (n = 4 rats in each group) staining with hematoxylin and eosin (H&E) was achieved based on our preceding work [12]. Neuronal loss and pyknotic nuclei were assessed by light microscopy in the CA3 region of hippocampus [12].
The mRNAs encoding for antioxidant transcription factor, Nrf2 and heme oxygenase
(HO)-1 were identified by real-time PCR in brain tissues. According to the
manufacturer’s instructions, we isolated the total RNA from brain tissue
specimens. RNA was quantified spectrophotometrically, and its quality was
determined by agarose gel electrophoresis and ethidium bromide staining. cDNA was
synthesized from 1
Sagittal section (40
GraphPad Prism version 5.0 (GraphPad Software Inc., CA, USA) was used for
statistical analysis. Statistical significance in behavioral parameters was
determined by two-way analysis of variance (ANOVA) with Bonferroni posthoc test.
Also, one-way ANOVA with Tukey’s posthoc test was applied to find the statistical
significance in biochemical, molecular, and histochemical parameters. Also,
Pearson correlations between IGF-1 expression and other studied parameters were
calculated. Statistical significance is determined when p
In comparison with PTZ group, rats with VA administration displayed significant
reduction in the PTZ-induced seizure score in trial 4 (p
Effects of VA on behavioral effects in PTZ-induced seizures.
(A) seizure score. (B) seizure latency (Sec). Two-way ANOVA test, results were
represented as mean
The concentration of MDA in the brain tissues showed a significant increase in
the PTZ group as opposed to the control group (p
Effects of VA on oxidative stress markers in PTZ-induced
seizures. (A) Malondialdehyde (MDA) (nmol/g brain tissues). (B) Catalase (CAT)
enzyme activity (U/g brain tissues). (C) Total antioxidant capacity (TAC).
One-way ANOVA test, results were represented as mean
In the hippocampus, the Nrf2 and HO-1 expression at the mRNA level showed
non-significant increase in the PTZ rats as opposed to the control ones
(p
Effects of VA in brain tissues. (A) The expression of Nrf2. (B)
The expression of heme oxygenase (HO)-1. Two-way ANOVA test, results
were represented as mean
Control rats displayed normally shaped neurons with normal number in the hippocampal CA3 subfield with histopathological examination (Fig. 4A–B), while neurons in the CA3 region in rats with PTZ were irregularly arranged, of diminished number and showed pyknosis (darkly stained nucleus and cytoplasm) (Fig. 4C–D). However, VA expanded the territory of normal neurons and decreased the number of abnormal ones in the hippocampus CA3 region (Fig. 4E–F).
Effects of VA on morphology of CA3 hippocampal region from
different groups. Brain specimens showing (A) normal structure and thickness of
hippocampal regions (CA3,2,1) (H&E, 100
Fig. 5A displays a significant rise in the GFAP positive cells number (a marker
of astrocytosis) in the hippocampus CA3 subfield in the PTZ group as opposed to
the control one (p
Moreover, the ROI of IGF-1 positivity in the hippocampus CA3 region
significantly decreased in the PTZ group as opposed to the control one (p
Effects of VA on GFAP (marker of astrocytes and astrocytosis)
expression in CA3 hippocampal region. (A) Represents the mean of GFAP-positive
cells per high power field (HPF) in the CA3 region in different groups. Brain
specimens from (B) normal control group shows normal expression (red arrows) of
GFAP in CA3 region of hippocampus (400
Effects of Stevia on IGF-1 expression in CA3 hippocampal region
by immunohistochemistry. (A) Represents the mean area of interest (ROI) of IGF-1
expression per high power field (HPF) in different groups. Brain specimens from
normal control group show (B) different regions of hippocampus (CA1, CA2, and
CA3) at low power (100
The expression of IGF-1 showed a negative significant correlation with the
PTZ-induced epileptic seizure score (p = 0.0067) (Fig. 7A), a
non-significant positive correlation with the seizure latency (p =
0.2401) (Fig. 7B). Also, the oxidative stress markers showed negative
correlations between IGF-1 and MDA (p = 0.0016) (Fig. 7C), and positive
correlation between IGF-1 and CAT enzyme activity (p
Pearson correlations of IGF-1 expression. (A) Seizure score.
(B) Seizure latency. (C) MDA. (D) Catalase enzyme activity. (E) Total antioxidant
capacity. (F) GFAP. (G) Nrf2. (H) HO-1. r = correlation coefficient, p =
probability significance (p
With an alternate day PTZ injection for two weeks, our study brought about a substantial increase in the PTZ-induced seizures, the GFAP expression, and the extent of neuronal loss. Also, PTZ worsened the oxidative state and decreased the IGF-1 level with insignificant changes in the Nrf2 and HO-1. Moreover, vanillic acid exhibited an antiepileptic and a neuroprotective role, since it markedly demoted the PTZ-induced seizures score, neuronal loss, and astrocytosis. Additionally, vanillic acid amended the oxidative state and elevated the Nrf2, HO-1, and IGF-1 expression.
In our study, high scored PTZ-induced seizures with a shortened latency and an extensive neuronal loss in the hippocampal region portrayed the existence of epileptogenesis. Also, PTZ mounted the GFAP expression signifying an expanded territory of astrocytes. A two weeks daily administration of 50 mg/kg vanillic acid decreased the score of PTZ-induced seizures, neuronal loss, and astrocytosis and prolonged the seizures latency. The apparent neuroprotective role of vanillic acid has been captured in various neurological disorders. Ullah et al. [29] reported that a 30 mg/kg vanillic acid protected against lipopolysaccharide-induced neuroinflammation, amyloidogenesis, memory deficit, and neurodegeneration in mice brain with a hopeful role in Alzheimer’s disease. Khoshnam et al. [30] used a transient bilateral common carotid artery occlusion model followed by reperfusion with a 14 days vanillic acid administration prior to the hypoperfusion. Their study revealed a protecting outcome of vanillic acid in ischemia reperfusion-induced injury in a rat brain since it diminished reactive hyperemia and improved the disruption of blood brain barrier (BBB). Also, in a rotenone-induced Parkinson’s disease rat model, Sharma et al. [31] displayed a protective effect of co-treatment of vanillic acid and levodopa-carbidopa, motor defects, and oxidative stress-induced neuronal damage indicating possible neuroprotection in Parkinson’s disease. In consistence with the results of the current study, Nesterkina, et al. [32]reported an anticonvulsant effect of vanillic acid against PTZ-induced seizures by minimum effective doses inducing clonic-tonic convulsions and tonic extension and against maximal electroshock induced seizures in mice. However, to the best of our knowledge, our study exclusively offers a novel dimension to the neuroprotective role of vanillic acid. We present vanillic acid not only as an anticonvulsant but also as a potential antiepileptic since it reduced the seizure score, neuronal loss, astrocytosis, and prolonged seizure latency.
Astrocytosis has a crucial role in the process of epileptogenesis intertwined
with oxidative stress which is a key hallmark in epilepsy. Puttachary et
al. [33] mentioned that reactive astrocytosis is accompanied by
hyperexcitability due to the down-regulation of glutamine synthase enzyme
resulting in high levels of un-metabolized glutamate. Moreover, this
hyperexcitability results in the overproduction of free radicals overwhelming the
antioxidant capacity and causing oxidative stress. This is well consistent with
our findings since PTZ exhibited a high GFAP level, a marker for astrocytosis,
and high oxidative stress markers. In the current study, vanillic acid reduced
the GFAP expression and the oxidative stress markers. The neuroprotective effect
of vanillic acid demonstrated in our study seems to be owing to its antioxidant
properties and slowing down astrocytosis. This is in harmony with Amin et
al. [24] who mentioned that 30 mg/kg vanillic acid administration
for 3 weeks after A
Nrf2 is a transcription factor that promotes the production of many antioxidant enzymes including glutathione peroxidases and HO-1 [13]. In the current study, a 50 mg/kg alternate day PTZ injection for 14 days showed an insignificant change in the expression of Nrf2 and HO-1 between the PTZ and the controls. This is consistent with Wang et al. [14] who used repeated administration of sub-convulsive electric stimulation for a 15 days amygdala kindling rat model. On the other hand, Li et al. [34] displayed a lowered expression of Nrf2 and HO-1 with a daily 37 mg/kg PTZ for 40 days in mice. The difference might emerge from the fact that Nrf2 is activated in epilepsy after oxidative stress as an endogenous adaptive mechanism to protect against the neuronal oxidant insult [14]. Vanillic acid upregulated the Nrf2/HO-1 expression which is in line with its antioxidant effect. The up-regulation of Nrf2/HO-1 expression has been a well-investigated notion as a potential therapeutic target for epilepsy [13]. Chen et al. [15] displayed amelioration of glutamate-induced oxidative damage by activating Nrf2/HO-1 Signaling Pathway in HT22 Cells. Moreover, Amin et al. [24] showed a neuroprotective role of vanillic acid through Nrf2/HO-1 up-regulation in a mice model of Alzheimer’s disease. So, up to the best knowledge, our study exclusively shows a neuroprotective role for vanillic acid in a PTZ-induced epilepsy model through up-regulation of Nrf2/HO-1. However, we did not measure the expression of Nrf2/HO-1 at the level of protein by western blotting which is considered as a limitation of the current study to be considered in further studies.
IGF-1 enhances neuronal differentiation, maturation, survival, and axon growth
[16]. This is well depicted in our findings since vanillic acid elevated the
PTZ-induced lowered levels of IGF-1. Also, there was a positive correlation
between IGF-1 and Nrf2 and a negative one between IGF1 and astrocytosis, neuronal
loss, seizures score, and oxidative stress markers. Our hypothesis of the
cross-talk between the two pathways of IGF-1 and Nrf2/HO-1 has been postulated in
various studies. Mahran [35] reported an improvement of cisplatin-induced
nephrotoxicity through growth hormone administration that increased IGF-1
expression along with up-regulation of Nrf-HO1 proposing IGF-1 as a mediator for
Nrf2/HO-1 which eventually opposes the oxidative damage in renal cells induced by
cisplatin. Bailey-Downs et al. [36] showed that IGF-1 deficiency
impaired the vascular anti-oxidant responses by impairing Nrf-2 expression and
its target genes. Also, Wang et al. [37] showed that IGF-1 treatment
promoted the nuclear translocation of Nrf2, and up regulated the expression of
its downstream gene (HO-1) in an Alzheimer’s disease model with improvement in
the oxidative state. Moreover, PI3-K inhibition abolished the protective effect
of IGF-1 on
In conclusion, vanillic acid demonstrated a neuroprotective and an anti-epileptic effect against PTZ-induced epilepsy. This outcome might be due to suppression of PTZ-induced astrocytosis, and oxidative stress. Vanillic acid seems to act through up regulation of IGF-1 and Nrf2. Moreover, the potential cross-talk between IGF-1 and Nrf2 could share in the neuroprotective and the anti-epileptic effect of vanillic acid in a PTZ-kindled rat model.
MAE, AY, AMH, EME and MAA conceived and designed the study. MAE, SS, WO, AMH performed all behavioral experiments and conducted biochemical and molecular experiments. AMH, SS, WO, EME, MAA, and WAA collected the data and performed statistical analysis. MAE, AY, EME, WO, AY, SS and AMH wrote the manuscript. All authors reviewed and approved the final draft.
The IRB-committee approved all experimental procedures (code #R.20.11.1098.r).
The authors would also like to express their gratitude to King Khalid University, Saudi Arabia, for providing administrative and technical support.
The authors would also like to express their gratitude to the Deanship of Scientific Research at King Khalid University, Saudi Arabia for funding this project through the General Research Program (grant # G.R.P/100/41).
The authors declare no conflict of interest.
The data will be available on reasonable request.