The current study investigated the effects of stevia extracts on a PTZ-induced
epileptic rat model and its potential mechanism. Thirty male Sprague-Dawley 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 2 weeks, and (3)
PTZ+ Stevia group: received PTZ and stevia (200 mg/kg orally daily) for 4 weeks
(2 weeks before the start of PTZ treatment and 2 weeks with PTZ administration).
The first jerk latency and the seizure score were assessed in rats. Also, brain
tissue samples were collected by the end of the experiment, and oxidative stress
markers (catalase, MDA, and total antioxidant capacity (TAC)) were measured by
biochemical analysis in hippocampal brain homogenates. Also, in the hippocampus,
the expression of IL6 and Bcl-2 at the mRNA level and expression of Sirt-1, P53,
caspase-3, GFAP, and NF-kB in CA3 hippocampal region by immunohistochemistry was
investigated. PTZ substantially increased the seizure score and decreased the
seizure latency. Also, PTZ significantly increased MDA, GFAP, IL-6, NF-kB,
caspase-3, and p53 and significantly reduced Sirt-1, TAC, and Bcl-2 in
hippocampal tissues compared to the control group (p
Epilepsy is a serious neurological disorder that impacts about 0.5%–2% of the world’s population . Intractable forms of epilepsy trouble approximately 30% of patients . Moreover, associated comorbidities such as cognitive impairment burden patients with epilepsy and markedly influence the quality of life . Therefore, a thorough grasp of the mechanisms behind epileptogenesis would open new avenues for novel anti-epileptic agents. Previous experimental studies have documented the role of oxidative stress, inflammatory cytokines, apoptosis, and autophagy in epileptogenesis, so therapeutic agents that target those mechanisms are considered good antiepileptic candidates [4, 5, 6].
Silent information regulator-1 (Sirt-1) is the first gene found in the mammalian
sirtuins family genes. Normally, it is located in the nucleus and acts as a
histone deacetylase of NAD
Nowadays, herbal supplements and alternative medicine represent a new era in the
prevention and treatment of diseases. Stevia Rebaudiana Bertoni
(Stevia R.) is a bush of the Asteraceae family local to South America,
particularly to the Northeast of Paraguay. Its leaves are commonly used as a
powerful alternative to artificial sweeteners . Besides their sweetening
properties, stevia extracts possess many potential therapeutic effects such as
hypotensive, hypoglycemic, anti-inflammatory, anti-tumor, and immunomodulatory
effects . Also, stevia contains several phenolic compounds such as
flavonoids, and phenolics which possess important antioxidant properties .
Few studies have investigated the neuroprotective effects of stevia extract and
its derivatives against scopolamine-induced learning and memory deficits  and
fructose-induced neuronal damage in the hippocampus, amygdala, and spinal cord
. Moreover, Rebaudioside A (derivative of stevia) significantly attenuated
the PTZ-induced convulsive and EEG changes . Mei et al. 
concluded that Isosteviol sodium (a derivative of stevia) protects against
oxidative stress and apoptosis in cardiomyocytes via activation of the
Thirty male Sprague-Dawley rats weighing 170–190 g were housed in standard cages at the experimental research center, Mansoura Faculty of Medicine, Egypt. Animals were fed on a standard diet and water ad libitum. Animal care was done in agreement with The Care and Use of Laboratory Animals (1996, distributed by the National Academy Press, 2101 Constitution Ave. NW, Washington, DC20055, USA). The IRB committee has approved all experimental procedures (code #R.20.11.1098).
The leaves of Stevia R. plants were purchased from Stevia International Company from Agro-industry Product (SICAP), Cairo, Egypt. All steps of isolation and methanolic extraction of S. Rebaudiana leaves were mentioned in a previous work by our research group . The methanolic extracts of Stevia R. were dissolved in 1.0 mL saline at a dose of 200 mg/kg orally via gastric gavage once daily for 4 weeks (2 weeks before PTZ injection and 2 weeks after PTZ injection) .
Rats were randomly (closed sealed envelopes) subdivided into 3 equal groups (each contains 10 rats) as follows: (a) Control group: normal animals received 0.5 mL saline via gastric gavage for 4 weeks with 0.2 mL saline via intraperitoneal injection (i.p.) for the last 2 weeks, (b) PTZ group: as the control group but rats received PTZ (50 mg/kg in 0.2 mL saline) via i.p. injection on an alternate day for the last 2 weeks of the experiment  and (c) Stevia R. extract (St)+ PTZ group: as the PTZ group but rats were pretreated with Stevia in a dose of 200 mg/kg daily for 4 weeks (2 weeks before the onset of PTZ treatment and 2 weeks with PTZ administration) . Stevia was given one hour before PTZ on the day of PTZ administration. Fig. 1 shows the study design.
Study design of the experiment. Saline 0.5 mL or stevia extracts dissolved in 0.5 mL was given daily via gastric gavage for 4 weeks (2 weeks before and 2 weeks after PTZ administration). But PTZ was given in 0.2 mL saline in PTZ and PTZ+ Stevia group every other day. On the day of PTZ injection, Stevia was given one hour before PTZ injection. Also, the rat behavior was recoded for 30 min after PTZ injection (trial or record), so we have 7 trials or records, trial 1 at day 1, trial 2 at day 3, trial 3 at day 5, trial 4 at day 7, trial 5 at day 9, trial 6 at day 11 and trial 7 at day 13.
After each PTZ injection, each rat was placed in a transparent Plexiglas cage to record its convulsive behavior for 30 min by a video camera for 7 records or trials. The latency of 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 = mouth and facial movements, 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) .
Once the study has ended, a high dose of Na
Hippocampal regions of the brain were homogenized in 1–2 mL of cold
phosphate-buffered saline (50 mM) in EDTA (1 mM) at pH 7.5 then, centrifuged at
4000 rpm at 4
The mRNA encoding for pro-inflammatory cytokine; interleukin-6 (IL-6) and
anti-apoptotic genes (Bcl-2) was identified by real-time PCR in hippocampal brain
tissue samples. In agreement with the manufacturer’s guidelines, total RNA from
hippocampal brain tissue samples was isolated, then quantified
spectrophotometrically, and its quality was determined by agarose gel
electrophoresis and ethidium bromide staining. cDNA was synthesized from 1
Brain sections of 20
Serial sagittal sections (40
Statistical analysis was done by GraphPad Prism version 5.0 (GraphPad Software,
Inc., California Corporation, USA). The statistical significance within groups
for behavioral parameters was calculated using two-way ANOVA with Bonferroni
post-hoc test, while in other parameters we used one-way ANOVA with Tukey’s
post-hoc test to find the statistical significance. Also, Pearson correlations
between Sirt-1 expression and other study parameters were calculated. p
The PTZ group showed an increase in the score of PTZ-induced seizures starting
from the 2nd trial compared to the 1st trial. Also, in comparison to the PTZ
group, the stevia group showed a significant reduction in the PTZ-induced seizure
score in trials 4 (day 7) and 5 (day 9) (p
Effect of Stevia R. on behavioral effects in
PTZ-induced seizures. (A) Seizure score and (B) seizure latency (Sec).
Data were expressed as mean
The concentration of MDA was significantly higher in hippocampal tissues of the
PTZ group than in the control group (p
|MDA (nmol/g tissues)||CAT (U/g tissues)||TAC (mmol/g tissues)|
|p (One-Way ANOVA)||F = 45.90||F = 27.49||F = 14.55|
|All data are expressed as mean |
The expression of Bcl-2 at the mRNA level in the hippocampal tissue showed a
significant decrease in the PTZ group compared to the control group (p
|p (One-Way ANOVA)||F = 27.95||F = 33.64|
|All data are expressed as mean |
The control group exhibited normal neuronal shape and number in the hippocampal
CA3 region on histopathological examination (Fig. 3A–B). However, PTZ disrupted
the neuronal regular arrangement, reduced their number and resulted in pyknotic
changes (darkly stained nucleus and cytoplasm) in the CA3 hippocampal region
(Fig. 3C–D). Conversely, stevia displayed a significant reduction in the number
of abnormal neurons in the in CA3 hippocampal region (Fig. 3E–F).
Moreover, the thickness, which is represented by black lines in
high power figures (400
Effect of Stevia R. on the morphology of the CA3
hippocampal region from different groups. The brain specimens from normal
control group show (A) normal structure and thickness of hippocampal regions
(CA3, 2, 1) (H&E, 100
Fig. 4A displayed a marked rise in the number of GFAP-positive cells (a marker
of astrocytosis) in the hippocampal CA3 region in the PTZ group in contrast to
the control group (p
Effect of Stevia on GFAP (marker of astrocytes and astrocytosis)
expression in the CA3 hippocampal region. Graph (A) represents the mean
of GFAP-positive cells per high power field (HPF) in the CA3 region in different
groups. Brain specimens from the (B) normal control group show normal expression
of GFAP in CA3 region of hippocampus (red arrows indicate cell bodies, while blue
arrows indicate cell processes) (400
Moreover, PTZ markedly lowered the mean ROI of Sirt-1 positivity in the
hippocampal CA3 region in contrast to the control group (p
Effect of Stevia on Sirt-1 expression in the CA3 hippocampal
region by immunohistochemistry. Graph (A) represents the mean area of
interest (ROI) of Sirt-1 expression per high power field (HPF) in different
groups. Brain specimens from the (B) normal control group show moderate
membranous expression of Sirt-1 (red arrows) in the CA3 region of the hippocampus
PTZ substantially increased the number of caspase-3 and p53-positive cells
(markers of apoptosis) in the hippocampal CA3 region in contrast to the control
Effect of Stevia on apoptotic marker (caspase-3) in the CA3
hippocampal region by immunohistochemistry. Graph (A) represents the mean of
caspase-3-positive cells per high power field (HPF) in different groups. Brain
specimens from the (B) normal control group show minimal nuclear expression of
caspase-3 in the different parts of the hippocampus (red arrows) (400
Effect of Stevia on apoptotic marker (p53) in the CA3
hippocampal region by immunohistochemistry. Graph (A) represents the mean of
p53-positive cells per high power field (HPF) in different groups. Brain
specimens from the (B) normal control group show minimal cytoplasmic expression
of p53 (red arrows) in the CA3 hippocampal region (400
Furthermore, the number of NF-kB-positive cells markedly increased in the PTZ
group compared to the control group in the CA3 hippocampal region (p
Effect of Stevia on NF-kB in the CA3 hippocampal region by
immunohistochemistry. Graph (A) represents the mean of NF-kB-positive cells per
high power field (HPF) in different groups. Brain specimens from the (B) normal
control group show minimal nuclear expression (red arrows) of NF-kB in the CA3
hippocampal region (400
The expression of sirt-1 showed a negative correlation with the PTZ-induced epileptic seizure score (Fig. 9A), a positive correlation with its latency (Fig. 9B), and a negative correlation with GFAP expression (Fig. 9C). Also, oxidative stress markers showed a negative correlation between Sirt-1 and MDA, and positive correlations between Sirt-1 and CAT and TAC correlations (Fig. 9D–F, respectively). Regarding, the apoptotic markers, the Sirt-1 showed a positive correlation with Bcl-2 and negative correlations with caspase-3 and p53 (Fig. 9G–I). Finally, the inflammatory markers, there were negative correlations between Sirt-1 and IL-6, and NF-kB (Fig. 9J–K).
Correlations of Sirt-1 expression with different studied parameters including; seizure score (A), seizure latency (B), GFAP expression (C), MDA concentration (D), catalase enzyme activity (E), total antioxidant capacity (F), Bcl-2 expression (G), caspase-3 expression (H), p53 expression (I), IL-6 expression (J), and NF-kB expression (K). Pearson correlations, r = correlation coefficient and p = probability significance.
The main findings of the current study included that: (a)administration of PTZ every alternate day for two weeks caused a significant increase in PTZ-induced seizures, which was associated with evident neuronal loss, astrocytosis, worsened oxidative stress, and a significant decrease in Sirt-1 and Bcl-2 and a significant increase in inflammatory mediators (NF-kB and IL-6) and apoptosis-related proteins (p53 and caspase-3) expressions in the hippocampal CA3 region and (b) pretreatment with stevia has neuroprotective and anti-epileptic effects accompanied by significant attenuation in PTZ-induced neuronal loss, astrocytosis, oxidative stress, and inflammatory cytokine IL-6 and apoptotic markers (p53 and caspase-3) with the upregulation of Sirt-1 and anti-apoptotic protein (Bcl-2) in the CA3 hippocampal region.
The study at hand demonstrated, with alternate day PTZ injections for two weeks, typical epileptic seizures with a significantly shortened latency. Additionally, PTZ exhibited evident hippocampal neuronal loss and escalation in astrocytes population, which is supported by an increase in GFAP. Also, the current study demonstrates the alleged neuro-protection of stevia rebaudiana in the PTZ-induced epilepsy model through decreased neuronal loss and reactive astrocytosis with a low GFAP expression. Stevia R. ameliorated the high Racine’s convulsion score and prolonged the latency of epileptic seizures. These findings conform to the neuroprotective actions of stevia extract and its derivatives displayed in previous studies such as scopolamine-induced memory loss , fructose-induced plurimetabolic changes in the hippocampus and amygdala  and the anti-convulsive effects for Rebaudioside A (derivative of stevia) on convulsions induced by a single high dose of PTZ (70 mg/kg) . However, in the current study, we demonstrated the role of Stevia R. during epileptogenesis induced by repeated administration of subconvulsive doses of PTZ suggesting not only anticonvulsive but also anti-epileptic effect for Stevia extracts.
Glial cells have been considered just a brain glue for neurons, however, a crucial role has been reported for glial cells especially astrocytes in the development of neurodegenerative diseases, including epilepsy. Upon brain insult, astrocytes become reactive and participate in hyper-excitation, neurotoxicity, and seizure spreading. After activation, astrocytes secrete pro-inflammatory mediators to initially protect, adapt, and return the central nervous system to its regular function. However, if the insult is maintained, persistent activation of inflammatory pathways contributes to the generation of seizures, activation of neuronal death pathways, and compromising the oxidative state . GFAP is an intermediate filament found specifically in astrocytes, and its accumulation indicates astrocyte activation, which is termed astrocytosis. The current study displays GFAP up-regulation with PTZ indicating astrocytosis alongside its ensuing detrimental effects, which is consistent with previous studies [25, 26, 27]. Moreover, the current work demonstrated down-regulation of GFAP with stevia rebaudiana treatment indicating regression of astrocytosis with a potential decline in its repercussions, inflammation, apoptosis, and oxidative stress. To the best of our knowledge, our study is the first study to demonstrate the effect of Stevia R. on astrocytosis in drug-induced epileptogenesis.
Inflammation stands as one of the three pillars of epileptogenesis along with
apoptosis and oxidative stress. Various studies have tied hippocampal neuronal
damage with an ongoing process of inflammation. IL-6, a pro-inflammatory
cytokine, is normally concentrated in low quantities within the brain and is
up-regulated with seizures. IL-6 up-regulation with PTZ decreases hippocampal
neurogenesis and increases neuro-degeneration and astrocytosis . The
activation of nuclear transcription factor NF-kB can efficiently induce extensive
gene expression and regulate the transcription of cytokine target genes, playing
a regulatory role in inflammation and immune responses. According to
Bertogliat et al.  high hippocampal expression of NF-kB was reported
to induce neuronal death and activation of glial cells, which promotes the
pathological changes with PTZ-induced seizures. Consistent with these studies, in
this study, we found an increase in IL-6 expression and NF-kB in hippocampal
neurons from the PTZ group. On the other hand, Stevia R. down-regulated
the hippocampal expression of both NF-kB and IL-6, implying an anti-inflammatory
role for stevia in epilepsy. In consistence with these findings, Latha et
al.  demonstrated that hydro-alcoholic extract of stevia leaves
(500 mg/kg) attenuates the inflammatory process and oxidative stress in the
lipopolysaccharides-induced acute liver injury mainly via the reduction of
pro-inflammatory cytokines, e.g., TNF-
Apoptotic hippocampal neuronal cell death is implicated in the process of epileptogenesis. PTZ-induced apoptosis is mediated by the activation of enzymes such as caspase-3, increased expression of apoptotic proteins such as p53, and decreased levels of anti-apoptotic Bcl-2 protein. The current study demonstrated a significant increase in caspase-3, p53 with a significant reduction in Bcl-2 in brain tissue of PTZ-treated rats, which is in agreement with previous studies by Hussein et al.  and Hamdy et al. . Moreover, Stevia R. exerted an anti-apoptotic action, as evidenced significant decline in caspase-3 and p53 with high expression of Bcl-2 with stevia-treated rats. These findings agree with the study of Hussein et al.  that reported anti-apoptotic effects for Stevia R. in diabetic cardiomyopathy by downregulating the p53 and caspase-3. Oxidative stress contributes to neuronal hyperexcitability and degeneration. Oxidative stress is intertwined with apoptosis in the pathogenesis of epilepsy since it induces the loss of the mitochondrial membrane potential and releases pro-apoptotic proteins causing downstream activation of caspase-dependent and caspase-independent cell death . Our findings showed that PTZ worsened the oxidative state, increasing the MDA level marking high lipid peroxidation along decreasing the total antioxidant capacity (TAC). These findings conform to our previous studies [6, 31, 33]. Alternatively, Stevia R. improved the oxidative stress state by increasing the activity of the catalase enzyme and the concentration of TAC and decreasing the MDA level, which confirms its antioxidant effects in the PTZ-induced epileptic rat model. The anti-oxidant effect of Stevia R. was demonstrated by our research group in diabetic cardiomyopathy .
The main objective in the current study was to test the role of Sirt-1 in the pathogenesis of epilepsy owing to its presumed neuroprotective role. Sirt-1 is highly expressed in both neuroglial and nerve cells in the brain. Kaewmool et al.  demonstrated that Sirt-1 exerts neuroprotective effects in rat models of microglial activation-induced neurodegenerative disease and Romeo-Guitart et al.  demonstrated that activation of Sirt-1 significantly improved motor nerve regeneration and recovery. Moreover, Dabke and Das  demonstrated that ketogenic diet metabolites protect the cultured cells via upregulation of Sirt-1 expression. In the current study, we found significant down-regulation of the expression of Sirt-1 expression in the PTZ group. It has been demonstrated that Sirt-1 was found in the cytoplasm of embryonic and adult neural precursor cells (NPCs) which was transiently translocated within 10 min to the nucleus in response to differentiation stimulus, then gradually retranslocated to the cytoplasm after several hours (~3 hrs) . Also, Teertam et al.  found a significant reduction in nuclear expression of Sirt-1 with a significant increase in immunopositivity the cytoplasm and cell processes of neurons in both young and aged experimental rats after ischemic insults. In the current study, the rats were euthanized 16 hrs. after the last stevia and PTZ injection, which might explain the membranous and cytoplasmic not nuclear expression of Sirt-1 in CA3 neurons in the current study. Also, we found that the expression of Sirt-1 showed negative correlations with seizure score, GFAP expression, apoptotic markers (p53, caspase-3), a marker of lipid peroxidation (MDA) and inflammatory mediators IL-6 and NF-kB, and positive correlations with Bcl-2 and seizure latency suggesting that the downregulation of Sirt-1 is linked to the process of the PTZ-induced astrocytosis, apoptosis, inflammation, and oxidative stress. In agreement with these findings, previous studies reported down-regulation of Sirt-1 during epileptogenesis [10, 11, 38]. Moreover, Zhao et al.  demonstrated up-regulation of Sirt-1 in a rodent model of traumatic brain injury (TBI), which protects against neuronal apoptosis in a rodent model of focal brain ischemia and Zhou et al.  demonstrated that suppression of Sirt-1 expression by sirtinol in a rat model of subarachnoid hemorrhage (SAH) worsened neurological deficits, brain edema, disruption of the blood-brain barrier (BBB) and endothelial cell apoptosis suggesting a neuroprotective role for Sirt-1. Also, Chen et al.  found that Sirt-1 upregulates the expression of anti-apoptotic protein, Bcl-2 and inhibits caspase-3 and Bax.
Moreover, we found that treatment of rats with Stevia R. upregulates
the expression of Sirt-1 in CA3 hippocampal region, suggesting that Sirt-1 could
be a mechanism for the neuroprotective effects of Stevia R. against
PTZ-induced epilepsy. This hypothesis was supported by the results of Mei
et al. , who reported that Stevia R. exhibited antioxidant
and anti-apoptotic actions in cardiomyocytes through up-regulation of Sirt-1.
Also, in agreement with these findings, Fu et al.  demonstrated that
an enhancement of Sirt-1 expression by alpha-lipoic acid (ALA) protects
against focal ischemia via up-regulation of the expression of PGC-1
In conclusion, Stevia R. exhibited neuroprotective and anti-epileptic effects on PTZ-induced epilepsy. This effect might be due to the suppression of PTZ-induced astrocytosis, inflammation (downregulating IL-6 and NF-kB), apoptosis (upregulating Bcl-2 and downregulating p53 and caspase-3), and oxidative stress (decreasing MDA and increasing the catalase activity and TAC). Moreover, the up-regulation of sirt-1 takes part in the neuroprotective and anti-epileptic effects of Stevia R. in PTZ-kindled rat model.
EMEN, AMH and MAA, WO conceived and designed the study. WO, SS, AY, AM and NAK performed all behavioral experiments and conducted biochemical and molecular experiments. AMH, EMEN, MAA, and WAA collected the data and performed statistical analysis. AMH, EMEN, WO, AY, SS, MAA wrote the manuscript. All authors reviewed and approved the final draft.
The IRB-committee approved all experimental procedures (code #R.20.11.1098).
The authors would also like to express their gratitude to King Khalid University, Saudi Arabia, for providing administrative and technical support. Also, we acknowledge Dr. Amira Awadalla, Center of Excellence for Genome and Cancer Research, Mansoura Urology and Nephrology Center, Egypt for helping us in molecular studies.
The authors would like to extend their appreciation to the Deanship of Scientific Research at King Khalid University, Saudi Arabia, for funding this project through the research group program under grant number (G.R.P/100/41).
The authors declare no conflict of interest.
The data will be available on reasonable request.