Anti-Inflammation Associated Protective Mechanism of Berberine and its Derivatives on Attenuating Pentylenetetrazole-Induced Seizures in Zebrafish

Article abstract of DOI:10.1007/s11481-019-09902-w

Epileptic seizures are characterized by synchronized discharges of neurons, leading to the activation of inflammatory responses that in turn contributes to seizure progression. Berberine (BBR), a bioactive constituent extracted from berberis, has been known to relieve seizures in rodent models. In this study, we synthesized two derivatives of berberine (BBR-D1 and BBR-D2) to compare their seizure reducing effect with BBR in pentylenetetrazole (PTZ)-induced seizures in zebrafish. We found a structure-activity relationship between hydrophilic/hydrophobic composition of the derivatives and their anticonvulsant activity. We also investigated the underlying mechanism related to their anti-inflammatory effect during seizures. BBR and its derivatives increased the seizure onset latency and suppressed the seizure-like behavior after PTZ treatment. Zebrafish larvae pretreated with BBR and its derivatives showed recovery on c-fos expression and neuronal discharges during seizures. The inflammatory responses occurred during the progression of seizures, including the recruitment of macrophages and neutrophils as well as an up-regulation of tumor necrosis factor alpha (TNFα), interleukin 1 beta (il1β), and interleukin 6 (il6). This effect was significantly suppressed by the pretreatment of BBR and its derivatives. Our results suggest that BBR and its derivatives attenuate PTZ-induced seizures and modulate anti-inflammatory effect to potentially protect zebrafish from the occurrence of further seizures. From the tested compounds, BBR-D1 (the hydrophilic berberrubine) showed the strongest seizure reducing effect. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s11481-019-09902-w

Journal of Neuroimmune Pharmacology
https://doi.org/10.1007/s11481-019-09902-w
ORIGINAL ARTICLE
Anti-Inflammation Associated Protective Mechanism
of Berberine and its Derivatives on Attenuating
Pentylenetetrazole-Induced Seizures in Zebrafish
Baoyue Zhang1,2 & Lizhen Wang1,2 & Xiuna Ji1,2 & Shanshan Zhang1,2 & Attila Sik3,4,5 & Kechun Liu1,2 & Meng Jin1,2
Received: 5 May 2019 /Accepted: 10 December 2019
#
Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Epileptic seizures are characterized by synchronized discharges of neurons, leading to the activation of inflammatory responses
that in turn contributes to seizure progression. Berberine (BBR), a bioactive constituent extracted from berberis, has been known
to relieve seizures in rodent models. In this study, we synthesized two derivatives of berberine (BBR-D1 and BBR-D2) to
compare their seizure reducing effect with BBR in pentylenetetrazole (PTZ)-induced seizures in zebrafish. We found a
structure-activity relationship between hydrophilic/hydrophobic composition of the derivatives and their anticonvulsant activity.
We also investigated the underlying mechanism related to their anti-inflammatory effect during seizures. BBR and its derivatives
increased the seizure onset latency and suppressed the seizure-like behavior after PTZ treatment. Zebrafish larvae pretreated with
BBR and its derivatives showed recovery on c-fos expression and neuronal discharges during seizures. The inflammatory
responses occurred during the progression of seizures, including the recruitment of macrophages and neutrophils as well as an
up-regulation of tumor necrosis factor alpha (TNFα), interleukin 1 beta (il1β), and interleukin 6 (il6). This effect was signifi-
cantly suppressed by the pretreatment of BBR and its derivatives. Our results suggest that BBR and its derivatives attenuate PTZ-
induced seizures and modulate anti-inflammatory effect to potentially protect zebrafish from the occurrence of further seizures.
From the tested compounds, BBR-D1 (the hydrophilic berberrubine) showed the strongest seizure reducing effect.
.
.
.
.
.
Keywords Epilepsy PTZ Berberine Anticonvulsant Anti-inflammation Zebrafish
Baoyue Zhang, Lizhen Wang and Xiuna Ji contributed equally to this
work.
Introduction
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s11481-019-09902-w) contains supplementary
material, which is available to authorized users.
One of the most common neuronal disorders is epilepsy,
which is characterized by the pathologically synchronized dis-
charges of neurons, termed seizures. Seizures can manifest in
convulsive behavior (Baraban et al. 2005; Stafstrom and
Carmant 2015). Neuronal loss, inflammation, and oxidative
stress induction are all observed in epileptic seizures (Choo
et al. 2018; Fontana et al. 2018; Jin et al. 2018a; Okuneva et al.
2016; Vezzani et al. 2011). Epilepsy is found in approximately
1% of the general population and its treatment mainly relies on
antiepileptic drugs (AEDs) (Devinsky et al. 2018).
Unfortunately, one third of epileptic patients fail to response
to existing AEDs, highlighting the need to find bioactive com-
pounds to defeat this disease (Franco et al. 2016).
* Meng Jin
mjin1985@hotmail.com
1
Biology Institute, Qilu University of Technology (Shandong
Academy of Sciences), 28789 East Jingshi Road,
Ji’nan 250103, Shandong Province, People’s Republic of China
2
Engineering Research Center of Zebrafish Models for Human
Diseases and Drug Screening of Shandong Province, 28789 East
Jingshi Road, Ji’nan 250103, Shandong Province, People’s Republic
of China
3
Institute of Physiology, Medical School, University of Pecs,
For centuries, berberis was widely used as traditional herb
in oriental countries to treat miscellaneous diseases
(Imenshahidi and Hosseinzadeh 2016; Siddiqui et al. 2017).
Berberine (BBR), the active compound of berberis, has hypo-
glycemic, antioxidant, and anti-inflammatory effects, thus it
Pecs H-7624, Hungary
4
Szentagothai Research Centre, University of Pecs, Pecs H-7624,
Hungary
5
Institute of Clinical Sciences, Medical School, University of
Birmingham, Birmingham B15 2TT, UK

could be used to treat neuronal disorders (Lin and Zhang
2018; Wang et al. 2017). BBR has shown the ability to protect
neurons from Parkinson’s disease-induced cell death in SH-
SY5Y cell line by reducing reactive oxygen species genera-
tion and up-regulating heme oxygenase-1 expression (Bae
et al. 2013). During the course of spinal cord injury rehabili-
tation in rats, injected berberine can relieve inflammation and
enhance oligodendrocyte autophagy contributing to primary
spinal neuronal recovery (Wang et al. 2017). In epilepsy stud-
ies BBR showed the ability to delay the onset of seizure-like
convulsions in pilocarpine-treated rats (Gao et al. 2014).
Additionally, BBR reduced the incidence of spontaneous
spasmodic seizures in rats and restored nuclear factor κB
(NF-κB), interleukin-1-beta (il1β), and tumor necrosis factor
alpha (TNFα) expression in hippocampus, suggesting that
BBR could ameliorate seizures mainly by suppressing inflam-
mation (Sedaghat et al. 2017).
spontaneous swimming of zebrafish larvae is relatively devel-
oped on 5 days post fertilization (dpf), making it a suitable
model for seizures related behavior research (Baraban et al.
2005).
Although the function of BBR on attenuating seizures has
been already revealed, the anticonvulsant effect of two newly
synthesized BBR derivatives remains unknown. This paper
compared the anticonvulsant effect of berberine with its two
derivatives, the hydrophilic berberrubine (hereafter referred to
as BBR-D1) and the hydrophobic ethyl-2-(9-
demethoxyberberine bromide-9-yl) hydroxyacetate (hereafter
referred to as BBR-D2) (Fig. 1). Since BBR has been reported
to suppress seizures in rodents mainly via restoring the anti-
inflammatory mediators (Sedaghat et al. 2017), we investigat-
ed whether the two derivatives also exert anticonvulsant effect
through regulating anti-inflammatory responses.
In clinical applications, the main problem of BBR is the
low bioavailability, probably routing from its rigid structure
and poor solubility in water (Lo et al. 2013; Maeng et al.
2002). To enhance its bioavailability and bioactivity, several
berberine derivatives were designed and synthesized includ-
ing 9-O-substituted and C-8,13-substituted berberine deriva-
tives, 9-OH or 10-OH pseudoberberines, and the
hydroxyberberines (Cheng et al. 2010; Ma et al. 2009;
Pongkittiphan et al. 2015; Shan et al. 2013; Wang et al.
2018). However, the structure–activity relationship between
the synthetic berberine derivatives and their anticonvulsant
activity is lacking.
Zebrafish are widely used to investigate the pathophysiol-
ogy and pharmacology of neurological diseases, including
Parkinson’s disease, motor neuron diseases, and epilepsy
(Babin et al. 2014; Barbalho et al. 2016; Cronin and Grealy
2017; Kundap et al. 2017; Rosa-Falero et al. 2014). Compared
with traditional rodent models, zebrafish has numerous advan-
tages including high number of offspring, low maintenance
cost, and fast brain development (Duy et al. 2017; Hortopan
et al. 2010; Stewart et al. 2014; Torres-Hernandez et al. 2015).
Although the structure of the nervous system of zebrafish is
less complex than in mammalian species, it contains similar
neurons to all higher vertebrate species enabling researchers to
investigate the generation of abnormal neuronal discharges
and the role of inflammation in seizures (Wullimann and
Mueller 2004; Wyckhuys et al. 2009). In addition, the
Materials and Methods
Animal Maintenance
All experimental manipulations concerning zebrafish were
performed in accordance with the NIH Guide for the Care
and Use of Laboratory Animals (No.8023, amended in
1996). Protocols and procedures were reviewed and approved
by the animal committee of the Biology Institute of Shandong
Academy of Sciences and were in accord with the guideline
for the Care and Use of Laboratory Animals of China. Adult
zebrafish was raised separately according to their gender un-
der a 14/10 h of light/darkness cycle photoperiod. Fish were
fed twice a day with live artemia nauplii from commercial fish
food supplier. Salinity of aquarium water was 450–500 μS,
with a pH maintained at 7.0–7.5. Fertilized eggs of zebrafish
were collected from natural mating of sexually mature
zebrafish bred, then washed and transferred to 6-wells cell
culture plates. 20 eggs per well were kept with 4 mL of aquar-
ium water. Wild-type zebrafish larvae were used for HPLC
analyses, seizure-like behavioral tests, macrophage migration
tests, quantitative real-time PCR (qPCR), and electroenceph-
alogram (EEG) recording. The larvae of neutrophil-specific
zebrafish line MPO: GFP strain (green fluorescent protein
expressed specifically in neutrophils driven by myeloperoxidase
Fig. 1 Structure and synthesis of BBR-D1 and BBR-D2. a Structure of BBR. b High temperature and vacuum were used to generate BBR-D1. c Ethyl
bromoacetate and K2CO3 at 80 °C were used to prepare BBR-D2.

promoter) was used for neutrophil migration assays (Renshaw
et al. 2006).
was maintained at 35 °C, and the flow rate was set to
0.8 mL/min. The mobile phase of this experiment was com-
posed by solvent A (2.4% acetic acid solution with 20 mM
ammonium acetate) and solvent B (acetonitrile). Elution con-
dition was: 72% A and 28% B from 0 to 30 min. The detection
wavelength is 345 nm. The retention time (RT; min) of BBR
and its derivatives (hereafter referred to as BBR&Ds) in the
environmental samples and experimental samples were
recorded.
Synthesis of BBR-D1 and BBR-D2
Berberine chloride (5 g, 13.48 mmol) was heated at 190 °C
under high vacuum (20–30 mmHg) for 30 min to give a crude
solid, which was purified by column chromatography
(CH2Cl2/CH3OH = 5/1) to provide BBR-D1 as a purple solid.
BBR-D1 (500 mg, 1.40 mmol), ethyl bromoacetate (701 mg,
4.20 mmol) and K2CO3 (580 mg, 4.20 mmol) in 5 ml of N,N-
Dimethylformamide (DMF) were stirred at 80 °C for 6 h. The
color of the mixture was turned from purple into yellow. The
reaction product was diluted with 10 mL of methanol and
filtered, then the filtrate was evaporated and the resulting solid
was purified by column chromatography to give BBR-D2
Pretreatment of BBR&Ds and PTZ Treatment
BBR (Berberine Chloride Hydrate; B0450, TCI; Tokyo,
Japan) and its derivatives (BBR-D1 and BBR-D2) were
solubilized in ddH2O to prepare stock solution. The final
concentration of BBR&Ds was 100 μM, which was select-
ed based on our preliminary studies. Zebrafish larvae were
incubated in 6-well plates (20 eggs per well), then treated
with BBR&Ds at six different concentrations (1, 10, 100,
1000, 2000, 5000 μM). Then the median lethal concentra-
tion (LC50) of BBR, BBR-D1, and BBR-D2 was deter-
mined. Since LC1 was usually considered as a no-
observed-effect concentration (NOEC) value, we tested an-
ticonvulsant activity of BBR at the concentration below
LC1 (LC1 of BBR is 308.9 μM). As a result, 100 μM
was found to be the minimum concentration required to
significantly inhibit the seizure-like behavior induced by
PTZ (data not shown). Since the LC50 value of BBR-D1
and BBR-D2 was higher than that of BBR, we selected
100 μM for the subsequent experiments to compare the
anti-seizure activity among these three compounds.
1
(490 mg, 79%) as a yellow solid. H NMR (400 MHz,
DMSO-d6): δ 9.95 (s, 1 H, CH=N), 8.95 (s, 1 H, ArH), 8.19
(d, J = 9.2 Hz, 1 H, ArH), 8.01 (d, J = 9.2 Hz, 1 H, ArH), 7.81
(s, 1 H, ArH), 7.10 (s, 1 H, ArH), 6.18 (s, 2 H, -OCH2O-), 5.06
(s, 2 H, -CH2COOCH2CH3), 4.94 (t, J = 6.0 Hz, 2 H, CH2),
4.19 (td, J = 7.2, 14.0 Hz, 2 H, -CH2COOCH2CH3), 4.04 (s, 3
H, OCH3), 3.21 (t, J = 6.0 Hz, 2 H, CH2), 1.21 (t, J = 7.3 Hz, 3
H, -CH2COOCH2CH3); 13C NMR (100 MHz, DMSO-d6): δ
169.30, 150.36, 149.68, 148.18, 146.19, 142.02, 138.07,
133.43, 131.16, 127.27, 123.95, 121.63, 120.88, 120.58,
108.91, 105.94, 102.57, 69.79, 61.26, 57.68, 55.91, 26.86,
+
14.52; ESI-TOF HRMS (m/z): calcd for C23H22NO6 , [M –
Cl]+, 408.1442; found, 408.1450.
Liquid Chromatography (HPLC) of BBR and its
Derivatives
For drug treatment, larvae at 6 dpf were exposed to
BBR&Ds in 6-well plates (20 eggs per well) for 24 h. At
7 dpf, larvae were exposed to 15 mM PTZ (Sigma-Aldrich;
USA) to induce seizures (Baraban et al. 2005). Based on
previous research results, migration of immune cells into
zebrafish brain can be observed within 1 h after PTZ ex-
posure (Jin et al. 2018b). In the present study, we found
that there was no significant difference in c-fos mRNA
expression levels and the number of migrated neutrophils
among 3 min, 30 min, and 60 min after PTZ exposure. In
order to investigate if the anti-seizure effect of BBR&Ds
were indeed rooted in their anti-inflammatory activity, we
chose two different PTZ exposure duration 3 min and
30 min for assays of inflammatory cells recruitment as well
as inflammatory cytokines and c-fos expression (Fig. 2a).
In addition, 100 μM of indomethacin (Sigma-Aldrich;
USA), an anti-inflammatory agent which has been reported
to have anti-seizure effect (Barbalho et al. 2016) was in-
cluded in our study to provide a solid support for the cau-
sality between BBR&Ds-mediated anti-inflammation and
their anti-seizure effect.
Samples of BBR, BBR-D1, and BBR-D2 (100 μM) contain-
ing aquarium water, which were used for pretreatment of 6 dpf
zebrafish were collected and termed “environmental sample”.
The experimental samples refer to the pretreated zebrafish
processed for HPLC investigation. By comparing the environ-
mental samples with the corresponding experimental samples,
we were able to test if BBR, BBR-D1, and BBR-D2 had been
absorbed by zebrafish. For preparing the experimental sam-
ples, pretreated larvae (at least 200 fish per group) were col-
lected into 1.5 mL tubes and immersed in methanol (1 mg per
20 μL) for ultrasonication. Both environmental samples and
experimental samples were centrifuged at 12,000 rpm for
20 min under 4 °C. Supernatant was collected before blow-
dried with nitrogen. The remaining residue was dissolved in
100 μL of 80% methanol, then centrifuged at 12,000 rpm for
15 min at 4 °C. 10 μL of the supernatant was used for HPLC
analysis. HPLC was performed with Agilent 1260 infinity
system (Agilent Technologies, USA) and carried out on
YMC HPLC ODS-A column (250 * 4.6 mmL, 5 μm,
12 nm). During HPLC process, the temperature of column

Fig. 2 The experimental work flow and LC50 evaluation of 6 dpf
zebrafish treated with BBR&Ds. a Larvae at 6 dpf were pretreated
with 100 μM BBR&Ds for 24 h. At 7 dpf, after 15 mM PTZ treatment
for 30 min, pretreated zebrafish were subjected to behavioral tests,
immune cell recruitment assays, and real-time qPCR. To investigate the
association between the anti-seizure and anti-inflammatory activities of
BBR&Ds, we also performed immune cell recruitment assays and tested
the expression of c-fos and inflammatory cytokines 3 min after PTZ
exposure. Untreated zebrafish and zebrafish pretreated with BBR&Ds
while without PTZ were considered as the control (Ctl) and BBR&Ds
alone groups, respectively. b The LC50 of BBR is 632.3 μM; c The LC50
of BBR-D1 is 3012 μM; d The LC50 of BBR-D2 is 994.5 μM. n ≥ 30 per
group.
Seizure Latency and Locomotor Activity Tests
whirlpool swimming; in stage III, larvae showed seizure-like
convulsions followed by a loss of posture. The time duration
from initial exposure to PTZ until zebrafish reach each con-
vulsion stage was recorded to assess the influence of
BBR&Ds pretreatment on seizure onset latency. At least 20
larvae in each group were tested individually.
Pretreated zebrafish larvae were transferred into 48-well plates
at 7 dpf (one larva per well), then left in aquarium water
(500 μL per well) for acclimation. After 30 min of acclima-
tion, aquarium water was replaced by 500 μL of 15 mM PTZ.
The locomotion activities of each larva were recorded imme-
diately after PTZ treatment by Zeblab video-tracking system
(Viewpoint, Lyon, France) for 30 min. Digital tracks and av-
erage speed were analyzed in every minute. At least 27 larvae
in each experimental group were included in this experiment.
Based on a previous report, latency period of PTZ-induced
seizures was characterized by three stages (Baraban et al.
2005; Siebel et al. 2015). In stage I, larvae showed an increas-
ing activity on swimming; in stage II, larvae presented a rapid
Zebrafish Neutrophil and Macrophage Recruitment
Assays
Larvae used for recruitment assays were treated with 200 mM
1-phenyl-2-thiourea (Sigma-Aldrich; USA) until performing
immune cell recruitment assays to avoid pigment formation.
At 6 dpf, zebrafish larvae were pretreated with BBR&Ds for
24 h. For neutrophil recruitment assays, BBR&Ds pretreated

MPO: GFP zebrafish larvae were exposed to 15 mM PTZ for
3 min and 30 min before imaging with a fluorescent micro-
scope (AXIO Zoom V16; ZEISS; Germany). We also moni-
tored neutrophil migration into the head of MPO: GFP
zebrafish exposed to 15 mM PTZ for 3 min. For macrophage
recruitment assays, pretreated zebrafish were exposed to
15 mM PTZ for 3 min and 30 min before neutral red staining
to visualize macrophages (Herbomel et al. 2001). Larvae were
washed, and then stained by 2.5 mg/mL neutral red dye for
1.5 h at 28.5 °C.
Enzyme Linked Immunosorbent Assay (ELISA)
In addition to the mRNA level measurement, ELISAwas per-
formed to test the protein levels of the inflammatory cytokine
in animals with PTZ-induced seizures. Pretreated larvae were
exposed to 15 mM PTZ for 3 min, washed, then the heads of
zebrafish (n = 50) were used for protein extraction. The con-
centration of total protein was determined by Enhanced BCA
Protein Assay Kit (Solarbio; China) according to the manu-
facturer’s protocol. Zebrafish ELISA kits 1608 and 1612
(ZGene Biotech Inc.; China) were used to detect the protein
levels of IL1β and TNFα, respectively. ELISAwas performed
according to the manufacturer’s protocol.
RNA Extraction and Real-Time Quantitative PCR
Larvae at 6 dpf were pretreated with BBR&Ds for 24 h. At
7dpf, larvae were exposed to PTZ for 3 min and 30 min,
then washed and collected for qPCR. Total RNA was ex-
tracted from 20 heads of larvae in each treatment using
EASY spin Plus RNA Mini Kit (RN2802; Aidlab
Biotechnologies; Beijing, China). Extracted RNA of each
group was reverse transcribed into cDNA according to the
manufacturer’s instructions of PrimeScript™ RT Master
Mix (Takara; Tokyo, Japan). qPCR was set up to investi-
gate the expression levels of four target gene (c-fos, il1β,
il6, and TNFα).
qPCR was performed using SYBR® Premix
DimerEraser™ (Takara; Tokyo, Japan) and the Light
Cycler® 96 System (Light Cycler® Instrument; Roche;
Switzerland). At the end of the PCR cycles, melting curve
analysis was performed to validate the specific generation of
the expected PCR product. All relative quantifications were
carried out in triplicates, and then normalized to housekeeping
gene rpl13a. Data were analyzed by the LC 96 Application
Software and calculated according to the 2-ΔΔCt method to
quantify the gene expression (Livak and Schmittgen 2001).
The primers used in this study were listed in Supplementary
Table 1.
Electroencephalogram (EEG) Recording
Zebrafish larvae were treated with BBR&Ds for 24 h before
EEG recording. Each larva was immobilized by 2% low melt-
ing point agarose (Solarbio; China). Then larvae were treated
with 2 mM tubocurarine (Tubocurarine hydrochloride
pentahydrate; Sigma-Aldrich; USA) in order to reduce the
artificial electric signal caused by occasional eye movements.
After acclimation for 30 min, 15 mM PTZ was added to the
prepared larva during EEG recording. 3% potassium acetate
filled glass micropipettes (diameter, 5 μm) were used as elec-
trodes. The signal was amplified (ELC-03XS; NPI Electronic;
Tamm, Germany) and recorded, then analyzed with Lab
Chart 7 (Lab Chart; ADInstruments, Australia).
Statistical Analysis
Data were analyzed using Graph Pad Prism 5.0 (GraphPad
Software; CA, USA) by one-way ANOVA followed by
Dunnett’s Multiple Comparison Test and were presented as
mean SEM. P < 0.05 was considered as significant.
Western Blot Analysis
Results
Heads of 7 dpf zebrafish (n = 50) from different groups
were collected in cold RIPA lysis buffer (Thermo Fisher
Scientific; USA). After centrifuging at 20,000 g for 10 min
at 4 °C, the supernatant was transferred to a new tube.
Protein concentration was determined by Enhanced BCA
Protein Assay Kit (Solarbio; China). Samples were boiled
in 5 × SDS-PAGE loading buffer (Beyotime, Beijing,
China), and western blotting was carried out according to
the Bio-Rad electrophoresis protocol with mouse anti-c-fos
(1:100, Santa Cruz biosciences; USA) (Pena et al. 2017)
and mouse anti-β-Actin (1:5000, Sigma-Aldrich)
antibodies.
Comparison of the Toxicity of BBR and its Derivatives
in Zebrafish
In order to compare the toxicity of BBR and its deriva-
tives in zebrafish, the LC50 of BBR&Ds was assayed.
Zebrafish larvae at 6 dpf were treated with various con-
centrations of BBR&Ds. As shown in Fig. 2b–d, the LC50
value of BBR, BBR-D1, and BBR-D2 were 632.3 μM,
3012 μM, and 994.5 μM, respectively. Our findings re-
vealed that BBR-D1 and BBR-D2 exhibited lower toxic-
ity than BBR.

The Absorption of BBR&Ds in Pretreated Zebrafish
Larvae
exposure (Fig. 4c), PTZ exposure duration was set to 3 min
and 30 min. In vivo, c-fos mRNA levels were increased during
seizures (Buenafe et al. 2013; Morgan et al. 1987). Similarly,
both 3 min and 30 min PTZ exposure periods generated a
significantly increased expression of c-fos, while BBR&Ds
markedly reduced this up-regulation (Fig. 4d, e). Moreover,
BBR&Ds pretreatments reversed the increased protein expres-
sion of c-fos induced by 3 min PTZ treatment (Fig. 4f, g).
Using an HPLC we investigated the absorption of BBR and its
derivatives 1 and 2 in zebrafish. As shown in Fig. 3a, the
adsorption peak of BBR, BBR-D1, and BBR-D2 was ob-
served at 18.0 min, 13.3 min, and 6.5 min, respectively.
Compared to the untreated zebrafish sample (Fig. 3b),
BBR&Ds treated zebrafish (experimental samples) had the
characteristic adsorption peaks (lower panels of Fig. 3c–e),
which correspond to the peak in the environmental samples
(upper panels of Fig. 3c–e).
Effects of BBR&Ds on PTZ-Induced Abnormal
Neuronal Discharge
Seizures are defined by the occurrence of abnormally synchro-
nous electrical discharges (Trebuchon and Chauvel 2016). We
performed EEG measurement to investigate the effect of
BBR&Ds on PTZ-induced seizure-like activity directly re-
corded form the surface of the skull (Fig. 5a). Abnormal neu-
ronal discharge manifested in the form of spikes was not ob-
served during baseline recording periods prior to PTZ expo-
sure. After the PTZ exposure, a long period of continuous
spiking was observed while reduced epileptiform events were
found in zebrafish pretreated with BBR&Ds (Fig. 5b). By
comparing the signals recorded among BBR&Ds pretreat-
ments, BBR-D1 showed the strongest ability to reduce the
frequency of spikes (Fig. 5c).
The Effects of BBR&Ds on PTZ-Induced Seizure-like
Behavior
To evaluate the effects of BBR&Ds on the development of
PTZ-induced seizures, the latency for larvae to reach each
seizure stage was recorded. In larvae pretreated with BBR-
D1 and BBR-D2, a significantly prolonged latency to reach
the stage III was found when compared to the larvae without
pretreatment. However, there was no significant difference in
latencies to reach stage I and II between larvae pretreated with
BBR&Ds and PTZ group (Fig. 4a). Our results revealed that
the pretreatment with two BBR derivatives delayed the occur-
rence of the first sign of seizure stage III, suggesting their
ability to relieve seizures.
To investigate the effect of BBR&Ds on PTZ-induced sei-
zure behavioral responses, locomotion plots were analyzed by
measuring the total distance travelled of each zebrafish larva
and the corresponding swimming speed. As a result, pretreat-
ment with BBR and BBR-D1 showed a significant reduction
on the increase of the total distance traveled triggered by PTZ
(Fig. 4b). The average speed of BBR and BBR-D1 pretreated
zebrafish was markedly decreased in comparison to that of
PTZ group (Fig. 4c). Notably, there was an apparent differ-
ence in larval moving speed between the PTZ group and
PTZ + BBR&Ds groups as early as 3 min after PTZ exposure,
suggesting that BBR&Ds had anti-seizure effect at this time
point. In contrast, no apparent change in the seizure-like be-
havior was found among the untreated zebrafish and those
treated with BBR&Ds alone, proving that BBR&Ds pretreat-
ments itself did not affect the locomotion activities of
zebrafish larvae.
The Effect of BBR&Ds on Neutrophil and Macrophage
Recruitment Following PTZ-Induced Seizures
In zebrafish inflammatory responses result in a recruitment of
immune cells, such as neutrophils and macrophages (Vezzani
and Viviani 2015). After PTZ exposure for 3 min and 30 min
an accumulation of neutrophils was observed in the brain re-
gion (Figs. 6a, b and 7a, b), indicating an inflammation action
occurred as the result of seizures. BBR&Ds showed a strong
ability to inhibit the neutrophil invasion into the brain induced
by 3 min and 30 min PTZ treatment. We also monitored the
migration of neutrophils into the head of zebrafish exposed to
PTZ for 3 min by video recording and found the number and
moving speed of neutrophils significantly decreased in
BBR&Ds pretreatment groups (Supplementary movies). In
addition, macrophage recruitment was performed to assess
inflammatory responses during seizures (Figs. 6c, d and
Fig. 3 HPLC analysis of BBR and its derivatives. a The standard
curves of BBR&Ds (loading amount 20 μL; BBR: RT = 17.953; BBR-
D1: RT = 13.349; BBR-D2: RT = 6.456); b The adsorption peak of
untreated zebrafish larvae sample; c BBR in the environmental sample
(upper panel; loading amount 10 μL; RT = 17.962) and in larvae (lower
panel; loading amount 10 μL; RT = 17.954). d BBR-D1 in environmental
sample (upper panel; loading amount 10 μL; RT = 13.448) and in larvae
(lower panel; loading amount 15 μL; RT = 13.738). e BBR-D2 in envi-
ronmental sample (upper panel; loading amount 10 μL; RT = 6.449) and
in larvae (lower panel; loading amount 10 μL; RT = 6.448).
b
Decreased c-fos Expression after BBR&Ds
Pretreatments in PTZ Treated Larvae
The mRNA levels of c-fos were analyzed after PTZ exposure
to investigate whether BBR&Ds pretreatments could affect
the expression of this neuronal activity marker. Since larval
travelling speed have shown drastic difference between PTZ
and BBR&Ds + PTZ groups as early as 3 min after PTZ

7c, d). 30 min of PTZ treatment increased the migration of
macrophages to the brain, which was significantly reversed by
BBR&Ds pretreatments. In contrast, there was no significant
migration of macrophages after the short time treatment with
PTZ.
numerous epilepsy focused studies (Buenafe et al. 2013;
Cho et al. 2017; Pisera-Fuster et al. 2017).
BBR&Ds Reverse PTZ-Induced Seizures-like Behavior,
c-fos Up-Regulation, and Neuronal Excitability
Inhibitory Effect of BBR&Ds on PTZ-Induced TNFα,
il1β, and il6 Up-Regulation
After PTZ treatment larvae showed an increase in distance
travelled along with an increasing swimming speed. In larvae
pretreated with BBR and BBR-D1, PTZ-induced seizure-like
behavior was suppressed, including a decrease of total dis-
tance and swimming speed. These results were consistent with
that have been found in BBR treated rat seizure models
(Ebrahimzadeh Bideskan et al. 2011). However, in zebrafish
pretreated with BBR-D2, there was no obvious difference in
total swimming distance whereas we observed significant
prolonged latency to stage III of seizures. These findings sug-
gested that although BBR-D1 and BBR-D2 are derived from
BBR, they may perform different anticonvulsant actions dur-
ing seizures. Based on our results of locomotor activity we
found zebrafish pretreated with BBR&Ds showed an obvious
reduced moving speed compared with PTZ group as early as
3 min after PTZ exposure, suggesting that BBR&Ds exerted
anti-seizure action at this time point. Therefore, we tested c-
fos expression and anti-inflammatory responses induced by
BBR&Ds after PTZ exposure for 3 min to further verify if
the anti-seizure effect of BBR&Ds were indeed rooted in their
anti-inflammatory activity. The expression of c-fos was found
to be associated with the hyperactivity levels of neurons dur-
ing seizures (Baxendale et al. 2012; Beer et al. 1998; Herrera
and Robertson 1996). We found that BBR&Ds pretreatments
markedly reversed the increased expression of c-fos following
PTZ treatment for 3 min and 30 min, respectively. EEG is
known as the most direct measurement for investigating neu-
ron discharge during seizures (Cho et al. 2017; Fujikawa et al.
2016; Lundt et al. 2016). In our study, BBR&Ds showed the
ability to reduce PTZ-induced synchronized neuron dis-
charges reflected in the decreased number of interictal spikes.
All these findings indicated that BBR&Ds attenuated PTZ-
induced seizures in zebrafish.
In zebrafish seizures increase the expression of TNFα, il1β,
and il6 when seizure-induced inflammation occurs (Choo
et al. 2018; Jin et al. 2018b; Vezzani and Viviani 2015). To
evaluate the effect of BBR&Ds on inflammation caused by
PTZ-induced seizures, the mRNA levels of TNFα, il1β, and
il6 were measured after seizure induction. The PTZ treated
group (both 3 min and 30 min) showed an increased level of
all three proinflammatory genes, whereas BBR&Ds pretreat-
ments were capable of significantly reducing the mRNA ex-
pression of TNFα, il1β, and il6 in comparison with PTZ group
(Fig. 8a–f). Accordingly, BBR&Ds pretreatments inhibited
the increased protein levels of TNFα and il1β, which were
induced by 3 min PTZ treatment (Fig. 8g, h). The inhibitory
effects of BBR&Ds on the up-regulation of TNFα, il1β, and
il6 caused by PTZ-induced seizures suggested that BBR&Ds
could reduce inflammation during seizures, and as a conse-
quence suppress seizure progression.
Discussion
In the present study we synthesized two hydrophilic BBR
derivatives, which were used to investigate the structure–
activity relationship between their hydrophilic/hydrophobic
structure and their action on seizure suppression. Our results
showed that BBR and its derivatives could relieve PTZ-
induced seizures in zebrafish via anti-inflammatory actions.
Notably, the hydrophilic berberrubine BBR-D1 exhibited the
strongest ability to attenuate PTZ-induced seizures.
The Zebrafish Seizure Model Used in this Study
Fig. 4 Effect of BBR&Ds on zebrafish locomotor activities and c-fos
expression after PTZ treatment. a The latency for larvae to reach each
seizure stage after PTZ treatment (n ≥ 20). *P < 0.05 vs PTZ. b The total
distance travelled (n ≥ 27 per group). ***P < 0.001 vs control; ##P < 0.01,
###P < 0.001 vs PTZ. In digital tracking map (at the bottom of B), high-
speed movement is represented in red lines; medium-speed movement is
depicted in green lines; low-speed movement is represented in black lines.
c The swimming speed of zebrafish larvae with different treatment.
Average speed in every 60 s was calculated. d Relative c-fos mRNA
expression levels after PTZ treatment for 30 min. e Relative c-fos
mRNA expression levels after PTZ treatment for 3 min. f Protein levels
of c-fos after PTZ treatment for 3 min revealed by western blotting. g
Quantitative measurements of the relative densities of c-fos protein.
*P < 0.05, **P < 0.01, ***P < 0.001 vs control; #P < 0.05, ##P < 0.01,
###P < 0.001 vs PTZ.
b
Unlike other neurodegenerative disorders for example
Parkinson’s and Alzheimer’s disease, the early symptoms of
epilepsy is the abnormal neuronal discharges without cell
death, which could not be modeled in cell culture
(Chakrabarti et al. 2016; Dauer and Przedborski 2003;
Zhang et al. 2017). Thus, in vivo animal models with no or
minimal ethical constrains are the ideal tools for investigating
the underlying mechanisms of epilepsy. In addition, the min-
imal amount of drugs needed for testing, the short test periods
used in assays, the manifestation of seizure-like behavior, the
possibility of EEG recordings, and the transparent nature
make zebrafish the in vivo animal model of choice in

Fig. 5 EEG recordings of 7 dpf zebrafish larvae. a Schematic
illustration of EEG recording methodology. b EEG recording of the
brain activity in control conditions and BBR&Ds pretreatments. No
abnormal activity was visible (in the control and BBR&Ds alone
groups). PTZ-induced interictal spikes were represented as single unidi-
rectional amplitude spikes. BBR&Ds reduced the number of spikes. Scale
bar, 2 mV; 2 s. c The frequency of abnormal synchronous neuronal dis-
charges. Spike with amplitude larger than 300% of the adjacent basal line
was counted as one abnormal neuronal discharge (blue arrows indicated
the distribution of these spikes), and the frequency was determined as the
number of abnormal neuronal discharges in 1 min. ***P < 0.001 vs PTZ.
BBR-D1 Showed the Strongest Ability to Attenuate
PTZ-Induced Seizures
unknown. Here we showed the seizure suppressing effect
of BBR-D1 and BBR-D2. By comparing the results of
seizure indexes, the hydrophilic BBR derivative BBR-
D1 showed stronger ability to attenuate PTZ-induced sei-
zures compared to the hydrophobic BBR-D2. This is pos-
sibly due to the high metabolic capability, good solubility
in water, and high stability in acid conditions of BBR-
D1, which play critical roles during drug absorption
(Huang et al. 2017; Yang et al. 2017). On the contrary,
the hydrophobic BBR-D2 has poor solubility in water
and most of the organic solvents, which may result in
negative effects on drug absorption in vivo. Indeed, as
shown in our HPLC assays, the content of BBR-D1 in
zebrafish larvae was close to that in its corresponding
environmental sample, suggesting its higher potential to
be absorbed.
In the present study BBR-D1 and BBR-D2 showed
higher LC50 value as compared to BBR, suggesting their
relatively low toxicity. Therefore, the bioactive effects of
BBR-D1 and BBR-D2 were worth exploring. BBR-D1
can be separated from the plant Berberis vulgaris L. or
obtained by pyrolysis of berberine, which has been re-
ported to have the same pharmacological activities in-
cluding antioxidant activity, antibacterial activity, and an-
ticancer activity as berberine but with higher bioavailabil-
ity (Kobayashi et al. 1995; Lo et al. 2013; Pongkittiphan
et al. 2015). On the other hand, BBR-D2 has been stud-
ied as an intermediary product in a cellulose material
research, while its biological activities are largely

Fig. 6 BBR&Ds reduce the
recruitment of neutrophils and
macrophages in the zebrafish
brain after PTZ treatment for
30 min a Migration of neutrophils
(green signal) into the head of
larvae after 30 min PTZ
treatment. b The number of
neutrophils in larvae heads after
30 min PTZ treatment. c
Infiltration of macrophages
(orange signal) into the head of
larvae after 30 min PTZ
treatment. d The number of
macrophages in larvae heads after
30 min PTZ treatment. n ≥ 8 per
group. ***P < 0.001 vs control;
#P < 0.05, ###P < 0.001 vs PTZ.
Scale bar, 100 μm.
BBR&Ds Inhibit Inflammatory Responses
during Seizure Progression
such as neutrophils and macrophages (Vezzani et al. 2011).
Although the migration of macrophages was not observed
after the short treatment with PTZ, the number of macro-
phages in the brain was significantly increased after 30 min
treatment with PTZ. One possible reason is that it takes at least
PTZ has been reported to cause inflammatory responses in the
brain of zebrafish, promoting a recruitment of immune cells,

Fig. 7 Effect of BBR&Ds on the recruitment of neutrophils and
macrophages in the zebrafish brain after PTZ treatment for 3 min.
a Migration of neutrophils (green signal) into the head of larvae after
3 min PTZ treatment. b The number of neutrophils in larvae heads after
3 min PTZ treatment. c Migration of macrophages (orange signal) into the
head of larvae after 3 min PTZ treatment. d The number of macrophages
in larvae heads after 3 min PTZ treatment. n ≥ 8 per group. ***P < 0.001
vs control; ###P < 0.001 vs PTZ. Scale bar, 100 μm
R
90 min to visualize macrophages by using neutral red staining,
by that time the number of macrophages in the brain had
returned to the normal level. It has been reported that inflam-
mation is activated by PTZ in rodents. Injection of PTZ in-
creased inflammatory cytokine levels in the cerebral cortex
and hippocampus of mice (Hoda et al. 2017; Temp et al.
2017). In PTZ-kindled rats, a markedly up-regulation in the
expression of hippocampal inflammatory cytokines was
Fig. 8 Effect of BBR&Ds on the expression of inflammatory
cytokines after PTZ-induced seizures. a–c Relative mRNA
expression levels of TNFα, il1β, and il6 after 30 min PTZ treatment. d–
f Relative mRNA expression levels of TNFα, il1β, and il6 after 3 min
PTZ treatment. g Protein levels of TNFα and il1β after PTZ treatment for
3 min revealed by ELISA assays. *P < 0.05, **P < 0.01, ***P < 0.001 vs
control; #P < 0.05, ##P < 0.01, ###P < 0.001 vs PTZ.

cytokines, such as il1β, il6, and TNF-α are expressed in acti-
vated astrocytes and microglia (Bonaventura et al. 2018;
Dupuis and Auvin 2015; Friedman and Dingledine 2011;
Vezzani and Granata 2005). Proinflammatory cytokines acti-
vate NF-κB, which in turn trigger inflammation in the brain
(Ali et al. 2018; Han et al. 2018; Liu et al. 2017). During
inflammatory responses, glia cells express chemokines to re-
cruit immune cells, such as macrophages and neutrophils, into
the brain (Fabene et al. 2008). Meanwhile, seizures also cause
blood–brain barrier (BBB) leakage and alter the permeability
of BBB, which may contribute to the migration of macro-
phages and neutrophils (Roch et al. 2002; van Vliet et al.
2015). In our study, BBR&Ds showed their ability to inhibit
the expression of proinflammatory cytokines and reverse the
recruitment of immune cells, which is possibly contribute to
seizure suppression (Fig. 9). These results are consistent with
the previous findings that indomethacin, an anti-inflammatory
agent also has therapeutic effect on seizure suppression
(Barbalho et al. 2016; Vieira et al. 2014). Although it has been
reported that BBR can penetrate through the BBB and accu-
mulate in hippocampus (Wang et al. 2005), BBB penetrating
ability of BBR-D1 and BBR-D2 needs confirmation.
Fig. 9 Schematic of anti-inflammation associated protective mecha-
nism of BBR&Ds on attenuating PTZ-induced seizures. PTZ-induced
seizures enhance the activation of microglias and astrocytes, promoting
the secretion of proinflammatory cytokines TNFα, il1β, and il6.
Proinflammatory cytokines activate NF-κB, which cause inflammation
responses and immune cell recruitment in the brain, thereby aggravating
seizures. BBR&Ds could relieve PTZ-induced seizures via:① reversing
the recruitment of immune cells and/or ② inhibiting the expression of
proinflammatory cytokines.
Acknowledgments We thank Ximin Wang for zebrafish maintenance.
This work was supported by the National Natural Science Foundation
of China (No. 81802629) and the International Science and Technology
Cooperation Program of Shandong Academy of Sciences (No.
2019GHZD10 and No. 2019GHPY13) for MJ. This work was also sup-
ported by the European Union’s Horizon 2020 research and Innovation
programme under the Marie Skłodowska-Curie (No. 734862) for AS and
Jinan High-end Foreign Experts Recruitment Program (No. 2019 T10)
for KCL.
revealed (Liu et al. 2018; Riazi et al. 2008; Xia et al. 2018).
Accordingly, we found that inflammatory biomarkers TNFα,
il1β, and il6 were increased after PTZ treatment in zebrafish.
We also found that this increase was reversed by BBR pre-
treatment, which is consistent with previous findings on glial
cells and in rat seizure models (Chen et al. 2014; Vieira et al.
2014). Similarly, BBR-D1 and BBR-D2 caused a significant
decrease in the recruitment of inflammatory cells and expres-
sion of inflammatory cytokines. Although BBR&Ds showed
similar anti-inflammatory effect during seizures, their inhibi-
tory effect on total swimming distance were variable, suggest-
ing that anti-inflammatory activity of BBR&Ds is one of the
contributors to seizure suppression. Since we did not demon-
strate that the anti-seizure effect of BBR&Ds was exclusively
due to their anti-inflammatory activity, further studies are
needed to clarify the mechanisms.
Author Contributions MJ, KCL, and AS conceived the project and de-
signed the experiments. MJ, BYZ, LZW, XNJ, and SSZ performed the
experiments and analyzed the data. MJ wrote the manuscript. AS helped
in the revision.
Compliance with Ethical Standards
Conflict of Interest The authors declare no conflict of interest.
References
Ali AE, Mahdy HM, Elsherbiny DM, Azab SS (2018) Rifampicin ame-
liorates lithium-pilocarpine-induced seizures, consequent hippo-
campal damage and memory deficit in rats: impact on oxidative,
inflammatory and apoptotic machineries. Biochem Pharmacol 156:
431–443. https://doi.org/10.1016/j.bcp.2018.09.004
Anti-Inflammation Associated Protective Mechanism
of BBR&Ds on Attenuating PTZ-Induced Seizures
Babin PJ, Goizet C, Raldua D (2014) Zebrafish models of human motor
neuron diseases: advantages and limitations. Prog Neurobiol 118:
36–58. https://doi.org/10.1016/j.pneurobio.2014.03.001
Bae J, Lee D, Kim YK, Gil M, Lee JY, Lee KJ (2013) Berberine protects
6-hydroxydopamine-induced human dopaminergic neuronal cell
death through the induction of heme oxygenase-1. Mol Cells 35:
151–157. https://doi.org/10.1007/s10059-013-2298-5
It has been reported that inflammatory responses play pivotal
roles in seizures progression (Jin et al. 2018b; Vezzani et al.
2012, 2016). When seizures occur, key proinflammatory

Baraban SC, Taylor MR, Castro PA, Baier H (2005) Pentylenetetrazole
induced changes in zebrafish behavior, neural activity and c-fos
expression. Neuroscience 131:759–768. https://doi.org/10.1016/j.
neuroscience.2004.11.031
Barbalho PG, Lopes-Cendes I, Maurer-Morelli CV (2016) Indomethacin
treatment prior to pentylenetetrazole-induced seizures
downregulates the expression of il1b and cox2 and decreases
seizure-like behavior in zebrafish larvae. BMC Neurosci 17:12.
https://doi.org/10.1186/s12868-016-0246-y
Fontana BD et al (2018) Taurine protects from Pentylenetetrazole-
induced behavioral and neurochemical changes in Zebrafish. Mol
Neurobiol. https://doi.org/10.1007/s12035-018-1107-8
Franco V, French JA, Perucca E (2016) Challenges in the clinical devel-
opment of new antiepileptic drugs. Pharmacol Res 103:95–104.
https://doi.org/10.1016/j.phrs.2015.11.007
Friedman A, Dingledine R (2011) Molecular cascades that mediate the
influence of inflammation on epilepsy. Epilepsia 52(Suppl 3):33–
39. https://doi.org/10.1111/j.1528-1167.2011.03034.x
Baxendale S et al (2012) Identification of compounds with anti-
convulsant properties in a zebrafish model of epileptic seizures.
Dis Models Mech 5:773–784. https://doi.org/10.1242/dmm.010090
Beer J, Mielke K, Zipp M, Zimmermann M, Herdegen T (1998) Expression
of c-jun, junB, c-fos, fra-1 and fra-2 mRNA in the rat brain following
seizure activity and axotomy. Brain Res 794:255–266
Bonaventura P, Lamboux A, Albarede F, Miossec P (2018) Differential
effects of TNF-alpha and IL-1beta on the control of metal metabo-
lism and cadmium-induced cell death in chronic inflammation. PloS
One 13:e0196285. https://doi.org/10.1371/journal.pone.0196285
Buenafe OE et al (2013) Tanshinone IIA exhibits anticonvulsant activity
in zebrafish and mouse seizure models. ACS Chem Neurosci 4:
1479–1487. https://doi.org/10.1021/cn400140e
Chakrabarti SS, Bir A, Poddar J, Sinha M, Ganguly A, Chakrabarti S
(2016) Ceramide and Sphingosine-1-phosphate in cell death path-
ways : relevance to the pathogenesis of Alzheimer's disease. Curr
Alzheimer Res 13:1232–1248
Chen CC, Hung TH, Lee CY, Wang LF, Wu CH, Ke CH, Chen SF (2014)
Berberine protects against neuronal damage via suppression of glia-
mediated inflammation in traumatic brain injury. PloS One 9:
e115694. https://doi.org/10.1371/journal.pone.0115694
Cheng Z et al (2010) 8,8-Dimethyldihydroberberine with improved bio-
availability and oral efficacy on obese and diabetic mouse models.
Bioorg Med Chem 18:5915–5924. https://doi.org/10.1016/j.bmc.
2010.06.085
Cho SJ, Byun D, Nam TS, Choi SY, Lee BG, Kim MK, Kim S (2017)
Zebrafish as an animal model in epilepsy studies with multichannel
EEG recordings. Sci Rep 7:3099. https://doi.org/10.1038/s41598-
017-03482-6
Choo BKM, Kundap UP, Kumari Y, Hue SM, Othman I, Shaikh MF
(2018) Orthosiphon stamineus leaf extract affects TNF-alpha and
seizures in a zebrafish model. Front Pharmacol 9:139. https://doi.
org/10.3389/fphar.2018.00139
Cronin A, Grealy M (2017) Neuroprotective and neuro-restorative effects
of minocycline and rasagiline in a zebrafish 6-Hydroxydopamine
model of Parkinson's disease. Neuroscience 367:34–46. https://doi.
org/10.1016/j.neuroscience.2017.10.018
Dauer W, Przedborski S (2003) Parkinson’s disease: mechanisms and
models. Neuron 39:889–909
Fujikawa DG, Pais ES, Aviles ER Jr, Hsieh KC, Bashir MT (2016)
Methamphetamine-induced neuronal necrosis: the role of
electrographic seizure discharges. Neurotoxicology 52:84–88.
https://doi.org/10.1016/j.neuro.2015.11.001
Gao F, Gao Y, Liu YF, Wang L, Li YJ (2014) Berberine exerts an anti-
convulsant effect and ameliorates memory impairment and oxida-
tive stress in a pilocarpine-induced epilepsy model in the rat.
Neuropsych Dis Treat 10:2139–2145. https://doi.org/10.2147/
NDT.S73210
Han K, Wang QY, Wang CX, Luan SY, Tian WP, Wang Y, Zhang RY
(2018) Ghrelin improves pilocarpineinduced cerebral cortex inflam-
mation in epileptic rats by inhibiting NFkappaB and TNFalpha. Mol
Med Rep 18:3563–3568. https://doi.org/10.3892/mmr.2018.9381
Herbomel P, Thisse B, Thisse C (2001) Zebrafish early macrophages
colonize cephalic mesenchyme and developing brain, retina, and
epidermis through a M-CSF receptor-dependent invasive process.
Dev Biol 238:274–288. https://doi.org/10.1006/dbio.2001.0393
Herrera DG, Robertson HA (1996) Activation of c-fos in the brain. Prog
Neurobiol 50:83–107
Hoda U, Agarwal NB, Vohora D, Parvez S, Raisuddin S (2017)
Resveratrol suppressed seizures by attenuating IL-1beta, IL1-Ra,
IL-6, and TNF-alpha in the hippocampus and cortex of kindled
mice. Nutr Neurosci 20:497–504. https://doi.org/10.1080/
1028415X.2016.1189057
Hortopan GA, Dinday MT, Baraban SC (2010) Zebrafish as a model for
studying genetic aspects of epilepsy. Dis Model Mech 3:144–148.
https://doi.org/10.1242/dmm.002139
Huang J, Sun R, Feng S, He J, Fei F, Gao H, Zhao Y, Zhang Y, Gu H, Aa
J, Wang G (2017) Sensitive analysis and pharmacokinetic study of
epalrestat in C57BL/6J mice. J Chromatogr B Analyt Technol
Biomed Life Sci 1055-1056:98–103. https://doi.org/10.1016/j.
jchromb.2017.03.040
Imenshahidi M, Hosseinzadeh H (2016) Berberis Vulgaris and Berberine:
An Update Review. Phytotherapy Research : PTR 30:1745–1764.
https://doi.org/10.1002/ptr.5693
Jin M, He Q, Zhang S, Cui Y, Han L, Liu K (2018a) Gastrodin Suppresses
Pentylenetetrazole-Induced Seizures Progression by Modulating
Oxidative Stress in Zebrafish. Neurochem Res 43:904–917.
https://doi.org/10.1007/s11064-018-2496-9
Devinsky O, Vezzani A, O'Brien TJ, Jette N, Scheffer IE, de Curtis M,
Perucca P (2018) Epilepsy. Nat Rev Dis Primers 4:18024–18024.
https://doi.org/10.1038/nrdp.2018.24
Dupuis N, Auvin S (2015) Inflammation and epilepsy in the developing
brain: clinical and experimental evidence. CNS Neurosci Ther 21:
141–151. https://doi.org/10.1111/cns.12371
Duy PQ, Berberoglu MA, Beattie CE, Hall CW (2017) Cellular responses
to recurrent pentylenetetrazole-induced seizures in the adult
zebrafish brain. Neuroscience 349:118–127. https://doi.org/10.
1016/j.neuroscience.2017.02.032
Ebrahimzadeh Bideskan AR, Hosseini M, Mohammadpour T, Karami R,
Khodamoradi M, Nemati Karimooy H, Alavi H (2011) Effects of
soy extract on pentylenetetrazol-induced seizures in ovariectomized
rats. Zhong xi yi jie he xue bao = Journal of Chinese integrative
medicine 9:611–618
Jin M, Sheng W, Han L, He Q, Ji X, Liu K (2018b) Activation of BDNF-
TrkB signaling pathway-regulated brain inflammation in
pentylenetetrazole-induced seizures in zebrafish. Fish Shellfish
Immun 83:26–36. https://doi.org/10.1016/j.fsi.2018.09.010
Kobayashi Y et al (1995) Inhibitors of DNA topoisomerase I and II
isolated from the Coptis rhizomes. Planta Med 61:414–418.
https://doi.org/10.1055/s-2006-958127
Kundap UP, Kumari Y, Othman I, Shaikh MF (2017) Zebrafish as a
model for epilepsy-induced cognitive dysfunction: a pharmacologi-
cal, biochemical and behavioral approach. Front Pharmacol 8:515.
https://doi.org/10.3389/fphar.2017.00515
Lin X, Zhang N (2018) Berberine: Pathways to protect neurons.
Phytotherapy Research : PTR 32:1501–1510. https://doi.org/10.
1002/ptr.6107
Fabene PF et al (2008) A role for leukocyte-endothelial adhesion mech-
anisms in epilepsy. Nat Med 14:1377–1383. https://doi.org/10.1038/
nm.1878
Liu T, Zhang L, Joo D, Sun SC (2017) NF-kappaB signaling in inflam-
mation. Signal Transduc Tar Ther:2. https://doi.org/10.1038/
sigtrans.2017.23

Liu Y et al (2018) Overexpression of zinc-alpha2-glycoprotein sup-
pressed seizures and seizure-related neuroflammation in
pentylenetetrazol-kindled rats. J Neuroinflammation 15:92. https://
doi.org/10.1186/s12974-018-1132-6
Biomedecine & pharmacotherapie 87:200–208. https://doi.org/10.
1016/j.biopha.2016.12.109
Shan YQ et al (2013) Berberine analogue IMB-Y53 improves glucose-
lowering efficacy by averting cellular efflux especially P-
glycoprotein efflux. Metab Clin Exp 62:446–456. https://doi.org/
10.1016/j.metabol.2012.09.009
Siddiqui FA, Ahmad M, Mehjabeen JN, Sajid MI (2017) Phytochemical
screening and assessment of analgesic, Anti-inflammatory and he-
matological properties of the fruit of Berberis baluchistanica. Pak J
Pharm Sci 30:1007–1012
Siebel AM, Menezes FP, da Costa SI, Petersen BD, Bonan CD (2015)
Rapamycin suppresses PTZ-induced seizures at different develop-
mental stages of zebrafish. Pharmacol Biochem Behav 139 Pt B:
163–168. https://doi.org/10.1016/j.pbb.2015.05.022
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data
using real-time quantitative PCR and the 2(−Delta Delta C(T)) method.
Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
Lo CY, Hsu LC, Chen MS, Lin YJ, Chen LG, Kuo CD, Wu JY (2013)
Synthesis and anticancer activity of a novel series of 9-O-substituted
berberine derivatives: a lipophilic substitute role. Bioorg Med Chem
Lett 23:305–309. https://doi.org/10.1016/j.bmcl.2012.10.098
Lundt A, Wormuth C, Siwek ME, Müller R, Ehninger D, Henseler C,
Broich K, Papazoglou A, Weiergräber M (2016) EEG
Radiotelemetry in small laboratory rodents: a powerful state-of-the
art approach in neuropsychiatric, Neurodegenerative, and Epilepsy
Research. Neural Plast 2016:8213878. https://doi.org/10.1155/
2016/8213878
Stafstrom CE, Carmant L (2015) Seizures and epilepsy: an overview for
neuroscientists. Cold Spring Harb Perspect Med:5. https://doi.org/
10.1101/cshperspect.a022426
Ma Y, Ou TM, Tan JH, Hou JQ, Huang SL, Gu LQ, Huang ZS (2009)
Synthesis and evaluation of 9-O-substituted berberine derivatives
containing aza-aromatic terminal group as highly selective telomeric
G-quadruplex stabilizing ligands. Bioorg Med Chem Lett 19:3414–
3417. https://doi.org/10.1016/j.bmcl.2009.05.030
Maeng HJ, Yoo HJ, Kim IW, Song IS, Chung SJ, Shim CK (2002) P-
glycoprotein-mediated transport of berberine across Caco-2 cell
monolayers. J Pharm Sci 91:2614–2621. https://doi.org/10.1002/
jps.10268
Morgan JI, Cohen DR, Hempstead JL, Curran T (1987) Mapping patterns
of c-fos expression in the central nervous system after seizure.
Science 237:192–197
Okuneva O, Li Z, Korber I, Tegelberg S, Joensuu T, Tian L, Lehesjoki AE
(2016) Brain inflammation is accompanied by peripheral inflamma-
tion in Cstb (−/−) mice, a model for progressive myoclonus epilepsy.
J Neuroinflammation 13:298. https://doi.org/10.1186/s12974-016-
0764-7
Pena IA et al (2017) Pyridoxine-dependent epilepsy in Zebrafish caused
by Aldh7a1 deficiency. Genetics 207:1501–1518. https://doi.org/10.
1534/genetics.117.300137
Pisera-Fuster A, Otero S, Talevi A, Bruno-Blanch L, Bernabeu R (2017)
Anticonvulsant effect of sodium cyclamate and propylparaben on
pentylenetetrazol-induced seizures in zebrafish. Synapse:71. https://
doi.org/10.1002/syn.21961
Pongkittiphan V, Chavasiri W, Supabphol R (2015) Antioxidant Effect of
Berberine and its Phenolic Derivatives Against Human
Fibrosarcoma Cells Asian Pacific. Journal of Cancer Prevention :
APJCP 16:5371–5376
Renshaw SA, Loynes CA, Trushell DM, Elworthy S, Ingham PW, Whyte
MK (2006) A transgenic zebrafish model of neutrophilic inflamma-
tion. Blood 108:3976–3978. https://doi.org/10.1182/blood-2006-
05-024075
Stewart AM, Braubach O, Spitsbergen J, Gerlai R, Kalueff AV (2014)
Zebrafish models for translational neuroscience research: from tank
to bedside. Trends Neurosci 37:264–278. https://doi.org/10.1016/j.
tins.2014.02.011
Temp FR, Marafiga JR, Milanesi LH, Duarte T, Rambo LM, Pillat MM,
Mello CF (2017) Cyclooxygenase-2 inhibitors differentially attenu-
ate pentylenetetrazol-induced seizures and increase of pro- and anti-
inflammatory cytokine levels in the cerebral cortex and hippocam-
pus of mice. Eur J Pharmacol 810:15–25. https://doi.org/10.1016/j.
ejphar.2017.05.013
Torres-Hernandez BA, Del Valle-Mojica LM, Ortiz JG (2015) Valerenic
acid and Valeriana officinalis extracts delay onset of
Pentylenetetrazole (PTZ)-induced seizures in adult Danio rerio
(Zebrafish). BMC Complement Altern Med 15:228. https://doi.
org/10.1186/s12906-015-0731-3
Trebuchon A, Chauvel P (2016) Electrical stimulation for seizure induc-
tion and functional mapping in stereoelectroencephalography.
Journal of Clinical Neurophysiology : Official Publication of the
American Electroencephalographic Society 33:511–521. https://
doi.org/10.1097/WNP.0000000000000313
van Vliet EA, Aronica E, Gorter JA (2015) Blood-brain barrier dysfunc-
tion, seizures and epilepsy. Semin Cell Dev Biol 38:26–34. https://
doi.org/10.1016/j.semcdb.2014.10.003
Vezzani A, Granata T (2005) Brain inflammation in epilepsy: experimen-
tal and clinical evidence. Epilepsia 46:1724–1743. https://doi.org/
10.1111/j.1528-1167.2005.00298.x
Vezzani A, Viviani B (2015) Neuromodulatory properties of inflamma-
tory cytokines and their impact on neuronal excitability.
Neuropharmacology 96:70–82. https://doi.org/10.1016/j.
neuropharm.2014.10.027
Vezzani A, French J, Bartfai T, Baram TZ (2011) The role of inflamma-
tion in epilepsy. Nat Rev Neurol 7:31–40. https://doi.org/10.1038/
nrneurol.2010.178
Riazi K, Galic MA, Kuzmiski JB, Ho W, Sharkey KA, Pittman QJ (2008)
Microglial activation and TNFalpha production mediate altered
CNS excitability following peripheral inflammation. Proc Natl
Acad Sci USA 105:17151–17156. https://doi.org/10.1073/pnas.
0806682105
Vezzani A, Balosso S, Ravizza T (2012) Inflammation and epilepsy.
Handb Clin Neurol 107:163–175. https://doi.org/10.1016/B978-0-
444-52898-8.00010-0
Vezzani A, Fujinami RS, White HS, Preux PM, Blumcke I, Sander JW,
Loscher W (2016) Infections, inflammation and epilepsy. Acta
Neuropathol 131:211–234. https://doi.org/10.1007/s00401-015-
1481-5
Vieira MJ, Perosa SR, Argaaaraz GA, Silva JA Jr, Cavalheiro EA, Graca
Naffah-Mazzacoratti M (2014) Indomethacin can downregulate the
levels of inflammatory mediators in the hippocampus of rats sub-
mitted to pilocarpine-induced status epilepticus. Clinics 69:621–626
Wang X, Wang R, Xing D, Su H, Ma C, Ding Y, Du L (2005) Kinetic
difference of berberine between hippocampus and plasma in rat after
intravenous administration of Coptidis rhizoma extract. Life Sci 77:
3058–3067. https://doi.org/10.1016/j.lfs.2005.02.033
Roch C, Leroy C, Nehlig A, Namer IJ (2002) Magnetic resonance imag-
ing in the study of the lithium-pilocarpine model of temporal lobe
epilepsy in adult rats. Epilepsia 43:325–335
Rosa-Falero C, Torres-Rodríguez S, Jordán C, Licier R, Santiago Y,
Toledo Z, Santiago M, Serrano K, Sosa J, Ortiz JG (2014) Citrus
aurantium increases seizure latency to PTZ induced seizures in
zebrafish thru NMDA and mGluR's I and II. Front Pharmacol 5:
284. https://doi.org/10.3389/fphar.2014.00284
Sedaghat R, Taab Y, Kiasalari Z, Afshin-Majd S, Baluchnejadmojarad T,
Roghani M (2017) Berberine ameliorates intrahippocampal kainate-
induced status epilepticus and consequent epileptogenic process in
the rat: underlying mechanisms. Biomedicine & Pharmacotherapy =

Wang H et al (2017) Berberine attenuated pro-inflammatory factors and
protect against neuronal damage via triggering oligodendrocyte au-
tophagy in spinal cord injury. Oncotarget 8:98312–98321. https://
doi.org/10.18632/oncotarget.21203
Wang YX et al (2018) Synthesis and biological evaluation of new ber-
berine derivatives as cancer immunotherapy agents through
targeting IDO1. Eur J Med Chem 143:1858–1868. https://doi.org/
10.1016/j.ejmech.2017.10.078
Medical Science Monitor : International Medical Journal of
Experimental and Clinical Research 24:6165–6177. https://doi.org/
10.12659/MSM.910352
Yang N et al (2017) In vitro assessment of the glucose-lowering effects of
berberrubine-9-O-beta-D-glucuronide, an active metabolite of
berberrubine. Acta Pharmacol Sin 38:351–361. https://doi.org/10.
1038/aps.2016.120
Zhang C et al (2017) Berberine protects against 6-OHDA-induced neu-
rotoxicity in PC12 cells and zebrafish through hormetic mechanisms
involving PI3K/AKT/Bcl-2 and Nrf2/HO-1 pathways. Redox Biol
11:1–11. https://doi.org/10.1016/j.redox.2016.10.019
Wullimann MF, Mueller T (2004) Teleostean and mammalian forebrains
contrasted: Evidence from genes to behavior. J Comp Neurol 475:
143–162. https://doi.org/10.1002/cne.20183
Wyckhuys T, Geerts PJ, Raedt R, Vonck K, Wadman W, Boon P (2009)
Deep brain stimulation for epilepsy: knowledge gained from exper-
imental animal models. Acta Neurol Belg 109:63–80
Xia J, Wang H, Zhang Q, Han Z (2018) Modulation of P2X Purinoceptor
3 (P2X3) in Pentylenetetrazole-induced kindling epilepsy in rats.
Publisher’s Note Springer Nature remains neutral with regard to jurisdic-
tional claims in published maps and institutional affiliations.