2014

http://informahealthcare.com/bijISSN: 0269-9052 (print), 1362-301X (electronic)

Brain Inj, 2014; 28(2): 227–234! 2014 Informa UK Ltd. DOI: 10.3109/02699052.2013.860469

ORIGINAL ARTICLE

Baicalin protects PC-12 cells from oxidative stress induced by hydrogenperoxide via anti-apoptotic effects

Wen-xia Zheng*, Feng Wang*, Xiao-lu Cao*, Hong-yan Pan, Xiao-ying Liu, Xia-min Hu, & Yu-ying Sun

Department of Pharmacy, College of Medicine, Wuhan University of Science and Technology, Wuhan, Hubei Province, PR China

Abstract

Primary objective: To examine the neuroprotection of baicalin, a flavonoid compound derivedfrom the dried root of Scutellaria baicalensis Georgi, on neurons.Research design: A rat PC12 cell line was used to study the neuroprotection and possiblemechanisms of baicalin on H2O2-induced neuron damage.Methods: Three anti- and one pro-apoptosis genes in PC12 cells were examined. Cell apoptosiswas induced by H2O2 and apoptotic rate was obtained by flow cytometry. MTT for cell viability,immunofluorescence microscopy for promoter activity and western blot for gene expressionwere also employed.Results: Data of MTT reduction assay and flow cytometry revealed that viability loss and apoptoticrate were reduced by pre-treatment of PC12 cells with baicalin for 24 hours. Baicalin was alsofound to increase SOD, GSH-Px activities and to decrease MDA level. Results from Western blotand immunofluorescence microscopy showed baicalin increased the expressions of survivin, Bcl-2 and p-STAT3 and decreased caspase-3 expression which were attenuated by AG-490.Conclusions: The results point to the possibility of the neuroprotective effects of baicalin onneuronal apoptosis induced by oxidative stress and indicate that activation of the JAK/STATsignalling pathway might involve the anti-apoptotic effect of baicalin.

Keywords

Apoptosis, baicalin, JAK/STAT3 signallingpathway, neuronal injury, PC12 cell

History

Received 28 December 2012Revised 17 October 2013Accepted 25 October 2013Published online 21 January 2014

Introduction

A growing body of evidence suggests that oxidative stress may

play a major role in the pathophysiology of stroke [1–4]. The

oxidative stress caused by redox balance disruption and the

over-production of reactive oxygen species (ROS) are followed

by dysfunction of important redox-sensitive enzymes, mem-

brane receptors and ion channels, leading to DNA damage,

membrane lipid peroxidation and cytochrome c release from

mitochondria, which then activate caspases that result in cell

death [5–7]. Thus, the strategies to reduce the oxidative injuries

of brain cells by ROS could have an enormous impact in

improving outcomes of ischaemic brain disease [8,9].

Flavonoids, which exist naturally in fruit, vegetables, nuts,

seeds flowers, etc., have effects of oxidation resistance, anti-

virus and anti-inflammation and thus play an important role

in the prevention of chronic cardiovascular or cerebrovascular

disease [10–12]. They are a group of polyphenolic compounds

diverse in chemical structure and characteristics. Scutellarin,

the major active principle (flavonoid) extracted from Erigeron

breviscapus, significantly reduced infarct volume, amelio-

rated the neurological deficit [13]. Baicalin (7-glucuronic

acid, 5, 6-dihydroxyflavone) (Figure 1(A)), is also a flavonoid

compound derived from the dried root of a traditional

Chinese medicine Scutellaria baicalensis Georgi. The previ-

ous studies have shown that baicalin has many effects, such

as anti-inflammation, antioxidation, anti-thrombosis, etc.

[14–18]. Therefore, it is of special interest to understand

whether baicalin has a positive influence on neuronal

apoptosis induced by hypoxia.

The rat pheochromocytoma (PC-12) cell line provides an

established neuron-like system that can be used as a model for

cell death due to oxidative stress. In the present study, PC12

cells were employed to detect the neuroprotective effect and

possible mechanisms of baicalin on hydrogen peroxide

(H2O2)-induced cell damage. Furthermore, whether these

neuroprotective effects were associated with the JAK/STAT

signalling pathway, one of the conserved signal transduction

pathways were also elucidated.

Materials and methods

Reagents

Baicalin was supplied by Wuhan Tobacco Group Co. Ltd

(Wuhan, China). It was derived from the dried root of

Scutellaria baicalensis Georgi and refined in the laboratory,

its purity was more than 96%, which was verified by

HPLC methods, and it was dissolved in PBS before use.

All other chemicals and solvents were of HPLC grade.

Ham’s F12K-Medium and antibiotics were purchased from

Gibco Invitrogen (Carlsbad, CA) and foetal bovine serum

*Wen-xia Zheng, Feng Wang and Xiao-lu Cao are co-first authors.

Correspondence: Xia-min Hu, MD, PhD, Professor, Department ofPharmacology, Medical College, Wuhan University of Science andTechnology, Wuhan 430065, Hubei Province, PR China. Tel: +86-27-68893640. Email: huxiamin@wust.edu.cn

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(FBS) was obtained from Hangzhou Sijiqing Biological

Engineering Materials Co. Ltd (Hangzhou, China). 3-(4,5-

Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

(MTT) were obtained from Sigma–Aldrich (St. Louis,

MO).The Detection Kit of Propidium Iodide (PI) and

Annexin V-FITC Apoptosis was purchased from Sigma

Chemical Co. (St. Louis, MO). The reagent kits for

determining Maleic Dialdehyde (MDA), Superoxide dismu-

tase (SOD), Glutathione (GSH) and Glutathione peroxidase

(GSH-Px) were purchased from Nanjing Jiancheng Institute

of Biological Engineering (Nanjing, China). Mouse mono-

clonal antibodies against survivin, Bcl-2, caspase-3, STAT3,

p-STAT3 and b-actin were obtained from Santa Cruz

Biotechnology (Santa Cruz, CA). Other general agents were

available commercially.

Cell culture

Rat pheochromocytoma cells (PC12 cells) were dissociated

and cultured at a density of 1.5� 105 cells cm�2 onto poly-

D-lysine-coated 96- or 6-well plates or coverslips. The cells

were cultured in Ham’s F12K–Medium with 15% horse

serum, 2.5% foetal bovine serum, 100 U ml�1 penicillin,

100mg ml�1 streptomycin and were maintained in a

humidified incubator in air with 5% CO2.

Assessment of cell viability

Cell viability was assessed by the MTT reduction assay. PC12

cells (in 96-well plates) were subjected to the appropriate

treatment. After incubation with baicalin (1 mM, 2 mM, 5 mM)

for 24 hours and exposure with H2O2 for 12 hours, the

cultures were incubated with MTT solution (5 mg ml�1) for

4 hours at 37 �C. Then, the medium was discarded and DMSO

was added to solubilize the reaction product formazan with

shaking for 5 minutes. Absorbance at 570 nm was measured

with a microplate reader (ELx800; Bio-Tek, Winooski, VT).

Cell viability of the vehicle-treated control group not exposed

to either H2O2 or baicalin was defined as 100%.

Apoptosis analysis by flow cytometry

Detection of apoptosis by flow cytometry was performed

using the AnnexinV-FITC/PI apoptosis detection kit. After

incubation with baicalin (1 mM, 2 mM, 5 mM) for 24 hours and

exposure with H2O2 for 12 hours cells cultured on 6-well

plates were washed twice with ice-cold PBS, suspended in

3 ml of PBS, followed by centrifugation at 1200 rpm for 5

minutes. After discarding the supernatant, the cells were

resuspended in 200 ml binding buffer and incubated with 10 ml

Annexin V-FITC and 10 ml PI at room temperature for 15

minutes in the dark. Then the cells were added 300 ml binding

buffer before the fluorescence analysis was performed using a

flow cytometer (BD-LSRII, New Haven, CT, USA).

Determination of superoxide dismutase (SOD),glutathione (GSH), glutathione peroxidase (GSH-Px)activities and malondialdehyde (MDA) content

PC12 cells were transferred to 6-well plates in 2 ml Ham’s

F12K-Medium and pre-incubated overnight. After pre-

incubation, the cells were incubated at 37 �C for 24 hours

without or with different concentrations of baicalin (1 mM,

2 mM, 5 mM). Then a final concentration of 400 mM H2O2 was

added to each well except the control for 12 hours. At the end

of the treatment, PC12 cells were washed with PBS and

homogenized in 300 ml 0.5% Triton X-100 (PBS, pH 7.4)

through sonication on ice for 10 seconds. After incubation at

4 �C for 10 minutes, the homogenates were centrifuged and

the supernatants were used for assay.

SOD, GSH, GSH-Px activities and MDA content in PC12

cells were measured by means of kit assays according to the

manufacturer’s instructions. SOD activity was determined

with xanthine oxidase method and MDA content was

determined with thiobarbituric acid method, with a spectro-

photometer at 550 nm (SOD) and 532 nm (MDA) wavelength,

respectively. The activities of GSH-Px and GSH were assayed

by quantifying the rate of oxidation of the reduced glutathione

to the oxidized glutathione by H2O2, with 420 nm (GSH) and

412 (GSH-Px) wavelength, respectively.

Immunofluorescence microscopy

A coverslip was added into a 6-well plate and cells were

grown in culture media until they reached 50% confluence.

After incubation with baicalin (1mM, 2 mM, 5 mM) for 24

hours and exposure with H2O2 for 12 hours, media was

aspirated from plates and washed twice with PBS. Cells were

fixed with 4% paraformaldehyde solubilized in PBS-0.1%

Triton-X 100 for 20 minutes at room temperature (RT). This

was blocked for 1 hour with 2 ml of 1�PBS-1% BSA-4%

goat serum. Antibodies were spun down for 5 minutes at

10 000 rpm before use, to remove small aggregates. This

was washed twice for 5 minutes with 2 ml of 1� PBS. It was

then stained with primary antibody for 45 minutes at room

Figure 1. (A) The chemical structure of baicalin. (B) Effect of baicalinon the viability of PC12 cells. PC12 cells were pre-incubated withdifferent concentrations of baicalin for 24 hours and then treated with400mM H2O2 for 12 hours. The viability of PC12 cells was detected byMTT assay. Results are presented as means� SD (n¼ 5). ##p50.01 vs.control group, ** p50.01 vs. H2O2-treatment group.

228 W.-X. Zheng et al. Brain Inj, 2014; 28(2): 227–234

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temperature in 40 ml of 1� PBS-1% BSA by forming a drop

on the coverslip. At least two dilutions (1:200 and 1:1000)

were used to start optimizing the staining. This was then

washed 5-times for 5 minutes with 1� PBS-0.2% BSA.

IT was then stained with conjugated secondary antibody

for 30 minutes at RT in 40 ml of PBS-1% BSA and washed

5-times for 5 minutes with 2 ml of PBS. The slide was

mounted with anti-fading agent.

Western blot analysis

PC12 cells were incubated with baicalin (1 mM, 2 mM, 5 mM) at

37 �C for 24 hours and then exposed with H2O2 for 12 hours.

The medium was removed and cells were washed twice with

cold PBS before lysed in ProteoJET� Mammalian Cell Lysis

Reagent (MBI fermentas). The lysate was sonicated for 20

minutes on ice, followed by centrifugation at 4 �C, 12 000� g

for 15 minutes. The protein concentration from the supernatant

was measured by the Lowry method [23]. The supernatants

(50mg protein/lane) were separated on 10% SDS-polyacryl-

amide gel and transferred to nitrocellulose membrane in

transfer buffer. After blocking with TBS buffer containing 5%

non-fat milk, the membrane was incubated with survivin, Bcl-

2, caspase-3, STAT3, p-STAT3 and b-actin monoclonal

antibody (sc-47750, sc-7382, sc-56055, sc-8019, sc-8059, sc-

47778, Santa Cruz, CA) at 4 �C overnight. After washing with

0.1% Tween-20 in TBS, a secondary antibody, horseradish

peroxidase-conjugated anti-mouse IgG antibodies (1:1000,

Santa Cruz Biotechnology, CA) was applied to the membrane

for 1.5 hours at room temperature. Finally, the membrane was

treated with the reagents in the enhanced chemiluminescence

detection kit (ECL system, Amersham Pharmacia Biotech,

Piscataway, NJ) according to the manufacturer’s instructions

and exposed using an X-ray film. The relative density of the

immunoreactive bands was quantitated by Quantity One

(Version 4.6.2, Bio-Rad Technical Service Department,

California, USA). The relative amount of survivin, Bcl-2,

caspase-3, STAT3, p-STAT3 in each lane was obtained after

normalization with the �-actin values in the same lane.

Statistical analysis

All values were expressed as mean�SD. Data were statis-

tically analysed by ANOVA. The Newman-Keuls compari-

sons were used to determine the source of significant

differences where appropriate. p values below 0.05 were

considered statistically significant.

Results

Effect of baicalin on cell viability againstH2O2-induced cytotoxicity

To examine the neuroprotective effects of baicalin against

H2O2-induced cytotoxicity, different concentrations of baica-

lin were added to the culture medium for 24 hours before

H2O2-treatment. As shown in Figure 1(B), compared with

the normal control group, the viability of the cells exposed

to 12-hour H2O2-treatment was reduced to 60.97%� 2.01.

Meanwhile, pre-treatment with baicalin significantly attenu-

ated H2O2-induced cell apoptosis in a dose-dependent manner

and the viability of these cells was significantly increased to

78.47%� 2.25, 90.81%� 1.65, 97.88%� 2.07 at the doses

of 1 mM, 2 mM and 5 mM, respectively.

Baicalin inhibited the apoptosis of PC12 cellexposed to H2O2

In this study, hydrogen peroxide (H2O2) is used to induce

oxidative injury and represents a valuable model for inves-

tigation of oxidative injury-driven neuronal death. After PC12

cells were treated with H2O2 for 12 hours, the average

apoptotic rate was 42.27%� 3.52, which was obviously

increased compared with the H2O2-untreated control group

(15.41%� 1.66, Figure 2). To determine the protective effect

of baicalin on apoptosis, cells were pre-treated with baicalin

(1 mM, 2 mM, 5 mM) for 24 hours and then exposure with H2O2

for 12 hours. The result showed that baicalin significantly

suppressed cell apoptosis induced by H2O2 at both the

moderate (2 mM, apoptotic rate: 24.8%� 1.96, p50.05) and

high doses (5 mM, apoptotic rate: 16.5%� 1.21, p50.01)

compared with the control group.

Effects of baicalin on SOD, MDA, GSH and GSH-Pxin PC12 cell exposed to H2O2

To determine whether the antipoptotic effect of baicalin is

associated with the activity of anti-oxidative enzymes, the

activities of SOD, GSH and GSH-Px and the content of MDA

were analysed. Compared to the normal group, the activities

of SOD and GSH-Px together with GSH content in PC12 cells

exposed to H2O2 for 12 hours were decreased significantly.

Pre-incubation with baicalin for 24 hours increased the

activities of SOD and GSH-Px along with GSH content

significantly; on the other hand, MDA level was increased

in PC12 cells exposed to H2O2, whilst pre-incubation with

baicalin (for 24 hours) decreased MDA level significantly

compared to the H2O2-treated group (Figure 3).

Baicalin down-regulated survivin expressionby immunofluorescence

Indirect immunofluorescence was used to determine survivin

expression in PC12 cells (shown in Figure 4). Survivin was

obviously expressed at endochylema in PC12 cells of the

control group (green flourescence). From the photomicro-

graphs it was found that survivin expression was reduced

by 12 hour H2O2-treatment. However, pre-incubation

with baicalin (for 24 hours) increased its expression. The

cytoprotective effect of baicalin was also confirmed by the

hoechst 33 342 staining. Condensed or fragmented nuclei

were shown in the cells exposed to H2O2 (12-hours). The

injury was also alleviated by pre-treatment with baicalin.

Effect of baicalin on the expressions of survivin, Bcl-2and caspase-3 partially in a JAK-STAT3 dependentmanner

The protein expressions of apoptosis-related genes including

survivin, Bcl-2, caspase-3, Cleaved Caspase-3, STAT3 and

p-STAT3 were assessed by western blot (Figure 5). As shown

in Figures 5(A–F) by western blot assay, survivin and Bcl-2

expressions were decreased obviously in H2O2-treated cells,

whiles caspase-3 and Cleaved Caspase-3 expression was

DOI: 10.3109/02699052.2013.860469 Baicalin protects PC-12 cells against apoptosis 229

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increased. However, pre-treatment with baicalin (1 mM, 2 mM,

5 mM) for 24 hours up-regulated survivin and Bcl-2 expres-

sions, but decreased caspase-3 and Cleaved Caspase-3

expression compared to the H2O2 -treated group, all these

effects being presented in a dose-dependent manner. It is

supposed that the above effects of baicalin on the expressions

of the apoptotic genes (survivin, Bcl-2, caspase-3 and Cleaved

Caspase-3) partially might via activating of the JAK-STAT

pathway. Although there was no significant difference on the

expression of the unphosphorylated enzyme (STAT3) among

the experimental groups, however, cells exposed to H2O2 only

resulted in a decreased level of p-STAT3 expression without

influencing the unphosphorylated form compared with the

normal group; on the other hand, pre-treatment with baicalin

(1mM, 2 mM, 5 mM) effectively elevated p-STAT3 expression

in a dose-dependent manner. At the presence of AG490

(the inhibitor for JAK/STAT pathway), there were no

differences on the expressions of survivin, Bcl-2, caspase-3,

p-STAT3 and STAT3 in all the experimental groups

(p50.05). As for the cells co-treated with baicalin (1 mM,

2 mM, 5 mM) and AG490, the increase of survivin, Bcl-2,

p-STAT3 and the reduction of caspase-3 expression in

baicalin-treated groups were inhibited by AG490.

Discussion

The intracellular ROS generation during ischaemia and

reperfusion contributes to a disturbed membrane function,

resulting in a critical intracellular calcium accumulation and

leading to cell apoptosis and death [19]. Rat pheochromocy-

toma PC12 cell line is a commonly used cell line that

retains the features of dopaminergic neurons. The main issue

addressed by this study is whether baicalin works against

H2O2-induced damage in PC12 cells and the possible

mechanisms underlying the process.

In the present study, H2O2, as an instrument, was

established the oxidative stress model of cerebral ischaemia

in PC12 cells. These studies have shown that baicalin

Figure 2. (A) Apoptotic rate in PC12 cells treated with baicalin by flow cytometry. PC12 cells were pre-treated with baicalin for 24 hours andthen exposed to H2O2 (400 mM) for 12 hours, the cells in the H2O2 (400 mM, 12 hours) treated group as a vehicle. The apoptotic rate was determinedby flow cytometry. (B) Data analysis depicted in (A). Results are presented as means� SD (n¼ 5). ##p50.01 vs. control group, *p50.05 or **p50.01vs. H2O2-treatment group.

230 W.-X. Zheng et al. Brain Inj, 2014; 28(2): 227–234

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(2mM, 5 mM for 24 hours) protected PC-12 cells from death

caused by H2O2-induced oxidative stress. Due to the key

role of ROS in hypoxic-ischaemic brain damage, it can be

hypothesized that the protective effect of baicalin on H2O2-

induced PC12 cells apoptosis is related to the activity of the

anti-oxidant system. As it is known that SOD is an important

antioxidant enzyme in mitochondria against oxidative stress

[20]; MDA, as a decomposition product of lipid hydroper-

oxides, is an indicator of oxidative damage to cells and tissues

and in vitro reacts with hydrogen peroxide to form undeter-

mined degradation products [21]. Also, the biochemical

function of glutathione peroxidase (GSH-Px) is to reduce

lipid hydroperoxides to their corresponding alcohols and

to reduce free hydrogen peroxide to water [22]. The findings

showed that the activities of SOD and GSH-Px were

decreased and MDA level was increased significantly after

PC12 cells were exposed to H2O2 for 12 hours. However,

baicalin could upregulate the activities of SOD and GSH-Px,

and down-regulate MDA level, which indicates that baicalin

improvesthe antioxidative defense of H2O2-treated PC12

cells.

Apoptosis can be triggered by numerous mediators

including receptor-mediated signals, withdrawal of growth

factors, and environmental agents. Direct exposure of cells to

ROS via H2O2 or superoxide (O�2 ) generating agents

can induce apoptosis. Caspases have been shown to be

specifically involved in the initiation and execution phases

of apoptosis. It has been reported that oxidative stress

induces the release of mitochondrial cytochrome c and

other apoptogenic proteins (e.g. apoptosis-inducing factor)

from the mitochondrial inter-membrane space into the

cytosol. These proteins then bind to Apaf-1 and activate

pro-caspase-3, which leads to mitochondrial dependent

apoptosis [23,24]. The Bcl-2 family, consisting of antiapop-

totic (e.g. Bcl-2) and proapoptotic (e.g. Bax and Bad)

members, plays an important role in the regulation of cell

death [25]. Survivin, an inhibitor of apoptosis, is a cytoplas-

mic protein highly expressed in cancer but hardly detectable

in normal differentiated tissues. The survivin pathway is

suggested to be crucial in controlling the initiation of the

upstream antiapoptotic mechanism that leads to the mito-

chondrial-dependent apoptosis [26]. This study showed that

baicalin could up-regulate the expression of survivin and

Bcl-2 obviously in PC12 cells exposed to H2O2 for 12 hours,

while caspase-3 expression was down-regulated. The above

results suggested that the anti-apoptotic effect of baicalin was

associated with its modulation on the expressions of these

genes.

Signalling through the JAK/STAT pathway is initiated

when a cytokine binds to its corresponding receptor. This

leads to conformational changes in the cytoplasmic portion of

the receptor, initiating activation of receptor-associated

members of the JAK family of kinases. The JAKs, in turn,

mediate phosphorylation at the specific receptor tyrosine

residues, which then serve as docking sites for STATs and

other signalling molecules [27]. STAT proteins bind to the

phosphorylated receptor by their SH2 domains and are

phosphorylated, then the phosphorylated STAT proteins

enter the nucleus to form an active transcriptional factor.

Among these STATs, STAT3 played an important role during

the activation of the JAK/STAT pathway, while AG490

(tyrphosin AG490, inhibitor of JAK kinase) can block the

Figure 3. Effect of baicalin on the activities of SOD and GSH-Px, the content of GSH and MDA in PC12 cells. PC12 cells were pre-treated withbaicalin for 24 hours and then exposed to H2O2 (400 mM) for 12 hours, the cells in the H2O2 (400 mM, 12 hours) treated group as vehicle. The activitiesof SOD and GSH-Px and the content of GSH and MDA were measured by means of kit assays according to the manufacturer’s instructions. Resultsare presented as means� SD (n¼ 6). #p50.05 and ##p50.01 vs. control group, *p50.05 or **p50.01 vs. H2O2-treatment group.

DOI: 10.3109/02699052.2013.860469 Baicalin protects PC-12 cells against apoptosis 231

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JAK/STAT pathway effectively. Previous studies have sug-

gested that Bcl-2 and caspase-3 are important STAT3 target

genes involved in the transformation. Recent investigations

have focused on the potential function of survivin as a down-

stream target of Stat3 signalling. Survivin, found on chromo-

some 17q25, represents one member of the inhibitor of

apoptosis family of proteins [28]. The previous study

indicates that ischaemia acts synergistically to promote

activation of STAT3 and STAT3-dependent transcription

of survivin in insulted CA1 neurons and identifies STAT3

and survivin as potentially important therapeutic targets in

an in vivo model of global ischaemia [29]. In order to

investigate whether the modulation of baicalin on the above

gene expressions was via the JAK/STAT3 pathway, the

protein expressions of STAT3 and p-STAT3 were determined

in the present study. It was found that baicalin increased

p-STAT3 expression; on the other hand, co-treated with

baicalin and AG490, the elevation of survivin and Bcl-2

expressions was attenuated while caspase-3 expression was

enhanced. Meanwhile, there was no significant difference

Figure 4. The effect of baicalin on survivinexpression in PC12 cells exposed to H2O2 byimmunofluorescence assay. PC12 cells werepre-treated with baicalin for 24 hours andthen exposed to H2O2 (400 mM, 12 hours),cells only exposed to H2O2 were as a vehiclegroup. The upper-layer showed the immuno-fluorescence morphology of survivin under afluorescence microscope, the down-layershowed the DNA counterstaining withHoechst 33342, merged images showed thecolocalization of survivin and nucleus, Scalebar¼ 100 mm.

232 W.-X. Zheng et al. Brain Inj, 2014; 28(2): 227–234

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Figure 5. (A, C, E, G, I) Western blot analysis for the expressions of survivin, Bcl-2, caspase-3, STAT3 and p-STAT3 in PC12 cells exposed to H2O2.(B, D, F, H, J) Quantitative analysis for survivin, Bcl-2, caspase-3, STAT3 and p-STAT3 protein levels in PC12 cells, �-actin serves as the house-keeping protein in western blot assay. Data are expressed as means� SD, $$ p50.01 vs control group, *p50.05 vs. H2O2-treatment group, **p50.01vs. H2O2-treatment group. D p50.01 vs. vehicle control group, #p50.05 or ##p50.01 vs. the same dosage of baicalin without AG490 groups,respectively.

DOI: 10.3109/02699052.2013.860469 Baicalin protects PC-12 cells against apoptosis 233

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on STAT3 expression among all the experimental groups, but

it was interested that p-STAT3 expression was upregulated

significantly by baicalin, indicating that the effect of baicalin

on PC12 cells apoptosis induced by H2O2 was partially via the

JAK/STAT3 pathway.

In summary, this study provided the first evidence

demonstrating that baicalin suppressed PC12 cell injury

induced by H2O2 via the mechanisms of anti-oxidation and

anti-apoptosis, in which the activation of the JAK/STAT3

pathway was possibly involved.

Declaration of interest

The work was supported by the National Natural Science

Foundation of China (no.31171327). The authors report no

conflicts of interest.

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