Neuroprotective Effects of Ginkgo Biloba Extract (GbE) on Oxygen-Glucose Deprivation (OGD) in PC12 Cells

doi:10.4236/eng.2013.510B030

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Engineering, 2013, 5, 142-145

http://dx.doi.org/10.4236/eng.2013.510B030 Published Online October 2013 (http://www.scirp.org/journal/eng)

Neuroprotective Effects of Ginkgo Biloba Extract (GbE)

on Oxygen-Glucose Deprivation (OGD) in PC12 Cells

Chunli Mei1, Xuelu Han1, Jing Zhang1, Ling Gao 1*, Huimin Liu1,2*

1Beihua University, Jilin, 132013, China

2Department of Neurology, Affiliated Hospital, Beihua University, Jilin, 132013, China

Email: [email protected]

Received April 2013

ABSTRACT

Ischemic cerebrovascular disease is a global health problem. According to the World Health Organization, ischemic

stroke is actually the most common cause of death in the world. Ginkgo biloba extract (GbE) is a traditional Chinese

medicine for angina pectoris. Ginkgo biloba plays a role in expanding blood vessels, increasing coronary and cerebral

blood flow, preventing platelet aggregation, inhibiting thrombosis, and improving the microcirculation. In the present

study, we investigated the mechanisms involved in the neuroprotective effects of GbE in a model of hypoxic-ischemic

brain disease. We used NGF (100 ng/ml for 6 days) and OGD (5% CO2 and 95% N2, 1 mmol/l NaS2O4 in sugar-free

DMEM for 16 h) to stimulate PC12 cells and convert them into neurons in order to establish an ischemia model. The

results showed that PC12 cells transformed into cells that looked like neurons and that MAP2 was up-regulated in

NGF-treated PC12 cells. Cell apoptosis was found to be up-regulated after NGF stimulation and OGD. The apoptosis

rate after 16 hours of OGD was 19.44%. GbE (50 ng/ml) reduces apoptosis rate to 11.35%. These results may help to

show that NGF treatment can be combined with OGD to establish an in vitro model of acute ischemic brain damage. In

the present study, we find that GbE effectively increases the survival rate of PC12 cells and relieves OGD damage.

These results suggest that GbE has the neuroprotective effects of ischemic brain damage.

Keywords: NGF; OGD; PC12 Cells; Ginkgo Biloba; Neuroprotective Effects

1. Introduction

Ischemic stroke occurs when the blood supply to the

brain is obstructed. Accumulating evidence suggests that

the cell death observed during the first few hours of ce-

rebellar ischemia is a result of apoptosis as opposed to

necrosis, which is considered the predominant form of

cerebellar damage generated by ischemia. Moreover,

effective methods of preventing and controlling ischemic

cerebrovascular disease have been a topic of great interest.

Ginkgo biloba extract (GbE) is obtained from green

leaves of the Ginkgo biloba tree according to a well-de-

fined procedure [1]. The GbE displays, mainly via its fla-

vonoid constituents, free radical scavenging and antioxi-

dant actions that are probably associated with its protec-

tive actions in animal models of hypoxia and ischaemia

[2]. Earlier studies have described its neuroprotetive and

neurotrophic activities in the hippocampal formation.

GbE is already recognized as a polyvalent therapeutic

agent in the treatment of disturbances of multifactorial

origin including cerebral insufficiency and mild cogni-

tive impairments in elderly patients [3]. Several clinical

studies support the potential usefulness of GbE in AD

and in vascular dementia.

In this study, the oxygen-glucose deprivation of PC12

cells was used to establish a cerebral hypoxia-ischemia

model to investigate the mechanism of GbE neuroprotec-

tion. In the following research, we measure the induc-

tion of sup eroxide dismutase (SOD) in an atte mpt to elu-

cidate possible mechanisms that underlie GbE-mediated

protection against OGD in PC12 cells.

2. Materials and Methods

2.1. Cell Culture and Differentiation by NGF

PC12 cells were purchased from the Cell Bank of the

Chinese Academy of Sciences. The cell line was main-

tained in DMEM medium supplemented with 10% (v/v)

fetal bovine serum and 5% horse serum (FBS, GIBCO),

100 IU/ml streptomycin, 100 IU/ml penicillin, pH 7.0,

and detached with 0.25% trypsin (Sigma, USA). PC12

cells were grown at 37˚C in 5% CO2. Cells were grown

in 5% horse serum containing media on collagen-coated

tissue culture dishes before differentiation [5]. After the

cells got attached, they were treated with 100 ng/ml

*Corresponding a uthor.

C. L. MEI ET AL.

143

nerve growth factor (NGF 2.5S; Promega, Madison, WI)

and Cultured with serum-free DMEM for 6 days. Ob-

served and photog ra phed.

2.2. MAP2 Immunocytochemical Analysis

The cells were fixed with 4% paraformaldehyde/PBS and

were permeabilized with 0.1% Triton X-100 in PBS for

10 min. The cells were then incubated in 5% goat se-

rum/PBS for 1 h at room temperature. Cells were washed

again then incubated at 4˚C overnight in the presence of

anti-MAP2 (1:1000 dilution, Santa SC-20172). After

washing twice with PBS, the cells were incubated with

fluorescently labeled secondary Cy3-goat anti-rabbit

(Santa ) for 1 hour at room temperature [5]. The results

were observed by a fluorescence microscope equipped

with a photomicrograph system.

2.3. OGD Model of PC12 Cells after NGF

Treatment

PC12 cells were treated with NGF for 6 days. Cells were

then washed 3 times with DMEM, and the cells were

cultured with DMEM in the presence of no sugar and 1

mmol/l Na2S2O4 in hypoxic conditions (37˚C, 5% CO2

and 95% N2) for 3 h, 6 h, 9 h, 12 h, 16 h, or 24 h.

2.4. Cell Viability Assay

The MTT method was used to assess the cytotoxic ef-

fects of GbE. The cells were grown to a density of 5 ×

104 cells/well and were then treated with 10 ng/ml, 20

ng/ml, 30 ng/ml, 50 ng/ml and 100 ng/ml GbE in a

96-well plate for 24 h. At the end of the treatment, the

GbE -containing medium was carefully removed and the

cells were treated with OGD for 16 h. The culture me-

dium was removed and 200 μl medium containing 20 μl

MTT (5 mg/ml in PBS) (St. Louis, MO, USA) was added

to each well. After 4 h of incubation at 37˚C, the medium

was removed and 100 μl DMSO was added to each well.

The optical absorbance (A) of each well was read at 490

nm. The percentage of viable cells was calculated as fol-

lows: (A of experimental group/A of control group) ×

100%.

2.5. Hoechst 33258/PI Staining

The cells were washed three times with DMEM and were

incubated in DMEM containing 50 ng/ml GbE for 24 h.

The cells were washed three times with DMEM and were

incubated for 16 h in DMEM containing 1 mmol/l

NaS2O4 under hypoxic conditions (37˚C, 5% CO2 and

95% N2) in the absence of sugar [5] The cells were

stained with PI (10 µg/ml) and Hoechst 33258 (10 µg/ml,

Sigma, USA) and then fixed by 4% paraformaldehyde.

For each cover slide, 1000 ~ 1500 cells were examined

under a fluorescence microscope (Olympus BX51, Japan)

and photographed with a digital camera (Olympus, Ja-

pan). The results were expressed as the percentages of

apoptotic cells and necrotic cells, respectively.

2.6. Western Blotting Analysis

After treatment with GbE and OGD for 16 h , the cells

were washed twice using cold PBS and 1 × 106 cells

were lysed using RIPA buffer (50 mmol/l Tris (pH 8.0),

150 mmol/l NaCl, 0.1% SDS, 1% NP40 and 0.5% so-

dium deoxycholate) containing protease inhibitors (1%

cocktail and 1 mmol/l PMSF). Total proteins were sepa-

rated using 15% SDS-PAGE and were transferred to a

PVDF membrane. The membrane was blocked using

Tris-buffered saline with 0.1% Tween 20 (pH 7.6, TBST)

for 1 h at room temperature and was incubated with the

primary antibody solution (1:1000) at 4˚C overnight.

After two washes in TBST, the membrane was incubated

with the HRP-labeled secondary antibody (Santa SC-

2073) for 1 h at room temperature and was washed three

times with TBST. The final detection was performed

using enhanced chemiluminescence (ECL) western blot-

ting reagents (Amersham Biosciences, Piscataway, NJ)

and the membrane was exposed to Lumi-Film Chemilu-

minescent Detection Film (Roche). Loading differences

were normalized using a monoclonal ß-actin antibody.

The antibodies used in the study included SOD (Santa

SC-18503) and ß-actin (Santa SC-2021).

2.7. Statistical Analysis

SPSS software was used for statistical analyses, and val-

ues are presented as means ± SD. An ANOVA was used

to compare the mean values. P values less than 0.05 were

considered to indicate statistically significant differences.

3. Results

3.1. Morphological Changes of PC12 Cells

The results show that treatment with NGF (100 ng/ml)

stimulates neuron-like differentiation of PC12 cells as

seen under the microscope. PC12 cells changed into

neurons after 1 day of NGF treatment and later formed

synapses. Synapses extended up the length of the cell

after 3 days of treatmen t. T he synaptic length increased 6

-to 8 -fold after 6 days of treatment. Bars = 20 um, Invert

microscope, Olympus IX71, Japan (×200) (Figure 1).

3.2. Immunoflu orescence Analysis

The results showed that PC12 cells cultured with NGF

for 6 da ys showed characteristic MAP2 immunofluores-

cence staining (Figure 2). Application plus pro 6.0 soft-

ware to add image fusion after confirm purple fluores-

C. L. MEI ET AL.

144

Figure 1. The morphological changes of PC12 cells (×200).

PC12 cells were pretreated with 100 ng/ml NGF for 1, 2, 3,

4, or 6d. The differentiated cells were photographed under

a phase contrast microscope.

Figure 2. Morphological changes captured with fluores-

cence microscopy following immunofluorescence staining

(×400).

cent were for PC12 cells transformation of neurons ap-

pearance cell. MAP2 immunofluorescence stain was

strong positive expression. PBS control was negative

fluorescence.

3.3. Effects of GbE on Cell Proliferation

The cytotoxicity of OGD for 16 h and GbE (Yabao

Shanxi China) plus OGD for 16 h treatments were de-

termined by examining their effects on the proliferation

of PC12 cells. PC12 cells were tr eated with 10 ng/ml, 20

ng/ml, 30 ng/ml, 50 ng/ml and 100 ng/ml GbE for 24 h

before OGD for 16 h (Figure 3). MTT assays showed

that treatment with OGD for 16 h effectively inhibited

the growth of PC12 cells by 19.44%. Compared to the

OGD for 16 h-treated group, the Ginkgo biloba plus

OGD for 16 h-treated group inhibited the growth of

PC12 cells by 17.82%, 13.31%, 10.36%, 4.29% and

3.92%, in dose-dependent manner, and the survival rates

were higher than in the OGD for 16 h -treated group. Be-

cause there was no significant difference (P > 0.05) be-

tween the survival rates of the 50 ng/ml and 100 ng/ml

GbE-treated cells, 50 ng/ml GbE was used in all subse-

quent experiments.

3.4. Hoechst 33258/PI Staining

The representative microphotographs show the PC12

cells as detected by PI staining after OGD-reperfusion

Figure 3. MTT assay showing the growth inhibition of PC12

cells treated with Ginkgo biloba for 24 h plus OGD for 16 h.

The cells were grown to a density of 5 × 104 cells per we ll in

a 96-well plate for 24 h. The results show the growth of

PC12 cells following incubation in 96-well plates for 24 h

with 10 ng/ml, 20 ng/ml, 30 ng/ml, 50 ng/ml and 100 ng/ml

Ginkgo bilob a plus OGD for 16 h, compared with OGD for

16 h. *denotes a significant difference from control, P < 0.05;

** denotes a significant difference from OGD for 16 h, P <

0.05; denotes a significant difference from 50 ng/ml Ginkgo

biloba plus OGD for 16 h, P < 0.05. The data represent the

means ± SD obtained from three separate experiments that

were performed in triplicate.

induced injury; The representative microphotographs

show the apoptotic cells as detected by Hoechst 33258

staining after OGD -reperfusion induced. GbE effectively

increases the survival rate of PC12 cells and relieves

OGD damage. The summarized data show percentage

changes in the numbers of normal and apoptotic cells.

Data are expressed as mean ± SD; n = 4 wells for each

group; *P < 0.05 and **P < 0.01, compared to control and

other gr ou p (Figure 4).

3.5. Effects of Ginkgo Biloba on SOD

SOD plays a protective role in ischemia following its

activation. To investigate the neuroprotective mechan-

isms of GbE, the expression of SOD were examined us-

ing western blots (Figure 5 ). Compared with the controls,

SOD expression levels in PC12 cells increased in the

OGD for the 16 h-treated group. Compared with this

group, the expression levels of OGD increased in the

groups treated with GbE for 24 h combined with OGD

for 16 h. In this group, as the concentration of Ginkgo

biloba increased, the expression levels of OGD increased

in a dose-dependent manner. GbE may mediate the neu-

roprotective effect by OGD.

4. Discussion

Rat adrenal pheochromocytomas have been made into

PC12 cell lines. PC12 rat pheochromocytoma cells are

one of the most widely used cell culture systems for

studying neuronal differentiation [4]. In this study, we

used physical and chemical methods to establish OGD

conditions, and we used sugar-free culture medium and

NaS2O4 to establish a liquid environment lacking oxygen

C. L. MEI ET AL.

145

Figure 4. The cell de ath was analyzed by double fluor escent

staining with Hoechst 33258 and propidium iodide (PI).

Figure 5. PC12 cells were treated with OGD for 16 h or

different concentrations of Ginkgo biloba for 24 h com-

bined with OGD for 16 h. OGD expression levels were de-

termined using western blots. The results shown are repre-

sentative of three independent experiments. NIH imaging

indicated that the protein signal densities increased in the

groups treated with Ginkgo biloba combined wi th OGD for

6 h compared with OGD for 6 h-tr e ate d groups.

and sugar [5]. The circulation of oxygen and glucose is

necessary to maintain neurons’ normal physiology func-

tion and survival. During the ischemic damage process,

neurons’ blood supply is disrupted. OGD is a model of

oxygen and glucose shortage. We used the OGD on neu-

rons to simulate the ischemic brain damage process that

occurs in the body. We used NGF combined with OGD

to set up an ischemia tolerance model. Our study shows

that PC12 cells treated with NGF form cells are neu-

ron-like in appearance. These results may help to estab-

lish NGF treatment followed by OGD as an in vitro

model of acute ischemic brain damage. The model pro-

vides a new tool for the identification of pathways that

are involved in cerebral ischemia. This cerebral ischemic

model is one example of the cell’s broader general-stress

response. Therefore, the present model could be applied

to the study of mechanisms that are involved in tolerance

to other stressful stimuli. Cell-based assays with high-

throughput capacity can be used as direct screens and

models to explore molecular mechanisms that are in-

volved in cellular function and pathology.

In the present study, we examined MAP2 expression

by immunoblotting. MAP2 is a neuron specific protein.

Microtubule stabilizing is critical for neurite outgrowth

and dendrite development. So MAP2 is widely used as a

marker and plays a critical role in neurite ou tgrowth. It is

a helpful diagnostic and prognostic feature in various

neurological disorders. We found that MAP2 is ex-

pressed in PC12 cells that had been exposed to NGF.

Tolerance to ischemia and hypoxia can be modeled in

vitro and has bee n de s c ribed i n c ultured PC12 cells.

An OGD-damage model is one of the more commonly

used models for the study of cerebral ischemia. The prin-

ciple of the OGD model is that Na2S2O4 quickly clears

the oxygen in the culture matrix. In this study, oxygen

and glucose deprivation are applied to PC12 cells and the

results show that the OGD for 16 h-treated group effec-

tively inhibits the growth of PC12 cells, indicating that

the cell model causes cell damage. When compared with

the OGD for 16 h-treated group, the GbE plus OGD for

16 h-treated groups effectively increased the survival rate

of PC12 cells (Figures 3 and 4). Therefore, ginkgo bilo-

ba has a protective effect on OGD-induced cell injury.

Many studies indicate that oxidative stress plays a key

role in ischemic cerebrovascular disease. SOD plays a

vital role in the body’s oxidant and antioxidant balance

by removing superoxide anion radicals and protecting

cells from damage. SOD is involved in oxidative damage.

The results show GbE increases SOD activity, and this

may be the mechanism by which GbE promotes the neu-

roprotective effect (Figures 4 and 5). In conclusion, our

results demonstrate that GbE protects PC12 cells in an

OGD-deprivation model though reducing apoptosis rate.

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