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Introduction
Alcohol abuse is associated with several neurological
disorders[1-3]. It is also closely associated with the pathogenesis
of several neurological diseases such as Korsakoff¡¯s Syndrome, Marchiafava-Bignami disease, pellagrous encephalopathy,
and acquired hepatocerebral degenera-
tion[4,5]. In addition, there is evidence that the brains of alcoholics undergo toxic changes including a reduction in brain
volume[6-8], which may be due to the loss of neurons, shrinkage of neuronal cell bodies, or the reduction in the number and
extent of dendrites. Recent studies have also shown that apoptosis is one of the main changes of neurodegenera-tion
induced by ethanol[9-11]. All these changes may result from the reduction of endogenous antioxidant levels, the formation of
reactive oxygen species (ROS), the depletion of GSH, and the DNA fragmentation induced by ethanol
exposure[12,13]. Thus, it is important to find a therapeutic drug that can prevent ethanol-induced neurotoxicity.
Tanshinone (Tan), a major active ingredient of
Salvia miltiorrhiza extract, is a mixture of many kinds of analogue
compounds[14]. Tan has been reported to have protective effects on neuron cells in some
in vitro models including hypoxia, hypoglucose, oxidant injury, calcium overload, nitric oxide neurotoxicity, and glutamic acid
injury[15]. Tan IIA is the most abundant and structurally representative of Tan and can reduce the brain infarct volume in transient focal cerebral ischemia
in mice[16]. It has been reported that miltir-one inhibits upregulation of the GAGA receptor alpha4
subunit mRNA by ethanol withdrawal in hippocampus
neurons[17]. In addition, recently published research suggests that Tan IIA inhibits serum
deprivation-induced apoptosis in PC12
cells[18]. However, whether Tan IIA can protect against ethanol-induced neurotoxicity is still
unknown.
The present study was designed to investigate whether Tan IIA can protect PC12 cells from the neurotoxicity induced by
ethanol. For this objective, we analyzed the cell survival, ROS formation, lactate dehydrogenase (LDH) release, cell apoptosis,
and levels of the apoptosis-related p53 protein expression in PC12 cells and explored the role of Tan IIA as a neuroprotective
drug.
Materials and methods
Materials Tan IIA, a gift from Dr Xiao-yan YANG (Department of Clinical Pharmacology, Wuhan Tongji Medical College),
was dissolved in dimethylsulfoxide (Me2SO). The concentration of
Me2SO in the final culture media was
£0.1% (v/v), and had no toxic effect on PC12 cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Fluka,
and Dulbeco¡¯s modified Eagle¡¯s medium was from Invitrogen (Carlsbad, California, USA). The annexin V/propidium iodide
(PI) kit was purchased from the Becton Dickinson (BD) Company (Rahway, New Jersey, USA).
Cell culture and ethanol treatment PC12 cells (American Type Culture Collection, Rockville, MD, USA) were cultured in
DMEM medium supplemented with 10% heat-inactivated horse serum, 5% fetal bovine serum (FBS), 100 kU/L of penicil-lin,
and 100 mg/L of streptomycin. They were split at a 1:3
ratio every 4 d, for up to 8 passages. The cells were
subjected to the following conditions: (1) control (C, normal culture media alone); (2) ethanol (E, the medium supplemented with 50, 100, or 150
mmol/L ethanol); (3) Tan IIA (T, medium supplemented with 10
µmol/L Tan IIA); and (4) ethanol+Tan IIA (E+T. medium
supplemented with 150 mmol/L ethanol and 10 µmol/L Tan IIA). The concentrations of ethanol were used as previously
described[19-21]. Tan IIA was added to the media 4 h before adding ethanol at 70% confluence of PC12 cells. To maintain
constant concentrations of ethanol in the experimental treatment, all cultures were placed in plastic containers with
tight-fitting lids. All cellular analysis procedures were performed 24 h after the experimental conditions.
MTT cell viability assay Cell viability was measured using MTT assay. Briefly, the MTT solution (5 g/L) was added to
each well and incubated at 37 ºC for 4 h. The culture medium was removed and 100 µL dimethyl sulfoxide was added to each
well to dissolve the formazan. The optical density of each well was measured at 570 nm using a microplate reader (Spectra
MAX 340, Molecular Devices Co, Sunnyvale, California , USA). One culture dish with PC12 cells was used for 1 experiment
condition. Thus, each plate contained multiple wells of a given experimental condition and multiple control wells. This
procedure was replicated for 2-4 plates/condition. The MTT data were converted to the percentage of the respective
controls prior to analysis.
LDH release assay Following incubation, the media were analyzed for LDH activity using a modification of the
calorimetric method[22]. Briefly, each cell culture supernate was added to fresh assay mixture (pyruvate substrate, NADH, Sigma color
reagent, and 8 mol/L NaOH), and absorbance at 450 nm was recorded. The values were expressed as the sample mean
absorbance normalized to the percentage of the control value.
Measurement of intracellular ROS formation
The dye 2¡¯,7¡¯-Dichlorodihydrofluorescein diacetate (DCFH2-DA), which
is oxidized to fluorescent 2¡¯,7¡¯-dichlorofluorescin (DCF) by hydroperoxides, was used to measure relative levels of cellular
peroxides. The treated PC12 cells were washed, suspended in serum-free DMEM, incubated with 50 mmol/L dye at 37 °C for
30 min, washed with PBS, centrifuged, and the medium was then removed. Cells were dissolved with 1% Triton X-100
(Zhongshan Biotech, Beijing, China) and fluorescence was measured at an excitation wavelength of 485 nm, and an emission
wavelength of 530 nm using a fluorescence microplate reader. The values were expressed as the sample mean absorbance
normalized to the percentage of the control value.
Annexin V/PI staining After treatment, the cells were washed in cold PBS 3 times and resuspended in binding
buffer (containing 10 mmol/L HEPES/NaOH pH 7.4, 140
mmol/L NaCl, 2.5 mmol/L
CaCl2). FITC-annexin V was added to a final
concentration of 1 mg/L and the cells were incubated in darkness for 10 min. The cells were then washed again in PBS, centrifuged
and resuspended in binding buffer (100 µL). PI was added at a final concentration of 2 mg/L and incubated for 5 min before
immediate analysis of the cells on the flow cytometer (Becton Dickinson, Rahway, New Jersey, USA). Viable cells were
negative for both PI and annexin V; apoptosis cells were positive for annexin V and negative for PI, whereas late apoptotic
cells displayed both high annexin V and PI labeling. Non-viable cells which underwent necrosis were positive for PI and
negative for annexin V.
Indirect immuno-fluorescence After the PC12 cells were treated with ethanol or Tan IIA for 24 h, they were fixed in
ethanol-acetone (1:1) for 15 min at 4 °C. After rinsing with phosphate-buffer saline, the cells were incubated with rabbit
anti-rat p53 antibody (Santa Cruz, California,USA) at 4 °C for 12 h. FITC-labeled goat anti-rabbit antibody (Zhongshan Biotech,
Beijing, China) was then added and further incubated for 45 min at 37 °C. Negative control was made by replacing the first
antibody with phosphate-buffer saline. After the slides were covered with glycerol buffer, the expression of p53 was
observed under fluorescence microscopy and photos were taken. The number of PC12 cells stained with p53 antibody was
determined by counting the number of positive-stained cells in a total of at least 600 cells under magnification of ×400 in each
group. Data from the 5 experiments were expressed as mean±SEM.
Flow cytometry The p53 protein expression was measured by flow cytometric analysis as previously describ-
ed[23,24]. Briefly, the cells were harvested and washed in PBS. The cells were then fixed in 1.0% paraformaldehyde for 15 min
and permeated with 0.2% Triton X-100. After washing, aliquots of
1×106 cells/mL were incubated at 37 °C for 30 min with
rabbit anti-rat p53 (1:1000 dilution, Santa Cruz,
California, USA). After washing with PBS, FITC-labeled goat anti-
rabbit IgG antibody (Zhongshan Biotech, Beijing, China) was added and incubated for 45 min. Following this,
1×106 cells in each group were subjected to a flow cytometric analysis. Quantitative changes in p53 expression were assessed by mean
fluorescence intensity (MFI). To correct for non-
specific binding, PBS solution (instead of the first antibody) was added to the blank control tube.
Statistical analysis All data were presented as mean± SEM. One-way analysis of variance followed by the Q test using
SPSS 11.0 Software (Chicago, Illinois, USA) was performed to determine statistical significance.
P<0.05 was considered statistically significant.
Results
Effects of Tan IIA on the cell viability induced by ethanol
The optical density in the control cultures exceeded that in the
cultures containing 50, 100, and 150 mmol/L ethanol, which suggested that ethanol significantly induced the death of PC12
cells by MTT analysis (P<0.05). Tan IIA alone did not influence the survival rate of PC12 cells
(P>0.05), but the exposure of PC12 cells to Tan IIA for 4 h before the addition of 150 mmol/L ethanol significantly increased PC12 cell viability
(P<0.05, Figure 1).
Necrosis always results in the disruption of the cytoplasmic membrane, and the necrotic cells release cytoplasmic LDH
and other cell contents into the medium. We therefore examined the presence of LDH in the cultured medium. Ethanol
induced an increased release of LDH, while Tan IIA attenuated these effects significantly (Figure 2).
Effects of Tan IIA on ethanol-induced intracellular ROS formation
After treatment with Tan IIA for 4 h, ethanol was
added the culture media at a final concentration 150 mmol/L. The final DCF concentration significantly increased in the
ethanol-treated cells. However, Tan IIA significantly suppressed DCF fluorescence intensity in the ethanol-exposed cells
(P<0.05; Figure 3).
Effects of Tan IIA on ethanol-induced apoptosis
Treatment with 150 mmol/L ethanol for 24 h resulted in an increase of the
annexin V-/PI+, annexin
V+/PI- and annexin
V+/PI+ cells. Treatment with Tan IIA for 4 h decreased the number of annexin
V-/PI+, annexin
V+/PI- and annexin
V+/PI+ cells, as compared with the control cells
(P<0.05; Figure 4).
Effects of Tan IIA on ethanol-induced p53 expression
Under fluorescence microscopy, almost no cell was stained with p53
under normal culture condition or when treated with Tan IIA alone. After the PC12 cells were cultured in medium containing
150 mmol/L ethanol, the percentage of p53 positive-stained cells was upregulated markedly
(P<0.05). Having been pretreated with Tan IIA before adding ethanol, the ratio of the p53 positive-stained cells significantly
decreased (P<0.05; Figure 5A).
As shown by flow cytometry, there was no difference in p53 protein expression between the control and the Tan
IIA-treated PC12 cells. A significant increase in the p53 protein expression was observed when the cells were treated with 150
mmol/L ethanol, which was significantly attenuated by Tan IIA pretreatment
(P<0.05; Figure 5B, 5C).
Discussion
PC12 cells, which are derived from rat phaeochromocy-toma, contain a more homogeneous population, develop a faithful
neuronal phenotype, and are available in large amounts for biomedical study. Thus, PC12 cells have been widely used in both
neurobiological and neurotoxicological
studies[25]. In the present study, cell viability was detected in the PC12 cells by MTT
assay. As expected, there were significantly less viable cells in the ethanol-treated cells as compared with the controls.
However, incubation with Tan IIA before adding ethanol increased cell viability and raised the survival rate.
The LDH enzyme release and the formation of ROS confirmed the results obtained from the MTT assay. A breakdown in
the integrity of the plasma membrane results in the release of LDH into the extracellular space. LDH activity in the extracellular
fluid of cells exposed to ethanol was greater than that observed in cells exposed to ethanol and Tan IIA. It has been shown
that chronic ethanol treatment can also cause the formation of ROS and nitrogen species and the activation of the
mitochondrial permeability
transition[26]. In the present study, the PC12 cells exposed to high concentrations of ethanol showed an
increased formation of ROS. However, incubation with Tan IIA reduced the formation of ROS. All corresponded to the
decrease in viability as assessed by MTT assay. These results favor the view that oxidative damage is mainly responsible for
the cytotoxicity of ethanol and that Tan IIA can reverse the effects.
It has been reported that ethanol can induce cell apoptosis and necrosis or
both[27-29], but whether Tan IIA can attenuate the
process is still unknown. Because programmed cell death follows an orchestrated sequence of events, temporal markers of
apoptosis occur in stages. Early apoptosis coincides with subtle membrane disruption leading to a change of topology in the
bilayer so that phosphatidylserine that mostly reside in the cytoplasmic leaflet can also be detected in the outer leaflet. PI can
permeate necrotic cell membrane, and then bind nucleic acids. Thus, in the present study, annexin-PI analysis, direct assay
of apoptotic death, was used to detect the effects of Tan II A on cell apoptosis and necrosis induced by ethanol. Figure 4
shows a bivariate annexin V/PI analysis of PC12 cells. From this, it is clear that ethanol induced massive apoptosis and a large
part of the cells were at late apoptotic stage. Although Tan IIA alone did not induce any increase of apoptosis, which was
essentially similar to the control cells, pretreatment with Tan IIA was effective in abrogating the apoptosis induced by
ethanol. This protective effect of Tan IIA against ethanol-induced apoptosis might be because of its radical scavenging
capacity. This view coincided with our previous findings that tanshinone attenuated aminoglycoside-induced free radical
formation both in vitro and in
vivo[14].
To further study the mechanism of Tan IIA on the apoptosis of PC12 cells induced by ethanol, we detected the protein
expression of p53 by immuno-fluorescence and flow cytometry. When cells are injured, p53 is one of the major mediators of
apoptosis, implicated in tumors of the central nervous system, neurological disorders, aging, as well as the death of
development neurons in the cerebellum, hippocampus, and cerebral cortex. Moreover, upregulation of p53 activates mediators of
apoptosis, such as caspase-3 and caspase-9 and is pro-apoptotic for neurons. In addition, the apoptosis in PC12 cells is
mainly controlled by p53 and the Bcl-2
family[30]. In the present study, the results of immuno-fluorescence and flow cytometry
show that Tan IIA can reduce the p53 protein expression, which coincides with the decreased apoptosis in the PC12 cells
treated with Tan IIA. Thus, our results indicate that p53 might mediate ethanol-induced PC12 cells apoptosis.
In summary, Tan IIA can protect the neurotoxicity induced by ethanol by increasing the cell viability and decreasing the
formation of ROS. It can also reduce cell apoptosis which may result from the inhibition of the pro-apoptosis p53 expression.
All these results suggest that Tan IIA might serve as a potential therapeutic drug for neurological disorders induced by
ethanol.
Acknowledgements
We are grateful to Prof Zhi-hui LIANG, Hui-fen ZHU, and the flow cytometry workgroup for their technical advice.
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