Protective effects of Ginkgo biloba extract on paraquat-induced apoptosis of PC12 cells.pdf

1、Protective eff ects of Ginkgo biloba extract on paraquat-induced apoptosis of PC12 cells Xiaogang Kang a, Jianzong Chena,*, Zhen Xub, Hailong Lia, Bairen Wangb a Research Center of Traditional Chinese Medicine, Laboratory of Integrated Traditional Chinese Medicine and Western Medicine on Elderly Enc

2、ephalopathy, Xijing Hospital, PR China b PLA Institute of Neurobiology, The Fourth Military Medical University, Xian, Shanxi 710032, PR China Received 17 March 2006; accepted 14 February 2007 Available online 21 February 2007 Abstract Previous studies have suggested that Ginkgo biloba extract (EGb76

3、1) has a protective potentiality against apoptosis of neurons or neuron-like cells induced by MPTP. In this study, the eff ects of EGb761 on PC12 cells injured by paraquat (PQ), a neurotoxin, were tested. The results showed that after incubation of PC12 cells with EGb761 prior to PQ exposure, the PQ

4、-induced decrease of cell via- bility was signifi cantly reversed, the collapse of mitochondrial membrane potential (MMP) was attenuated and the percentage of apop- totic cells was reduced. Moreover, EGb761 pretreatment evidently increased the numbers of tyrosine hydroxylase (TH) positive and bcl-2

5、positive cells and degraded the number of caspase-3 positive cells in PQ-injured PC12 cells, in comparison to the treatment with PQ alone. This study indicates that EGb761 has a neuroprotective eff ect on paraquat-induced apoptosis of PC12 cells. The mechanism underlying the protective eff ects of E

6、Gb761 in PQ-injured PC12 cells might be related to the increase of bcl-2 activation, maintenance of MMP stability and decrease of caspase-3 activation through mitochondria-dependent pathway. The results from this study provide an experimental basis for the potential use of EGb761 in treatment of Par

7、kinsons disease. ? 2007 Elsevier Ltd. All rights reserved. Keywords: Parkinsons disease; Paraquat; Ginkgo biloba extract; Apoptosis; PC12 cells; Neuroprotection 1. Introduction Ginkgo leaf is a widely used herbal medicine. The stan- dardized Ginkgo biloba extract is termed as EGb761 which contains 2

8、4% fl avonol glycosides, 6% terpene trilactones (ginkgolides, bilobalide), 7% proanthocyanidins and other constituents (De Feudis, 1991). It has been proved that EGb761 can protect neurons against damages induced by a variety of injuries in diff erent experimental paradigms. Therefore, Ginkgo leaf h

9、as been proposed as a potential reagent for treatment of Parkinsons disease (PD) (Christen, 2001). Researches have indicated that EGb761 adminis- tered before or after 1-methyl-4-phenyl-1,2,3,6-tetrahy- dropyridin (MPTP) treatment could eff ectively protect against MPTP-induced nigrostriatal dopamin

10、ergic neuro- toxicity (Wu and Zhu, 1999) and prevent PC12 cells from apoptosis induced by 1-methyl-4-phenyl pyridinium cation (MPP+) (Yang et al., 2001a,b), an active metabolite of MPTP. However, there has yet been no report concerning possible eff ects of EGb761 against neurotoxicity induced by par

11、aquat (1,10-dimethyl-4,40-bipyridinium, Paraquat, PQ), a widely used nonselective herbicide which has a sim- ilar structure to MPP+ (Shimizu et al., 2001). PQ has been demonstrated to induce oxidative stress, cause toxicity to human and animals, and produce PD symptoms (Houze et al., 1990). The stru

12、ctural similarity between PQ and MPP+ suggests its possible role as a poten- tial etiologic factor in PD. Previous studies have shown that there is a strong correlation between the incidence of PD and the level of PQ exposure (Liou et al., 1996; Liou et al., 1997; Morano et al., 1994). Research usin

13、g animal 0887-2333/$ - see front matter ? 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2007.02.004 * Corresponding author. Tel.: +86 029 84775955. E-mail address: (J. Chen). Toxicology in Vitro 21 (2007) 10031009 model also indicates that PQ induces toxic eff ect in nigro- striatal dopa

14、minergic cells (Thiruchelvam et al., 2003). PC12 cells, a cell line established from rat adrenal pheo- chromocytoma cell (Greene and Tischler, 1976), possess intracellular substrates for dopamine (DA) synthesis, metabolism and transport (Hatanaka, 1981; Rebois et al., 1980; Tuler et al., 1989). The

15、cells contain the enzymes required for synthesis and decomposition of DA such as tyrosinehydroxylase(TH)andmonoamineoxidase (MAO). The membrane receptors and synthesized trans- mitters in PC12 cells are similar to dopaminergic neurons located in midbrain. Therefore, PC12 cell line has been used as a

16、 cellular model in PD studies (Timothy and Wil- liam, 1991). Investigations are done in this study to test the possible eff ects of EGb761 on the neurotoxicity induced by PQ in PC12 cells in order to provide experimental basis for the potential use of EGb761 in PD clinic. 2. Materials and methods 2.

17、1. Reagents G. biloba extract 761 (EGb761) was obtained from Dr. Willmar Schwabe of German. Acetone/water extraction was performed with standard criteria. The ratio of herb/ extraction was 35:167:1. The product consisted of two major groups of substances, the fl avone glycosides (fl avo- noid fracti

18、on, 24%) and the terpene lactones (terpenoid fraction, 6%). Annexin V-FITC, propidium iodide (PI), PQ, 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT), rhodamine 123 and hoechst 33342 were from Sigma. Dulbeccos modifi ed Eagles medium (DMEM), fetal calf serum (FCS) and horse seru

19、m were obtained from Gibco (USA). Lactate dehydrogenase (LDH) assay kit was from Nanjing Jiancheng Bioengineering Institute, China. The TH antibody, caspase-3 antibody, bcl-2 antibody and the terminal deoxynucleotidyl transferase (TdT)-medi- ated dUTP biotin nick end-labeling (TUNEL) kit were obtain

20、ed from Roche (Switzerland). 2.2. Cell culture and treatment PC12 cells were cultured in DMEM supplemented with 5% (v/v) FCS and 10% (v/v) heat-inactivated horse serum, 50 U/mL penicillin, and 50 lg/mL streptomycin, and grew at 37 ?C in a humidifi ed atmosphere of 5% CO2. Cells were seeded at a dens

21、ity of 2.0 105cells/mL. They were seeded on poly-l-lysine-coated plates and passaged at 6070% con- fl uence. Twenty four hours after replating, the medium was substituted with the presence or absence of diff erent concen- trations of EGb761 (fi nal concentrations: 10, 20, 40 lg/mL) for 2 h. Then PQ

22、(fi nal concentration: 300 lmol/L) was added for an additional 24 h. PQ was dissolved in DMEM. For those experiments, control cultures were also given the same amount of DMEM. All experiments were repeated three times for each treatment condition in each experiment. 2.3. Analysis of cell viability C

23、ell viability was measured by using the MTT assay, which is based on the conversion of MTT to formazan crystalsbymitochondrialdehydrogenases.Following PC12 cells (2 104 cells/100 lL) were treated with diff erent concentrations of PQ for 24 h or exposed to 300 lmol/L PQ at diff erent time points, 20

24、lL MTT (5 mg/mL) were added to each cell culture well containing 100 lL medium. After 4 h incubation at 37 ?C, the medium was carefully removed and the formazan crystals were lysed in 150 lL DMSO by gently shaking the plate. Absorbance was mea- sured at 570 nm by using a micro plate reader (Dynatech

25、). Cell viability was expressed as a percentage of the value in the control cultures. 2.4. LDH release assay Cytotoxicity was quantitatively assessed by measuring the activity of LDH released from the damaged cells into the culture medium. At the end of treatments, PC12 cells were treated with 10% T

26、riton X-100, and the media which contained detached cells were collected and centrifuged at 800g at 4 ?C for 2 min. The supernatant was used for the assay of LDH activity. The enzyme was determined by using an assay kit according to the manufacturers proto- col. The absorbance of the samples was rea

27、d at 440 nm. The LDH release was in proportional to the number of damaged PC12 cells. Reagent blanks were subtracted. 2.5. Flow cytometry (FCM) detection of cell death ApoptoticandnecroticcellswerequantitatedbyAnnexin V binding and PI uptake. Annexin V has a high affi nity for phosphatidylserine whi

28、ch is translocated from the internal to the external surfaces of the cell membrane when PC12 cells undergo apoptosis. Necrotic cells take up PI as a result of the increased permeability of their damaged cell wall. In this study, the cells were harvested after the treatment by EGb761 and PQ for diff

29、erent time, and then washed twice with PBS, and the concentration was adjusted to 1 106cells/mL. The pellets were resuspended in 70% ice cold ethanol and fi xed at 4 ?C for 24 h. The cells were then centri- fuged, and ethanol was removed by washing thoroughly with PBS. The cell pellets were resuspen

30、ded in 1 mL bind- ing buff er (10 mM Hepes/NaOH, pH 7.4; 140 mM NaCl; 2.5 mM CaCl2) and incubated for 15 min in the dark with Annexin V-FITC (20 lg/mL) and PI (50 lg/mL) at 4 ?C. Fluorescence was analyzed with an FCM. 2.6. Assessment of nuclear changes Cells were fi xed with 4% paraformaldehyde for

31、45 min at 4 ?C. Washed with PBS (pH 7.2), cells were permeabilized with 0.4% Triton X-100 for 30 min at room temperature. A 10 lg/mL (fi nal concentration) solution of Hoechst 33342 was added to the cultures at room temperature for 1004X. Kang et al. / Toxicology in Vitro 21 (2007) 10031009 5 min in

32、 the dark. Stained cells were observed and photo- graphed with a fl uorescence microscope (Leica, German). 2.7. Measurement of mitochondrial membrane potential (MMP) Cells were incubated in Hanks solution containing 10 lg/mL rhodamine 123 for 30 min at 37 ?C. Unbounded dye was removed by washing the

33、 cells twice with pre- warmed Hanks solution (37 ?C). Ten thousand cells per sample were analyzed with FCM. The mean fl uorescence intensity (MFI) in the cells represented the state of depolar- ization of MMP. 2.8. Determination of DNA fragmentation With designated treatment, PC12 cells were washed

34、with PBS, fi xed with 4% paraformaldehyde and then permeabili- zed with 0.1% Triton X-100 in 0.1% sodium citrate. Modifi ed nucleotides (dNTP) were incorporated at the free 30-OH ends of DNA fragments by using terminal deoxynu- cleotidyl transferase (TdT) and the dNTP was detected by using ABC-HRP K

35、its (Roche, Switzerland). Stained cells were observed and analyzed under the light microscope (Olympus, Japan). Positive cells were counted in three random microscopic fi elds at 100 magnifi cation. 2.9. Immunocytochemical staining PC12 cells were seeded on poly-lysine coated coverslips. For immunoc

36、ytochemical detection of TH, caspase-3 and bcl-2, cells were fi xed with 4% paraformaldehyde in PBS for 20 min at room temperature and treated with 1% hydrogen peroxide for 10 min. Rinsed in PBS, the fi xed cells were permeabilized and nonspecifi c antigenic sites were blocked for 1 h in the blockin

37、g solution (0.5% BSA/ 5% normal goat serum/0.1% Triton X-100). All antibodies were diluted in the blocking solution. Cells were exposed to the TH antibody (1:1000), caspase-3 antibody (1:200) or bcl-2 antibody (1:200) overnight at 4 ?C. Subsequently, cells were washed for three times 10 min in PBS a

38、nd incu- bated with biotinylated secondary antibody for 2 h at room temperature, and then incubated with the PBS containing peroxidase-conjugated avidinbiotin complex (ABC) for 1 h. The immunoreactivities were developed with a solution containing 0.05% diaminobenzidine and 0.03% H2O2in PBS (pH 7.2).

39、 Cells were examined under the light microscope. Six representative areas per cover slip were counted. 2.10. Statistical analysis The results were expressed as means SD. Data were evaluated for signifi cance with one-way ANOVA by using the SPSS13.0 software. P 0.05 was considered signifi cant. 3. Re

40、sults 3.1. Eff ect of EGb761 on PQ-induced decrease of cell viability After incubation with various concentrations of PQ (50 600 lmol/L) for 24 h, the cell viability of PC12 cells was determined by using MTT assay. Compared with the control group, PQ reduced the cell viability in a concentra- tion-d

41、ependent manner (Fig. 1a). At 300 lmol/L of PQ, cell viability reached 53.3 5.1%. When PC12 cells were treated with 300 lmol/L PQ for diff erent times, cell death increased with prolongation of exposure time (Fig. 1b), reaching 57 5.4% at 24 h. As shown in Fig. 1c, EGb761 signifi cantly protected PC

42、12 cells against the neurotoxic eff ect of PQ. The increase of cell viability by EGb761 was in a concentration- and time-dependent manner. It was noticed that the cytotoxic eff ects were attenuated by prein- cubations with 10, 20 and 40 lg/mL EGb761 in PC12 cells. EGb761 raised the cell viability to

43、 65.8 4.6%, 71.6 5.6% and 82.4 6.2%, respectively, compared with the control group. 3.2. Eff ect of EGb761 on PQ-induced LDH release To further investigate the protective eff ect of EGb761, LDH assay, another indicator for cell toxicity, was per- formed. The results were similar to those determined

44、by MTT assay. Treatment with 300 lmol/L PQ resulted in an increase of LDH release in the medium (236 8.8%) (Fig.1d). Preincubation with 10,20 and 40 lg/mL EGb761 blocked LDH release in the PC12 cell system to 189 10.3%, 168 9.8% and 135 8.6%, respectively. 3.3. Eff ect of EGb761 on PQ-induced increa

45、se of apoptotic percentage PC12 cells were stained with both PI and FITC-labeled Annexin V (AV-FITC) to analyze the percentage of apop- totic cells with FCM. Necrotic cells were demonstrated by AV?/PI+ staining, since when membrane integrity was lost PI entered cells and combined with nucleic acids.

46、 Apoptotic cells were demonstrated by AV+/PI-staining, since AV combined to phosphatidylserine that had trans- located to the outer leafl et of the plasma membrane dur- ing apoptosis. AV+/PI+ stained cells were likely to be late apoptotic or necrotic cells, and AV?/PI-cells repre- sented viable cell

47、s. In the control group, most cells were viable (92.9%) (Fig. 2a). When the PC12 cells were exposed to 300 lmol/L PQ, the number of AV+ cells was increased signifi cantly (41.6%) (Fig. 2b). Simulta- neous incubation with (10, 20 and 40 lg/mL) EGb761 and PQ markedly reduced the number of cells labele

48、d with AV. The percentage of apoptosis cells was signifi - cantly decreased to 33.8%, 25.7% and 19.4%, respectively (Fig. 2ce). X. Kang et al. / Toxicology in Vitro 21 (2007) 100310091005 3.4. Eff ect of EGb761 on PQ-induced MMP collapse MMP in PQ-induced PC12 cells preincubated with EGb761 was asse

49、ssed by the retention of rhodamine 123, a specifi c fl uorescent cationic dye that was readily aggre- gated by active mitochondria. After PQ treatment, rhoda- mine 123 fl uorescence decreased in PC12 cells, compared with the control group (P 0.05). Pretreatment with EGb761 (10, 20, and 40 lg/mL) dos

50、e-dependently reversed the reduction of MMP resulted from PQ (Fig. 3a). 3.5. Eff ect of EGb761 on PQ-induced DNA fragmentation During the process of apoptosis, DNA fragmentation was caused by activation of endonucleases. The appearance of intensely stained nucleus is indicative for terminal incor- p

51、oration of labeled dUTP into the 30-OH ends of frag- mented DNA derived from apoptotic nuclei. Quantitative analysis of TUNEL positive cells is shown in Fig. 3b. The exposure of PC12 cells to 300 lmol/L PQ signifi cantly enhanced the ratio of TUNEL-positive cells to the total Fig. 1. PQ-induced cell

52、 death and neuroprotective eff ects of EGb761 in PC12 cells. (a) Neurotoxic eff ect of PQ at concentrations from 0 to 600 lmol/L for 24 h, detected by MTT assay. (b) Time-dependent neurotoxicity of 300 lmol/L PQ from 0 to 72 h. (c) Neuroprotective eff ects of EGb761 against PQ- induced neurotoxicity

53、 of PC12 cells detected by MTT assay. (d) EGb761 prevents PQ-induced cell damage in PC12 cells detected by LDH assay. The data are represented as means SD of three experiments. *P 0.05 vs. control group.#P 0.05 vs. group treated with PQ alone. Fig. 2. Flow cytometric histograms of EGb761 eff ects on

54、 apoptotic rate of PC12 cells treated with PQ. After incubation, cells were labeled with Annexin V-FITC and PI. (a) Control; (b) 300 lmol/L PQ only; (c) 300 lmol/L PQ + 10 lg/mL EGb761; (d) 300 lmol/L PQ + 20 lg/mL EGb761; (e) 300 lmol/L PQ + 40 lg/mL EGb761. 1006X. Kang et al. / Toxicology in Vitro

55、 21 (2007) 10031009 count (20.5 1.02%), compared with that in the control group(5.6 0.93%).Pretreatmentswith10,20and 40 lg/mL EGb761 lowered the proportion of TUNEL- positive cells to 19.4 0.73%, 13.7 0.95%, and 9.1 0.64%, respectively. 3.6. Eff ect of EGb761 on expressions of TH, caspase-3 and bcl-

56、2 in PQ-treated PC12 cells In order to elucidate the possible molecular mechanisms associated with the protective eff ect of EGb761 on PQ- induced apoptosis, immunocytochemical staining for TH, caspase-3 and bcl-2 was performed. Quantifi cations of immunostained cells demonstrated that signifi cant

57、loss of TH-positive and bcl-2-positive cells and marked increase of caspase-3-positive cells were observed in PQ-incubated cells, compared with the control group. In contrast, prein- cubation with 10, 20 and 40 lg/mL EGb761 showed a sig- nifi cant increase in TH- and bcl-2-positive cells and a decre

58、ase of caspase-3-positive cells, in comparison to the group treated with PQ only (Table 1). 4. Discussion In recent years increasing researches focus on the rela- tionship between PQ exposure and neurodegenerative dis- eases.IthasbeenfoundthatPQselectivelykills nigrostriatal dopaminergic neurons in

59、the experimental animals (Corasaniti et al., 1992; Corasoniti et al., 1998; McCormack et al., 2002) or causes dopaminergic neurons damage in vitro midbrain culture (Keiko et al., 2003). Apoptosis is thought to play an important role in the neu- ronal loss in PQ-induced neurological disorders. However, the mechanism of PQ neurotoxicity is still unclear. There have been studies suggesting that PQ-induced apoptosis is mediated by the oxidative stress (Cappelletti et al., 1998; Fabisiak et al., 1998; McCarthya et al., 2004; Mollace et al., 2003). The fi ndings of the