Henghui Wang, Weifeng Luo, Xiaoxia Wang, Xiaoling Qin, Shiyao Bao

1Department of Neurology, the Second Affiliated Hospital of Soochow University, Suzhou 215004, Jiangsu Province, China

2Department of Oncology, the Second Affiliated Hospital of Soochow University, Suzhou 215004, Jiangsu Province, China

lNTRODUCTlON

6-hydroxydopamine (6-OHDA) exhibits a toxic effect on dopaminergic neurons and is detected in the brain and urine of Parkinson’s disease patients. Therefore,6-OHDA has been generally used to establish Parkinson’s disease animal models via stereotaxis injection[1]. Reduced glutathione (GSH), a clinically and commonly used antioxidant, demonstrates free radical scavenging and mitochondrial protection[2-13]. As the most important protective antioxidant of the substantia nigra,GSH reduction in Parkinson’s disease patients is correlated to disease severity[14-15].

Although GSH treatment in early stages of Parkinson’s disease shows some therapeutic effect[16], it is unclear whether GSH promotes the survival of bone marrow stromal cells (BMSCs).

Our study investigates whether GSH can alleviate 6-OHDA toxicity, and the effect of 6-OHDA and GSH on BMSCs in vitro.

RESULTS

GSH improves BMSC proliferation in vitro

The effect of various GSH concentrations on BMSC proliferation was determined by MTT assay. Passage 3 BMSCs were seeded into a 96 well plate at 2 × 105cells/mL in 100 μL culture medium per well. After 3 days, cells were divided into eight groups consisting of a PBS control group and GSH 0.5, 1, 1.5,2.25, 3, 6, 12 mg/mL groups. After a further 12 and 36 hours of culture, MTT assays were performed, and cell viability ratios were calculated for statistical analysis. BMSCs proliferation was higher in all GSH groups compared with that in the PBS control group(P ＜ 0.05). With the exception of the GSH 12 mg/mL group, BMSC proliferation at 12 hours was higher in GSH groups compared with that in 36 hours (Table 1).

Table 1 Viability of bone marrow stromal cells treated with GSH

Toxic effect of 6-OHDA on BMSCs in vitro

The toxic effect of various 6-OHDA concentrations on BMSC viability was determined by MTT assay. Passage 3 BMSCs were seeded into a 96 well plate at 2 × 105cells/mL in 100 μL culture medium per well. After 3 days, cells were divided into three groups: PBS control, 6-OHDA 0.05 and 0.1 mg/mL groups. After a further 12 and 36 hours of culture, MTT assays were performed, and cell viability ratios were calculated for statistical analysis. BMSC viability in 6-OHDA groups were lower compared with that in the PBS control group (P ＜ 0.05). Moreover, 6-OHDA 0.1 mg/mL resulted in a lower BMSC viability compared with 6-OHDA 0.05 mg/mL (P ＜ 0.05; Table 2).

Table 2 Cell viability ratio of bone marrow stromal cells treated with 6-OHDA

GSH alleviates the toxic effect of 6-OHDA on BMSCs in vitro

Passage 3 BMSCs were seeded into a 96-well plate at 2 × 105/mL in 100 μL medium per well. After 3 days, cells were divided into seven groups consisting of a PBS control, 6-OHDA 0.05 mg/mL, GSH 0.5 mg/mL +6-OHDA 0.05 mg/mL, GSH 1 mg/mL + 6-OHDA 0.05 mg/mL, 6-OHDA 0.1 mg/mL, GSH 0.5 mg/mL +6-OHDA 0.1 mg/mL and GSH 1 mg/mL + 6-OHDA 0.1 mg/mL groups. After a further 12 and 36 hours of culture, BMSC proliferation in GSH + 6-OHDA groups was significantly higher compared with that in the 6-OHDA groups (P ＜ 0.05; Table 3).

Table 3 Viability of bone marrow stromal cells treated with both GSH and 6-OHDA

Morphology of BMSCs treated with both GSH and 6-OHDA

BMSC proliferation increased after passaging and reached confluence at 1 week (Figure 1).

Figure 1 Morphology of bone marrow stromal cells under a normal culture condition (× 100). (A-D) Primarily cultured cells, 1st, 2nd and 3rd passages cells exhibit simple morphology with most cells being spindle-shaped, while a a small number of cells are flat, triangle or irregular shaped with two or multiple processes.

Passage 3 BMSCs were treated with GSH for 36 hours.

Upon the GSH concentration increasing to more than 2.25 mg/mL, BMSC morphology obviously changed compared with that of the PBS control group, and BMSCs appeared to differentiate into neuron-like cells.

BMSCs treated with GSH and 6-OHDA showed little to no changes by retaining spindle, flat, triangle or irregular morphologies. However, BMSCs treated with 6-OHDA only significantly changed morphology with an obvious retraction of the cell body, enlarged intercellular space,and an increase in differentiated bipolar and multipolar cells (Figures 2-4).

Figure 2 Morphological changes of bone marrow stromal cells treated with GSH 0.5 (B,× 100), 1 (C, × 100) and 2.25 (D, ×400) mg/mL, and cultured for 36 hours. (A, × 100) Cells in the PBS control group exhibit no changes. (D) Cells in the GSH 2.25 mg/mL group differentiate into neuron-like cells with long neurites. GSH: Reduced glutathione.

Figure 3 Morphological changes of bone marrow stromal cells treated with 6-OHDA, and cultured for 36 hours (× 100). (A) Cells in the PBS control group exhibit no changes. Cells in 0.05 (B) and 0.1 (C) mg/mL 6-OHDA groups show significant cell body retraction and an enlarged intercellular space. 6-OHDA: 6-hydroxydopamine.

Figure 4 Morphological changes of bone marrow stromal cells treated with both 6-OHDA and GSH, and cultured for 36 hours(× 100). (A) GSH 0.5 mg/mL + 6-OHDA 0.05 mg/mL; (B) GSH 1 mg/mL + 6-OHDA 0.05 mg/mL; (C) GSH 0.5 mg/mL + 6-OHDA 0.1 mg/mL; (D) GSH 1 mg/mL + 6-OHDA 0.1 mg/mL. Cell morphology is similar in each group. Most cells are spindle-shaped and a small number of cells exhibit two or more processes. 6-OHDA: 6-hydroxydopamine; GSH: reduced glutathione.

DlSCUSSlON

Passage 3 BMSCs were used in this experiment and as demonstrated in our previous study, the purity exceeded 92%[17]. The present study showed that BMSC proliferation significantly decreased after 6-OHDA treatment for 36 hours, which suggests that 6-OHDA has a toxic effect on BMSCs in a dose-dependent manner. In addition, GSH demonstrated a protective effect on BMSCs and alleviated 6-OHDA toxicity.

6-OHDA has a similar molecular structure to dopamine and is taken up by dopaminergic neurons. The hydroxy free-radical that is produced induces oxidative stress and inhibits the mitochondrial oxidation-respiration chain complex I and IV, and results in the selective cell death of dopaminergic neurons[18-20]. Zhang et al[21]found that treatment with 6-OHDA 0.2 mmol/L (0.033 8 mg/mL) for 24 hours decreases PC12 cell viability by 27%. In this study, we also demonstrated a viability reduction of approximately 25% after BMSCs were treated with 6-OHDA 0.05 mg/mL for 36 hours.

The underlying mechanism by which GSH increases the cell viability of BMSCs remains unclear. A low GSH concentration (≤ 1.5 mg/mL) performs an anti-oxidant function, clears free radicals, enhances the activity of the respiratory chain enzyme complex I and protects the normal function of mitochondria. A GSH concentration of more than 2.25 mg/mL resulted in significant BMSC morphology changes compared with that of the PBS control group, and BMSCs appeared to differentiate into neuron-like cells. The proliferation of differentiated neuron-like cells was significantly higher compared with that of undifferentiated cells, thus, the cell viability ratio was used in place of cell viability in this study. BMSCs treated with GSH 0.5, 1 mg/mL resisted the toxic effect of 6-OHDA to ensure the homogeneity of the cells.

In conclusion, further research is needed to elucidate the toxic effect of 6-OHDA on BMSCs and the protective mechanism of GSH.

MATERlALS AND METHODS

Design

An in vitro cytology study.

Time and setting

Experiments were performed from March 2007 to March 2008 at the Central Laboratory of the Second Affiliated Hospital, Soochow University, China.

Materials

Eighteen Sprague-Dawley rats of clean grade, aged 2-3 weeks and weighing 60-90 g were provided by the Animal Center of the Medical College, Soochow University, China (License No. SYXK (Su) 2007-0035).

Experimental procedures complied with the Guidance Suggestions for the Care and Use of Laboratory Animals,formulated by the Ministry of Science and Technology,China[22]. GSH was purchased from Lvye Pharmaceutical Co., Ltd., Shandong Province, China. 6-OHDA was purchased from Sigma Corporation (St. Louis, MO,USA).

Methods

Rat BMSC isolation and culture

Rats were sacrificed and bone marrow was harvested under sterile conditions from the bilateral humerus,femurs and tibias. The bone marrow cavity was rinsed with L-DMEM (Gibco, New York, NY, USA) and then the bone marrow was centrifuged at 1 000 r/min(111 × g) for 5 minutes. Cells were resuspended in complete culture medium consisting of L-DMEM supplemented with 42.5% F12 (Gibco), 15% fetal bovine serum (Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin. Then, cells were plated in culture flasks at 1 × 107cells/mL and incubated at 37°C in a humidified atmosphere with 5%CO2for 3 days.

Non-adherent cells were removed by replacing the medium. Culture medium was replaced every 3-4 days.Cells were harvested with 2.5 g/L trypsin at 80%confluency[14]. BMSC morphology was observed under a light microscope (Olympus, Tokyo, Japan).

BMSC viability after GSH and/or 6-OHDA treatment as detected by MTT assay

Passage 3 BMSCs in each group were cultured for 12 and 36 hours followed by three PBS washes. Then,10 μL MTT solution (5 mg/mL MTT dissolved in 0.01 mol/L PBS, pH 7.2) was added to culture plates,and cells were incubated for an additional 4 hours. The supernatant was removed and each well received 200 μL DMSO at 37°C for 5-10 minutes with agitation.

The absorbance value at 570 nm of each well was recorded[23-24]. The cell viability ratio = (absorbance value of drug treatment group-absorbance value of the blank control group) / (absorbance value of the PBS control group-absorbance value of the blank control group) × 100%. BMSC morphology was observed under an inverted phase-contrast microscope (Olympus).

Statistical analysis

Data were expressed as mean ± SD. and analyzed using SPSS 11.0 software (SPSS, Chicago, IL, USA). One-way analysis of variance and a SNK-q test were used to identify significant differences between groups and in multiple comparisons. A value of P ＜ 0.05 was considered statistically significant.

Author contributions:Weifeng Luo was responsible for the research concept and design, and performed statistical analyses as well as validated the manuscript. Henghui Wang was responsible for the research concept and design,implemented the experiments, performed statistical analyses,and drafted the manuscript. Xiaoxia Wang, Xiaoling Qin and Shiyao Bao participated in the research concept, statistical analyses and paper reviews.

Conflicts of interest:None declared.

Funding:This study was supported by Jiangsu Ordinary University Science Research Project, No. 06KJB320097.

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