The protective effect of salidroside on oxidative stress injury induced by hydrogen peroxide in PC 12 cell

The protective effect of salidroside (SD) on oxidative stress injury induced by hydrogen peroxide in PC12 cell was investigated. The PC 12 cell was cultured in RPMI-1640 medium supplemented with 10% newborn calf serum at 37°C in a 5% CO2 incubator. 40 μM of H2O2 was conducted to establish an oxidative-stress injury model in vitro. The effect of SD was evaluated by using the model. The LDH release rates of PC12 cells in different treatment group was compared. The cell apoptosis was detected by using flow cytometry (FCM) and the cell proliferation by MTT assay. This paper found that pretreatment with SD could improve cell growth and proliferation in a concentration dependent manner, reduce the release of LDH and suppresses the H2O2-induced apoptosis of PC12 cells, Therefore, these results significantly suggested that SD possessed the protective activity of PC12 cells injury by H2O2 in vitro.


INTRODUCTION
Oxidative stress is a pathological process of cell toxicity resulting from the loss of balance of oxygen, free radical production and scavenging, or excessive intake of exogenous oxidants leading to reactive oxygen species (ROS) accumulation in the body (Simonian and Coyle, 1996).Some studies have shown that various central nervous system diseases were closely related to oxygen free radicals, such as cerebral ischemia, Alzheimer's disease, Parkinson's disease and multiple sclerosis (Weber, 1994).Therefore, developing novel agents for anti-oxidative damage to improve the processes of these diseases and aging, and then exploring their protection of cellular mechanisms have become important research topic in the medicinal fields.
In our present study, we investigate the effect of SD on oxidative stress injury induced by hydrogen peroxide in PC12 cell.We compared the LDH release rate of PC12 cells in different treatment group.The cell apoptosis was detected by using flow cytometry (FCM) and the cell proliferation by MTT assay.

Cell culture and treatment
PC12 cells (rat adrenal pheochromocytoma cells) were gotten from Experimental Animal Center of Northwest University (Xi'an, China) and then cultured in RPMI-1640 medium supplemented with 10% newborn calf serum at 37°C in a 5% CO2 incubator.When cells were near 80% confluence, new media with newborn calf serum were added before the compounds treatment.40 μM of H2O2 was used to establish an oxidative-stress injury model in vitro, and evaluate the effect of SD by using this model.H2O2 was added in final concentrations ranging from 5 to 80 μM in the pilot study, and the concentration (40 μM) was chosen by determining doseresponse curves.When needed, cells were incubated for 30 min with different concentrations of SD and then exposed to 40 μM of H2O2 for 24 h.

Detection of LDH activity of PC12 cells
When PC12 cells were near 80% confluence, new media with newborn calf serum was added before the compounds treatment.
Cells were treated with SD for 30 min followed by the addition of H2O2 to a final concentration of 40 μM, and incubated for 24 h.The medium was kept in a 1 mL EP tube and then the LDH activity was detected by multifunction biochemistry analysator.

Flow cytometry (FCM) with propidium iodide (PI) staining
The PC12 cells were treated in the same way as described above.Cells were collected, digested with 0.25% trypsin and made into a single cell suspension by RPMI-1640 medium supplemented with 10% newborn calf serum.The single cell suspension was centrifuged at 1000 rpm for 5 min at 4°C.The supernatant was removed, washed with cold PBS, centrifuged at 1000 rpm for 5 min at 4°C.Cells were resuspensed as a single cell suspension with 50 μl of PBS, fixed with l ml of cold 70% ethanol, stained with propidium iodide (PI) and then cell apoptosis detected using flow cytometry.

MTT assay
Logarithmic growth phase cells were seeded in 96-well plates at a density of 5 ×10 3 / mL.Cells were cultured for 24 h at 37 o C with 5% CO2, treated in the same way as described above.Each group was set up in three parallel holes.Cells were cultured for 24 h, followed by incubation with 0.5 mg/ml of MTT, 200 μL of serum-free medium for 4 h.Finally, 100 μl of DMSO was added and absorbance at 570 nm wavelength (A570) was measured by means of enzyme-linked immunosorbent instrument.Relative cell proliferation inhibition rate (IR) = (1 -average A570 of the experimental group/average A570 of the control group) × 100%.

Statistical analysis
The database was set up with the SPSS 16.0 software package for analysis.Data were represented as Mean ± S.D. The means of multiple groups were compared with One-Way ANOVA, after the equal check of variance, and the two-two comparisons among the means were performed by Student's t-test.P<0.05 was considered as statistically significant.

SD decreased the LDH release of PC12 cells
After PC12 cells were treated with 40 μM of H 2 O 2 for 24 h, the release of LDH significantly increased from 27.12 ± 1.10 U/ml to 98.23 ± 4.35 U/ml.When 0.1, 1.0, 2, 5, 10 and 20 μM of SD were added to the assay, the release of LDH decreased to 67.12 ± 3. 83, 58.58 ± 3.39, 53.34 ± 3.31, 49.27 ± 3.19, 42.17 ± 3.08 and 39.56 ± 2.21 U/ml in a concentration dependent manner.The LDH release results showed that SD could effectively reduce the release of LDH H 2 O 2 -induced PC12 cells in a concentration dependent manner (Table 1).

DISCUSSION
Numerous central nervous system diseases are closely related to oxygen free radicals, such as cerebral ischemia, Alzheimer's disease, Parkinson's disease and multiple sclerosis (Weber, 1994;Burton, 1995;Irani et al., 1997).PC12 cells are extremely similar to neurons in cell morphology, structure and function.Therefore, it has been widely used as a cell model for study of nerve cells (Saito et al., 2003).Growing studies have shown that oxygen free radicals and their derivatives are also closely Huang et al. 2975  The LDH release is a classic indicator of cell function.An increased in LDH release, signify injury of cell function.Our study found that SD could reduce LDH release of PC12 cells injured by oxidative stress.MTT assay confirmed that cell growth and proliferation were suppressed when PC12 cells were treated with H 2 O 2 for 24 h, while SD could effectively reduce the suppression.FCM with PI staining showed that SD could reduce the H 2 O 2-induced apoptosis of PC12 cells in a concentration dependent manner.It is probable that SD could provide protection against PC12 cells injury caused by oxidative stress.

Conclusion
In our present work, we established the model of PC12 cells injury and apoptosis induced by H 2 O 2, using genistein as control group to evaluate different concentration of SD against oxidative stress injury in PC12 cells.The results in our study demonstrated that SD possesses the protective activity of PC12 cells injury by H 2 O 2 in vitro.However, further study in this field is still needed.

Table 1 .
PC12 cells were seeded and treated as described in the materials and methods.The culture medium from each treatment was collected and the LDH release was analyzed.

Table 2 .
FCM has shown that all genistein derivatives can decrease apoptosis rate of H2O2induced PC12 cells.

Table 3 .
MTT assay has shown that all genistein derivatives can increase proliferation rate of H2O2induced PC12 cells.