Protective Effect Of Three Plant Extracts On H₂O₂ Treated Oxidative Damage In PC12 Cells

Excessive reactive oxygen species（ROS） can cause imbalance of oxidative system and antioxidant system in the body, inducing cellular oxidative damage. The oxidative damage will trigger radical chain reaction and result in lipid peroxidation and cell membrane structure damage. And the oxidative damage will give rise to excessive oxidation of protein or enzyme which resulting in the function loss of protein or enzyme. Also, the oxidative damage will cause DNA, RNA crosslinking and gene expression abnormalities. Thereby, the oxidative damage lead to a series of oxidative damage associated with chronic diseases such as aging, inflammation,neurodegenerative diseases.Pine pollen extract, grape seed extract and flavone of bamboo leaves, are three plant extracts with natural, non-toxic side effects features. Among them, the pine pollen is the essence of life of plants, known as "the best health products". Pine pollen is rich in nutrients with antioxidant, immune, anti-inflammatory effects. Grape seed extract has a good effect on against oxidative stress, anti-inflammatory, anti-bacterial, prevention of cardiovascular disease, and other aspects of the neuroprotective effect. Flavone of bamboo leaves, its function factor is water soluble flavonoid glycosides which have anti-radical, anti-lipid peroxidation, and anti-radiation,anti-bacterial effects, and also can be used for oil and other foods as a food additive. Therefore,because of their function in defense oxidative damage, these plant extracts as the good natural active substances are gained more and more attention.In this paper, we investigated the possible protective effects of the pine pollen extract,grape seed extract and flavone of bamboo leaves against oxidative damage of PC12 rat pheochromocytoma（PC12） cells treated by hydrogen peroxide（H₂O₂）.Followings are the main contents of this study:1. Using MTT assay established H₂O₂ oxidative injury model. The PC12 cells were exposured in different concentrations（100, 150, 200, 300, 400, 500 μM） of H₂O₂. After 4 h,compared with the control group, the cell viability separately decreased to 87.56%, 55.38%,26.38%, 9.26%, 4.05%, 3.37%.Therefore, we choose the H₂O₂（150 μM, 4 h） as the oxidativedamage model.2. Using the MTT assay choosed the concentration gradient of pine pollen extract, grape seed extract and flavone of bamboo leaves. PC12 cells were treated with three plant extracts 24 h.Then the PC12 cells were exposured in 150 μM H₂O₂. After 4 h, the cell viability was determined. According to cell survival, we define the concentrations of pine pollen extract were25, 50,100 μg/mL, the concentrations of grape seed extract were 5, 25, 50 μg/m L, the concentrations of flavonoids of bamboo leaves concentration were 50,100,200 μg/m L.3. Cell viability was measured using the MTT assay to investigate the effect of pine pollen extract, grape seed extract and flavone of bamboo leaves on cytotoxicity in the concentration range. At the same time, we analyzed the protective effect of three extracts on oxidative damage in PC12 cells. The results showed that three extracts can improve the survival rate of oxidative damage in PC12 cells. And within the selected concentration range, the three plant extracts have no toxicity on the PC12 cells.4. Lactate dehydrogenase（LDH） release in extracellular, intracellular malondialdehyde（MDA） and ROS were detected to investigate the effect of pine pollen extract, grape seed extract and flavone of bamboo leaves on cell injury treated by H₂O₂ in PC12 cells. It contained the degree of cell membrane damage, lipid peroxidation as well as cellular oxidative level. The results showed that within the concentration range, three plant extracts were able to prevent cell membrane damage according to cell extracellular LDH release, scavenge ROS formation and decrease MDA.5. Superoxide dismutase（SOD）, catalase（CAT） and glutathione（GSH） were detected to investigate the effect of pine pollen extract, grape seed extract and flavone of bamboo leaves on antioxidase and antioxidant in PC12 cells treated by H₂O₂. The results showed that three extracts can increase SOD, CAT activity and increase intracellular GSH levels. This shows that the three plant extracts can exert antioxidant effects by increasing the antioxidant enzyme activity and antioxidant content of body.6. Mitochondrial membrane potential（MMP） was measured to investigate the effect of pine pollen extract, grape seed extract and flavone of bamboo leaves on mitochondrial membrane damage in PC12 cells treated by H₂O₂. The results showed that the three plant extracts could improve the MMP in damage PC12 cells and protect the stability of themitochondrial membrane.7. The cell cycle and cyclin was measured to investigate the effect of pine pollen extract,grape seed extract and flavone of bamboo leaves on the cell cycle in PC12 cells treated by H₂O₂.We investigate the protective mechanism of the three plant extracts preliminary. The results showed that PC12 cells were arrested in S phase after H₂O₂ treatment and the cell arrest were improved after three extracts treated.All the above results of this study showed that the PC12 cells were treated oxidative damage by H₂O₂（150 μM, 4 h）. However, after PC12 cells were treated 24 h with pine pollen extract, grape seed extract and flavone of bamboo leaves, it can improve the oxidative damage of cells and had good protection to the cells. Thus, these results suggest that these plant extracts provide potential to combat the disease associated with oxidative damage.