Protection Of Gynostemma Pentaphyllum Polysaccharides Against MPP~+-Induced PC12 Cell Damage And The Underlying Mechanisms

Objective: The aim of this study was to investigate the neuroprotective effect of GP against 1-methyl-4-phenylpyridinium(MPP+)-induced PC12 cell damage and the underlying mechanisms, so as to provide experimental evidence for the prevention and treatment of Parkinson’s disease. Methods: MPP+-damaged PC12 cells were used to establish the cell model of Parkinson’s disease. The cultured PC12 cells were assigned into 3 groups. Cells in the blank control group were treated with RPMI 1640 medium, cells in the MPP+group were treated with 1mM MPP+, while cells in the MPP+and GP group are incubated with 50 μg/ml GP for 2 h, followed by incubation in 1mM MPP+for 24 h. Following drug treatment, the cell morphology and cell count of PC12 cells was used inverted microscope. The cell cycle analysis of PC12 cells was determined using flow cytometry.the viability of PC12 cells was measured using MTT assay. The release of lactate dehydrogenase(LDH) from PC12 cells was detected using colorimetry. The apoptosis of PC12 cells following drug treatment was determined using flow cytometry. Cytosolic cytochrome c level was determined using the Quantikine® rat/mouse cytochrome c assay kit, while caspase-3 and caspase-9 activities in PC12 cells were determined using caspase-3/9 assay kit. In addition, Western blotting analysis was performed to detect the protein expression of Cleaved caspase-3, Bcl-2, Bax and PARP in PC12 cells. Results:(1) Treatment with 1m M MPP+caused reduced the number and synapse of PC12 cells, while treatment with 50μg/ml GP resulted in rise of PC12 cells number and synapse.(2) Treatment with 1mM MPP+caused reduced cell density and growth rate of PC12 cells, while treatment with 50μg/ml GP resulted in rise of PC12 cells cell density and growth rate.(3) Treatment with 1m M MPP+caused reduced Proliferation index(PI) of PC12 cells, while treatment with 50μg/ml GP resulted in rise of PC12 cells Proliferation index(PI).(4) Treatment with 1m M MPP+caused reduced survival rate of PC12 cells, while treatment with 50μg/ml GP resulted in rise of PC12 cells survival.(5) MPP+treatment caused a remarkable release of LDH from PC12 cells, while the addition of 50μg/ml GP reduced LDH release from PC12 cells.(6) Treatment with 1mM MPP+significantly increased apoptotic rate of PC12 cells, while the addition of 50μg/ml GP significantly reduced apoptotic rate of PC12 cells.(7) A 24-h treatment of 1mM MPP+significantly increased the activity of caspase-3(P<0.01) and caspase-9(P<0.01) and protein expression of Cleaved caspase-3(P<0.01) in PC12 cells, while the activity of caspase-3(P<0.01) and caspase-9(P<0.01) and protein expression of Cleaved caspase-3(P < 0.01) was significantly lower in the GP+MPP+group than in the MPP+group.(8) Compared with the blank control group, treatment with 1mM MPP+for 24 h decreased Bcl-2/Bax ratio(P<0.01), and a higher Bcl-2/Bax ratio was found in the GP+MPP+group than in the MPP+group(P<0.01).(9) Compared with the blank control group, treatment with 1mM MPP+for 24 h increased the level of cleaved PARP protein, and a lower level of cleaved PARP protein was found in the GP+MPP+group than in the MPP+group(P<0.01). Conclusion: GP protects MPP+-induced PC12 cell damage, and GP inhibits MPP+-induced apoptosis of PC12 cells through mediating Bcl-2 and Bax protein expression and via a mitochondrial pathway.