The Effects Of Puerarin On P53and Its Related Genes Expression About MPP~+-induced SH-SY5Y Apoptosis Cells

Objective To explore changes of p53and its related genes on1-methyl-4-phenylpyridinium iodide(MPP+)-induced apoptosis in SH-SY5Y cells and the protectiveeffects of puerarin.Methods The SH-SY5Y cells were cultured in the DMEM including13%fetal bovineserum. The cells were randomly divided into six groups according to the experimentdesign: control group, MPP+model group,50μmol/L puerarin+MPP+treatment group,100μmol/L puerarin+MPP+treatment group,150μmol/L puerarin+MPP+treatmentgroup and Pifithrin-ɑ+MPP+treatment group. Cell morphology were observed with aninverted microscope; cell viability were assayed by3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT). The apoptosis rates were detected by flowcytometry with Annexin-V/PI fluorescence staining. The expression of p53protein wereassayed by Western blotting.The expression of p53mRNA and its related genes p21,MDM2, Bax, Bcl-2, Caspase-3mRNA were assayed by real-time quantitative reversetranscription-PCR(RT-PCR).Results1morphology Changes: in control group, cells presented a spindle shape andhad a relatively long axons. In MPP+model group, cell neurites vanished and cell bodiesbecame smoother, the number of adherent cells had reduced. However, puerarin andPifithrin-ɑ could significantly prevent MPP+-induced cell neurites from vanishing.2MTT results: in control group, cell viability rates is100.0%±0.0%, in MPP+Modelgroup, cell viability rates is36.0%±0.5%, in puerarin and Pifithrin-ɑ treatment group,cell viability rates is52.0%±3.0%,76.6%±4.8%,86.0%±2.0%and92.0%±2.0%.The pretreatment puerarin and Pifithrin-ɑ could apparently increase the viability rates ofcells comparing with MPP+model group (P<0.05).3. Annexin-V/PI fluorescence stainingresults: in control group, the cell apoptosis rates is12.01%±3.31%, in MPP+Modelgroup, the cell apoptosis rates is31.78%±1.50%, in puerarin and Pifithrin-ɑ treatmentgroup, cell viability rates is25.34%±1.06%,21.70%±3.2%,16.28%±0.90%and14.23%±2.43%. The pretreatment puerarin and Pifithrin-ɑ negatively regulated theapoptosis ratio, compared with the MPP+group.(P<0.05).4Western blotting resultsshowed that MPP+can increase the expression of p53protein(P<0.05). The pretreatmentpuerarin and Pifithrin-ɑ could apparently inhibit the expression of p53protein(P<0.05). In control group, the relative expression of p53protein is0.12±0.01, in MPP+Modelgroup, the relative expression of p53protein is0.84±0.03, in puerarin and Pifithrin-ɑtreatment group, relative expression of p53protein is0.72±0.02,0.51±0.03,0.32±0.02and0.21±0.02.5. Real-time PCR results showed that the MPP+can increase theexpression of p53mRNA(P<0.05), but the expression of p53, p21, Bax, Caspase-3mRNA were down-regulated in pretreatment puerarin groups and the expression ofMDM2and Bcl-2mRNA was up-regulated simultaneously(P<0.05), Pifithrin-ɑ allowsp53and its related genes, such as p21, Bax, Caspase-3, MDM2, Bcl-2,mRNA expressionchanges and this change similar to the results of after giving Puererin. In control group,MPP+model group,50μmol/L puerarin+MPP+treatment group,100μmol/L puerarin+MPP+treatment group,150μmol/L puerarin+MPP+treatment group and Pifithrin-ɑ+MPP+treatment group, the relative expression of p53mRNA is1.00±0.00,1.79±0.08,1.66±0.03,1.35±0.10,1.19±0.04and1.10±0.02；The relative expression of p21mRNA is1.00±0.00,1.91±0.04,1.80±0.02,1.67±0.07,1.43±0.12and1.24±0.07；The relative expression of MDM2mRNA is1.00±0.00,0.62±0.11,0.88±0.08,1.27±0.18,1.50±0.18and1.69±0.36；The relative expression of Bax mRNA is1.00±0.00,2.53±0.14,1.90±0.39,1.61±0.32,1.16±0.43and1.03±0.12；Therelative expression of Bcl-2mRNA is1.00±0.00,0.18±0.06,0.74±0.07,2.19±0.16,2.30±0.30and2.47±0.20；The relative expression of Caspase-3mRNA is1.00±0.00,1.54±0.09,1.29±0.11,1.09±0.06,1.04±0.18and1.01±0.11.Conclusion It was such p53activated by MPP+-induced as to activate a series of targetgenes of p53and to induce cell apoptosis, it showed that p53has an important role in SH-SY5Y cells apoptosis by MPP+-induced. The cell apoptosis can be inhibited to someextent by pretreatment puerarin and Pifithrin-ɑ. It showed that puerarin can regulate theexpression of p53and its related genes and play a protective role against the death ofMPP+-induced SH-SY5Y cells.