The Apoptosis Of PC12 Cells Induced By Aβ_25-35 And The Protections Of Lychee Seed Saponins

Objective:To study the effect of Lychee Seed Saponin on apoptosis of pc12 cells induced by Aβ25-35, and to investigate initially the mechanism.Methods:By Hoechst33342/PI fluorescence double staining and TUNEL, the apoptosis of PC 12 cells induced by AP25-35 was observed respectively under fluorescence microscope and light microscope. By AnnexinV-PI dubule staining, apoptosis of PC 12 cells apoptosis of PC 12 cells induced by AP25-35 was evaluated with flow cytometry. The apoptotic percentage and the apoptotic index of PC 12 cells was reckoned with SPSS Software. By means of marking PC 12 cell with rhodamine123, the change of mitochondria membrane potentials (Ψm) was observed under fluorescence microscope. The expression of NF-κB P65 mRNA of PC 12 cell was detected by RT-PCR and Agarose Gel Electrophoresis.Results:(1)the level of apoptosis:①Hoechst33342/PI double staining:Compared with control group, there was more obvious apoptosis characteristics of PC 12 induced by Aβ25-35, for example, anachromasis, granular fluorophore, caryolobism, nuclear fragmentation and so on. Number and rate of apoptotic cells were increased obviously(P <0.05), and many dead cells were observed (red). Compared with model group, the number of apoptotic and dead cells and apoptotic rates decreased obviously in preconditioning group with 0.938-7.500mg·L-1 Lychee Seed Saponin(P< 0.05).②TUNEL:Compared with control group, apoptotic characteristic of PC 12 cell induced by AP25-35, for example, pyknosis, compact nucleus, brownness of anachromasis and so on. The number of apoptotic cell and index of apopotosis are increased(P <0.01). Compared with model group, they are decreased preconditioning group with 0.938-7.500mg·L-1 Lychee Seed Saponin(P<0.01).②flow cytometer measured:On the scatter diagram, there are many PC 12 cells on the left lower quadrant control group, and few apoptotic cell on the right quadrant. Compared with control group, the apoptotic and dead cells obvious wered increased on the right lower or upper quadrant, and the apoptotic rate of PC 12 cell are increased significantly(P<0.05). Compared with model group, all of them were reduced significantly. (2)Mitochondria membrane potentials(ΔΨm):Under fluorescence microscope, there are obvious fluorescence quenching, and the attenuated fluorescence intensity, in other words, dissipation ofΔΨm was attenuated in the control group.In the model group, the fluorescence intensity was enhanced, and there was little fluorescence quenching. In the pretreatment group with 0.938-7.500mg·L-1 Lychee Seed Saponin, the fluorescence intensity was attenuated,in other words, dissipation ofΔΨm was attenuated. (3)Expression of NF-KBP65mRNA:In every group,there was obvious amplified strap of glyceraldehyde-3-phosphate dehydrogenase(GAPDH) in the 141bp, and there was amplified strap of NF-κBP65mRNA which is different intensity but sharply demarcated in the 237bp. Compared with control groups, The intensity of NF-κBP65mRNA amplified segmental are more obvious, also the relative gray value of NF-κBP65mRNA was increased significantly in the model group(P<0.05). Compared with model group, the intensity of NF-κBP65mRNA amplified segmental are attenuated, and the relative gray value of of NF-κBP65mRNA were decreased significantly in the pretreatment group with 0.938-7.500mg·L-1 Lychee Seed Saponin(P<0.05).Conclusion:(1) Apoptosis was induced by exposure of PC12 cell to 20μmol·L-1Aβ25-35 for 12h. (2) 0.938-7.500mg·L-1 Lychee Seed Saponin can inhibit the apoptosis of PC12 cells induced by Aβ25-35. (3) The mechanism of LSS aganist the apoptosis of PC12 cells induced by Aβ25-35 include:①there is relevant to inhibitting the descend of mitochondria membrane potentials and improving the functional disorder of mitochondria;②The mechanism is associated with inhibitting the activation of NF-κB and attenuating the apoptotic cascade reaction caused by NF-κB.