| Objective:To study the effect of Lychee Seed Saponins on the expression of Bcl-2 and Bax in PC 12 cells induced by AP25-35, and to investigate mechanism of Lychee Seed Saponins inhibitting apoptosis in PC 12 cells. Methods: By means of percolation and segregation with 70% alcohol and D101 macroreticular resin, Lychee Seed Saponins were got. By means of CCK-8, the optimum Aβ25-35 concentration and time of injured in PC 12 cells induced by Aβ25-35 were defined, the maxium safe concentration of Lychee Seed Saponins action on normal PC 12 cells and the optimum concentration to protect PC 12 cells from apoptosis induced by Aβ25-35.IC50 of Aβ25-35 inhibitting PC 12 cells activity was worked out by LOGIT. AD cells model was duplicated by 20μmol·L-1 AP25-35 acting on PC 12 cells for 12h. The expression of Bcl-2 and Bax in PC 12 cells induced by Aβ25-35 was detected by Western-blot. Result:①Extractionof Lychee Seed Saponins:the yield of Lychee Seed Saponins was 3.15%.②The inhibitive effect of Aβ25-35 on PC 12 cells:There was a positive correlation between Aβ25-35 concentrations and action time(24h>12h>6h). At 24h, the inhibition of Aβ25-35 on PC 12 cells was increased along with different concentration of Aβ25-35, and the inhibition ratio of Aβ25-35 is 160μmol-L-1>80μmol-L-1>40μmol·L-1>20μmol·L-1> 10μmol·L-1>5μmol·L-1>2.5μmol·L-1, IC50 of PC12 cell induced by Aβ25-35 was 23.74μmol·L-1, and the maximum inhibition ratio of Aβ25-35 appeared at 12h.③The toxicity and the safest concentration measurement of Lychee Seed Saponins action on PC 12 cells:Compared with control group, OD decreased obviously in the groups in which the Lychee Seed Saponins concentration were 15mg·L-1 and above it(P<0.05), and a negative correlation was found between the concentration and OD; the RGR of PC 12 cells in these groups were below 75%, the toxicity of these concentration LSS were above grade 2; from 7.5mg·L-1 to the lowest concentration, no obvious change was found(P>0.05); and these RGR were above 75%, the toxicity ranged from 0 to 1.④The optimal effective concentration measurement of Lychee Seed Saponins inhibiting injury of PC 12 cells induced by Aβ25-35. Compare with model group, OD increased in treatment group with 0.938-7.500mg·L-1 Lychee Seed Saponins, the relationship was 7.5 mg·L-1>3.75 mg·L^1.875 mg·L-1>0.938 mg-L-1.⑤The expression of Bcl-2 and Bax in PC12 cells induced by AP25-35:Compared with control group, the stripe of Bcl-2 weakened, and the stripe of Bax was strengthened, the relative gray value of Bcl-2 decreased obviously(P<0.05), the relative gray value of Bax increased obviously(P<0.05). The ratio of Bcl-2/Bax decreased obviously(P<0.05).⑥The effect of the Lychee Seed Saponins on expression of Bcl-2 and Bax in PC 12 cells induced by Aβ25-35:Compared with model group, the stripe of Bcl-2 strengthened, and the stripe of Bax weaked obviously. The relative gray value of Bcl-2 increased obviously(P<0.05), and the relative gray value of Bax decreased obviously(P<0.05), the ratio of Bcl-2 and Bax increased obviously in treatment group with 0.938-7.500mg·L-1 Lychee Seed Saponins(P<0.05). Conclsution:(1) The selective routine to Lychee Seed Saponins was practicable, and the macroporous resin could be used for emundation and disassociation of Lychee Seed Saponins. (2)The optimum condition of PC12 cell to be injured was 20μmol·L-1 Aβ25-35 exposed on PC12 cells for 12h; there were time-effect relationship and concentration-effect relationship to the influence of Aβ25-35 on PC 12 cells activity. (3)Concentration of LSS below 7.5mg·L-1 were safe to PC 12 cells, the safest concentration of Lychee Seed Saponins on PC 12 cells was 7.5mg·L-1, and 7.5mg·L-1 Lychee Seed Saponins as well as drug concentration by double dilution was reliability in pharmacodynamic studies; (4) 0.938-7.500mg·L-1 Lychee Seed Saponins had obvious protective effect on injuries of PC12 cells induced by A(325-35, among 0.938-7.500mg·L-1 concentration, the studies about Lychee Seed Saponins inhibiting apoptosis of PC12 and mechanism were practical. (5)Apoptosis of PC12 cells induced by Aβ25-35 was relative to increasing Bax, decreasing Bcl-2 and reducing the ratio of Bcl-2/Bax. (6)The mechanism of Lychee Seed Saponins inhibitting apoptosis of PC 12 cells was relative to decreasing Bax, increasing Bcl-2 and improving the ratio of Bcl-2/Bax. |
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