Protective Effects of Procyanidins on 6-OHDA-induced PC12 Cells Injury and its Mechanism Exploration

Objective:To investigate the protective effect of procyanidins(PC)on PC12 cells injured induced by 6-hydroxydopamine(6-OHDA),and then to explore the underlying mechanism. Methods:PC12 cells were treated with 6-OHDA for 24h to establish cell injury model,PC were added in 2 h before 6-OHDA and the PI3K inhibitor LY294002 was added in 0.5 h before PC to detect the mechanism of protection. Pharmacodynamic study of PC in this model were divided into 5 groups:control group,model group(75 μmol·L-16-OHDA),PC-L,M,H group(75μmol·L-16-OHDA+12.5, 25,50 μmol/L);mechanism study of PC in this model were divided into 5 groups:control group,model group(75 μmol/L 6-OHDA),PC-H group,LY group(75 μmol·L-16-OHDA+20 μmol/L LY294002), PC-H+LY group(75μmol·L-16-OHDA+50μmol·L-1 PC +20 μmol·L-1 LY294002). MTT assay was used to detect cell viability,colorimetric method was used to detection of lactate dehydrogenase(LDH) leakage,the total superoxide dismutase(SOD)activity was measured by WST-8 method,JC-1 staining was used to detect the mitochondrial membrane potential. The expression of Akt and p-Akt(Ser473) protein was assayed by Western Blot. Results:OD570 of 1.5×105/mL PC12 cells at 0.82±0.05 was in a good linear range that for the appropriate subculturing concentration;PC12 cells were treated with 6-OHDA for 24 h and its viability decreased with the increase of 6-OHDA concentration,compared with control group,6-OHDA(75 μmol·L-1)damaged cell 49.55±5.32％(P<0.01)for the appropriate model concentration;cells were treated with PC alone for 24 hours without significant toxicity at each concentration,the cell viability was slightly increased at 50 μmol·L-1. Compared with control group, the cell viability,total SOD activity,mitochondrial membrane potential and protein of Akt Ser473 phosphorylation levels decreased in model,while the leakage rate of LDH increased. Compared with the model group,PC(12.5,25,50 μmol·L-1)treatments could improve the morphological damage of PC12 cells induced by 6-OHDA and increase the cell viability,significantly reduce the leakage rate of LDH(P<0.01),increase the activity of total SOD(P<0.01),inhibit the decrease of mitochondrial membrane potential,and increase the expression of p-Akt protein(P<0.01). All of these effects were antagonized by LY294002. Conclusion:Under the experimental conditions,PC has protective effect on 6-OHDA-induced PC12 cells injury,and its mechanism may be related to up-regulation of PI3K/Akt pathway.