The Protective Effect And Mechanism Of Procyanidins Extracted From The Lotus Seedpod On Damage Of PC12 Induced By ?-amyloid 25-35

Part 1 The protective effect of LSPC on damage of PC12 induced by A?25-35Objectives: A?25-35 were added to the PC12 cell to induce its damage and establish cell injury model, observing the effect of LSPC on cellular morphology changes, cell damage and apoptosis.Methods:(1) Determination of the intervention level and time of A?25-35 and LSPC.Cells were divided into several groups and cultured with different A?25-35 concentration including 0, 5, 10, 20 and 40 ?M. The inhibit rate of each group was detected by CCK-8 method after cultured for 0, 6, 12, 24, 48 h, respectively. Afterwards, we detected vitality of cell with different LSPC concentration(1, 2.5, 5, 10, 20 and 40?g/m L) to explore the best intervention dose.(2) The protective effect of LSPC on cell damage induced by A?25-35.Cells were divided into five groups: the control group(without any interventions), the A? group(treated with 20 ?M A?25-35 for 24 h), and three LSPC groups(treated with 10, 20,40 ?g/m L LSPC, respectively). After incubation, cells and culture medium were collected to detect T-SOD, MDA and LDH.(3) The protective effect of LSPC on cell apoptosis induced by A?25-35.Cells were divided into three groups: the control group, the A? group, and the LSPC groups(treated with 10 ?g/m L LSPC). Cellular morphology was observed by inverted microscope. Cell apoptosis was observed by inversed fluorescent microscopeafter Hoechst staining, then apoptosis rate was measured using flow cytometry after Annexin V/PI staining.Results:(1) Compared with control group, vitality of other groups decreased in different degrees. According to inhibit rate of each group, IC50 was about 20 ?M for 24 h or40 ?M for 12 h. Therefore, 20 ?M and 24 h were chosen as the exposure dose and time, respectively. The pre-incubation of LSPC increased cell vitality in a dose-dependent way. Cell vitality was the highest when treated with 10 ?g/m L LSPC(P < 0.05).(2) Exposure of A?25-35 caused decrease in activity of intracellular T-SOD and increase in level of intracellular MDA and extracellular LDH(P < 0.05), which suggested A?25-35 may reduce antioxidant capacity of PC12, induce lipid peroxidation and further damage the cell membrane. The pre-incubation of LSPC alleviated the damage in PC12. MDA and LDH were more in 10?g/m L group than in 20 and 40?g/m L group, and activity of T-SOD had a trend to enhance with augmented dose(P< 0.05).(3) Observation by microscope suggested that the pre-incubation of LSPC reversed drop in cell count, morphology degeneration and retraction of cell processes on PC12. The number of apoptotic cell in A? group was the most among three groups.Results of flow cytometry showed that apoptosis rate of A? group had climbed to(33.36±3.05)% which was attenuated by LSPC(P < 0.05).Conclusions: Under the condition of this research, exposure of A?25-35 results in damage in PC12, causing changes in cell morphology and eventually leading to cell apoptosis. The pre-incubation of LSPC can alleviate the damage and show protective effect.Part 2 The effect of LSPC on CREB/BDNF signal pathway in PC12Objectives: To determine the effect of LSPC on CREB/BDNF signal pathway in PC12 through detection of upstream signal pathways, including PI3K/AKT and Raf/ERK1/2 signal pathway.Methods: Cells were divided into seven groups: control group, A? group, LSPCgroup, inhibitor groups(LY group and PD group) and inhibitor + LSPC groups. The expression of CREB/BDNF and upstream signal pathways were measured by western blotting analysis, and level of BDNF m RNA was detected by real time-PCR.Results: Compared with control group, the expression of P-CREB decreased in A? group(P<0.05), which reducing the expression of BDNF and its genes(P<0.05).Compared with A? group, the use of LSPC obviously increased the expression of CREB/BDNF(P<0.05). The level of P-AKT and P-ERK1/2 showed a reduction in accord with P-CREB(P<0.05). Compared with inhibitor groups, LSPC alleviated inhibiting effect by LY and PD(P<0.05), suggesting that LSPC may regulate CREB/BDNF through PI3K/AKT and Raf/ERK1/2 signal pathways.Conclusions:(1) Under the condition of this research, A?25-35 inhibits the activation of CREB/BDNF pathway, LSPC can reverse the inhibiting effect.(2) The results of this research show that LSPC may promote the activation of CREB/BDNF pathway through PI3K/AKT and Raf/ERK1/2 signal pathways, which contribute to its protective effects.Part 3 The effect of LSPC on PERK/e IF2? signal pathway in PC12Objectives: To investigate the effect of LSPC on ERS status in PC12 through detection of the PERK/e IF2? signal pathway, and further explore mechanism underlying cell damage by ERS through detection of CHOP/Caspase-3 and GSK-3?/Tau signal pathways.Methods: Cells were divided into six groups: control group, A? group, LSPC group, inhibitor group(GSK), GSK+A? group and GSK+LSPC group. The expression of PERK/e IF2? and downstream signal pathways were measured by western blot method.Results: The expression of P-PERK and P-e IF2? greatly increased in A? group than in control group(P<0.05), and the pre-incubation of LSPC inhibited the pathway,decreasing the level of P-PERK and P-e IF2?(P<0.05). The use of PERK inhibitor and A? suggested that A? induced ERS in PC12 which was alleviated by LSPC(P<0.05). Subsequently, level of CHOP, Caspase-3, P-GSK-3? and P-Tau weresimilar with P-PERK and P-e IF2?.Conclusions:(1) Under the condition of this research, A?25-35 can induce ERS in PC12, and LSPC is likely to protect PC12 cells by alleviate ERS.(2) LSPC may inhibit the activation of apoptosis signal pathway by alleviate ERS; LSPC can decrease the phosphorylation of GSK-3?, thus reducing the phosphorylation of Tau though regulation of PERK/e IF2? signal pathway.