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

Part 1 The protective effect of LSPC on damage of PC12 induced by Aβ25-35Objectives: Aβ25-35 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 Aβ25-35 and LSPC.Cells were divided into several groups and cultured with different Aβ25-35 concentration including 0, 5, 10, 20 and 40 μM. 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 40μg/m L) to explore the best intervention dose.(2) The protective effect of LSPC on cell damage induced by Aβ25-35.Cells were divided into five groups: the control group(without any interventions), the Aβ group(treated with 20 μM Aβ25-35 for 24 h), and three LSPC groups(treated with 10, 20,40 μg/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 Aβ25-35.Cells were divided into three groups: the control group, the Aβ group, and the LSPC groups(treated with 10 μg/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 μM for 24 h or40 μM for 12 h. Therefore, 20 μM 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 μg/m L LSPC(P < 0.05).(2) Exposure of Aβ25-35 caused decrease in activity of intracellular T-SOD and increase in level of intracellular MDA and extracellular LDH(P < 0.05), which suggested Aβ25-35 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 10μg/m L group than in 20 and 40μg/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 Aβ group was the most among three groups.Results of flow cytometry showed that apoptosis rate of Aβ 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 Aβ25-35 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, Aβ 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 Aβ group(P<0.05), which reducing the expression of BDNF and its genes(P<0.05).Compared with Aβ 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, Aβ25-35 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 IF2α signal pathway in PC12Objectives: To investigate the effect of LSPC on ERS status in PC12 through detection of the PERK/e IF2α signal pathway, and further explore mechanism underlying cell damage by ERS through detection of CHOP/Caspase-3 and GSK-3β/Tau signal pathways.Methods: Cells were divided into six groups: control group, Aβ group, LSPC group, inhibitor group(GSK), GSK+Aβ group and GSK+LSPC group. The expression of PERK/e IF2α and downstream signal pathways were measured by western blot method.Results: The expression of P-PERK and P-e IF2α greatly increased in Aβ 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 IF2α(P<0.05). The use of PERK inhibitor and Aβ suggested that Aβ induced ERS in PC12 which was alleviated by LSPC(P<0.05). Subsequently, level of CHOP, Caspase-3, P-GSK-3β and P-Tau weresimilar with P-PERK and P-e IF2α.Conclusions:(1) Under the condition of this research, Aβ25-35 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-3β, thus reducing the phosphorylation of Tau though regulation of PERK/e IF2α signal pathway.