Mechanism Of Protective Effect Of Ginsenoside Rb1 On Amyloid-β-peptide Induced Neuron Apoptosis

ObjectiveAlzheimer's disease (AD) is a neurodegenerative disease characterized by progressive loss of cognitive ability in eldly people. As more people live to old age, the morbidity of AD increases year by year and the incidences increase from 5% per year at the age of 65 years to 8% per year at the age of 85 years. AD has become a serious medical and social problem. Due to lacking the effective treatment, AD has become one of the deadly diseases. The neuropathological features of AD include senile plaques (SP) formed by pathological deposition ofβ-amyloid (Aβ), neurofibrillary tangles (NFT) and neurons loss induced by neuronal apoptosis. It is well known that the deposition of Aβin brain tissue is a key factor contributing to the development of AD. The mounting evidence indicated that the abnormal extra-cellular Aβdeposition might lead to oxidative stress and might initiate a series of cascade reactions of apoptosis which lead to the progressive decline of cognition functions in patients. But at present the proteins induced by Aβwhich are involved in neuronal apoptosis and the regulation mechanism of the interaction between the proteins are not clearly understood. Therefore, it is very important to further study the mechanism of Aβ-induced neuronal apoptosis and choose the effective medicines for AD treatment.Ginsenoside-Rb1 (GSRb1) makes up 0.37-0.5% of ginseng extracts among 26 identified ginsenosides. It has been suggested to have pharmacological activities in central nervous systems, but a detailed mechanism has not yet to be determined. In the study, Mice were intracerebroventricularly injected with the aggregated Aβto mimic the AD. In vivo we aimed to examine SOD,GSH-PX,MDA and the protein expression of Bc1-2,Bax,Caspase-3 and to determine whether ginsenoside-Rb1 is effective on Aβ-induced neuronal apoptosis. PC12 cells exposed to Aβwere cultivated to mimic damaged neurons and then were treated with ginsenoside Rb1. In vitro we aimed to examine the cell viability, the cell apoptosis percent, intracellular ROS and the protein expression of p-P38/ P38,p-ERK/ ERK,p-JNK/ JNK and to determine whether ginsenoside-Rb1 is effective on Aβ-induced cell apoptosis.Materials and Methods1,In vivo(1) Aβmodel rats and experimental groups: Male, Sprague-Dawley (SD) 8～12 weeks Rat (n=60) were enrolled in the study, and they were equally randomly divided into six groups. There were experiment groups (5mg/kg/d, 10mg/kg/d, 20mg/kg/d), model control group, sham control group, normal control group. To induce AD, model control group and experiment groups (5mg/kg/d, 10mg/kg/d/, 20mg/kg/d) received icv. injection of 10μg of Aβ_25-35. Normal control group and sham control group received icv injection of the same dose of NS. Experiment groups was ip injection daily for 28 days with GSRb1 (5mg/kg/d, 10mg/kg/d, 20mg/kg/d) after three days of AD induction.(2) The learning and memory abilities were evaluated by the escape latency in Morris Water Maze performance and the times to find a hidden platform.(3) Biochemical analysis for detecting the activity of SOD and GSH-PX and the level of MDA in the brain tissue.(4) Western blotting for detection of Bcl-2 and Bax expression in rat hippocampal formation.(5) Immunohistochemical staining for detection of Caspase-3 protein expression in rat hippocampal formation.2,In vitro(1) Cell culture and groups: PC12 cells were cultivated in RPMI-1640 medium containing 5% fetal calf serum and 10% heat-inactivated horse serum at 37°C in a 5% CO₂ atmosphere. Prior to experiment, the medium was replaced with serum-free RPMI medium and the experiment groups were incubated with GSRb1 and the aggregated Aβ_25-35 for 24h in all studies. PC12 cells were divided into five groups, normal control group ( PC12 ), Aβmodel group, experiment groups ( 0.01mM GSRb1, 0.1mM GSRb1, 1mM GSRb1).(2) MTT assay for the determination of cell viability.(3) Flow cytometry assay for measurement of cell apoptosis. (4) Fluorescence spectrophotometer for detection of the fluorescence intensity of the ROS product.(5) Western blotting for the expression of p-P38/P38,p-ERK/ ERK,p-JNK/ JNK.3,Statistical analysisImage collection analysis system was applied. The data were expressed as Mean±SD and were analyzed by SPSS 16.0 statistical package. The differences among groups were compared with one-way analysis of variance. P<0.05 was regarded as the significant difference and P<0.01 was regarded as the extremely significant difference.Results1,In vivo(1) Morris water maze test: In place navigation test, the mean escape latencies of the model group were obviously increased compared with control groups (P<0.01). In spatial probe test, the frequency of passing through the platform of the model group were obviously decreased as compared with the control groups (P<0.05). In place navigation test, the mean escape latencies of experiment group ( 5mg/kg/d, 10mg/kg/ d/, 20mg/kg/d) were obviously decreased compared with model group (P<0.05 ) and were not statistically significant compared with the control groups (P>0.05). In spatial probe test, the frequency of passing through the platform of experiment group (5mg /kg/d, 10mg/kg/d/, 20mg/kg/d) were obviously increased as compared with the model groups (P<0.05) and were not statistically significant difference compared with the control groups (P>0.05).(2) Biochemical analysis: The SOD activity from the model group (58.73±2.67 U/mg) was significantly lower than that in the control groups (P<0.05), and that from the experiment groups(10mg/kg/d/, 20mg/kg/d) (respectively 73.64±3.21U/mg, 78.42±2.89U/mg) was significantly higher than in the model group (P<0.05).The MDA from the model group (6.81±0.45nmol/mg) was significantly higher than that in the control groups(P<0.05), and that from the experiment groups (10mg/kg/d/, 20mg/kg/d) (respectively 5.53±0.67nmol/mg, 4.93±0.12nmol/mg) was significantly lower than that in the model group(P<0.05). The GSH-PX activity from the model group (4.81±0.45U/mg) was significantly higher than that in the control groups (PO.05), and that from the experiment groups (10mg/kg/d/, 20mg/kg/d) (respectively 6.73±0.71U/mg, 6.93±0.79U/mg) was significantly lower than that in the model group (P<0.05).(3) Immunohistochemistry: The positive reaction product of Caspase-3 was stained buffy or brown. They mainly deposited in pyramidal cell layer of hippocampal CA1 and CA3 regions and granular cell layer of dentate gyrus. Compared with the control groups, the mean optical density (MOD) of Caspase-3 positive reaction product in model group was significantly increased (P<0.01). The MOD of Caspase-3 positive reaction product in the experiment groups was significantly decreased compared with model group (P<0.05).(4) Western blotting: The expression of Bcl-2 protein in the model group was extremely significantly lower than that in the control groups (P<0.01), and that from the experiment groups was significantly higher than that in the model group (PO.05). The expression of Bax protein in model group is extremely significantly higher than that in control groups (P<0.01), and that from the experiment groups was significantly lower than that in the model group (PO.05).2,In vitro(1) MTT: The analyses were done after 24 hours of treatment. The cell viability (48.55±1.39%) in the model group was significantly lower than that in the controlgroup (99.23±1.35%) (P<0.05), and that from the experiment groups (64.45±1.67%,72.34±1.34%,81.67±1.09%) was significantly higher than that in the model group (P<0.05).(2) Flow cytometry: The analyses were done after 24 hours of treatment. The percent of cell apoptosis (54.09±2.85%) in the model group was significantly higher than that in the control group (0.29±0.01%) (P<0.05), and that from the experiment groups (46.17±2.94%, 32.34±2.68%, 20.50±1.23%) was significantly lower than that in the model group (P<0.05).(3) Fluorescence spectrophotometer: The analyses were done after 24 hours of treatment. The fluorescence intensity of the ROS product in the model group (370±24.58) was significantly higher than that in the control group (80±5.49) (P<0.05), and that from the experiment groups (330±21.78, 310.34±32.81, 150.50±17.45) was significantly lower than that in the model group (P<0.05).(4) Western blotting: The analyses were done after 24 hours of treatment. The expression of p-ERK protein in the model group was significantly higher than that in the control group (P<0.05), and that from the experiment groups was significantly lower than that in model group (P<0.05). The expression of p-P38 protein in model group is significantly higher than that in control group (P<0.05), and that from the experiment groups was significantly lower than that in the model group (P<0.05).Conclusions1,GSRb1 decreased MDA level and increased SOD activity and GSH-PX activity, which can increase the potential of the hippocampal formation against oxidation and can improve learning and memory deficits.2,GSRb1 upregulated Bcl-2 expression and downregulated Bax expression which can alter Bcl-2/Bax ratio and downregulated Capase-3 expression which can decrease neuronal apoptosis in rat hippocampus formation.3,GSRb1 alleviated ROS generation and inhibited the expression of p-ERK and p-P38, which can protect neurons against apoptosis induced by Aβand exert a neuroproctive action.