| OBJECTIVE To explore the effect of Icariin(ICA) combined with Panax notoginseng saponins (PNS), under the guideline of Kidney-reinforcen and Blood-activating therapy method, on the behavior of senescence-accelerated mice P8(SAMP8), which is considered as an generally accepted mouse model of Alzheimer’s disease (AD), thus to elucidate the therapeutic effect of ICA combined with PNS on SAMP8and the practicability of this method in the prevention and treatment of AD; To probe the potential remedial protein targets of AD by analyzing the differential expression of hippocampus proteins in SAMP8with the intervention of ICA combined with PNS, thus to analyze the therapeutic mechanicsm of the relevant target proteins from the angle of Multi-target treatment strategies in complex disease.METHODS50six-month-old male SAMP8were randomly divided into3groups, which are the follows:combination group (n=30), model group (n=10) and medicine control group (n=10). Another10six-month-old male SAMR1were prepared as normal control group (n=10). Mice in combination group were individually ig administrated with ICA combined with PNS [(2.5+20),(5+40), and (10+80) mg/kg], Mice in medicine control group were treated with Donepezil HCL, and mice in model group and normal control group were administered intragastrically with the same dose of Double-distilled water. After8weeks, using Morris water maze for mice in each group to analyze their behavior to detect changes in learning and memory ability. After behavioral experiments, the total proteins of hippocampus tissue of SAMP8in treatment group (optimal dose group in behavioral experiments), model group and medicine control group were separated by two-dimensional gel electrophoresis respectively. The gels of the three groups were stained by Coomassie brilliant blue, scanned by ImageScanner and analyzed in PDQuest software. Taking the gel of model group as reference object, part of differential expression protein spots in treatment group and medicine control group were identified by peptide mass fingerprint based on the technology of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and MSDB or NCBInr database searching. Using Western blot and Reverse transcription-Polymerase chain reaction (RT-PCR.) techniques to testify the expression differences of some identified protein spots. In the meanwhile, we also detected the expression levels of these proteins in the hippocampus tissue of SAMR1in mormal control group.RESULTS:1. In Morris water maze test, SAMP8in the model group had longer escape latency compared with SAMR1in the normal control group (P<0.05); For SAMP8in the model group, the stay time in the former platform quadrant and former platform crossing times were much less than SAMR1mice in the normal control group (P<0.05); With the intervention of ICA with PNS combined, we can significantly shorten the escape latency and increase the stay time in the former platform quadrant and former platform crossing times for SAMP8in the treatment group (P<0.05); There was no significant diffenrence between the combination group (ICA+PNS5+40mg/kg, ig) and the combination group (10+80mg/kg, ig)(P>0.05).2. Taking the2-DE map of model group as reference object, we compared the map of treatment group with it of model group to obtain the differentially expressed protein spots. We found that there were23diferentialy expressed protein spots in the treatment group, of which12proteins were up-regulated and11proteins were down-regulated; For identification, we selected12diferentialy expressed protein spots in the treatment group and finally identified7points were identified;15diferentialy expressed protein spots in the model group were selected and finally9protein points were identified. According to the information provided in the protein database, we made analysis of these proteins as potential remedial targets for its direct or indirect remedial effects in the treatment of AD and found that the therapeutic mechanism of these proteins in AD involved in regulating Aβ metabolism, regulating phosphorylation of tau protein, anti-oxidative stress, inhibiting neuroin-flammation, regulation of mitochondrial energy metabolism and decreasing the neuron apoptosis, etc.3. Validation of some differentially expressed proteins:(1) The results of western blot:we detected the expression levels of flotillin-1and HMGB1in western blot experiment, and found that the expression levels of flotillin-1and HMGB1significantly increased in SAMP8in the model group compared with SAMR1in the normal control group (P<0.05); both flotillin-1and HMGB1significantly decreased in SAMP8in treatment group and medicine control treatment group compared with SAMR1in model control group (P<0.05), which is in accordance with the results in the proteomic experiment.(2) The results of RT-PCR:we also detected HMGB1and Ngb in RT-PCR experiment, and found that the expression levels of HMGB1significantly increased in SAMP8in the model group compared with SAMR1in the normal control group(P<0.05), the results consistent with the research in the western blot experiment; the expression levels of Ngb significantly decreased in SAMP8in the model control group compared with SAMR1in the normal control group (P<0.05); and found that the expression levels of Ngb can be significantly enhanced with the intervention of ICA combined with PNS (P<0.05), which is consistent with the results in the proteomic experiment.CONCLUSIONS:1. The intervention of ICA combined with PNS can significantly improve the learning and memory ability as well as the ability of memory consolidation and reproduction in AD model mice SAMP8.2. Hippocampus tissue protein expression profiles of SAMP8can be regulated by ICA combined with PNS, showing which has multitarget mechanism of AD.3. ICA combined with PNS may have therapeutial effect on AD via regulating the protein expression such as flotillin-1, HMGB1and Ngb, which may be the target proteins against AD. |
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