| OBJECTIVE To explore the effect of a chinese herbal compound (the Concentrated Fluid of YiZhiJianNao Decoction,CFYD) and acupunc-ture (Kidney-reinforcing and Blood-activating Acupuncture Method, KBAM), both under the guideline of Kidney-reinforcing and Blood-acti vating therapy method, on the behavior of senescence-accelerated mice P8(SAMP8) which is considered as an generally accepted mouse model of Alzheimer's disease, thus to elucidate the therapeutic effect of Kidney-reinforcing and Blood-activating therapy on SAMP8 and the practicability of this method in the prevention and treatment of Alzheimer's disease from different aspects; To probe the potential remedial protein targets of Alzheimer's disease by analyzing the differential expression of hippocampus proteins in SAMP8 with the intervention of Kidney-reinforcing and Blood-activating therapeutic principle(CFYD and KBAM),thus to analyze the therapeutic mechanism of the relevant target proteins from the angle of Multi-target treatment strategies in complex disease.METHODS 30 five-month-old male SAMP8 were randomly divi-ded into three groups:chinese herbal treatment group (n=10), acupunctu-re treatment group (n= 10) and model control group (n=10). Another 10 five-month-old male SAMR1 mice were prepared as normal control group (n= 10). Mice in chinese herbal treatment group were treated with intragastric administration of CFYD, Mice in acupuncture treatment group were intervened by KBAM and intragastric administration of Double-distilled water with the same dose simultaneously, and mice in model control group and normal control group were administered intra-gastrically with the same dose of Double-distilled water. In order to red-uce bias between groups as far as possible, make sure that the grasping time of the mice,the degree of the grasping and intragastric adminis-tration were consistent between groups, and this job was completed by the same skilled experimenter. After 4 weeks, using Morris water maze for mice in each group to analyze its behavior to detect changes in learning and memory ability. After behavioral experiments, the total proteins of hippocampus tissue of SAMP8 in chinese herbal treatment group, acupuncture treatment group and model control group were separated by two-dimensional gel electrophoresis(2-DE) 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 control group as reference object, part of differential expression protein spots in chinese herbal treatment group and acupuncture treatment group were identified by peptide mass fingerprint(PMF) 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 immunohistochemical 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 SAMR1 mice in mormal control group.RESULTS1. In Morris water maze test, SAMP8 mice in the model control group had longer escape latency compared with SAMR1 mice in normal control group (p<0.05); For SAMP8 mice in the model control group, the stay time in the former platform quadrant and former platform crossing times were much less than SAMR1 mice in normal control group (p<0.05); With the intervention of Kidney-reinforcing and Blood-acti vating therapy, for CFYD and KBAM, both of them can significantly shorten the escape latency and increase the stay time in the former platform quadrant and former platform crossing times of SAMP8 mice (p<0.05).2. Taking the 2-DE map of model control group as reference object, we compared the map of chinese herbal treatment group with model control group, then acupuncture treatment group with model control group to obtain the differentially expressed protein spots. We found that there were 19 diferentialy expressed protein spots in chinese herbal treatment group, of which 11 proteins were up-regulated and 8 proteins were down-regulated; There were 13 diferentialy expressed protein spots in acupuncture treatment group, of which 7 proteins were up-regulated and 6 proteins were down-regulated.3. For identification, we selected 14 diferentialy expressed protein spots in chinese herbal treatment group and finally identified 11 points, of which 6 proteins were up-regulated and 5 proteins were down-regulated; 10 diferentialy expressed protein spots in acupuncture treatment group were selected and finally 9 protein points were identified, of which 6 proteins were up-regulated and 3 proteins were down-regulated: 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 Aβmetabolism, regulating phosphorylation of tau protein, anti-oxidative stress, inhibiting neuroinflammation, regulation of mitochondrial energy metabolism and glucose metabolism, etc.4. Validation of some differentially expressed proteins:(1) The results of western blotting:we detected the expression levels of flotillin-1 and GFAP, two differentially expressed proteins which were present in both chinese herbal treatment group and acupuncture treatment group. We found that the expression levels of flotillin-1 and GFAP significantly increased in the model control group compared with SAMR1 mice in normal control group(p<0.05); With the intervention of Kidney-reinforceing and Blood-activating therapy, both flotillin-1 and GFAP significantly decreased in SAMP8 mice of chinese herbal treatment group and acupuncture treatment group compared with model control group (p<0.05), which is in accordance with the proteomic results.(2) The results of immunohistochemical experiments:we also detecte-d flotillin-1 and GFAP in immunohistochemical experiments and found the results consistent with the research of proteomics and western blot-ting; Besides, we detected the expression levels of Pin l and Ngb, differ-enttially expressed in chinese herbal treatment group and acupuncture tre- atment group respectively, and found that the expression levels of Pin l and Ngb significantly decreased in SAMP8 mice of model control group compared with SAMR1 mice of normal control group(p<0.001); CFYD can significantly enhance the expression levels of Pin l (p<0.001), which is in accordance with the proteomic results; KB AM can significantly enhance the expression levels of Ngb (p<0.001), which is consistent with the proteomic results.CONCLUSIONS1.The intervention of Kidney-reinforceing and Blood-activating ther-apy(represented by CFYD and KBAM) in traditional chinese medicine can significantly improve the learning and memory ability as well as the ability of memory consolidation and reproduction in AD model mice SAMP8.2.Kidney-reinforceing and Blood-activating therapy in TCM may exzert a remedial effect of AD model mice SAMP8 through a multi-target, multi-channel and multi-mechanism approach. The function of relevant potential therapeutic target proteins involved in regulating Aβmetabolism, regulating phosphorylation of tau protein, anti-oxidative stress, inhibiting neuroinflammation, regulating mitochondrial energy metabolism and glucose metabolism, and so on. Treatment mode like this highly fits with not only the complexity of the pathogenesis of AD on one hand but also multi-target theraputic strategy of complex diseases on the other hand, which could provide a useful reference for the treatment of AD.3.Find some potential therapeutic target proteins of Kidney-rein forceing and Blood-activating therapy, thus to lay a foundation for further determination of the target proteins and their mutual connections in the fight against AD. |
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