| 1. BackgroundAlzheimer Disease (AD) is one of central nervous system degenerative diseases, which has a main performance gradual cognitive dysfunction, bad memory and learning ability, sometimes it may also be accompanied by anxiety, depression, language barriers and other many personality changes. So far, the pathogenesis of AD is not yet clear. But the main pathological characteristics can include:neurofibrillary tangles (NFTs), gathered by the abnormal tau protein in the cell; Age spots (senile plaques, SP), by beta amyloid protein (Aβ) outside the cell formed by the large amounts of precipitation and the loss of neurons.2. PurposeThe study is based on the theory of Traditional Chinese Medicine, we choose the internationally recognized SAMP8rapidly aging dementia model mice. The selection is closely related to the AD neuron loss of PI3K/AKT signal transduction pathway, exploring the different electro-acupuncture intervention difference in the protection of neurons. Seeking new targets to reveal the mechanism of action of acupuncture treatment of senile dementia may, provide a certain theoretical basis for clinical treatment of AD.3. MethodThere were70adult male mice at8month, including10SAMR1mice and60SAMP8mice. The60SAMP8mice were randomly divided into six groups:Model group, Non-acupuncture group, Music group, Donepezil Hcl group, Pulse electric acupuncture(EA) group and Music electric acupuncture group (N=10). The EA group, the needles handle connected the HANS-LH202electric acupuncture apparatus, selecting "Bai Hui","Yin Tang" and "Ren Zhong" acupoints,20min/day,15days. The non-acupuncture group was given the same stimulus in the tail root1/3of mice. The music electric acupuncture group, selecting the same acupoints and choosing dementia music prescription, the needles handle connected the ZJ-12H musical electro-acupuncture apparatus. The Music group, in order to ensure under the same conditions, mice were catched once a time while listening to music. The Donepezil HCL group, mice were used intragastric administration for15days. The normal group and model group mice only were be catched at a time as same as other groups. After15days, using mirrors water maze(MWM) to judge the behavior of groups of mice. Choosing Nissl’s staining and HE staining to observe number of neurons and pathological changes of morphology in mice’s hippocampus CA1area. Using enzyme-linked immunosorbent (ELISA) method to detect each group mice serum A beta40 and A beta42protein expression levels. Using immunohistochemistry, western bolt and Real-time PCR test groups of mice hippocampus Tau, P-Tau (ser199/202), AKT, P-AKT (ser473), GSK-3beta, P-GSK-3beta (ser9) and GFAP protein and gene expression level.4. Results(1) MWM Results:Compared with normal group, the SAMP8mice were significantly poor in the ability of learning and memory, reflecting the escape latency period extended, swimming distance increased, the reduced the number of cross-platform and target quadrant stay a shorter time (P<0.01). Compared with the SAMP8model group mice, the learning and memory ability in non-acupuncture group is similarity to the model group (P>0.05). In music group, the escape latency time and swimming distance have a downward trend, through the platform number and the target quadrant residence time has increasing trend, but no statistical difference (P>0.05). The Donepezil HCL group, the pulse electric acupuncture group and music electric acupuncture group had a significantly improve the ability of learning and memory, this is reflected in escape latent period and reduce the swimming distance (P<0.05), the cross-platform and target quadrant time frequency increases (P<0.05, P<0.05, P<0.01).(2) HE Staining results:In model group, the disordered arrangement of cells, layer number of cells significantly reduced, cell nucleolus is not clear and the phenomenon of nuclear pyknosis in hippocampus CA1area. In non-acupuncture group and music group, cells arranged disorder, loss of neurons, the cell layers reduced in hippocampal CA1region. The Donepezil HCL group, the pulse electric acupuncture group and the music electric acupuncture group, arrangement of cells rules and clear nucleoli assumes the circular or elliptic, layer number of cells increased significantly.(3) Nissl’s staining results:Compared with normal group, the model group, austenite shallow dye, the neuron cell body form is irregular, number of neuron were decreased significantly in hippocampal CA1area. Compared with SAMP8model group, number of hippocampus CA1area neurons and the pathological morphological changes are similarity in the non-acupuncture and music group. In Donepezil HCL group, the pulse electric acupuncture group and music electric acupuncture group, it could improve neurons decrease and loss phenomenon to protect the neurons in hippocampus CAl area(4) ELISA Results:Compared with normal group, A beta40and42A beta protein in serum expression were significantly increased in model group (P<0.01). Compared with model group, the serum in non-acupuncture group is similar to model group, there was no significant difference (P>0.05). A beta protein expression in serum showed A trend of decrease in music group, but there was still no significant difference (P>0.05). Donepezil HCL, pulse electric acupuncture and music electric acupuncture group, the A beta40is reduced in serum, which has a statistically significant (P<0.05), and the decreasing A beta42protein level had significant difference (P<0.05, P<0.05, P<0.01). Compared with the pulse electric acupuncture group, the A beta42protein levels in serum dropped significantly in music electric acupuncture group, which has statistically significant (P<0.05).(5) Different electric acupuncture intervention SAMP8mice hippocampus’PI3K/AKT signal transduction pathway:1) The immune histochemical method (HTC):the content of total AKT and GSK-3beta protein changed little within these seven groups, there were no significant difference (P>0.05). Compared with the normal mice, the express of P-AKT (ser473), P-GSK-3beta (ser9) protein were reduced rapidly in SAMP8(P<0.01). Compared with model of SAMP8mice, the express of P-AKT (ser473), P-GSK-3beta (ser9) protein expression there was no significant difference in the non-acupuncture and music group (P>0.05). In Donepezil HCL, pulse electric acupuncture and music electric acupuncture group, the express of P-AKT (ser473), P-GSK-3beta (ser9) protein increased (P<0.05);2) The Western Bolt results were in accordance with the immunohistochemical’s;3) Real-time PCR results:Compared with normal group, the expression AKT mRNAand GSK-3beta mRNA was significantly lower in model group (P<0.01); The non-acupuncture had no significant difference with model group (P>0.05); The expression of these genes has a rising trend in music group, but also has no statistical difference (P>0.05); In Donepezil HCL, the pulse electric acupuncture and music electric acupuncture group, there had a certain degree of improvement expression in mice hippocampal’s AKT mRNA and GSK-3beta mRNA, which had a statistically significant (P<0.05, P<0.05, P<0.01).(6) The different electric acupuncture influence of tau protein in hippocampus region:1) The immune histochemical method (HTC):the content of total tau protein changed little within these seven groups, there were no significant difference (P>0.05). Compared with the normal mice, the express of P-tau (ser199/202) protein were increased rapidly in SAMP8(P<0.01). Compared with model group, the express of P-tau (serl99/202) protein expression there was no significant difference in the non-acupuncture and music group (P>0.05). In Donepezil HCL, pulse electric acupuncture and music electric acupuncture group, the express of P-tau (ser199/202) protein decreased (P<0.05);2) The Western Bolt and Real-time PCR results were in accordance with the immuno histochemical’s.(7) The different electric acupuncture influence of GFAP protein in hippocampus region:1) Compared with the normal mice, the express of GFAP protein were increased rapidly in SAMP8(P<0.01). Compared with model group, the express of GFAP protein expression there was no significant difference in the non-acupuncture and music group (P>0.05). In Donepezil HCL, pulse electric acupuncture and music electric acupuncture group, the express of GFAP protein decreased rapidly (P<0.05, P<0.05, P<0.01).2) The Western Bolt and Real-time PCR results were in accordance with the immunohistochemical’s.5. Conclusion(1) The SAMP8mice had a obvious cognitive impairment of learning and memory, the pathological form of tau protein and A beta protein increased significantly, accompanied by neurons damaged and reduced, which is one of the ideal model of AD.(2) Electric acupuncture Have a good adjustment function for neuron protection, including pulse electric acupuncture and music electric acupuncture can increase the number of neurons in hippocampal CA1area, improving SAMP8mice on neuronal damage, and the music electric acupuncture is superior to the pulse.(3) Electric acupuncture could obviously improve cognitive impairment of learning and memory in the SAMP8rapid aging mice. In terms of improving cognitive impairment, the music electric acupuncture group is superior to the pulse; But the music electric acupuncture on learning memory keep better than the pulse.(4) One of the curative mechanism of action of dementia may be through the PI3K/AKT signal transduction pathway, increasing the activity of AKT, promoting the phosphorylation of GSK-3beta (ser9), thus to restrain the activity of GSK-3beta.-and thus A beta protein in deposition and abnormal tau protein phosphorylation, neurotoxicity, reduced finally protect the neurons. The music electric acupuncture in regulating the PI3K/AKT signal transduction pathways is superior to the pulse.(5) Improving the aspect of hippocampal astrocytes GFAP in SAMP8mice, the music electric acupuncture can significantly reduce its expression, inhibiting the release of inflammatory cytokines, relieving neuron damage, which is obviously better than plus. This may be one of the mechanisms of music electric acupuncture treating Alzheimer’s disease. |
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