The Effect Of “Tongdu Qishen” Fashao On Brain Glucose Metabolism And Activation Of Microglia In Hippocampus Of APPswe/PS1dE9 Mice

1.ObjectiveThis research aimed to observe the therapeutic effects and differences on 7-month-old APPswe/PS1dE9 mice induced by electro-acupunctures(electro-acupuncture and musical electro-acupuncture),positive control drug and drug combined with electro-acupuncture treatments.We applied Morris water maze,Micro-PET,HE staining,immunohistochemistry and Western blot to examine the perspectives of animal behavior,brain glucose metabolism,as well as key proteins and inflammatory cytokines,which related to microglia activation on Alzheimer's disease(AD)mice.Exploring the potential mechanism of "dredging Governing Vessel and awaking mind" method for treating AD,and interpret its possible efficacy on rejuvenating the brain,resolving the puzzle and beneficial to intelligence.2.MethodsSixty seven-month-old,male,APPswe/PS1dE9 mice were randomly divided into AD Model group(AD),medicine group(M),electro-acupuncture group(EA),EA combined medicine group(EA+M),musical electro-acupuncture group(MEA)and medicine combined with MEA group(MEA+M),n = 10 in each group.Ten seven-month-old,male,C57BL/6 mice were used for Normal control group(N).Mice in N and AD groups didn't receive any treatment but grabbing for restriction for 20min,while mice in other groups receive treatments.Mice in M group received donepezil intervention by gavage with dose at 0.92 mg/kg,as well as grabbing for restriction for 20min.EA(HANS-202 electro-acupuncture device,with 2Hz of the frequency,2V of the voltage and 0.1mA of the current intensity on sparse wave)was applied at Baihui(GV20)and Yintang(GV29)acupoints for 20 min,as well as manually pricked at Shuigou(GV26)acupoint before the treatment.In the MEA group,mice were administered a same acupoints prescription as the EA group,while the needle handles(GV20 and GV29)were connected on ZJ-12H musical electro-acupuncture device(with music prescription "curing dementia prescription" in a clear rhythm,moderated speed and intensity)and conducted for 20 min,as well as manually pricked at Shuigou(GV26)acupoint before the MEA treatment.Mice in EA+M and MEA+M groups received medicine combined with EA and MEA treatment,respectively.All treatments administrated once a day for 15 consecutive days.After the 15 days treatment,Morris water maze(MWM)was performed to observe the behavioral changes in spatial learning and memorize ability in mice;then hippocampal,frontal lobe,temporal lobe and parietal lobe glucose metabolism levels were tested by Micro-PET imaging;HE staining was used to observe the changes in hippocampal neuron morphology characteristics;immunohistochemistry and Western blot were used to observe and analyze the key proteins TREM2?DAP12?Syk?PI3K?Akt and PLC-? in microglia activation pathway,as well as inflammatory cytokines TNF-??IL-1??IL-6?IL-12 produced by microglia activation in the hippocampus.3.Results3.1 Results from the test of gaining concealed platform:the escape latency of mice in each group showed varying extends of declining with the increase of training time.Compared with N group,from the second day to fifth day,other groups showed a longer latency(p<0.05,p<0.01,p<0.01,p<0.01).On the third day of the training,EA,EA+M and MEA+M groups showed a shorter latency compared with AD group(p<0.05).On the fourth and fifth days of the training,M,EA,EA+M and MEA+M groups showed a significant shorter latency compared to AD group(p<0.01).Results of spatial probe trial:compared with N group,other groups showed a significant lower ratio of swimming distance in the target quadrant.M,EA,EA+M and MEA+M groups showed a significant higher ratio of swimming distance in the target quadrant compared to AD group(p<0.01).In addition,mice in MEA+M group performed a better spatial memory ability than mice in other treatment groups.3.2 Micro-PET imaging showed that mice in AD group showed a lower glucose metabolic rate in hippocampal,frontal lobe,temporal lobe and parietal lobe compared with N group(p<0.01).Compare with N group,M group showed a significant lower injected dose of 18F-FDG rate in hippocampal,frontal lobe,temporal lobe and parietal lobe(p<0.01,p<0.05,p<0.01,p<0.05),EA group showed a significant lower injected dose rate in frontal lobe and temporal lobe(p<0.05).EA group presented a significant higher injected dose of 18F-FDG rate in hippocampal,frontal lobe,temporal lobe and parietal lobe compared to AD group(p<0.01,p<0.05,p<0.01,p<0.01).3.3 Results from HE staining:N group:hippocampal neuron cell layer of granular cells arranged closely and in order,cell's structure was round and pale-stained nuclei,the molecular layer and pleomorphic cell layer were also arranged neatly.AD group:cells arranged scattered and irregularly,neuronal cells exhibited large or small condensed,fragmented and dark nuclei combined with apoptotic cell bodies.3.4 The results from immunohistochemical and Western blot tested in mice hippocampal indicated that:M,EA,EA+M,MEA,MEA+M treatment could increase the relative expression levels of microglia activation pathway key proteins TREM2,DAP 12,Syk,PI3K,Akt and PLC-y,as well as decrease the relative expression levels of inflammatory cytokines TNF-?,IL-1?,IL-6,IL-12 produced by microglia activation in the hippocampus.Compared with N group,AD group showed a significant lower protein relative expression level of DAP12 and PLC-y(p<0.01,p<0.05).AD,M,EA,EA+M,MEA and MEA+M groups presented a lower expression level of Syk(p<0.01,p<0.01,p<0.05,p<0.05,p<0.05,p<0.05)compared to N group.Compared with N group,AD group showed a significant higher protein relative expression level of TNF-a and IL-12(p<0.01),while AD,MEA,MEA+M groups presented a significant higher level of IL-1?(p<0.01,p<0.05,p<0.01).The protein level of IL-1? and IL-12 decreased significantly in M,EA and EA+M groups compared to AD group(p<0.01,p<0.05,p<0.01 for IL-1? and p<0.05,p<0.05,p<0.01 for IL-12).Compared with N group,AD,EA,EA+M and MEA+M groups presented a significant higher level of IL-6(p<0.01,p<0.05,p<0.05,p<0.05),while M,EA,EA+M and MEA decreased significantly compared to AD group(p<0.01,p<0.05,p<0.05,p<0.01).4.Conclusion4.1 The electro-acupuncture and musical electro-acupuncture treatment based on "dredging Governing Vessel and awaking mind" method,positive drug(donepezil),as well as drug combined with electro-acupuncture interference can effectively improve the spatial learning and memory ability of AD model mice,while donepezil combined with musical electro-acupuncture presented the most prominent therapeutic effect.4.2 Seven-month-old APPswe/PS1dE9 mice showed a lower glucose metabolic rate in hippocampal,frontal lobe,temporal lobe and parietal lobe.After 15 days' interference,Donepezil treatment can not improve the glucose metabolic in AD model mice,while the electro-acupuncture treatment based on "dredging Governing Vessel and awaking mind"method could significant increase the glucose metabolic rate in hippocampal,frontal lobe,temporal lobe and parietal lobe.4.3 The electro-acupuncture and musical electro-acupuncture treatment based on "dredging Governing Vessel and awaking mind" method,positive drug(donepezil),as well as drug combined with electro-acupuncture interference can effectively inhibit neuron apoptosis in hippocampus of AD model mice.To a certain extent,increase the relative expression levels of microglia activation pathway key proteins TREM2,DAP 12,Syk,PI3K,Akt and PLC-?,as well as decrease the relative expression levels of inflammatory cytokines TNF-a,IL-1?,IL-6,IL-12 produced by microglia activation.