The Effect Of "Tongdu Qishen" Electrotherapy On Microglia And TLR4 Pathway In The Hippocampus Of AD Mice

1ObjectiveAlzheimer’s disease(AD)is the most common neurodegenerative disease,posing a huge threat to human health in today’s aging society and placing a heavy economic burden on society.AD is mainly caused by amyloid β(Aβ)deposition,abnormal tau phosphorylation and intracerebral neuroimmunological inflammatory reactions.A large number of studies have shown that under the guidance of traditional Chinese medicine theory,electroacupuncture therapy has shown certain advantages in improving the clinical symptoms of Alzheimer’s disease patients and AD model animals in experimental research.This study aims to explore the effects of different electroacupuncture type(pulse electroacupuncture and musical electroacupuncture),under the guidance of “dredging DU meridian and awake mind”,on 7-month-old APPswe/PS1δE9 transgenic AD mice,and to explore the possible mechanism of its function from the perspective of microglia activation.2MethodsSixty mice of APPswe/PS1δE9 were randomly divided into six groups: AD model group(M group),Donepezil group(D group),pulse electroacupuncture group(DZ group),pulse electroacupuncture combined with donepezil group(DZ+D group),musical electroacupuncture group(YZ group)and musical electroacupuncture combined with donepezil group(YZ+D group),with each group containing nine mice.Ten male C57BL6 mice with the same genetic background at 7 months of age were used as normal control group(N group).Mice in the D group were given 0.92 mg/kg of donepezil hydrochloride dissolved in distilled water by intragastric administration.The D group received this treatment once per day for 15 consecutive days,while the N and M groups received no treatments.All of the mice in the M,N and D groups were immobilized in bags for 20 min to ensure the same treatment conditions as the other groups.Each mouse in the DZ group was immobilized in a bag during electroacupuncture(EA)treatment.Three acupoints were selected based on our previous findings: Yintang(GV 29),Baihui(GV 20)and Shuigou(GV 26).The GV 29 and GV 20 acupoints were transversely punctured to a depth of 2-3 mm using 0.25 mm×13 mm disposable sterile acupuncture needles.Following the insertions,the needle handles were connected to electrodes on an EA apparatus,with a density wave set to 2/50 Hz and 0.1 mA,for 20 min.Following the EA treatments at GV 20 and GV 29,a fast insertion into GV 26 was performed.In the DZ+D group,the intervention was combined EA and donepezil treatment.In the YZ group,the acupoints and acupuncture methods were the same as those in the DZ group.The musical electroacupuncture was connected to the mice at the GV 20 and GV 29,and the “Dementia” music therapy prescription was selected.In YZ+D group,the intervention was combined musical electroacupuncture and donepezil treatment.The above interventions were administered 20 min once a day for 15 consecutive days.After 15 days of intervention,each group of mice was placed in the Morris water maze for 1 day of visable platform test,followed by a 5-day hidden platform test and a probe trial test conducted within 24 hours after the end of hidden platform test.The structure and pathological changes of dentate gyrus in hippocampus of each group were observed by HE staining.The amount of microglia activation was observed by immunohistochemical labeling of hippocampal microglial surface receptor Iba1.The immunofluorescence method was used to observe the coexpression of activated microglia and Toll-like receptor 4(TLR4).The protein and mRNA expression levels of TLR4,MyD88,IRAK4,TRAF6,TAK1,NF-κB,iNOS and CD40 were analyzed by immunohistochemistry,Western blot(WB)and RT-PCR.3Research results 3.1Morris water maze test results:Visible platform test: There was no significant difference in escape latency and swimming speed between the groups(p>0.05).Hidden platform test: Different intervention methods changed the escape latency of each group with time(p<0.05).On the 2nd-5th day,the escape latency of M group was higher than that of N group(p(day 1-5)< 0.05),compared with the M group,the escape latency of the D group,DZ group,DZ+D group,YZ group and YZ+D group was shortened(both P<0.05),and the difference was statistically significant.Probe trial test: There was a significant difference between platform crossover numbers and the percentage of time spent in the target quadrant(WS)in each group(both P<0.05).The platform crossover numbers and the percentage of time spent in the target quadrant(WS)in M group was significantly lower than that in N group(P <0.01).Compared with M group,the crossover numbers in D group,DZ group,DZ+D group,YZ group and YZ+D group(both P <0.05)and the percentage of time spent in the target quadrant(WS)increased(P <0.01),which was statistically significant.Compared with D group,the time spent of target quadrant stagnation of YZ+D group was increased,and the difference was statistically significant(p<0.05).3.2The morphology of dentate gyrus and the number of activated microglia in hippocampus of each groupAfter hematoxylin-eosin(HE)staining,the neuronal structure of N group was densely arranged,the shape of neurons was normal,the nucleolus was clear,the nucleolus was visible,the cell cluster boundary was clear,and the cytoplasm was reddish.The histopathological morphology of group M was significantly different from that of group N.The structure of hippocampus was loose,the arrangement of cells was disordered,the nucleus of neurons was pyknosis,nuclear translocation,and the boundaries between cytoplasm and nucleus were not obvious.The cellular space between neurons and glial cells is enlarged,and extensive vacuole changes occur.After treatment,the pathological changes of D group,DZ group,YZ group,DZ+D group and YZ+D group were all improved.Immunohistochemically labeled microglia surface receptor(Iba1)results showed that the numbers of Iba1-labeled microglia in the hippocampi of normal mice were low,and the observed microglia appeared to be in a resting state,with small cell bodies,light staining,and long,slender processes.In the M group,the microglia appeared to be in the typical activation state,with enlarged cell bodies,shortened cell processes and round or amoebic cellular morphology.The numbers of Iba1-positive cells were also greater in the M group than in the N group(p < 0.01),and Iba1-positive cells appeared to cluster around senile plaques.In comparison,the microglia in the D group,DZ group,YZ group,DZ+D group and YZ+D group tended to be at rest.The numbers of Iba1-positive cells in these five groups were fewer than in the M group(both p<0.01).In the DZ group(p<0.05),YZ group(p<0.01),DZ+D group(p<0.05)and YZ+D group(p<0.01),Iba1-positive cells were fewer in number than in the D group.3.3Expression levels of TLR4 pathway-associated proteins and genes in each group of miceThe results of immunofluorescence expression of CD11 b and TLR4 in each group were observed by laser confocal microscopy.Compared with N group,M group had strong CD11 b and TLR4 fluorescence expression.The co-expression of CD11 b and TLR4 fluorescence was attenuated in the D group,DZ group,DZ+D group,YZ group and YZ+D group.The results of immunohistochemistry showed that the TLR4,MyD88,IRAK4,TRAF6,TAK1,NF-κB,iNOS and CD40 positive cells in the hippocampus of the 7-month-old APPswe/PS1δE9 transgenic mice were dark brown compared with the N group.More,the coloration was deeper,and the integrated optical density values of the M groups were significantly higher than those of the N group(p<0.01);after 15 days of intervention,compared with the M group,the integral optical density values of the positive expression of each protein in the D group,the DZ group,the DZ+D group,the YZ and the YZ+D group were decreased to different degrees(p<0.01 or p<0.05).Among them,the integral optical density values of TLR4 and CD40 protein were positively expressed in the YZ+D group decreased significantly(p < 0.05),compared with the D group.The results of WB and RT-PCR showed that the relative expressions of TLR4,MyD88,IRAK4,TRAF6,TAK1,NF-κB,iNOS and CD40 in the hippocampus of group M were significantly higher than those in group N(p<0.01 or p<0.05).Compared with M group,the relative expression levels of protein and mRNA in D group,DZ group,DZ+D group,YZ group and YZ+D group were decreased to different degrees(p<0.01 or p<0.05).Compared with D group,the relative expression levels of protein and mRNA in DZ group,DZ+D group,YZ group and YZ+D group were also decreased to different degrees(p<0.01 or p<0.05).Among them,the decline in YZ+D group was the most significant.4Conclusion(1)The intervention measures based on "dredging DU meridian and awake mind " method improved the spatial learning ability and memory retention ability of 7-month-old APPswe/PS1δE9 mice,and the effect was better than that of donepezil hydrochloride alone: music electro-acupuncture combined with donepezil was the most significant;(2)The number of neurons in hippocampus of 7-month-old APPswe/PS1δE9 mice decreased and activated microglia increased.The interventions based on Tongdu Qishen method could reduce the number of activated microglia in hippocampus,improve the morphology of neurons in hippocampus and reduce the apoptosis of neurons,which may be the mechanism of improving learning and cognitive impairment in AD model mice.(3)Interventions based on "dredging DU meridian and awake mind " method can reduce the co-expression of microglia and its TLR4 pattern recognition receptor,and can also significantly regulate TLR4 and downstream proteins MyD88,IRAK4,TAK1,TRAF6 and NF-κ B,oxidative stress product iNOS and costimulatory molecule CD40.It may be that "dredging DU meridian and awake mind " method can inhibit microglia activation and slow down microglia activation.Neuronal apoptosis may play a role in improving learning and memory ability of AD mice.(4)Interventions based on the method of "dredging DU meridian and awake mind " can reduce the gene expression of TLR4 and downstream proteins MyD88,IRAK4,TAK1,TRAF6 and NF-κ B,oxidative stress product iNOS and costimulatory molecule CD40,and explain the possible mechanism of TLR4 in improving AD-like symptoms from the perspective of genetics.(5)In improving the learning and memory ability of 7-month-old APPswe/PS1δE9 mice,protecting neurons in hippocampus,reducing the number of activated microglia,and reducing the expression of TLR4 pathway-related proteins and inflammatory factors,the effect of different electroacupuncture combined with positive drug donepezil was better than that of single drug,pulse electroacupuncture and musical electroacupuncture.