Acupuncture Improves Cognitive Function Of APP/PS1 Mice By Regulating Mitophagy

ObjectiveThe incidence rate of Alzheimer’s disease(AD)is increasing year by year,and it is accompanied by a high disability rate and mortality rate.However,the clinical effects of the currently marketed drugs and the drugs under research are not ideal,so it is urgent to explore effective treatments.The damage of synaptic function caused by abnormal accumulation and deposition of pathological proteins such as amyloid is the core problem of AD.Mitophagy is the key factor to regulate synaptic plasticity and maintain synaptic function homeostasis.Mitophagy is one of the key targets in the research and development of AD prevention and control measures.Jin’s three-needles therapy is an acupuncture treatment system summarized by Professor Jin Rui in combination with the clinical laws of Lingnan famous acupuncture and moxibustion experts in the past dynasties.As an important group of acupoints,the mindregulating acupuncture has the characteristics of good clinical efficacy,less side effects,and high compliance in improving memory function.However,the mechanism of its clinical effect is still unclear.This study aims to verify whether the mind-regulating acupuncture can improve the cognitive function and synaptic plasticity of APP/PS1 mice by regulating mitophagy.MethodsExperiment 1:Retrieve the national knowledge infrastructure data(CNKI),the Chinese academic journal database(CSPD),the Chinese scientific and technological journal database(CCD),the Chinese biomedical literature database(CBM disc),EMbase,PubMed,Web of Science and Cochrane Library databases to obtain relevant research,and retrieve the randomized controlled trial of acupuncture treatment of Alzheimer’s disease.Cochrane risk bias assessment tool was used to evaluate the methodological quality of the included literature.The quality evaluation and data extraction of the inclusion study were conducted by two researchers independently and strictly in accordance with the inclusion and exclusion criteria.Meta analysis was conducted using Stata 16.0 software,and sequential analysis(TSA)was conducted using TSA 0.9.5.10 Beta software.Experiment 2:C57 mice were selected as the normal control group,and APP/PS1 mice were randomly divided into model group,positive drug group,and acupuncture intervention group.(1)The effects of acupuncture on APP/PS1 cognitive function were observed by water maze and dark avoidance behavioral experiments.(2)The presenilin protein PSEN was detected by Western blot,β-Secretory enzyme BACE1,amyloid precursor protein APP andβ Amyloid A β 1-42 protein level.(3)Immunofluorescence was used.(4)ImmunohistochemistryExperiment 3:C57 mice were selected as the normal control group,and APP/PS1 mice were randomly divided into model group and acupuncture intervention group.The difference of metabolites in the hippocampus of mice in each group was studied by ultra-high throughput liquid chromatography-mass spectrometry(LC-MS),combined with multivariate statistical analysis,univariate statistical analysis,cluster analysis and other methods.Experiment 4:With the addition of mitophagy activator Ruxolitinib and mitophagy inhibitor U0126,APP/PS1 mice were randomly divided into model group,acupuncture intervention group,activator group,inhibitor+acupuncture intervention group.(1)The effects of mind-acupuncture,mitophagy activator and mitochondrial autophagy inhibitor on the cognitive function of APP/PS1 were observed by water maze and dark avoidance behavioral experiments.(2)Western blot was used to detect ubiquitin protein USP30,PTENinduced putative kinase 1PINK-1,Parkin,mitochondrial outer membrane protein HK2,TOMM40,TOMM20,autophagy substrate protein P62,autophagy structural protein LC3Ⅱ/Ⅰ,autophagy key regulatory protein complex Beclin-1,autophagy receptor protein NDP52,lysosomal transport protein RAB7,lysosomal membrane protein Lamp1 β Amyloid A β 142 protein,synaptophysin Syn,synaptic dense protein PSD95,PSD93 protein expression.(3)The ubiquitin protein USP30,PTEN-induced putative kinase 1PINK-1,Parkin,mitochondrial outer membrane protein HK2,TOMM40,TOMM20,autophagy substrate protein P62,autophagy structural protein LC3Ⅱ/Ⅰ,autophagy key regulatory protein complex Beclin-1,autophagy receptor protein NDP52,lysosome transport protein RAB7,lysosome membrane protein Lampl β Amyloid A β MRNA content of 1-42 protein,synaptophysin Syn,synaptic dense protein PSD95 and PSD93.(4)The autophage,mitochondrial structure and synaptic structure were observed by transmission electron microscopy.(5)Detection of APP and APP of amyloid precursor protein by ELISA β Amyloid A β 1-42 content.(6)The co-expression of autophagic structural protein LC3Ⅱ/Ⅰ and lysosomal transporter RAB7 was detected by immunofluorescence method,and the co-expression of synaptic dense protein PSD95 and PTEN-induced putative kinase 1PINK-1 was detected.(7)ImmunohistochemistryResults1.Acupuncture can effectively improve the cognitive function of patients with Alzheimer’s diseaseFinally,32 articles were included,with a total of 2221 patients.The results of metaanalysis showed that acupuncture could improve the MMSE score of the treatment group compared with the control group,and there was a statistically significant difference in clinical efficacy between the two groups[WMD=2.90,95%CI(2.15-3.64),P<0.01];To improve MoCA score,there was statistically significant difference in clinical efficacy between the two groups[WMD=4.27,95%CI(2.98-5.55),P<0.01];There was statistically significant difference in clinical efficacy between the two groups by reducing ADAS-log score[WMD=4.37,95%CI(-6.77-1.97),P<0.01];To improve Barthel index,there was statistically significant difference in clinical efficacy between the two groups[WMD=8.36,95%CI(4.67-12.04),P<0.01].To improve HDS-R score,there was statistically significant difference in clinical efficacy between the two groups[WMD=1.34,95%CI(0.67-2.00),P<0.01];There was statistically significant difference in clinical efficacy between the two groups in reducing NPI score[WMD=-3.11,95%CI(-3.87-2.35),P<0.01];TSA shows that the study on the effectiveness of acupuncture treatment for Alzheimer’s disease has crossed both the traditional and TSA boundaries.2.The changes of differential metabolites in the hippocampus of APP/PS1 mice caused by acupuncture are mainly related to mitophagy.Through the analysis of differential metabolites and differential metabolic pathways,it was found that the differential metabolites in hippocampus caused by the improvement of APP/PS1 mice by acupuncture were mainly related to the metabolic products of glutamate cycle and sphingolipids.The function of the differential metabolism substances in the hippocampus caused by the improvement of APP/PS1 mice by the method of regulating the nerves is mainly related to the sphingomyelin signal pathway,D-amino acid metabolism pathway and mitochondrial autophagy related pathway.3.Acupuncture can improve the cognitive dysfunction of APP/PS1 mice by activating mitophagyThe results of the water maze experiment showed that from the third day to the fifth day of the training period,compared with the APP/PS1 mice in the model group,the latency of the APP/PS1 mice treated with the method of acupuncture and the APP/PS1 mice treated with the mitohagy activator Ruxolitinib was shortened.However,the latency of the APP/PS1 mice treated with both U0126 and acupuncture intervention was not significantly different from that of the APP/PS1 mice in the model group.After 5 days of training,compared with the APP/PS1 mice in the model group,the APP/PS1 mice that received the intervention of acupuncture method and the intervention of the mitochondrial autophagy activator Ruxolitinib significantly increased the time of staying in the target quadrant and the times of crossing the platform.Compared with APP/PS1 mice in the model group,APP/PS1 mice receiving both U0126 intervention and mind-regulating acupuncture intervention had no significant difference in target quadrant residence time and times of crossing the platform.The results of dark avoidance experiment showed that compared with APP/PS1 mice in the model group,APP/PS1 mice after receiving the intervention of acupuncture and the intervention of mitochondrial autophagy activator Ruxolitinib had significantly reduced the number of times of passing through the dark room and significantly increased the dark avoidance latency.Compared with APP/PS1 mice in the model group,APP/PS1 mice who received the intervention of both the acupuncture method and the intervention of the mitochondrial autophagy inhibitor U0126 had no statistical difference in the number of times of passing through the dark room and the dark avoidance latency.It is suggested that the results of acupuncture intervention are similar to those of mitophagy activator intervention,which can improve the learning and memory ability of APP/PS1 mice,while mitophagy inhibitor inhibits the improvement of acupuncture on the learning and memory ability of APP/PS1 mice,suggesting that acupuncture improves the learning and memory ability of APP/PS1 mice by activating mitophagy.4.Acupuncture promotes the degradation of A β in hippocampal of APP/PS1 mice by activating mitophagy,and interferes with APP processing.(1)Acupuncture promotes the degradation of A β in hippocampal of APP/PS 1 mice by activating mitophagyCompared with C57 mice in the control group,APP and Aβ1-42 in hippocampus of APP/PS1 mice was significantly increased.Compared with APP/PS 1 mice,acupuncture group and mitophagy activator group significantly decreased APP and Aβ1-42 expression,but after receiving acupuncture and U0126 intervention at the same time,There was no significant difference between the expression of APP,Aβ1-42 in hippocampus of APP/PS 1 mice.(2)Acupuncture can interfere with APP processing in hippocampus of APP/PS 1 mice and limit synthesis rate of A β.Compared with C57 mice,The protein expression of γ-secreted enzyme constituent proteins PSEN1 and PSEN2 was significantly increased in hippocampus of APP/PS 1 mice.Compared with APP/PS1 mice,acupuncture group significantly reduced the protein expression of PSEN1 and PSEN2.Compared with C57 mice,protein expression of βsecretory enzyme BACE1 and amyloid precursor protein APP was significantly increased in hippocampus of APP/PS1 mice.Compared with APP/PS1 mice,acupuncture group significantly reduced the protein expression of BACE1 and APP.It is suggested that acupuncture can reduce expression of y-Secretase and β-secretase in hippocampus of APP/PS 1 mice,interferes with the processing of APP and limits synthesis rate of A β.5.Acupuncture can improve the cognitive function and synaptic plasticity of APP/PS 1 mice by activating mitophagy in hippocampus of APP/PS 1 mice.(1)Acupuncture Activates ubiquitination of mitochondrial outer membrane protein mediated by PINK-1/Parkin pathway.Compared with C57 mice,ubiquitin protein USP30 in hippocampus of APP/PS 1 mice increased significantly,and the protein expression of PINK-1 and Parkin decreased significantly.Compared with APP/PS 1 mice,acupuncture group and the mitophagy activator Ruxolitinib group significantly decreased the protein expression of USP30,and increased the protein expression of PINK-1 and Parkin.However,the contents of USP30,PINK-1 and Parkin were not significantly different from those of APP/PS 1 mice after receiving acupuncture and U0126 at the same time.Compared with C57 mice,protein expression of mitochondrial outer membrane proteins HK2,TOMM40 and TOMM20 in the hippocampus of APP/PS1 mice was significantly increased;Compared with APP/PS1 mice,protein expression of HK2,TOMM40 and TOMM20 were significantly decreased in acupuncture group and the mitophagy activator Ruxolitinib group.After receiving acupuncture and U0126 simultaneously,the contents of HK2,TOMM40 and TOMM20 were not significantly different from those of APP/PS1 mice.(2)Acupuncture activates mitochondrial autophagy by promoting the fusion of autophagy and lysosomeCompared with C57 mice,expression of autophagy substrate protein P62 and autophagy structural protein LC3Ⅱ/Ⅰ were significantly increased in the hippocampus APP/PS 1 mice,key regulatory protein complex of autophagy Beclin-1 was significantly decreased,autophagy receptor protein NDP52 and lysosomal transport protein RAB7 were significantly decreased,expression of lysosomal associated membrane protein LAMP1 were significantly increased.Compared with APP/PS1 mice,the expression of P62,LC3Ⅱ/Ⅰ,LAMP1 and the expression of Beclin-1,NDP52 and RAB7 were significantly decreased in acupuncture group and mitophagy activator Ruxolitinib group.However,after receiving acupuncture and U0126 intervention at the same time,the protein expression of P62,LC3Ⅱ/Ⅰ,Beclin-1,NDP52,RAB7,LAMP1 had no significant difference with APP/PS1 mice.The results of immunofluorescence co-localization showed that the co-expression of LC3Ⅱ/Ⅰ and RAB7 in the hippocampus of APP/PS 1 mice decreased compared with C57 mice,while the coexpression of LC3Ⅱ/Ⅰ and RAB7 was significantly increased in acupuncture group and the activator group.After receiving acupuncture and U0126 simultaneously,there was no significant difference between the co-expression of LC3Ⅱ/Ⅰ and RAB7 and APP/PS 1 mice.(3)Acupuncture promotes the synaptic plasticity of the hippocampus of APP/PS 1 mice by activating mitophagyCompared with C57 mice,expression of Syn,PSD95 and PSD93 in APP/PS 1 mice were significantly decreased.Compared with APP/PS 1 mice,expression of Syn,PSD95 and PSD93 were significantly increased in acupuncture group and the mitophagy activator Ruxolitinib group.After receiving acupuncture and U0126 simultaneously,the expression of Syn,PSD95 and PSD93 was not significantly different from APP/PS 1 mice.The results of immunofluorescence co-localization showed that compared with C57 mice,the co-expression of PSD95 and PINK-1 in the hippocampus of APP/PS 1 mice was decreased,while the co-expression of PSD95 and PINK-1 was significantly increased in acupuncture group and the mitophagy activator Ruxolitinib group.After receiving acupuncture and U0126 simultaneously,there was no significant difference in the coexpression of PSD95 and PINK-1 in hippocampus of APP/PS 1 mice.ConclusionAcupuncture can significantly improve the cognitive function of APP/PS 1 mice and improve the learning and memory ability.At the molecular level,acupuncture can activate mitophagy by activating the ubiquitination of mitochondrial outer membrane protein mediated by PINK-1/Parkin and fusion of autophagy and lysosome in hippocampus of APP/PS1 mice,and reduce the level of Aβ in hippocampus of APP/PS1 mice and improve the cognitive function and synaptic plasticity of APP/PS1 mice.