| Objective In recent years,study on the prevention of Alzheimer's disease(AD)has gradually become an international and mainstream trend.Mounting evidence indicate that there is high correlation between aging,epigenetic modification and the early occurrence of AD pathology.And dysregulated GSK3?/m TOR signaling pathway is involved in the formation and autophagy disorder of the neurofibrillary tangles(NFTs)in dorsal raphe nucleus(DRN),which is considered as the starting brain area of NFTs pathology.Based on this,in this study we investigated whether preventive acupuncture could inhibit the NFTs deposition in DRN via targeting epigenetic modification on GSK3?/m TOR signaling pathway,and attenuate cognitive deficits in D-galactose-induced rats with AD-like pathology.We hope our findings can provide scientific evidence for prompting the application of preventive treatment by acupuncture in the prevention and treatment of AD.Methods A total of 72 3-month-old Sprague Dawley male rats were randomly divided into 6 groups,namely the normal group(N),model group(M),preventive electroacupuncture(EA)+ inhibitor group(PEA+I),inhibitor group(I),preventive acupuncture group(PA)and(PEA)with 12 rats in each group.And all rats had free access to food and water.Rats in each group except for the N group were injected intraperitoneally with Dgalactose(120mg/kg/ day)continuously for 7 weeks.And on the beginning day of D-galactose injection,rats in PEA+I,PA and PEA were administrated with acupuncture intervention at the points Shenshu and Baihui.At Baihui,the needle was inserted 15° obliquely to a depth of 3–5-mm.At Shenshu,the needles were inserted perpendicularly to a 3–5-mm depth.EA was delivered using an EA apparatus(HANS-100A).Stimulation in a continuous wave at 50 Hz in EA group for 20 minutes each day for consecutive 7 weeks was applied to the pair of acupuncture points(Baihui and alternating unilateral Shenshu).The intensity(1 m A)was adjusted to induce light muscle contractions.Besides,rats in PEA+I and I group were injected intraperitoneally with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine(0.4mg/kg/day)on the 7th week,rats in N and M group were wrapped in the same way but received no other interventions.Open-field test and Morris water maze test were performed after the interventions.After the behavior tests the fresh hippocampus and DRN tissues as well as perfusion brain sections were prepared.Transmission electron microscopy(TEM)was adopted to observe the neuronal microtubules,the width of synaptic clefts and the postsynaptic density changes in hippocampal CA1 area.Golgi staining was performed to observe the dendritic spine density change in hippocampal CA1 area.Western-Blot(WB)assay was performed to detect GSK3? ? GSK3?-pTyr216?GSK3?-p Ser9?m TOR?m TOR-p S2448?LC3I/LC3II?Tau-5?Taup S262?PHF-1?DNMT1 levels in DRN and Tau-5?Tau-p S262?PHF-1 levels in hippocampus.Immunohistochemistry(IHC)was performed to detect the PHF-1 expression and location in DRN area.Immunofluorescence(IF)was performed to detect the expression and location of DNMT1?GSK3?in DRN area.Real-time quantitative PCR(RT-PCR)was performed to detect GSK3?m RNA level.Bisulfite sequencing PCR(BSP)method was adopted to detect the methylation levels of Cp G island in GSK3? gene promoter region.Results 1.Both PEA and PA can attenuate cognitive impairments in D-galactoseinduced Alzheimer's –like pathology rats,and PEA showed stronger protective effects than PA.(1)Open-field test results:The moving distance,up-right times and residence time in central area in model group were significantly decreased compared with that of normal group(P<0.01).Compared with model group,DNMT1 inhibitor aggravated this depression-like behaviors(P<0.01),while depression-like behaviors in PEA+I and PA group were obviously alleviated(P<0.01).In addition,the moving distance,up-right times and residence time in central area in PEA group were significantly higher than that of PEA+I group(P<0.01).The moving distance,up-right times and residence time in central area in PA group were significantly lower than that of PEA group(P<0.05,P < 0.01)but higher than that of(P < 0.01),indicating that PEA treatment showed stronger anti-depressive effects than PA treatment in D-galactose-induced rats.(2)Morris water maze test results:Compared with the N group,escape latency in M group were significantly longer(P<0.01).Compared with the M group,escape latency in I group were significantly longer than M group(P<0.01),while rats in PA and PES group had shorter escape latency(P < 0.01).Besides,escape latency in PEA group were significantly shorter than PA group(P<0.01).In spatial probe trial,the time spent in target quadrant of rats in M group were significantly decreased compared with the N group(P<0.01).While,the time spent in target quadrant of rats in PEA+I,PA and PEA group were significantly increased compared with the M group(P<0.01),and the swimming paths were mainly adjacent to the target platform,time in N group were significantly decreased(P<0.01).In addition,PEA showed better protective than PA intervention.2.Both PEA and PA can alleviate synapse and neuronal microtubule injuries in hippocampal CA1 area in D-galactose-induced Alzheimer's –like pathology rats,and PEA exerted better neuroprotective effects than PA.(1)Golgi staining results: The dendritic spine density in hippocampal CA1 area of rats in N group was higher than other groups(P <0.01).Compared with M group,rats in I group had lower density(P<0.01).While,No statistical difference was observed between PEA+I and M group(P>0.05).In addition,the dendritic spine density in PA and PEA group were both higher than that of M group(P<0.05,P<0.01),and rats in PEA group had higher density than PA group(P<0.01).(2)The width of synaptic clefts and the postsynaptic density changes: No statistical difference was observed among the 6 groups regarding to the width of synaptic clefts(P>0.05).Compared with N group,the postsynaptic density in M group was significantly decreased(P<0.01).Compared with M group,the postsynaptic density in I group was significantly decreased(P<0.01),while PA and PEA group showed thicker postsynaptic density(P<0.01).In addition,PEA+I intervention reversed the inhibitor-induced synaptic injury and increased the density(P<0.01).However,no statistical difference was observed between PA and PEA group(P>0.05).(3)Neuronal microtubules changes: Microtubules in axon in N group were compact and formed a crossed and compact network structure without fractured microtubules.Microtubules in axon in M group were sparse and fractured,and autophagosomes can be observed.Microtubules in I group were tangled and accumulated intensely.And mitochondrials were in pyknosis and degeneration,autophagosomes can be observed.Though microtubules in PEA+I group were sparser than that of N group,it presented compacter and longer microtubules without tangled shapes compared with M and I group.In addition,PEA and PA group showed compacter,evenly arranged and less fractured microtubules,and autophagosomes were aslo observed.3.Both PEA and PA can inhibit GSK3?/m TOR signaling pathway and NFTs deposition in DRN in D-galactose-induced Alzheimer's –like pathology rats,but statistical difference was only observed in tau phosphorylation levels between two groups.(1)NFTs levels in DRN: As detected by IHC,compared with the N group,NFTs levels in DRN in M group were significantly increased(P<0.01).Compared with the M group,both PEA+I,PA and PEA group showed decreased NFTs levels(P<0.01),but NFTs levels in I group were significantly increased(P < 0.01).However,no statistical difference was observed between PA and PEA group(P>0.05).(2)Tau phosphorylation levels in DRN and hippocampus area: Compared with N group,the relative expression levels of tau-5,PHF-1and taup S262 were significantly increased in rats of M group(P <0.01).Compared with M group,the levels of tau-5,PHF-1and tau-p S262 were significantly decreased in PEA+I,PA and PEA groups(P<0.01)but were still higher than that of N group(P < 0.01).And tau phosphorylation levels in I group were significantly increased compared with N group(P<0.01),but no statistical difference was observed when compared with M group(P > 0.05).Besides,tau phosphorylation levels in PEA were significantly lower than that of PA group(P<0.05,P<0.01).(3)GSK3?/m TOR signaling pathway proteins levels in DRN: Compared with N group,the relative expression levels of GSK3? and GSK3?-p Tyr216 were significantly increased(P < 0.01),while the level of GSK3?-p Ser9 were significantly decreased(P<0.01).Compared with M group,expression levels of GSK3? and GSK3?-p Tyr216 were significantly decreased in PA,PEA groups(P<0.05,P<0.01)but were still higher than that of N group(P<0.01),but no significant changes of GSK3?-p Ser9 levels were observed when compared with M group(P>0.05).No significant changes regarding to GSK3?,GSK3?-p Tyr216 and GSK3?-p Ser9 levels were observed between PA and PEA group(P>0.05).Compared with N group,the relative expression levels of m TOR and m TOR-p S2448 in DRN in M group were significantly increased(P<0.01),the ratio of LC3I/LC3 II were increased,too(P<0.01).Compared with M group,the levels of m TOR and m TOR-p S2448 in PA,PEA group were significantly decreased(P<0.01)and the ratio of LC3I/LC3 II were lower(P<0.01).While,no significant changes in m TOR and m TOR-p S2448 levels between I and M group(P >0.05).Besides,m TOR and m TOR-p S2448 levels in PA group were higher than that of PA group(P<0.05,P<0.01),and PEA+I intervention can increase the expression levels of m TOR and m TOR-p S2448 compared with M group(P<0.01,P<0.05).4.Both PEA and PA can decrease the methylation levels of Cp G island in GSK3? gene promoter region,inhibit the transcription and expression of GSK3? in DRN in D-galactose-induced Alzheimer's –like pathology rats,but statistical difference was only observed in GSK3?m RNA levels between two groups.(1)DNMT1 levels in DRN detected by WB:Compared with N group,the relative expression levels of DNMT1 in M group were significantly decreased(P<0.01).Compared with M group,the levels in I group were significantly decreased(P<0.01),but the levels of DNMT1 in PEA+I,PA and PEA were significantly increased(P<0.05,P<0.01)and were still lower than that of N group(P<0.01,P<0.05).In addition,the inhibitive effects on DNMT1 inhibitor of PEA were stronger than that of PA(P<0.05).(2)DNMT1 levels in DRN detected by IF:Compared with the N group,the percentages of DNMT1 positive cells in M group were significantly decreased(P < 0.01).Compared with M group,the percentages of DNMT1 positive cells in I group were decreased(P<0.01),but the percentages of DNMT1 positive cells in PEA+I,PA and PEA groups were significantly increased(P<0.01)and still lower than that of N group(P<0.01).Besides,PEA showed stronger inhibitive effects on DNMT1 inhibitor than PA(P<0.05).(3)The methylation levels of Cp G islands in GSK3? gene promoter region in DRN:Compared wiith N group,the methylation levels of Cp G islands in GSK3? gene promoter region in M group were significantly decreased(P<0.01).Compared with M group,the methylation levels of Cp G island in I group were decreased(P < 0.05),and were significantly increased in PEA+I,PA and PEA groups(P<0.05,P<0.01) but were still lower than that of N group(P<0.01).No statistical difference of methylation levels were observed between PA and PEA groups(P>0.05).In addition,the loci 118,215,284,26,57,221 and 205 in GSK3? gene promoter region were the target locies of preventive acupuncture effects.(4)GSK3?RT-PCR results:Compared with the N group,the relative levels of GSK3?m RNA in M group were significantly increased(P <0.01),indicating that the GSK3? gene transcription levels in DRN were increased in D-galactose-induced Alzheimer's –like pathology rats.Compared with M group,the GSK3?m RNA in I group were increased(P < 0.05),and were decreased in PEA+I,PA and PEA groups(P<0.01)but were still higher than that of N group(P<0.01).In addition,the GSK3?m RNA levels in PEA group were lower than PA(P <0.01).(5)GSK3? levels in DRN detected by IF:Compared with N group,the percentages of GSK3? positive cells in M group were increased(P<0.01).Compared with M group,the percentages of GSK3? positive cells in PEA+I,PA and PEA groups were decreased(P<0.05,P<0.01)but were still higher than that of N group(P<0.01,P<0.05).No statistical differences were detected between I and M group(P>0.05),or between PA and PEA group(P>0.05).Conclusion: 1.Both PEA and PA can attenuate spatial learning and memory impairments and depressive-like behaviors in D-galactose-induced Alzheimer's –like pathology rats,and PEA exerted stronger protective effects than PA.Inhibiting NFTs deposition,alleviating neuronal microtubules and synapse dysfunction could be one of the underlying mechanisms.2.Both PEA and PA can inhibit NFTs deposition in DRN via inhibiting GSK3?/m TOR signaling pathway involved in the formation and autophagy clearance process of NFTs in D-galactose-induced Alzheimer's –like pathology rats.However,statistical difference regarding the NFTs levels between the two groups was not observed.3.Both PEA and PA can increase the methylation levels of Cp G islands in GSK3? gene promoter region in DRN in D-galactose-induced Alzheimer's –like pathology rats,which leads to a downregulation of GSK3? transcription and expression as well as the following inhibition on GSK3?/m TOR signaling pathway,thus inhibiting the NFTs formation and prompting its autophagy clearance.But PEA intervention exerted stronger inhibitory effects on GSK3? gene transcription as evidenced by lower GSK3? m RNA levels in PEA group than PA,indicating that other epigenetic modulation may involved in the inhibition of GSK3? gene transcription by PEA. |
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