| ObjectiveThis study aimed to investigate the interventional efficacy of acupuncture on the behavior and cerebral blood flow(CBF)of SAMP8 mice,and further clarify the underlying mechanism of astrocytic phospholipase A2(PLA2)-arachidonic acid(AA)pathway.Based on this,we provided the objective imaging evidence of against Alzheimer’s disease(AD)with acupuncture and explored the scientific connotation of "dredging DU meridian and awaking mind".Method66 male SAMP8 mice were divided into three groups(n=22 per group):the AD group,the acupuncture(A)group,and the medicine(M)group.22 male SAMR1 mice were used as the normal control(N)group.Both types of mice were 8 months old.In the A group,the mice were immobilized in mouse bags.Acupuncture on Baihui(GV20)and Yintang(GV29)was applied for 20 min,with transverse puncturing at a depth of 3 mm.During the acupuncture,twirling manipulation was applied every 5 min and lasted 15 s each time.Each needle was rotated bidirectionally within 90° at a speed of 180°/s.For the M group,donepezil hydrochloride tablets were crushed and dissolved in distilled water and were delivered to mice by oral gavage at a dose of 0.65μg/g.The above interventions were administered once a day for 26 consecutive days,with no intervention of the N or AD groups.The mice in the N,AD,and M groups received the same 20 min restriction as the A group.On day 16,10 mice in each group were selected randomly and used for the Morris water maze test.The above intervention lasted throughout the Morris water maze test period for 11 days.The visible platform trial,hidden platform trial,probe trial and reversal trial were conducted in order.On day 27,6 mice in each group were randomly selected and used for the arterial spin labeling MRI.The CBF images were reconstructed with paravision software.The bilateral CBF of the prefrontal lobe and hippocampus were calculated and compared between groups.On day 28,the mice were enthanized in order to harvest their brain.HE staining were used to observe microcirculatory vessel in the hippocampus,the vascular internal diameter(ID),vascular external diameter(ED),ID/ED and microvessel density were calculated and compared between groups.On day 27,6 mice in each group were randomly selected and used for immunofluorescence(IF)staining.Through confocal laser scanning microscope,the co-expression of glial fibrillary acidic protein(GFAP)and PLA2,cyclooxygenase-1(COX-1)and cytochrome P450 proteins 2C23(CYP2C23);a smooth muscle actin and cytochrome P450 proteins 4A(CYP4A)were examined.The mean optical density of PLA2,COX-1,CYP2C23 and CYP4A in astrocytes of the hippocampus was calculated by image J and compared in each group.On day 27,6 mice in each group were randomly selected and used for western blotting(WB),and ultra performance liquid chromatography-mass spectrometry/mass spectrometry(UPLC-MS/MS).The relative expression of PLA2,COX-1,CYP2C23 and CYP4A and the contents of AA,prostaglandin E2(PGE2),epoxyeicosatrienoic acids(EETs)were compared in each group.Results1.In the visible platform trial,there were no significant differences in the escape latency or swimming speed among the groups.In the hidden platform trial,there were no significant group differences in the escape latency on day 2.The escape latency in the AD group significantly higher than the N group from day2-5(p<0.01).The escape latency in the A and M groups were notably higher than the N group on day 4 and from day 2-5,respectively(p<0.01 or p<0.05).Compared with the AD group,the escape latency in the A and M groups were substantially decreased from day2-5and from day 4-5,respectively(p<0.01).In the reversal trial,the escape latency in the AD group significantly higher than the N group(p<0.01).The escape latency in the A and M groups were notably higher than the N group on day 3 and from day 1-4,respectively(p<0.01 or p<0.05).Compared with the AD group,the escape latency in the A and M groups were substantially decreased from day 1-4 and from day 2-4,respectively(p<0.01).The platform crossover number and the percentage of time and swimming distance in the SW quadrant in the AD group was significantly lower than in the N group(p<0.01),and the percentage of time and swimming distance in the NE quadrant were significantly increased(p<0.01).The platform crossover number,the percentage of time and swimming distance in the SW quadrant in the A and M groups were higher compared to the AD group(p<0.01or p<0.05),whereas the platform crossover frequency was still lower than the N group(p<0.01).The percentage of time and swimming distance in the NE quadrant in the A and M groups were significantly lower than the AD group(p<0.01).No significant difference on the swimming speed among the groups was observed in the hidden platform,probe and reversal trial.2.The bilateral CBF of the prefrontal lobe and hippocampus in the AD group was significantly lower compared to the N group(p<0.01).Compared with the AD group,CBF of the right prefrontal lobe and bilateral hippocampus was increased in the MA and M groups(p<0.01 or p<0.05),yet levels in these groups were still substantially lower compared to the N group(p<0.01).3.The vascular ID,ED and ID/ED in hippocampus of the AD,A and M groups were significantly lower compared to the N group(p<0.01 or p<0.05).The vascular ID in hippocampus of the A group was markedly lower than the AD and M groups(p<0.05),the vascular ED was lower than the AD group(p<0.05).4.The mean optical density of astrocytic PLA2,COX-1,and CYP2C23 in the AD,A,and M groups was significantly higher than it was in the N group(p<0.01),the mean optical density of CYP4A was markedly lower(p<0.01).The mean optical density of PLA2,COX-1,and CYP2C23 in the A and M groups was markedly lower than in the AD group(p<0.01),whereas the mean optical density was still higher than the N group(p<0.01 or p<0.05).The mean optical density of CYP4A in the A and M groups was markedly higher than in the AD group(p<0.01).5.The relative expression of PLA2,COX-1,and CYP2C23 in the AD group was significantly higher than in the N group(p<0.01 or p<0.05),the relative expression of CYP4A was markedly lower(p<0.01).Compared with the AD group,the relative expression of PLA2,COX-1 and CYP2C23 in the A and M groups decreased drastically(p<0.01 or p<0.05).The relative expression of PLA2 in the M and A groups was higher than that of the N group(p<0.05).The relative expression of CYP4A in the A and M groups increased significantly(p<0.05),yet levels in these groups were still substantially lower compared to the N group(p<0.05).6.Compared with the N group,the contents of AA,PGE2,5,6-EET,8,9-EET,11,12-EET,and 14,15-EET in the AD group increased significantly(p<0.01 or p<0.05).The contents of AA,PGE2,8,9-EET,11,12,EET,14,15-EET in the A group and AA,PGE2,8,9-EET,11,12-EET in the M group were remarkably lower than in the AD group(p<0.01 or p<0.05).The contents of PGE2,5,6-EET,8,9-EET in the A group and PGE2,8,9-EET,11,12-EET,14,15-EET in the M group were drastically higher than in the N group(p<0.01or p<0.05).Conclusion1.Acupuncture based on "dredging DU meridian and awaking mind" has the beneficial effects on spatial learning,reversal learning,and memory in SAMP8 mice.These effects of acupuncture were equivalent to donepezil.Acupuncture tends to have a greater effect in improving spatial learning ability,and presents therapeutic advantages of high efficacy.2.Acupuncture based on "dredging DU meridian and awaking mind" can effectively enhance CBF in the prefrontal lobes and hippocampus,and was equivalent to donepezil.Improving hemoperfusion of the brain and protecting against neuronal damage in the prefrontal lobe and hippocampus could be one important mechanism for the effects of acupuncture against AD.3.Acupuncture has a negative regulatory effect on the PLA2-AA pathway,as reflected in its down-regulation of the expression of PLA2,COX-1,CYP2C23 and the contents of AA,PGE2,and EETs,up-regulation of the expression of CYP4A in the hippocampus of SAMP8 mice.With the inhibition of vasodilatory pathway,we speculate that the increased cerebral blood flow velocity induced by the "abnormal" vasoconstriction might be the key reason for its beneficial effect of acupuncture based on "dredging DU meridian and awaking mind" on CBF.It is worth noting that this kind of vasoconstriction is an "abnormal" intervention mode under the pathological status of AD.Further in vivo study should be encouraged to observe and confirm the modulatory effect of acupuncture on cerebral vessels. |
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