| Objective:Alzheimer's disease(AD)is a neurodegenerative disease characterized by cognitive impairment,which seriously affects the quality of life of middle-aged and elderly people and brings heavy burden to family and society.A large number of studies have shown that moxibustion has significant characteristics and advantages in preventing and improving the cognitive function of AD patients and animal experimental research of AD model.However,there are few reports about the phenomenon of heat sensitivity in the process of moxibustion treatment of AD.Therefore,this study is to observe the effects of heat sensitive moxibustion on the behavior,brain ultrastructure,hippocampal glial cell markers,inflammatory factors and neurovascular unit related proteins of AD model rats,and to explore the anti-inflammatory effect of heat sensitive moxibustion on cognitive function of AD model rats and its possible mechanism.Methods:First,clean grade male SD rats were randomly divided into control group and sham operation group(the same operation procedure as model group,replaced by normal saline).The other rats were injected A?1-42 into bilateral lateral ventricle to establish AD model rats,and the AD model rats were randomly divided into model group and positive control group(0.5mg/kg/d of donepezil was given).The remaining AD model rats were treated with moxibustion(moxibustion was carried out at“Dazhui”point,40 minutes per time,once a day for 28 days).According to the fluctuation of tail temperature during moxibustion,AD model rats were divided into moxibustion heat sensitive group(the average tailtemperature of rats increased by more than 1?),moxibustion continuous non heat sensitive group(moxibustion operation is the same as moxibustion heat sensitive group,the average tail temperature of rats increased by less than 1?)and moxibustion delayed heat sensitive group(moxibustion operation is the same as moxibustion heat sensitive group,the average tail temperature of rats increased less than 1?in the first 7 days after moxibustion treatment,and with the extension of time,the average tail temperature of rats gradually increased more than 1?).After treatment,Morris water maze navigation test and spaceexploration test were performed to observe the learning and memory ability of rats in each group.Immunofluorescence was used to observe the positiveexpression of M1 marker CD68 and M2 marker cd206 in activated microglia.Immunohistochemistry was used to observe the expression of IL-6,TNF-?,NF-?B and GFAP in hippocampus.Transmission electron microscope was used to observe the ultrastructural changes of brain tissue of rats in each group.Western blot was used to observe the expressions of neurovascular unit related protein Neu N,GFAP,Laminin,VWF,Iba-1,Occludin,HO-1 and VEGF in brain tissue.Results:1.Results of Morris water maze experiment:Compared with the control group,the escape latency of the model group was significantly increased,and the times of crossing platform and the ratio of platform quadrant to total time were significantly decreased(P<0.01).Compared with the model group,the escape latency of moxibustion heat sensitive group and positive control group were significantly decreased,and the times of crossing platform and the ratio of platform quadrant to total time were significantly increased(P<0.01,P<0.05);There was no significant change in escape latency and times of crossing platform in moxibustion persistent non heat sensitive group(P>0.05),but the ratio of platform quadrant to total time was significantly increased(P<0.05);Inmoxibustion delayed heat sensitive group,the times of crossing platform and the ratio of platform quadrant to total time were significantly increased(P<0.01),but there was no significant change in escape latency(P>0.05).Compared with the positive control group,there were no significant changes in escape latency,times of crossing platform and the ratio of platform quadrant to total time in moxibustion heat sensitive group(P>0.05);The escape latency was significantly increased,and the number of crossing platform and the ratio of platform quadrant to total time were significantly decreased in moxibustion persistent non heat sensitive group and moxibustion delayed heat sensitive group(P<0.01,P<0.05).Compared with the moxibustion continuous non heat sensitive group,the escape latency of moxibustion heat sensitive group was significantly decreased,and the times of crossing platform and the ratio of platform quadrant to total time were significantly increased(P<0.01,P<0.05),but there was no significant change in moxibustion delayed heat sensitive group(P>0.05).2.The results of immunofluorescence:Compared with the control group,the fluorescence intensity of CD68 and CD206 protein in the model group wassignificantly increased(P<0.01).Compared with the model group,thefluorescence intensity of CD68 and CD206 protein in moxibustion heat sensitive group,moxibustion persistent non heat sensitive group,moxibustion delayed heat sensitive group and positive control group were significantly decreased(P<0.01).Compared with the positive control group,the fluorescence intensity of CD68 protein in moxibustion heat sensitive group and moxibustion delayed heat sensitive group had no significant change(P>0.05),but the fluorescenceintensity of CD206 protein was significantly increased(P<0.01);Thefluorescence intensity of CD68 and CD206 protein increased significantly in the moxibustion group(P<0.01,P<0.05).Compared with the moxibustion continuous non heat sensitive group,the fluorescence intensity of CD68 and CD206 protein in moxibustion heat sensitive group and moxibustion delayed heat sensitive group were significantly decreased(P<0.05).3.The results of immunohistochemistry:Compared with the control group,the expression of IL-6,TNF-?,NF-?B and GFAP in the model group were significantly increased(P<0.01).Compared with the model group,the expression of IL-6,TNF-?,NF-?B and GFAP in moxibustion heat sensitive group,moxibustion persistent non heat sensitive group,moxibustion delayed heat sensitive group and positive control group were significantly decreased(P<0.01,P<0.05).Compared with the positive control group,the expression of IL-6,TNF-?,NF-?B and GFAP in moxibustion heat sensitive group had no significant change(P>0.05);Theexpression of IL-6,TNF-?and NF-?B in moxibustion delayed heat sensitive group had no significant change(P>0.05),and the expression of GFAPsignificantly increased(P<0.05);The expression of IL-6,TNF-?,NF-?B and GFAP in moxibustion persistent non heat sensitive group significantly increased(P<0.01).Compared with moxibustion persistent non heat sensitive group,the expression of IL-6 and GFAP in moxibustion heat sensitive group was significantly decreased(P<0.01,P<0.05),there was no significant change in expression of TNF-?and NF-?B(P>0.05);There was no significant change in the expression of IL-6,TNF-?,NF-?B and GFAP in moxibustion delayed heat sensitive group(P>0.05).4.The results of transmission electron microscope:In the model group,the perivascular diffuse dissolved edema,microvascular endothelial cell edema,basement membrane loose,vesicular changes,uneven thickness,severe lumen stenosis,large perivascular dissolved and structure disappeared,large blank area existed,edema was obvious,neuronal cell chromatin degradation,mitochondria swelling,vesicular changes.Compared with the model group,the degree of diffuse dissolved edema around blood vessels of rats in moxibustion heat sensitive group,moxibustion persistent non heat sensitive group,moxibustion delayed heat sensitive group and positive control group was significantly reduced,the structure of endothelial cells was relatively complete,and the surrounding space was reduced.5.The results of Western blot:Compared with the control group,the protein expressions of Neu N,Laminin,VWF,Occludin,HO-1 and VEGF in the model group were significantly decreased(P<0.01),the protein expressions of Iba-1 and GFAP were significantly increased(P<0.01).Compared with the model group,the protein expressions of Neu N,Laminin,VWF,Occludin,HO-1 and VEGF inmoxibustion heat sensitive group,moxibustion delayed heat sensitive group and positive control group were significantly increased,the protein expressions of Iba-1 and GFAP were significantly decreased(P<0.01,P<0.05);In themoxibustion persistent non heat sensitive group the protein expressions of VWF,Occludin,Laminin,HO-1 and VEGF were significantly increased(P<0.01,P<0.05),the protein expression of Iba-1 was significantly decreased(P<0.05),the protein expressions of Neu N and GFAP had no significantly change(P>0.05).Compared with the positive control group,the protein expression of Neu N,GFAP,Iba-1,Occludin,Laminin,HO-1 and VEGF in the moxibustion heat sensitive group had no significant change(P>0.05),but the protein expression of VWF was significantly decreased(P<0.05);The protein expressions of Neu N,VWF,Occludin,HO-1 and VEGF were significantly decreased,the protein expressions of Iba-1 and GFAP were significantly increased(P<0.01,P<0.05),the protein expression of Laminin had no significant change(P>0.05);The proteinexpressions of Neu N,VWF and Occludin in moxibustion delayed heat sensitive group were significantly decreased,the protein expression of GFAP wassignificantly increased(P<0.01,P<0.05),the protein expressions of Iba-1,Laminin,HO-1 and VEGF had no significant changes(P>0.05).Compared with the moxibustion persistent non heat sensitive group,the protein expressions of Neu N,VWF,Occludin,HO-1 and VEGF in the moxibustion heat sensitive group were significantly increased,the protein expressions of Iba-1 and GFAP weresignificantly decreased(P<0.01,P<0.05),the protein expression of Laminin had no significant change(P>0.05);In the moxibustion delayed heat sensitive group,the protein expressions of HO-1 and VEGF were significantly increased(P<0.01),and the protein expression of Iba-1 was significantly decreased(P<0.05),the protein expressions of Neu N,GFAP,Laminin,VWF and Occludin had no significantly change(P>0.05).Conclusion:(1)Heat sensitive moxibustion can significantly improve the cognitive function of AD model rats,and has obvious therapeutic advantages over non heat sensitive moxibustion.(2)Heat sensitive moxibustion can improve the cognitive function of AD model rats by inhibiting the activation of microglia and astrocytes,reducing the expression of inflammatory factors,inhibiting the neuroinflammatory reaction,repairing the neurovascular ultrastructure of brain tissue,improving thepermeability of blood-brain barrier and alleviating neuron damage. |
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