| Objective:This experiment is intended to investigate the mechanism of the effect of reverse moxibustion on improving cognitive dysfunction in Alzheimer ’s disease(AD)from the perspective of mitochondrial autophagy,taking the relationship between Pink 1/Parkin signaling pathway and cognitive function as the entry point,and to provide some theoretical basis and reference for the clinical prevention and treatment of AD The results of the study was presented.Methods:1.Model preparation:Amyloid beta-peptide25-35 was injected into the bilateral hippocampal region of rats to prepare the AD model under a stereo stereoscope.After injection of 20%ratan(2.5mL/kg)into anesthetized rats,the head of the rats was fixed on the brain stereoscope to make the front and back independent.After sterilization,the scalp was cut open to expose the forehead,and the hippocampus was located(3.5 mm behind the forehead,2.0 mm on both sides of the median line,hard2.7mm under the membrane),A small hole(diameter 1 mm)was drilled with A bench skull,and the head of the rat was fixed,so that the microinjector was vertically inserted into the head of the rat,slowly injected Aβ25-35,1μm(5 μg/μL)into the left and right sides of the head,slowly injected Aβ25-35,and the needle was left for 5min after injection.Aβ25-35 was fully penetrated into the local tissue,and then slowly extracted at A speed of 1.0 mm/min.The scalp was sutured immediately after surgery,and penicillin was intraperitoneally injected 10000 U/piece per day within 5 days after surgery.On the fifth day after the completion of modeling,all rats were subjected to the Morris water maze test,and unqualified rats were excluded and supplemented according to the random principle.2.Grouping and treatment methods:3 6 qualified rats were weighed and separated into four groups according to the random number table method:Reverse moxibustion group(9rats):normal rats were first treated with moxibustion,each point lasted for15 min,once a day,6 days a course of treatment,and 1 day of rest between the course of treatment,a total of 3 courses of treatment.Model group(9):After 3 weeks of normal feeding,the model was constructed according to the above method;All indexes were measured by feeding for one week after modeling.Sham operation group(9):The hippocampus of both sides were injected with the same amount of normal saline,the rest of the model group;Normal group(9):Feeding at the same time,no other treatment.3.Moxibustion method:Based on Experimental Acupuncture and Moxibustion Science edited by Yu Shuguang,special small moxa sticks were used for 15 min at each point,once a day,a 6-day course of treatment,with a rest of 1 day in the middle,a total of 3 courses of treatment were carried out on the "Baihui" point(the middle of the parietal bone),"Shenshu point(both sides of the lower waist),and Zusanli point(the posterior lateral side of the knee joint)of normal rats.4.Index detection:Behavioral differences of each group were observed by water maze test;HE staining was used to observe the neuropathological changes in the hippocampus of rats.The ultrastructural changes of nerve cells in hippocampus were observed by transmission electron microscopy.Beclin-1 and LC3Ⅱ in hippocampus were detected by immunofluorescence labeling.The relative expression values of PINK1 and Parkin related proteins in the mitochondrial autophagy pathway were detected by Western blot.The mitochondrial membrane potential of the hippocampus was detected by flow cytometry with fluorescent dye JC-1.Results:1.Comparison of behavioral test results in the hippocampal area of rats in each groupThe rats in the normal group and the sham group had a shorter movement track,and could quickly find the platform in the water maze.The latency time of escaping and the number of crossing the platform were unchanged,and there was no statistical,significance between the two groups(P>0.05).Compared with the normal group,the escape latency time of rats in the model group was prolonged,and the number of crossing the platform was reduced,with statistical significance(P<0.01).Compared with model group,the escape latency time of rats in inverse moxibustion group was shortened,and the number of crossing platforms was increased,with statistical significance(P<0.01).2.Comparison of hippocampal pathology in each group2.1 HE staining was used to observe the pathological changes in the hippocampus of rats in each groupUnder the light microscope,the neurons in the hippocampus of the normal and sham groups were closely arranged,the cell outline was clear and complete,and no obvious degeneration,necrosis,and other pathological changes were observed.In the model group,the number of neurons in the hippocampal region decreased significantly,the nuclei shrank,the nucleoli were not clear,and the boundary of the nuclear envelope was blurred.Compared with the model group,the number of neurons in the inverse moxibustion group increased to a certain extent,and the cell structure was improved,but not completely restored to the normal level.2.2 Comparison of the ultrastructure of hippocampal neurons in each groupIn the normal group and the sham group,the nucleus structure of neurons was obvious,the nuclear membrane was continuous and complete,and organelles such as mitochondria,Golgi bodies and endoplasmic reticulum were visible in the cytoplasm.The morphology was complete and clearly arranged,and the structure of medullated nerve fibers was tight.In the model group,the morphology of neurons was irregular,chromatin increased with slight edge set,cytoplasmic matrix dissolved and disappeared,cavitation was serious,mitochondrial swelling and mitochondrial crest disappeared.In the anti-moxibustion group,the pathological changes of neurons were alleviated,the nuclei basically tended to be normal,the nuclear membrane was clear,the cytoplasmic organelles were obvious,the endoplasmic reticulum and mitochondria showed slight swelling and ridge fracture,and the nerve fibers were slightly denaturated,but no other significant changes were observed.3.Comparison of the relative expression levels of PINK1 and Parkin in the hippocampus of rats in each group.There were no significant differences in the relative expressions of PINK1 and Parkin between the normal and sham groups(P>0.05).Compared with the normal group,the expressions of PINK1 and Parkin in the model group were significantly decreased,with statistical significance(P<0.01).Compared with the model group,the expression levels of PINK1 and Parkin in the inverse moxibustion group were increased,and the difference was statistically significant(P<0.01).4.Comparison of Beclin-1 and LC3II positive cells expression in hippocampal CA1 region of rats in each groupThere were no significant differences in Beclin-1 and LC3Ⅱ positive expression cells between the normal group and sham group(P>0.05).Compared with the normal group,Beclin-1 and LC3Ⅱ positive cells in the model group were significantly decreased,and the difference was statistically significant(P<0.01).Compared with the model group,positive expressions of Beclin-1 and LC3Ⅱ in the inverse moxibustion group were significantly increased,and the difference was statistically significant(P<0.01).5.Comparison of the relative expression levels of PINK1 and Parkin in the hippocampus of rats in each groupThere were no significant differences in the relative expressions of PINK1 and Parkin between the normal and sham groups(P>0.05).Compared with the normal group,the expressions of PINK1 and Parkin in the model group were significantly decreased,with statistical significance(P<0.01).Compared with the model group,the expression levels of PINK1 and Parkin in the inverse moxibustion group were increased,and the difference was statistically significant(P<0.01).6.Comparison of hippocampal mitochondrial membrane potential results among all groups.There was no significant difference in mitochondrial membrane potential between the normal and sham groups(P>0.05).Compared with the normal group,mitochondrial membrane potential in the model group was extremely decreased,the difference was statistically significant(P<0.01).Compared with the model group,mitochondrial membrane potential increased in the inverse moxibustion group,the difference was statistically significant(P<0.01).Conclusion:Reverse moxibustion can prevent and delay the occurrence of AD,and its mechanism may be related to the activation of the PINK1/Parkin signaling pathway,regulation of Beclin-1,LC3Ⅱ factor expression,enhancement of hippocampal mitochondrial autophagy function,improvement of neuron structure,and improvement of cognitive function. |
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