| Objective:With the increase of aging population,senile dementia in modern society is a serious health,social,and economic burden.Chronic cerebral hypoperfusion(CCH)induced Alzheimer's and other neurological degenerative diseases.Although the CCH induced cognitive impairment pathogenesis research gradually proceded,but the current clinical treatment is limited by availability and usage.The interaction of the development of the CCH damage is more targets,study on the curative effect of the treatment of single targets have not yet received a chronic.Traditional Chinese medicine compound has multiple targets for intervention,adjust the action of the disease pathology as a whole.Therefore,it is necessary to study the traditional Chinese medicine.Explore the mechanism of Modified Dioscorea Pills against chronic cerebral hypoperfusion model rats with Alzheimer's lesions.From the energy regulation and stress signals mediated autophagy,explore its mechanism against beta amyloid deposition,and from the function of the microRNA-132 signal to explore the mechanism againt Tau protein phosphorylation.Methods:Select 60 male Wistar rats were randomly divided into four groups,respectively is the normal group,model group,western medicine and Chinese medicine group,each group of 15.In addition to the normal group,the rest of the group rats were performed bilateral common carotid artery ligation points times and cut,make the rat model with chronic cerebral hypoperfusion,7 days after the building began to fill the stomach.Chinese traditional medicine group to fill the stomach to Modified Dioscorea Pills daily(10 g/kg);Western medicine group daily lavage give nim horizon(10 g/kg);Physiological saline model group and normal group was given,such as capacity,both for 4 weeks continuously.4 weeks after Morris water maze behavior test,observation of chronic cerebral ischemia rats learning and memory ability.Behavior after the test,take the whole brain section wood grain-eosin stain(HE)to observe the histopathological changes,NISSL neurons were observed,in situ apoptosis detection(TUNEL)at the end of the neuron apoptosis;In hippocampal CA1 area of fresh tissue,fixed embedding production of ultra-thin sectioning,uranium lead after double staining tem observation hippocampal autophagosome formation;Immunohistochemical detection LC3 B,A?42,AMPK,p-AMPK,eIF2?,ATF4;QRT-PCR to detect miR-132 and eIF2?,ATF4,GSK-3? mRNA level;Western blot detection eIF2?,ATF4,LC3B-?,LC3B-?,Tau,p-Tau,GSK-3? protein levels.Results:1.Morris water maze test: In the escape latency,the 1st and 2nd days,model group has no obvious difference was found in the time spent with western medicine and Chinese medicine group,on the third day of western medicine has obvious difference was found in the time spent with western medicine and Chinese medicine group(P < 0.05),In the 4th day,the escape latency time of traditional Chinese medicine,western medicine group and model group were reduced,the western medicine group showed the most close to normal the escape latency time.5th day of water maze space exploration experiment results show that the swimming speed between the four groups have no obvious difference.Normal group rats swim to area often and from the original shuttle or stay,residence time ratio increases,pass the platform area often;Compared with normal group,the number of model group rats spent in original residence time decreased significantly(P < 0.05),western medicine and Chinese medicine group rats after drug intervention,time scale and the number of passing the original residence have improved significantly in the model group(P < 0.05).2.HE staining: Normal group of hippocampus CA1 area neurons neatly,clear nuclei,and after 2VO model group rats hippocampal nerve cells decreased,loosely arranged,nuclear pyknosis neuropathy rational change,in traditional Chinese medicine and western medicine after the intervention,the hippocampus nerve pathology results improved,nerve cell number increase,more rules,suggest to Modified Dioscorea Pills for chronic cerebral hypoperfusion rats hippocampal neurons plays a protective role.3.The Nissl staining: Normal group rats hippocampal neurons contained with more Nissl,2VO after chronic cerebral hypoperfusion model group rats decreased in the number of Nissl,the traditional Chinese medicine and western medicine intervention,Nissl amount recovered.4.TUNEL staining: Normal group of TUNEL fluorescent dye cell number is less,less tip apoptosis of nerve cells;Model group hippocampal CA1 area TUNEL fluorescence staining cells increased obviously,prompt apoptosis nerve cell populations;By the western medicine and traditional Chinese medicine(TCM)after the intervention,western medicine and Chinese medicine group decrease in the number of TUNEL staining cells in hippocampal CA1 area.5.Transmission electron microscopy(TEM): TEM in normal group was not observed in the formation of autophagosome,a large number of observed in model group autophagosome,morphological diversity,including visible is the parcel autophagosome cytoplasm components;In traditional Chinese medicine and western medicine group autophagosome can also be observed.6.Real-time fluorescent quantitative PCR detection: Rat hippocampal CA1 region after chronic cerebral hypoperfusion ATF4,eIF2 alpha mRNA expression level was significantly increased(P<0.001),the traditional Chinese medicine and western medicine after the intervention,ATF4,eIF2 alpha mRNA expression levels;After chronic cerebral hypoperfusion rats hippocampal miR-132 were significantly reduced(P<0.001),GSK-3 beta mRNA level increased significantly(P<0.001);Traditional Chinese medicine and western medicine after the intervention,microRNA-132 levels(P < 0.01),GSK-3 beta mRNA levels were lower(P < 0.01).7.Western blot detection: each group of rat hippocampal CA1 area AMPK,Tau protein expression had no significant difference.Compared with the normal group,model group after induced by chronic hypoperfusion,p-AMPK proteins were significantly increased(P<0.01),significantly increased the protein levels of ATF4,eIF2 alpha(P<0.001),model group LC3 B ?/? ratio increased significantly(P<0.01),GSK-3? and p-Tau(Ser396)protein levels,but does not affect the Tau protein level,traditional Chinese medicine and western medicine after the intervention,reduced protein levels of p-AMPK,ATF4,eIF2 alpha,LC3 B ?/? ratio,GSK-3? and p-Tau(Ser396)protein levels.8.Immunohistochemical detection: Compared with normal group,model group after induced by chronic hypoperfusion,p-AMPK proteins were significantly increased(P<0.01),significantly increased the protein levels of ATF4,eIF2 alpha(P<0.001),A?42 protein deposition and LC3 B protein levels were significantly elevated,nerve in plaque deposits,traditional Chinese medicine and western medicine after the intervention,reduced protein levels of p-AMPK,ATF4,eIF2 alpha,LC3 B,GSK-3?,the traditional Chinese medicine and western medicine group no significant difference.Conclusion:1.Modified Dioscorea Pills regulated energy stress mediated autophagy aganist A? deposition induced by CCH.2.Modified Dioscorea Pills regulated miR-132/GSK-3? signal aganist Tau protein phosphorylation induced by CCH. |
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