| Based on the observation of microtubule associated protein1light chain3(mi-crotubule-associated protein1light chain3, MAP1-LC3, LC3), autophagy protein Beclin-1, autophagic vacuole, apoptosis index (Apoptotic, Index, AI) and blood rheology in vascular dementia (Vascular dementia, VD) model rats, to study the effect of Qufeng Tongqiao Decoction on autophagy^apoptosis and blood rheology, and research the protection mechanism of Qufeng Tongqiao Decoction on VD ratS。Methods:1) Animal grouping: With the Morris water maze test as a screening criteria, the requirements of SD rats in ninety male SD rats selected, preparing VD model by permanent ligation of bilateral common carotid artery (2-VO), were randomly divided into sham operation group, model group, nimodipine group (Western medicine group), Qufeng Tongqiao Decoction high dose group (high dose group), group Qufeng Tongqiao Fang (middle dose group), Qufeng Tongqiao the low dose group (low dose group) with12rats in each by random table method。Sham operation group only isolated bilateral common carotid artery (Common carotid artery, CCA) without ligation。2) Administration:The western medicine group was given nimodipine concentration of6.25mg/kg/d solution by intragastric administration, In the High, medium, low dose group respectively, to concentrate the equivalent of Qufeng Tongqiao Decoction of crude drug 26.8g/kg/d,13.4g/kg/d,6.7g/kg/d gavage, sham operation group and model group were given normal saline,1times a day,3ml each time, continuous feeding for30days。3) Specimen collection:The heart blood (5ml) is injected in to anticoagulant tube after1%sodium pentobarbital intraperitoneal anesthesia (40mg/kg), used for blood rheology examination; five rats were randomly selected were killed，its fresh brain tissue were reserved in the PE-70℃;7rats cardiac perfusion fixation, its brains were removed, fixed, dehydrated, embedded sections。4) Observation indexes:the Morris water maze test for learning and memory ability of VD rats; Electron microscopic observation of neuronal cell ultra fine structure; Western Blot method was used to detect the expression of autophagy-related protein LC3; immunohistochemical detection of Beclin-1protein expression; in situ end labeling (TUNEL) detection of AI in hippocampal neurons; rheological fast detection indexes of blood rheology。 Results:1) Morris water maze test:Compared with the sham operation group, the escape latency (escape latency, EL) of model group and each treatment group prolonged, platform quadrant dwell time shortened, times across virtual platform decreased, the difference was statistically significant (P<0.05); Compared with the model group, western medicine group, high、middle dose group EL gradually shortened, platform quadrant dwell time extended, across virtual platform number is increased, the difference was statistically significant (P<0.05); low dose group EL shortened, platform quadrant dwell time is longer, the number of spanning the virtual platform has increased, but the difference comparison with the model group had no statistical significance (P>0.05); Compared with the control group, high dose group EL shortening, platform quadrant extended residence time, across virtual platform number is increased, but the difference was not statistically significant (P>0.05). Compared with the low dose group, western medicine group, high, middle dose group EL shortening, platform quadrant extended residence time, times across virtual platform increases, the difference was statistically significant (P<0.05)。2) The electron microscope:hippocampal neurons under electron microscope: model group showed cell vacuoles and irregular shape nucleus, nuclear membrane structure is not clear even nuclear fragmentation, organelle decreased, mitochondria edema, mitochondrial crista bilayer structure is not clear even pyknosis necrosis; The western medicine group, high、middle dose group showed nuclear morphology rules, the nucleus was round, rich organelles, mitochondria, endoplasmic reticulum and mild edema, mitochondrial crista visible, a small amount of lysosome; neuronal cell structure of low dose group has improved, but still visible nuclear condensation and fragmentation, fewer organelles, mitochondria, endoplasmic reticulum edema。Frontal neurons in cortex under electron microscopic:model group neurons were irregular in shape and fragmented, organelle decreased, mitochondrial pyknosis, double membrane structure cristae disappeared; The western medicine group, high、 middle dose group nuclei are round, clear karyotheca, rich organelles, mitochondria, endoplasmic reticulum slightly edema, lysosome obviously. Low dose group compared with the model group has improved, but still see nuclear pyknosis, organelle decreased, mitochondria, endoplasmic reticulum edema。 Hippocampus and cerebral cortex were autophagosome structure。3) LC3protein detection:Compared with model group, each treatment group, the expression level of LC3protein increased, the difference was statistically significant (P<0.05). Compared with the control group, high dose group of LC3protein expression was significantly increased, the difference was statistically significant (P<0.05); the expression of LC3protein in dose group has increased, but the difference was not statistically significant (P>0.05). Compared with the low dose group, high, middle dose group of LC3protein expression increased, the difference was statistically significant (P<0.05).4) Beclin-1protein detection:Compared with the sham operation group, model group and each treatment group, the average optical density of Beclin-1protein (Mean optical density, MOD) values were increased, the difference was statistically significant (P<0.05)o Compared with the model group, western medicine group, high、middle dose group of Beclin-1protein was significantly higher MOD, the difference was statistically significant (P<0.05); low dose group Beclin-1protein MOD level was increased, but the difference was not statistically significant (P>0.05)。Compared with the control group, high dose group of Beclin-1protein MOD value increased, no significant difference (P>0.05). Compared with the low dose group, western medicine group, high, middle dose group Beclin-1protein MOD value increased, the difference was statistically significant (P<0.05)。5) AI of the hippocampal neurons detection: Compared with the sham operation group, model group and the drug intervention group AI increased, the difference was statistically significant (P<0.05)。Compared with the model group, western medicine group, high、middle dose group AI decreased, the difference was statistically significant (P<0.05); low dose group AI decreased, but the difference was not statistically significant (P>0.05)。Compared with the control group, high dose group, AI decreased, but the difference was not statistically significant (P>0.05). Compared with the low dose group, western medicine group, high, middle dose group AI decreased, the difference was statistically significant (P<0.05)。6) The detection of blood rheology index:Compared with the sham operation group, model group and each drug intervention group whole blood viscosity, plasma viscosity increased, the difference was statistically significant (P<0.05)。Compared with model group, each drug intervention group whole blood viscosity, plasma viscosity decreased, the difference was statistically significant (P<0.05); Compared with the control group, high dose group, the whole blood viscosity, plasma viscosity decreased, the difference was statistically significant (P<0.05), low dose group increased, but the difference was not statistically significant (P<0.05)。 Compared with the low dose group, high dose group, the whole blood viscosity, plasma viscosity decreased, the difference was statistically significant (P<0.05)。Conclusion:1) It has autophagy activation in VD,and autophagy can inhibit the neuronal apoptosis.2)Qufeng Tongqiao decoction can improve learning memory function and protective the ultrastructure of hippocampal and cortical neurons of VD rats.3) Qufeng Tongqiao decoction can up-regulated expression of autophagy-related proteins LC3and Beclin-1, effectively improve the apoptosis and blood rheology of VD rats.4) There has some dose-effect relationship of Qufeng Tongqiao Decoction on VD. |
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