| Objectives :A rat model of vascular dementia(VD)was prepared by the modified permanent ligation of bilateral common carotid arteries(2-VO),and the effect of the traditional Chinese medicine Rhizoma Corydalis Decumbentis(RCD)on the learning and memory abilities of VD rats was observed.The influence of hippocampus and cortex morphology and cell apoptosis.From the perspective of the HIF1-VEGF-Notch signaling pathway,we further explore the possible mechanism of "angiogenesis" in the treatment of VD by RCD,aiming to promote blood circulation and remove blood stasis drugs in the treatment of VD.Provide experimental basis.Methods :1.Grouping,modeling and administration: Healthy male SD(Sprague-Dawley)rats aged 10-12 weeks were purchased as the research object.The rats were bred adaptively for one week before modeling,and 14 rats were randomly selected as the sham operation group(Sham),and the remaining rats were all used the modified 2-VO method to establish the VD model.After modeling,the VD model rats were randomly divided into model group(Model),nimodipine group(Nim),low-dose RCD group(RCD-L),and middle-dose RCD group(RCD-M)and high-dose RCD group(RCD-H).The Tianwu and nimodipine tablets were prepared into suspensions of corresponding solubility,and each Tianwu group was given 0.2 g/kg,0.4 g/kg,and 0.8 g/kg Tianwu suspension by gavage according to the weight of the rats.The nimodipine group was given 0.01 g/kg nimodipine suspension by gavage,and the sham operation and model groups were given the same amount of normal saline gavage for 40 days.2.Neurobehavioral testing: Morris water maze records the evasion latency of each group of rats,the stay time in the target quadrant,the time to reach the original platform quadrant for the first time,the number of times to cross the platform and the trajectory diagram to observe the neurobehavioral differences of VD rats in each group.3.Morphological observation: HE staining to observe the pathological changes of neurons in the hippocampus and cortex of each group of rats,Nissl staining to observe the changes in the number of neurons in the hippocampus CA1 and cortex,the distribution of Nissl bodies and their protrusions Variety.4.Cell apoptosis detection: TUNEL method was used to observe the apoptosis of neurons in the hippocampus CA1 area and cortex of each group in situ.The Motic counting analysis software counted the number of positive cells and evaluated the level of apoptosis.5.Measurement of microvessel density(MVD): immunohistochemistry(SABC)method was used to detect the expression of CD34 protein in hippocampal CA1 area and cortex area of rats in each group.Three visual fields were selected for microvessel count using Motic counting analysis software,and the average value was taken for evaluation Angiogenesis in ischemic area.6.VEGF/VEGFR-2 protein expression detection: The SABC method was used to detect the average optical density(AOD)of VEGF and VEGFR-2 in the hippocampal CA1 area and cortex area of each group of rats,and the Image pro plus image analysis system was used to analyze it.7.HIF-1α/ VEGF/ Notch pathway protein expression detection: Western blot was used to detect the expression levels of HIF-1α,Notch and VEGF proteins in the hippocampal CA1 area and cortex area of each group of rats,and the Image J image analysis system was used to determine the bands Gray value and data analysis on it.8.Data statistics and analysis: record and count all the data.The data uses SPSS21.0 software for statistical analysis.The measurement data is expressed as the mean ± standard deviation((?)±s).The positioning navigation test uses repeated measurement and multi-factor analysis of variance,and other experiments The results were analyzed by one-way analysis of variance,with P<0.05 as the difference was statistically significant,and Graph Pad Prism 9 was used to draw statistical charts.Results:1.The effect of RCD no on the neurobehavior of VD rats: The next day the positioning navigation experiment was carried out,the Model had significantly longer escape latency than the Sham(P<0.05),and its latency continued to be higher for the next 3 days Horizontal state(P<0.01),and the running track is long and messy.In the space exploration experiment,compared with the Sham,the rats in the Model had significantly fewer times of crossing the original platform position(P<0.01),the staying time in the target quadrant was significantly shortened(P<0.01),and the first time to reach the original platform was prolonged(P<0.01)).Compared with the Model,each treatment group’s escape latency and the time to reach the original platform for the first time were relatively shorter(P<0.05,P<0.01),among which there was a significant difference between the RCD-M,the RCD-H and the Nim(P<0.01),And the number of crossing the original platform position is significantly increased(P<0.01),the stay time in the target quadrant is significantly increased(P<0.01),and the running trajectory is significantly simpler.2.The effect of RCD on the morphology of neurons in the hippocampus CA1 and cortex of VD rats: HE staining showed that the pyramidal cells in the hippocampus CA1 of the Sham were clearly organized,arranged tightly,and had obvious nucleoli;hippocampal CA1 in the Model The cell level in the area decreases,pyramidal cells undergo pyknosis and necrosis,cortical neurons are deeply stained,and necrosis is obvious;compared with the Model,the pathological changes in hippocampal CA1 area and cortex area of each treatment group are reduced to varying degrees,and the cell level is clearer and arranged Closer,especially in the RCD-M and the RCD-H and the Nim.3.The effect of RCD on the number of neurons in the hippocampal CA1 and cortical areas of VD rats: Nissl staining showed that the pyramidal cells in the hippocampal CA1 and cortical areas of the Sham had obvious protrusions,abundant Nissl bodies and complete morphology.In comparison,the number of pyramidal cells in the CA1 and cortical areas of the Model was significantly reduced(P<0.01),and Nissl bodies were significantly reduced.Compared with the Model,the number of pyramidal cells in the CA1 area and cortex area of the RCD-M and RCD-H and the Nim increased significantly(P<0.01),the number of Nissl bodies increased,and the protrusions were more obvious.Tunnel experiment results showed that the number of apoptotic neurons in the RCD-M was significantly higher than that in the Sham(P<0.01).Compared with the Model,the RCD-L can alleviate neuronal apoptosis(P<0.05),and the positive apoptotic cells in the RCD-M,the RCD-H and Nim were significantly reduced(P<0.01).4.The effect of RCD on MVD in hippocampal CA1 and cortex of VD rats: Compared with Sham,MVD in hippocampal CA1 and cortex of Model increased significantly(P<0.05,P<0.01).Compared with the Model,the MVD of hippocampal CA1 area and cortex area of each treatment group increased to different degrees(P<0.05,P<0.01).Among them,the MVD of the RCD-M and RCD-H and the Nim increased significantly(P<0.01)MVD in the cortical area of the RCD-H was significantly higher than that of the Nim(P<0.05).5.The effect of RCD on the AOD value of VEGF/VEGFR-2 protein in VD rats: The results showed that the expression levels of VEGF and VEGFR-2 protein in hippocampal CA1 and cortex areas of the Model were significantly higher than those of the Sham(P<0.01);and Compared with the Model,the RCD-L can increase the expression level of VEGF and VEGFR-2 protein,and the difference is statistically significant(P<0.05).The RCD-M,the RCD-H and Nim can significantly increase the expression level of VEGF and VEGFR-2 protein(P<0.01),and the expression level of VEGFR-2 protein in cortical area of the RCD-H was significantly higher than that of the Nim(P<0.05).6.The effect of RCD on the expression of HIF-1α/ VEGF/ Notch protein in VD rats: Western blot experiments showed that compared with the Sham,the contents of HIF-1α,VEGF and Notch protein in the hippocampal CA1 area of the Model were all Increase(P<0.05);Compared with the Model,the RCD-L showed an overall upward trend in HIF-1α,VEGF and Notch protein content(P<0.05).The RCD-M and RCD-H and the Nim had significant HIF-1α,VEGF and Notch protein content.Increase,showing a very significant difference(P<0.01).Conclusions:1.RCD has a certain effect on improving the learning and memory ability of VD rats.2.RCD can reduce the pathological damage of brain tissue to a certain extent,reduce neuronal apoptosis,and play a neuroprotective effect.3.RCD can improve the blood supply of VD rats by increasing the microvessel density in the brain tissue.4.The neuroprotective effect of RCD on VD rats may be related to its intervention in HIF1-VEGF-Notch signaling. |
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