Effect Of Naoluo Xintong And Its Mechanism On Cognitive Impairment In Vascular Dementia Rats

Background:Vascular dementia is defined as a group diseases of acquired cognitive impairment by brain damage which is caused by various types of cerebrovascular diseases.In recent years,VD has become a common disease that seriously affects the physical and mental health of the elderly.At present,the treatment of the disease is still based on Western medicine,but the efficacy is not satisfactory.Traditional Chinese medicine has many advantages such as multiple links and multiple targets.It has shown great features and advantages in the prevention and treatment of various types of cerebrovascular diseases.It has important social value and significance to deeply discuss the treatment of VD by traditional Chinese medicine.Naoluo Xintong is a clinical prescription initiated by the old and famous doctor of Xin'an School of Medicine,Wang LeTao,and is composed of astragalus,angel-ica,amaranth,notoginseng,chuanxiong,safflower,and gastrodia.It is used for the treatment of ischemic stroke.Many reliable modern experimental studies have proven that this prescription can improve cerebral blood circulation,reduce neuronal apopto-sis,and promote synaptic structure regeneration in ischemic cerebrovascular disease,and can significantly improve vascular cognitive impairment.However,its mechani-sm of effect on VD has not been further studied.This experiment further explored the protective effect of Naoluo Xintong on the cognitive function of VD and its possible mechanism from animal experiments,providing reliable evidence for the future treat-ment of VD with Naoluo Xintong.Objectives:By preparing VD rat models,The effects of NaoLuo XinTong on the behavioral parameters,hippocampal pathology,expression of SYN,ChAT,CGRP and its receptors,and Ca²⁺concentration in hippocampal neurons of model rats were examined.From the perspective of animal experiment,it provides a reliable basis for the trea-tment of VD with Naoluo Xintong.Method:In this study,the modified bilateral carotid artery ligation?2-VO?method was used to construct VD rat model.Then,rats were randomly assigned to four groups:sham operation group,model group,Naoluo Xintong group?8.54 g/kg?and Huperzine A group?0.06 mg/kg?,and were administered intragastrically for 28 days.Subsequently,the effects of Naoluo Xintong on the learning and memory ability of VD model rats were observed by Morris water maze test and step-down experiment.The pathological changes of hippocampal CA1 region were observed by HE staining.The expression levels of synapses?SYN?and Acetylcholine transferase?ChAT?of the hippocampus were detected by immunohistochemistry.Enzyme-linked immunosorbent assay?ELISA?was used to measure the expression levels of calcitonin gene-related peptide?CGRP?and its receptors.The changes of Ca²⁺concentration in hippocampal neurons were determined by flow cytometry.Results:1)Effect on behavior of ratsThe navigation experiment of Morris Water Maze Experiment showed that the escape latency of each group of rats gradually decreased with the increase of training times.Compared with the sham-operated group,the escape latency of the model group increased significantly?P<0.01?.Compared with the model group,the escape latency of Naoluo Xintong group and Huperzine group were significantly shortened?P<0.05 or P<0.01?.The water maze space exploration experiment results show that compared with the sham group,the number of VD rats crossing the virtual platform in the 120 s reduced significantly?P<0.01?,and the time spent in the fourth quadrant reduced significantly?P<0.01?.Compared with the model group,the time of Naoluo Xintong group and Huperzine group spent in the fourth quadrant and the number of times crossed the virtual platform increased significantly?P<0.01?.The results of the platform-skipping test showed that the number of basic errors and response time of the model rats increased significantly compared with the sham-operated group?P<0.01?.The number of basic errors and reaction time of the Naoluo Xintong group and Huperzine group decreased significantly compared with the model group?P<0.01?.Compared with the sham-operated group,the latency of the model rats was significantly shorter?P<0.01?,and the number of errors was significantly increased?P<0.01?.The latency of the rats in Naoluo Xintong group and Huperzine group was significantly higher than the model group?P<0.05 or P<0.01?.But the number of errors was significantly reduced?P<0.01?.2)Effect on hippocampal morphology in ratsThe results of HE staining showed that the number of pyramidal cells in the hippocampal CA1 region in the sham operation group was larger,well-arranged and well-structured,the cells were uniform in size,the shape was regular,the structure was clear,the cytoplasm was cool,and the cytoplasm was round,nucleoli was clear.The model group of rat hippocampal CA1 pyramidal cell number was sparse,disorderly disorder arrangement,cell was irregular shape,structure was disorder,cytoplasmwasconcentrated,nuclearwasdeepstained,nucleuswas ambiguous.Compared with the model group,the number of pyramidal cells in the hippocampal CA1 area of Naoluo Xintong group and Huperzine group was more,the arrangement was more orderly,the morphology and structure was improved,and the nucleus tended to be normal.3)Effect of expression of SYN and ChAT in rat hippocampal CA1 regionUnder the light microscope,SYN immunoreactive products showed brownish-yellow granules or dot-like patches,mainly distributed in the cytoplasm.The results of immunohistochemistry showed that compared with the sham group,the mean optical density of SYN protein in hippocampal CA1 region was significantly reduced in the model group?P<0.01?.After treatment with drugs,the average optical density of SYNprotein in the CA1 area of the hippocampus of Naoluo Xintong group was significantly increased?P<0.01?.However,there was no significant change in Huperzine group?P>0.05?.Under the light microscope,the ChAT immunoreactive products were brown precipitates,which were mainly found in the cytoplasm and more common in the pyramidal cell layer and granular cell layer.Compared with the sham-operated group,the expression of ChAT in hippocampal CA1 region was significantly decreased in the model group?P<0.01?.Compared with the model group,the expression of ChAT in the CA1 region of the hippocampus of Naoluo Xintong group and Huperzine group was significantly increased?P<0.05 or P<0.01?.4)Effect of CGRP and its receptor expression in hippocampus of ratsCompared with the sham group,the expression of CGRP and its receptor in the hippocampus of model rats was significantly decreased?P<0.01?.However,compared with the model group,the expression of CGRP and its receptor in the hippocampus of Naoluo Xintong group was significantly increased?P<0.05?.However,there was no significant change in Huperzine group?P>0.05?.5)Effect of Ca²⁺concentration on hippocampal neurons in ratsCompared with the sham group,the Ca²⁺fluorescence intensity in the hippocampal neurons of the model group was significantly increased?P<0.01?;The Ca²⁺fluorescence intensity in hippocampal neurons of NaoluoXintong group was significantly decreased?P<0.01?.However,there was no significant change in Huperzine group?P>0.05?.Conclusion:1)The learning and memory ability of VD rats were impaired.The number of pyramidal neurons in hippocampal CA1 region was significantly reduced,the abnormal morphological structure was abnormal,and the expression of SYN and ChAT protein was significantly decreased.The expression of CGRP and its receptor in hippocampus decreased.The concentration of Ca²⁺in neurons was overloaded.2)Naoluo Xintong can significantly improve the behavioral parameters of VD rats and protect the neuronal cell morphology of hippocampal CA1 region in VD rats.3)The effect of Naoluo Xintong on learning and memory ability of VD rats may be related to the following mechanisms:?1?To improve the expression of SYN in the hippocampus of VD rats and restore the function of synaptic remodeling.?2?To increase the ChAT expression in the hippocampus of VD rats and promote the recovery of central cholinergic function.?3?The expression level of CGRP and its receptor in the hippocampal tissue of VD rats was up-regulated,and the Ca²⁺concentration in the hippocampal neurons of VD rats was reduced.