Mechanism Of Shen-ma-yi-zhi Granule On Treating SHR Rats With Vascular Dementia Based On The Multi-omics Of Protein Metabolism

Experiment Ⅰ:The effect of Shen Ma Yi Zhi granules on cognition and synaptic plasticity of rats with hypertension combined with endothelial injury-induced vascular dementia modelVascular cognitive impairment(VCI)and vascular dementia(VaD)refer to a group of syndromes ranging from mild cognitive impairment to dementia caused by risk factors for cerebrovascular disease,overt or non-dominant cerebrovascular disease.Under the trend of accelerating the aging process of China’s population,the prevalence,morbidity and mortality of VCI are increasing,which has become one of the focuses of today’s medical industry.VaD refers to a chronic acquired degenerative intellectual impairment syndrome caused by or associated with cerebrovascular disease,which is clinically manifested as intellectual disability,including cognitive ability,memory,judgment and thinking ability,calculation ability,and social life ability,accompanied by emotional and personality changes.Hypertension is not only the primary cause of stroke in China,but also an independent risk factor for cognitive impairment such as VaD and Alzheimer’s disease(AD).Proteomics is based on protein as the research object,through mass spectrometry technology to detect and analyze proteins in biological samples,through the study of VaD model animals protein expression level,post-translational modification,protein-protein interaction,etc.Metabolomics technology refers to the analysis of all low molecular weight metabolites of a certain organism or cell in a specific physiological period,and the study of diseases leading to changes in the pathophysiological processes of the body,and finally causing corresponding changes in metabolites Key substances.On the basis of previous research,the research group applied the traditional Chinese medicine compound for the prevention and treatment of VaD.Shen Ma Yi Zhi granules,the national new drug invention patent(ZL201610565191.5),and has been approved as a traditional Chinese medicine preparation in Beijing medical institutions(Z20200005000).It is composed of Panax ginseng C.A.Mey,Gastrodiaelata Bl,Euonymus alatus(Thunb.)Sieb.,and Ligusticum chuanxiong Hort,which has the ability to invigorate qi and dissolve stasis,flatten the liver and latent yang.The main treatment is qi deficiency and blood stasis,liver and yang disturbance,symptoms include forgetfulness,sluggish expression,mental decline and other vascular dementia treatment.Previous animal experiments and clinical studies have shown that Sam Nootropic Formula can improve the pharmacological effects and multiple mechanisms of action of VCI rats,and also has a good therapeutic effect on patients’ cognition,behavior and neurological function.The research group intends to analyze the differences in protein expression and metabolites of VaD model animals with hypertension combined with endothelial injury through proteomics and metabolomics techniques,and carry out intervention research on traditional Chinese medicine compound-ginseng nootropic formula,so as to obtain an overall and comprehensive understanding of the process of disease occurrence and cell metabolism at the protein level of VaD.Through the analysis of VaD model and brain tissue metabolites after intervention in Samma Nootropic Formula,the biomarkers of VaD were searched,and the overall research concept of Chinese medicine and the concept of more accurate standardization and refinement of proteomics and metabolomics techniques were explained.ObjectiveEstablished a Vascular dementia(VaD)mode by compound endothelial injury in rats with spontaneous hypertension rats(Spotaneously hypertensive rats,SHR).Observing the effects of SMYZG on learning and memory ability,hippocampal synaptic plasticity,serum cholinergic and β-amyloid content in VaD model.Applying proteomics and metabolomics techniques to find SMYZG intervention in VaD-involved metabolic pathways and differential proteins and metabolites with the same changing trend,and systematically describe the molecular regulatory mechanism of SMYZG in organisms.MethodsExperiment 1.Effects of SMYZG on learning,memory and synaptic plasticity in cognitive dysfunction model rats.Spontaneous hypertensive rats(SHR),no specific pathogen level(SPF),male,13～14 weeks old.VaD models were prepared by microcurrent stimulation of SHR bilateral carotid artery thrombosis,and randomly divided into:VaD model(Model)group,donepezil hydrochloride group(DNPQ),SMYZG high-dose group(SMYZG-H),SMYZG low-dose group(SMYZG-L),10 in each group,and given the corresponding drug volume gavage.Wistar-Kyoto rats(WKY)sharing the same genetic background with normal blood pressure were used as control group and gavaged with the same volume of distilled water with Model for 8 weeks.Morris water maze,brain histopathological HE staining,Nissl staining,and Golgi silver staining were used to observe the effects of SMYZG on learning and memory function,hippocampal(CA1,CA3)pathological morphology and synaptic plasticity morphology in rats.Using ELISA to detect serum inflammatory factors:interleukin,IL-1 β,IL-6,tumor necrosis factor(TNF)-α levels;Vasoactive substances:Angiotensin(Ang)Ⅱ,endothelin-1(ET-1),calcitonin gene related peptide(CGRP)levels.Dementia-specific markers:Amyloid β-protein(A β).Colorimetric and chemical fluorescence methods were used to detect oxidation-related indexes,inducible nitric oxide synthase(iNOS)and Reactive oxygen species(ROS)levels.Cholinergic related indexes including acetyl-choline(Ach),acetyl-cholinesterase(AchE)levels were detected by colorimetric method to reflect.Experiment 2.Proteomics study of cognitive dysfunction model rats intervened by SMYZG.The method of quantitative proteomic research was conducted with application of liquid chromatography-mass chromatography and related techniques.All proteins expressed in the genome of SMYZG intervention model rats were labeled by TMTpro 16 standard reagent.The ion abundance reported by the marker was used to accurately identify and quantify the proteins.Data analysis uses Sequest HT,the search engine of Proteome Discoverer software,to retrieve the Uniport proteome database for protein sequence information to find differential proteins and drug targets.Experiment 3.Metabolomics study of cognitive dysfunction model rats intervened by SMYZG.TM1000 targeted metabolomics technique was used to analyze the metabolite changes and mechanism of metabolites in brain tissues of rats in the VaD model from the metabolite level.Experiment 4.Joint omics study of cognitive dysfunction model rats intervened by SMYZG.The proteomics and metabolomics data were performed through R 4.0.1 and cytoscape 3.8.2 for bioinformatics analysis and protease correspondence table,metabolite-protein combination analysis KEGG metabolic pathways enrichment,shared KEGG analysis,network analysis etc..Results1.Effect of SMYZG on model rats’ memory learning ability.Compared with the WKY group,the passes number of Model group was significantly reduced(P<0.05),the target quadrant search time was reduced(P<0.05),and the total distance in the target quadrant was reduced(P<0.05).Compared with the model,the number of stage penetrations of SMYZG-L and smyzg-h increased significantly(P<0.05,P<0.05),the target quadrant search time increased(P<0.05,P<0.05),and the total distance of the target quadrant increased significantly(P<0.05,P<0.05).2.Effect of SMYZG on model rats’ synaptic plasticity in CA1-CA3 regions.Compared with the WKY group,the cell arrangement was irregular,the nucleus was deformed and solidified,and the staining was deepened in each group by HE staining.Nystinite powder changes,decreased number,cell edema and lysis of cells were observed in each group by Nistinite staining.The length of the synapsis was shortened and the density of synaptic spines decreased,and immature neurons were observed.The pathological changes of Model group were the worst,while the SMYZG-H were the least.3.Effect of SMYXG on model rats’serological indexes.Compared with the WKY group,the levels of IL-1 β,IL-6,INOS,ROS,TNF-αand the contents of AngⅡ,ET-1,A β,AchE were significantly increased,while the content of CGRP,Ach decreased in Model group.Compared with the Model group,the levels of IL-1 β,IL-6,iNOS,ROS,TNF-α and the content of AngⅡ,ET-1,A βwas reduced,and the content of CGRP,Ach,AchE increased.4.Effect of SMYZG on proteomics in VaD model ratsThe potential signaling pathways and biomarkers of SMYZG to improve VaD were screened,and its potential targets were:epidermal growth factor receptor(Egfr),associated coil-containing helix-binding protein kinase 1(Rock1),proto-oncogene Pik3ca,RAS related virus(Rras),Tubulin beta-3 chain(Tubb3),cytochrome C oxidase subtype 5A(Cox5a),non-metastatic cell 1 gene(Nmel),that can improve vascular endothelial function,protect neurons and improve cognitive function.The key pathways of neurodegenerative diseases and Alzheimer’s disease are:AMPK/PGC-1α/UCP2/ATP5A,HIF/VEGF,VEGF/Flk-1/p8 mapk,which has vascular endothelial protection and neuronal protection functions.5.Effect of SMYZG on metabolomics in rats with VaD modelThe study found that SMYZG could improve the differential metabolite profile of cognitive function in rats in the VaD model,and the results showed that significant markers at the metabolite level were:tyrosine(Tyr),ergothioneine(ET),herringidine(AGM),methylnicotinamide(MNA),vitamin B6(VB6),and "arginine and proline metabolism","cofactor biosynthesis","amino acid biosynthesis" pathways.The main metabolic markers of SMYZG and DNPQ in the treatment of VaD are:cytosine,nitric oxide synthase inhibitor(L-NMMA),indoxyl sulfate,and "neuroactive ligands and receptors" and "cofactor biosynthesis".6.Joint omics study of VaD model rats by SMYZGStudies have found that the common signal pathways between the onset of VaD and the effect of SMYZG are neurodegenerative diseases-multi-disease pathway,gap junction,ErbB signaling pathway.ConclusionsSMYZG can improve the learning and memory function of rats with vascular dementia caused by hypertension and vascular endothelial injury.Proteomic and metabolomics studies have shown that differential proteins involving "Egfr,Rock1,Pik3ca,Rras,Tubb3,Cox5a,Nmel" and "Tyr,ET,AGM,MNA,VB6" differential metabolites are potential biomarkers of SMYZG to improve vascular dementia.The characteristics of hypertension and endothelial injury in the VaD model were elucidated from the molecular perspectives of integrity,spatiotemporality,and complexity,which provided a new perspective for exploring the mechanism of traditional Chinese medicine compound prevention and treatment of VaD.