| Purpose: Based on the theory of “the holistic concept and the image thinking”,this study used the network pharmacology and the transcriptome sequencing technology to construct a "bioinformatics analysis-effector molecule" network of the anti-AS mechanism of Huayu Qutan prescription and its “Yi Qi-Qu Tan-Hua Yu” disassembled prescriptions.At the same time,combined with experimental verification,in order to analyze the pathogenesis of AS and the intervention mechanism of Huayu Qutan prescription from the perspective of modern biology,it can provide research strategies and experimental basis for similar research on traditional Chinese medicine compounds and the pathogenesis of atherosclerosis.Material and method:1.The first partFirst of all,this study explores the guiding role of "holistic concept" in the treatment of AS with "Xu-Tan-Yu",and uses the technology of bioinformatics analysis to construct a network of TCM pathogenesis and "bioinformatics analysis-effector molecules" with "image thinking".It is helpful to analyze the pathogenesis of AS from the perspective of modern biology,and provide the basis for the diagnosis and treatment of AS.2.The second partIn this study,TCMSP or BATMAN-TCM was used to obtain the active ingredients and targets of Huayu Qutan prescription,and disease-related genes were obtained from Genecards and OMIM databases.The Draw Venn Diagram online program was used to obtain the intersection of Huayu Qutan prescription and atherosclerosis target genes.Cytoscape 3.7.1was used to draw the visual network diagram of components,AS and target genes.At the same time,we make a PPI visualization diagram on the String website.The key genes were extracted by perl language,and the statistical bar graph of connectivity protein was made.GO and KEGG enrichment analysis was performed using R3.6.0 software.3.The third part10 C57BL/6J mice were used as normal control group and were fed with normal mouse chow.20 Apo E-/-mice were randomly divided into 10 model group and 10 Huayu Qutan prescription group.Model replication was carried out by feeding with high-fat diet,half male and half female.Mice were reared for 16 weeks.Beginning from the 13 th week,the Huayu Qutan Fang group was given a traditional Chinese medicine decoction with a crude drug content of 0.91 g/m L by gavage,0.5 m L each time,once a day,and the normal control group and the model group were given an equal volume of distilled water by gavage.At the end of the 16 th week,the mice were fasted for 24 hours.The mice were killed by de-neck method.The abdominal aorta to the thoracic aorta of the mice were separated.One part was immediately frozen in liquid nitrogen for future use,and the other part was cut to the appropriate size and placed in 4% paraformaldehyde solution.The histomorphological changes of the aorta of the mice in each group were observed by hematoxylin-eosin staining.The collected three groups of aortas were sent to Shanghai Tianhao Biotechnology Co.,Ltd.for transcriptome sequencing.4.The fourth partExperimental animal feeding,grouping,modeling,drug administration and specimen collection are the same as the third paper.Using the online program Draw Venn Diagram,239 targets obtained from network pharmacology were intersected with 1607 differential m RNA genes obtained from transcriptome sequencing.With the help of R3.6.0,the intersection gene results were analyzed by GO and KEGG enrichment respectively.The GO and KEGG enrichment results were analyzed to screen out the biological processes,molecular functions and signal transduction pathways that may be related to the pathogenesis of atherosclerosis in traditional Chinese medicine.We build a network of "disease-TCM pathogenesis-bioinformatics analysis-effect molecules-ingredients-traditional Chinese medicine(preparation)-traditional Chinese medicine treatment method" guided by "image thinking".Elisa method was used to detect the changes of PGE2 content in the aorta of mice in each group.The expressions of PTGS2,FASN,PTGES,EGF and PAI-1 in the aorta tissue of mice in each group were detected by immunohistochemistry.Realtime RT-PCRmethod was used to detect the m RNA levels of PTGS2,AKT,FASN,PTGES,EP4,CCL2,EGF and PAI-1 genes in the aorta of mice in each group.Wes was used to detect the protein expression levels of PTGS2,P-AKT,FASN,PTGES,EP4,CCL2,EGF and PAI-1 in mouse aorta.Results:1.The first part provides theoretical basis and research ideas for the second,third and fourth parts of the thesis by discussing the theory of “the holistic concept and the image thinking” in treating AS with “deficiency,phlegm and blood stasis” and "bio-information analysis-effector molecule" network construction.2.The results of the second part2.1 Huayu Qutan prescription may play a role in the treatment of AS through multiple components,multiple targets,multiple biological processes and signaling pathways.2.2 113 active ingredients were obtained,412 possible targets of Huayu Qutan prescription,3785 atherosclerotic disease-related genes,and 239 common targets of Huayu Qutan prescription and atherosclerosis were predicted.2.3 The network pharmacological mechanism of Huayu Qutan prescription for prevention and treatment of atherosclerosis may include biological processes such as lipid metabolism,inflammatory response,angiogenesis,oxidative stress and apoptosis.2.4 The network pharmacological mechanism of Huayu Qutan Recipe for prevention and treatment of atherosclerosis may include IL-17 signaling pathway,fluid shear stress and atherosclerosis,HIF-1 signaling pathway,TNF signaling pathway,apoptosis and other signaling pathways.3.Results of the third part3.1 HE staining results showed that the aorta of mice in the normal control group had uniform thickness,no lipid deposition,neatly arranged cells,and complete endothelial structure.On the aorta of mice in the model group,there were thickened endothelium and vessel wall,many fat vacuoles,and infiltrated with a large number of foam cells.After the intervention of Huayu Qutan prescription,the thickening of the aortic endothelium of the mice was reduced,and the fat vacuoles and foam cells were reduced.3.2 The results of differential m RNA expression in transcriptome showed that compared with the model group,the normal control group had 2754 differential m RNAs,including 2162up-regulated genes and 592 down-regulated genes.Compared with the model group,the Huayu Qutan prescription group had 1607 different m RNAs,including 572 up-regulated genes and 1035 down-regulated genes.3.3 Transcriptome analysis showed that the mechanism of Huayu Qutan prescription in preventing and treating atherosclerosis may be closely related to biological processes such as T cell activation,lipid metabolism,inflammatory response,angiogenesis,oxidative stress,and apoptosis.3.4 Transcriptome analysis showed that the mechanism of Huayu Qutan prescription in preventing and treating atherosclerosis includes cytokine-cytokine receptor interaction,B cell receptor signaling pathway,cell adhesion molecules,T cell receptor signaling pathway and other signaling pathways.4.Results of the fourth part4.1 The interaction analysis of transcriptomics and network pharmacology showed that 34 interactive target genes were obtained by taking the intersection of the differential genes of network pharmacology and transcriptomics.4.2 Interaction analysis of transcriptomic and network pharmacology showed that the mechanism of Huayu Qutan prescription in preventing and treating atherosclerosis may be closely related to biological processes such as cellular responses to ATP,positive regulation of apoptotic processes,lipid metabolism processes,angiogenesis,cellular responses to platelet-derived growth factor stimulation,inflammatory responses.4.3 Interaction analysis of transcriptomic and network pharmacology showed that the mechanism of Huayu Qutan prescription in preventing and treating atherosclerosis includes regulation of lipolysis,vascular endothelial growth factor signaling pathway,PI3K-Akt signaling pathway,HIF-1 signaling pathway,tumor necrosis factor signaling pathway,calcium signaling pathway,AMPK signaling pathway,insulin signaling pathway.4.4 In this study,a network of "disease-TCM pathogenesis-analysis of life and information-effect molecules-components-traditional Chinese medicine(preparation)-traditional Chinese medicine treatment method" guided by “the holistic concept and the image thinking”was constructed.This is helpful to analyze the TCM pathogenesis of anti-AS of Huayu Qutan prescription and its disassembled prescription from the perspective of modern biology.4.5 PTGS2/AKT/FASN signaling pathwayRealtime RT-PCR results showed that compared with the normal control group,the m RNA levels of PTGS2,AKT and FASN genes in the aorta of the model group were significantly increased(P<0.01).After drug intervention,the m RNA levels of PTGS2,AKT and FASN genes in the Huayu Qutan prescription group were significantly decreased(P<0.05 or P<0.01).Wes results showed that compared with the normal control group,the expression level of FASN protein in the aorta of mice in the model group was significantly decreased,while the protein expression levels of PTGS2 and P-AKT were significantly increased(P<0.01).The expression level of P-AKT protein in the aorta of mice in Huayu Qutan prescription group was significantly decreased(P<0.01),the expression level of FASN protein was increased,and the expression level of PTGS2 protein was decreased,but there were no statistical significance.The results of immunohistochemistry showed that compared with the normal control group,the expression of PTGS2 tan granules in the aorta of the model group was significantly increased,while the expression of FASN brown granules was not significantly different.After drug intervention,the expression of PTGS2 brown granules in the aorta of mice in Huayu Qutan prescription group was significantly decreased,but the difference in the expression of FASN tan granules was not significant.4.6 PTGS2/PTGES/PGE2 signaling pathwayThe results of Realtime RT-PCR showed that compared with the normal control group,the m RNA levels of PTGS2,PTGES and EP4 genes in the aorta of the mice in the model group were significantly increased(P<0.01).After drug intervention,the m RNA levels of PTGS2 and PTGES genes in the aorta of mice in Huayu Qutan prescription group were significantly decreased(P<0.05),and the m RNA level of EP4 gene showed a decreasing trend,but there was no statistical significance.The results of Elisa showed that compared with the normal control group,the aortic PGE2 content of the mice in the model group was significantly increased(P<0.01).After drug intervention,the content of PGE2 in the aorta of mice in Huayu Qutan prescription group was significantly decreased(P<0.01).Wes results showed that compared with the normal control group,the expression level of PTGS2 protein in the model group was significantly increased(P<0.01),and the protein expression levels of PTGES and EP4 were significantly decreased(P<0.01).After drug intervention,the expression level of EP4 protein in Huayu Qutan prescription group was significantly increased(P<0.01),the expression level of PTGS2 protein showed a downward trend,and the expression level of PTGES protein increased,but there were no statistical significance.The results of immunohistochemistry showed that compared with the normal control group,there was no significant difference in the expression of PTGES brown granules in the aortic tissue of the mice in the model group.After drug intervention,there was no significant difference in the expression of PTGES brown granules in the aortic tissue of mice in Huayu Qutan prescription group.4.7 Ccl2/EGF/PAI-1 signaling pathwayThe results of Realtime RT-PCR showed that compared with the normal control group,the m RNA levels of CCL2,EGF and PAI-1 genes in the aorta of the mice in the model group were significantly increased(P<0.05 or P<0.01).After drug intervention,the m RNA levels of CCL2 and EGF genes in the Huayu Qutan prescription group were significantly decreased(P<0.05).Among them,the m RNA levels of PAI-1 gene showed a downward trend,but there was no statistical significance.Wes results showed that compared with the normal control group,the relative expression levels of CCL2,EGF and PAI-1 proteins in the aorta of the model group were significantly increased(P<0.01).After drug intervention,the relative expression levels of CCL2,EGF and PAI-1 proteins in the aorta of mice in Huayu Qutan prescription group were significantly decreased(P<0.01).Immunohistochemical results showed that compared with the normal control group,the expression of EGF and PAI-1yellow-brown granules in the aortic tissue of the mice in the model group were significantly increased.After drug intervention,the number of EGF and PAI-1 brown granules in the aortic tissue of mice in Huayu Qutan prescription group was significantly reduced.Conclusion:1.Huayu Qutan prescription can reduce the plaque of aortic vessel wall in Apo E-/-atherosclerosis model mice.2.The anti-AS mechanism of Huayu Qutan prescription may be related to biological processes such as lipid metabolism,inflammatory response and angiogenesis.3.The anti-AS mechanism of Huayu Qutan prescription may be related to the regulation of the expression of related m RNA and proteins in the PTGS2/AKT/FASN,PTGS2/PTGES/PGE2,Ccl2/EGF/PAI-1 signaling pathways.4.Huayu Qutan prescription may exert its anti-AS effect through multiple components,multiple targets,multiple biological processes and signaling pathways. |
PDF Full Text Request