Study On The Anti Atherosclerosis Mechanism Of Active Components Of Prunella Vulgaris

Background:Atherosclerosis(as)is one of the common clinical diseases,which is an important cause of cardiovascular and cerebrovascular diseases.Coronary heart disease,stroke,peripheral vascular disease and other related diseases are seriously harmful to human health,which is becoming increasingly serious in China.With the development of social economy,profound changes have taken place in the national lifestyle,especially the acceleration of population aging and urbanization.The prevalence trend of cardiovascular risk factors in China is obvious,which leads to the continuous increase of the number of cardiovascular diseases.Traditional Chinese medicine has unique advantages in the prevention and treatment of as.Prunella vulgaris,as a kind of soft and hard medicine,is one of the common Chinese medicines in the treatment of as,but its pharmacological action and mechanism need to be further studied.Objective:To predict the active components and pathways of Prunella vulgaris drugs which can interfere with the course of AS,and to establish the cell hypoxia model and foam cell model in vitro,to study the therapeutic effect of active ingredients of Prunella vulgaris on cell hypoxia and lipid metabolism disorders,and to further study its mechanism.Method:1.Study on the target and related pathway of active components of Prunella vulgaris in the treatment of as based on network pharmacologyTcmsp and literature retrieval were used to determine the effective active components and target information of Prunella vulgaris.OMIM database and HPO database were used to summarize as disease target.using string database and network analyzer plug-in of cycloscape 3.7.1 to draw PPI,and using cluego plug-in and David online analysis platform to analyze KEGG pathway enrichment and go function enrichment.To explore the multiple pharmacological mechanism of the active components of Prunella vulgaris and the pathogenesis of as.It lays the foundation for the next experimental verification.The docking results of active components and key targets of Prunella vulgaris were verified by moleculardocking method,and the key targets of stable docking with active components were selected by virtual screening.2.Study on the effect of active components of Prunella vulgaris on cell hypoxia model.293T cells were cultured,and the anoxic model of 293 T cells was established to test the activity of the active components of Prunella vulgaris in vitro.The four active components of Prunella vulgaris obtained through the network pharmacology analysis were respectively configured into a concentration gradient of 0.1-100 μM.through CCK-8 and LDH detection,the non-toxic concentration of each active component to 293 T cells was determined.Through the cell transfection experiment,pgl-hif-1 α was constructed Eukaryotic expression plasmids were used to establish the hypoxia model of cells in vitro,and then the selected active components of Prunella vulgaris intervened the cell model according to the appropriate concentration gradient.Through the detection of double fluorescent reporter gene,it was clear whether there was inhibition on the expression of HIF-1 α.Finally,Western blot was used to detect the expression of HIF-1 α protein.3.Establishment of foam cell model in vitro and inhibition of active ingredients of Prunella vulgaris on lipid droplet formation.using RAW264.7 macrophages and OX-LDL to establish an in vitro foam cell model,0.01-10μM of Prunella vulgaris active ingredients 24 h were given respectively.The inhibitory effect of four active ingredients of Prunella vulgaris on foam droplet formation was observed by using the optimized oil red O staining method.4.Study on the mechanism of active components of Prunella vulgaris regulating lipid metabolism disorderusing RAW264.7 macrophages and OX-LDL,an in vitro foam cell model was established.After giving 0.01-10 μM active ingredient 24 h,the content of total cholesterol in foam cells was detected,and the expression of cholesterol transport related protein was detected by Western blot.Finally,the effect of HIF-1α protein inhibitor on lipid droplets in foam cells was detected.Result:1.Study on the target and related pathway of Prunella vulgaris in the treatment of as based on network pharmacologyThrough the network pharmacology analysis and literature search,it was determined that Prunella vulgaris contains 14 active components,such as quercetin and luteolin,which correspond to EGFR,EGF,IL-6,VEGFA,MMP9 and other targets,including age / range,TNF,HIF-1 and other signal pathways."Active component target" network reveals the close interaction between multiple active components and multiple targets of Prunella vulgaris.Through the PPI network analysis of Prunella vulgaris,we can know the key targets in the action targets,KEGG pathway enrichment analysis and go function enrichment analysis to clarify the biological pathway and gene function involved in the active components of Prunella vulgaris;PPI network analysis of disease-related targets of as We know the key target of as disease.In addition,the overlapping parts of Prunella vulgaris and disease targets were screened.In this part,the enrichment analysis of KEGG pathway and go function was carried out to preliminarily explore the biological pathway and gene function of Prunella vulgaris in the treatment of as.According to the network pharmacology screening analysis,melilotine,caffeic acid,quercetin and β-sitosterol are the four active components that have the highest correlation with as disease.The stable connection between the four active components and the overlapping targets was verified by molecular docking technology.2.Study on the effect of active components of Prunella vulgaris on cell hypoxia modelCompared with contorl group,luteolin at the concentration of 100 μM significantly inhibited cell proliferation,while luteolin at the concentration of 10 μM,1μM and 0.1 μM did not significantly inhibit cell proliferation;luteolin at the concentration of 100μMsignificantly promoted LDH release,but at the concentration of 10 u m,1 u m and 0.1 μM did not significantly promote cell proliferation;caffeic acid,quercetin and β-sitosterol at the same concentration of 100 μM significantly inhibited cell proliferation,The concentration of10 μM,1 u m and 0.1μM did not significantly inhibit the LDH release of cells,and the concentration of 10μm,1 u m and 0.1 u m did not significantly promote the LDH release of cells.In the transfection experiment,the transfection efficiency was the highest in the environment of 3 × concentration of transfection solution,and the concentration of 10μM of four active components of Prunella vulgaris was given to cells for stem prognosis Western blot was used to detect the expression of HIF-1 α protein in the model cells.The results showed that the four active components of Prunella vulgaris inhibited the expression ofHIF-1 α protein in model cells.3.Establishment of mouse foam cell model and inhibition of active components of Prunella vulgaris on lipid droplet formationIt is proved by experiments that the foam cell model established by using 120 μg/ml OX-LDL is the most stable.Stay in the cytotoxic experiment,it was found that the concentration of luteolin 100 μM in the four active components of Prunella vulgaris had a significant inhibitory effect on the cell viability,and the concentration of 0.1-10μM had no significant inhibition;and the concentration of 100 μM had a significant promoting effect on LDH release,0.1-10μM The concentration of caffeic acid,quercetin and β-sitosterol were all the same.The concentration of caffeic acid,quercetin and β-sitosterol were 100 μm and0.1-10 μM,respectively.The concentration of caffeic acid,quercetin and β-sitosterol were100 u m and 0.1-10 μM,respectively.In the experiment of inhibiting lipid droplet formation in foam cells,the active ingredient of Prunella vulgaris at 10 μM concentration had the most obvious inhibition effect on lipid droplets.4.Study on the mechanism of active components of Prunella vulgaris regulating lipid metabolism disorderThe effect of active ingredients of Prunella vulgaris on cholesterol content in foam cell model was tested.Compared with the Control group,the cholesterol deposition in the model group increased significantly.The active ingredients and the active ingredients of Prunella vulgaris,luteolin,caffeic acid,quercetin and beta sterol were used to intervene.The results showed that the positive drugs could significantly reduce the deposition of cholesterol in the foam cells,and the inhibition of the active ingredients of Prunella vulgaris on cholesterol deposition.The concentration of 10 μM had significant inhibition.With the gradual decrease of concentration,the inhibition effect also gradually decreased.The active ingredients of Prunella vulgaris can reduce the deposition of lipid in foam cells,and reduce the degree of foam formation.In regulating cholesterol transport protein,luteolin,caffeic acid,quercetin and beta gesween 10 μM significantly regulate the secretion of cholesterol transporter related proteins HIF-1α,PPAR-γ,LXR-α,ABCA1 and ABCG1 in foam cells.Finally,HIF-1αprotein inhibitor was added to detect the effect of lipid droplets on foam cells,and whether the active components of Prunella vulgaris could affect the formation of foam cells throughHIF-1 pathway.Conclusion:(1)The active components of Prunella vulgaris may have therapeutic effect on as through HIF-1 pathway(2)Luteolin,caffeic acid,quercetin and β-sitosterol,the active components of Prunella vulgaris,can inhibit HIF-1 α protein content in hypoxic cells.(3)the active constituents of Prunella vulgaris,luteolin,caffeic acid,quercetin and beta glutamol can inhibit lipid deposition in foam cell models and regulate the expression of cholesterol transporters.