Study On The Mechanism Of Erchen Decoction And Taohong Siwu Decoction In Regulating P53/SLC7A11-mediated Oxidative Damage And Ferroptosis Against AS

Purpose:1.Objective to predict the potential molecular mechanism of Erchen decoction and Taohong Siwu Decoction in the prevention and treatment of atherosclerosi.2.Objective to observe the intervention effect of Erchen decoction and Taohong Siwu Decoction on oxidative stress injury and Ferroptosis mediated by p53/SLC7A11 in the aorta of Apo E-/-mice and the cells oxidized by ox-LDL,to explore the potential mechanism of Erchen decoction and Taohong Siwu Decoction in the treatment of atherosclerosis from both in vivo and in vitro,so as to provide a new experimental basis for the prevention and treatment of traditional Chinese medicine.Material and method:This research was divided into two parts: 1.Network data analysis of the potential molecular mechanism of Erchen decoction and Taohong Siwu Decoction in the prevention and treatment of AS.2.To explore the intervention effect of Erchen decoction and Taohong Siwu Decoction on oxidative damage and Ferroptosis in vivo and in vitro.1.Network data analysis of the potential molecular mechanism of Erchen decoction and Taohong Siwu Decoction in the prevention and treatment of AS.Screened the components and targets of Erchen decoction and Taohong Siwu Decoction by tcmsp database.Screened the targets of atherosclerosis with TTD,Dis Ge NET,and Drugbank databases.The target genes were standardized with Uni Prot,and then obtained the common targets with VENNY.The results summed with the targets from the literature related to AS and the prescription of traditional Chinese Medicine,then analyzed the PPI network with STRING database,and saved the result as ‘tsv’.At the same time,GO function and KEGG signaling pathway analysis were carried out by using David database,and data visualization is carried out by using Cytoscape software and Omic Share cloud platform.2.To explore the intervention effect of Erchen decoction and Taohong Siwu Decoction on oxidative damage and iron death in vivo and in vitro.2.1 In vivo experiment:10 C57BL/6J wild mice served as normal group were fed with normal diet,and 50 C57BL/6J background Apo E-/-mice were fed with high-fat diet to establish atherosclerosis model.Then the Apo E-/-mice were divided into model group,Chinese medicine low,Chinese medicine medium,Chinese medicine high dose group and simvastatin group after adaptive feeding.Normal group and model group were given the same amount of normal saline,Each Chinese medicine dose group was given different concentrations of decoction twice a day,and the pharmacognostic dosage in the three groups of low,medium and high Chinese medicine was 0.72g/m L,1.44g/m L,2.89g/m L.The simvastatin group was given simvastatin tablets dissolved in water 0.26mg/ml by gavage once per night.After 4weeks of administration,blood and aortic samples were collected 2 hours after the last administration.Observing the pathological changes of the aorta in mice by HE staining.Observing the levels of anti-oxidation and oxidation index(SOD,MDA,GSH)in serum of mice by colorimetry.Immunohistochemical techniques were used to detecte the expression of COX2 and FTH1 in aorta.Western blot and q RT-PCR were used to detect the expression of proteins and genes related to oxidative stress and Ferroptosis in aorta.2.2 In vitro experiment:First,20 SPF SD rats were divided into two groups.One group was given 2.0g/m L(pharmacognostic dosage)Erchen decoction and Taohong Siwu Decoction twice a day,2 m L each time,twice a day,and the other group was given the same amount of normal saline.After continuous gavage for one week,the blood was taken from abdominal aorta 2 hours after the last administration,then the centrifugation is carried out and the supernatant was separated.and the serum of the group given Chinese medicine decoction by gavage was medicated serum,and the serum of the group given normal saline by gavage was control serum.The EA.hy926 cell strain was cultured in DMEM high sugar medium containing 10% fetal bovine serum.After a certain period of cell passage,divide cells into three groups DMEM high sugar cultured in medium containing 10% fetal bovine serum.The control group medium contains 10% control serum,10% control serum and 50mg/L ox-LDL were added to the medium in the ox-LDL group,and 10% medicated serum and 50mg/L ox-LDL were added into the medium in the medicated serum group.The three groups of cells were cultured for 12 hours,and then the next step of index detection was carried out.Fluorescence in situ probe was used to detect the level of ROS,and the levels of SOD,MDA and GSH were observed by colorimetry.Western blot and q RT-PCR were used to detect the expression of proteins and genes related to oxidative damage and Ferroptosis in each cell group.Results:1.Network data analysis of the potential molecular mechanism of Erchen decoction and Taohong Siwu Decoction in the prevention and treatment of AS.1.1 Obtained 208 components of Erchen decoction and Taohong Siwu Decoction from TCMSP database,mainly including sitosterol,quercetin,baicalein,β-carotene,β-sitosterol,etc.;319 targets including TNF,NOS3,SOD1,etc.;obtained 175 potential targets related to AS from TTD,Dis Ge NET and Drugbank database,including SLC7A11,PTGS2,etc.A total of 54 proteins were summed with the targets from literature and 60 related proteins were obtained.1.2 Obtained a total of 262 BP from the GO biological function results,including oxidation-reduction process,response to oxidative stress,cholesterol metabolic process,etc.;28 CC,including organelle membrane,mitochondrion,etc.;42 MF,including glutathione binding,peroxidase activity,etc.Obtained a total of 53 signaling pathways from KEGG results,including glutathione metabolism and NF-κ B signaling pathway,etc.2.The results of in vitro and in vivo experiments2.1 In vivo experiment:2.1.1 HE staining results showed that,Compared with the normal group,the aortic lumen of the model group was narrowed,with larger plaque formation,and a large number of inflammatory cells and foam cells were found inside the model group.After drug intervention,all the treatment groups were improved.Chinese medicine medium and high dose group and simvastatin group improved significantly,inflammatory and foam cells were significantly reduced,plaque tissue reduced,the intima was relatively complete and smooth.2.1.2 The results of serum SOD,MDA and GSH showed that,Compared with the normal group,the levels of SOD and GSH in the model group were significantly decreased(P<0.01),while the level of MDA in the model group was significantly increased(P<0.01).Compared with the model group,the levels of SOD and GSH in the treatment groups were increased(P<0.01 or P<0.05),and the level of MDA in the serum was decreased(P<0.01 or P<0.05),especially in the Chinese medicine medium and high dose groups and the simvastatin group(P<0.01).2.1.3 The expression of COX2 and FTH1 in aorta was detected by immunohistochemistry showed that,compared with the normal group,the expression of COX 2 in model group increased significantly,while FTH1 expression decreased significantly.Compared with the model group,the expression of COX2 in each treatment group was decreased in varying degrees,while the expression of FTH1 was on the contrary,and there were significant differences in the expression among the Chinese medicine medium dose group,high dose group and simvastatin group.2.1.4 Western blot test showed that,compared with the normal group,the protein levels of COX2,NOX1 and p53 in the aorta of mice in the model group were significantly increased(P<0.01),while the protein levels of GPX4,FTH1 and SLC7A11 were on the contrary(P<0.01).Compared with model group,the expression levels of COX2,NOX1 and p53 in aorta of mice decreased with different degrees in each treatment group(P<0.01 or P<0.05),while GPX4,FTH1 and SLC7A11 protein levels were opposite(P<0.01 or P<0.05).The expression of FTH1 protein in the simvastatin group was not statistically significant.2.1.5 q RT-PCR test showed that,Compared with the normal group,the levels of PTGS2 m RNA,p53 m RNA and NOX1 m RNA in aorta were significantly up-regulated in model group(P<0.01),while the levels of GPX4 m RNA,FTH1 m RNA and SLC7A11 m RNA were significantly down regulated(P<0.01).Compared with the model group,the levels of PTGS2 m RNA,p53 m RNA and NOX1 m RNA in the aorta of mice in each treatment group decreased in varying degrees after drug intervention(P<0.01 or P<0.05),while the changes of GPx4 m RNA,FTH1 m RNA and SLC7A11 m RNA showed the opposite trend(P<0.01 or P<0.05).2.2 In vitro experiment:2.2.1ROS expression in each group of cells observed by fluorescence microscope showed that,compared with the control group,the fluorescence luminance of ox-LDL group was improved and the number of cells increased.Compared with the ox-LDL group,the number of cells in the medicated serum group was decreased and the fluorescence luminance was weakened.2.2.2 The results of SOD,MDA and GSH in cells observed by colorimetry showed that,compared with the control group,the levels of SOD and GSH in the ox-LDL group were significantly decreased(P<0.01),while the expression of MDA was significantly increased(P<0.01).Compared with the ox-LDL group,the levels of SOD and GSH in the medicated serum group were significantly increased(P<0.01),while the expression of MDA was on the contrary(P<0.01).2.2.3 Western blot test showed that,compared with the control group,the expression of COX2,NOX1 and p53 in ox-LDL group was significantly up-regulated(P<0.01),while the expression of FTH1,GPX4 and SLC7A11 in the ox-LDL group was on the contrary(P<0.01).Compared with the ox-LDL group,the protein levels of COX2,NOX1 and p53 in the medicated serum group were significantly decreased(P<0.01),while the protein levels of FTH1,GPX4 and SLC7A11 were increased(P<0.01).2.2.4 q RT-PCR test showed that,compared with the control group,the levels of PTGS2 m RNA,NOX1 m RNA and p53 m RNA in the ox-LDL group were significantly increased(P<0.01),while the levels of FTH1 m RNA,GPX4 m RNA and SLC7A11 m RNA were significantly decreased(P<0.01).Compared with the ox-LDL group,the expressions of GPX4 m RNA,FTH1 m RNA and SLC7A11 m RNA in the medicated serum group were significantly increased(P<0.01),while the expressions of PTGS2 m RNA,NOX1 m RNA and p53 m RNA were opposite(P<0.01).Conclusion:1.The components and targets of Erchen decoction and Taohong Siwu Decoction could play a role in the prevention and treatment of atherosclerosis through multiple pathways and biological processes,and the mechanism could be related to the pathological processes such as oxidative stress,glucose and lipid metabolism,inflammatory reaction and so on.Erchen decoction and Taohong Siwu Decoction2.The mechanism of Erchen decoction and Taohong Siwu Decoction against atherosclerosis may be related to the regulation of oxidative damage and Ferroptosis mediated by p53/SLC7A11 based on the theory of phlegm and blood stasis.3.Both in vivo and vitro experiments showed that p53/SLC7A11 signaling pathway could promote the pathological process of oxidative damage and Ferroptosis in vascular endothelial cells,while Erchen decoction and Taohong decoction could interfere with the signaling pathway.