Study On The Clinical And Mechanism Of Modified Erzhi Pill In Regulating Lipid Metabolism In AS Based On NCOA4 Mediated Iron Autophagy And Iron Death Pathway

ObjectiveAtherosclerosis(AS)is a major cause of cardiovascular disease,and atherosclerotic plaque is the main pathological feature of AS.Clinically,the effect of Modified Erzhi Pill on regulating lipid metabolism in AS is significant.In this study,we intend to further explore the mechanism of Modified Erzhi Pill on regulating lipid metabolism in AS through network pharmacology,clinical observation research and basic experiments.We tend to provide a reliable theoretical basis for the prevention and treatment of AS with traditional Chinese medicine.Methods1.Exploration of the potential mechanism of Modified Erzhi Pill in treating AS by network pharmacologyIn this study,the known components of Modified Erzhi Pill(Fructus ligustrum,Epimedium,and Eclipta)were screened by means of systematic pharmacological analysis platform of traditional Chinese medicine.Target prediction of the components and atherosclerotic diseases were carried out respectively,and the two targets were mapped to obtain potential prediction targets of Modified Erzhi Pill in treating AS.Finally,the potential targets of Modified Erzhi Pill for treating AS were predicted by protein-protein correlation,GO and KEGG analysis.2.Study on correlation of iron deficiency with atherosclerosis risk factors and syndrome of TCMBased on the network pharmacology,we conducted a literature study to develop a clinical study on the correlation between iron metabolism and atherosclerosis disease.In this study,313 patients admitted to the Cardiovascular Department of the The First Affiliated Hospital of Guangzhou University of Chinese Medicine,from March 2021 to March 2022,were retrospectively included who were diagnosed with coronary artery atherosclerosis and had completed relevant examinations.They were divided into two groups according to whether they had iron deficiency or not.Collected and recorded the information of patients,including basic information,laboratory tests,electrocardiogram,ultrasound and contrast records.By comparing iron metabolism indexes,lipid metabolism indexes and AS risk factors between the two groups of patients,logistic regression analysis was used to preliminarily explore the correlation between ID and AS risk factors and anemia risk factors.Then relevant risk factor variables were screened out for multivariate logistic regression analysis in order to obtain the risk factors of ID.Moreover,through the correlation analysis between the different syndrome types of ID and AS,the differences of the three groups of patients with syndrome type AS were compared,and the correlation between ID and different syndrome types of AS was preliminarily explored3.Study on the mechanism of Modified Erzhi Pills in regulating lipid metabolism of AS based on the iron autophagy and ferroptosis pathway mediated by NCOA4According to the results of network pharmacology and clinical studies,we will further conduct experimental research to demonstrate the results,in order to further explore whether Modified Erzhi Pills regulate lipid metabolism of AS by inhibiting the iron autophagy-ferroptosis pathway mediated by NCOA4 on AS mice.(1)Experimental study on regulation of lipid metabolism by Modified Erzhi Pills in atherosclerotic miceIn this study,60 male ApoE-/-mice were fed with a high-fat diet for 12 weeks to induce vascular lesions and establish an atherosclerosis model.ApoE-/-mice were randomly divided into 5 groups:model group,simvastatin group,ferroptosis inhibitor group,lowdose Chinese medicine group and high-dose Chinese medicine group.12 male C57BL/6 mice were fed with normal diet for 12 weeks as normal control group.After 10 weeks of drug or saline intragastric administration,the weight of mice was measured and compared.The changes of serum lipid levels were measured by automatic biochemical analyzer.The histopathologic changes of aorta and liver were detected by HE staining.Masson staining was used to observe the hyperplasia of collagen fibers in aorta.Lipid deposition in aorta,aortic valve and liver tissues was detected by oil red O staining.(2)Experimental study on the effects of Modified Erzhi Pill on oxidative damage and iron death pathways in the liver of atherosclerotic miceIn this study,the levels of Fe2+ and oxidation index MDA levels in liver tissues of each group of mice were detected by the kits.The relative expressions of SLC7A11,GPX4 and Nrf2 mRNA were detected by RT-qPCR.The expressions of ferroptosis related proteins were detected by Western blot.The expression of GPX4,a negative regulator of ferroptosis,in liver tissues of mice in each group was detected by immunohistochemistry.ROS expression in liver tissues of each group were detected by immunofluorescence assay.(4)Based on the AMPK/NCOA4/FTH1 pathway,to explore the mechanism of Modified Erzhi Pill inhibiting lipid metabolism in ASRT-qPCR was used to detect the relative expressions of AMPK,NCOA4,FTH1,LC3B mRNA of iron autophagy related genes.Western blot was used to detect the expression of iron autophagy pathway related proteins.The co-expressions of NCOA4 and LC3B proteins in liver tissues of mice in each group were detected by immunofluorescence assay.Results1.Explore the potential mechanism of Modified Erzhi Pills in treating AS by network pharmacologyBased on the network pharmacology,46 kinds of active compounds in Modified Erzhi Pills and 97 targets for AS were obtained,including 9 key proteins TNF,ESR1,NCOA4,PTGS2,GSH and ROS.Potentially related signaling pathways include AMPK pathway,lipid peroxidation pathway,autophagy pathway,TNF inflammatory signaling pathway,VEGF pathway,etc.2.Study on correlation between iron deficiency and atherosclerosis risk factors and TCM syndrome types(1)A total of 313 patients were included in this study,including 147 patients with ID or anemia(47%),101 patients with ID(32%)and 104 patients with anemia(33%).These results indicated that ID and anemia were common complications of AS,and iron metabolism was related to the progression of AS.(2)Patients in this study were divided into 101 in the ID group and 212 in the nonID group according to iron deficiency.It was found that there were significant differences between the two groups of patients in blood lipid index(TC,LDL-C),iron metabolism index(hemoglobin,serum ferritin,transferrin,transferrin saturation),inflammation index(CRP),anemia risk factors(anticoagulant or antiplate drug use,GFR),BMI,heart rate,gender,smoking history,drinking history,hypertension history and diabetes history(P>0.05).(3)The results of univariate and multivariate logistic regression analysis showed that the risk factors for ID in AS patients included anemia(OR=4.11,P<0.001,95%Cl:1.94-8.73),heart rate(OR=1.02,P=0.020,95%CI:1.00-1.05),LDL(OR=2.65,P=0.039,95%Cl:1.05-6.68),CRP(OR=5.94,P<0.001,95%CI:3.09-11.42).(4)In the correlation study between the probability of ID and the TCM syndrome types of AS patients,it was found that the rate of ID in patients with deficiency of liver and kidney was 39.2%,that in patients with qi stagnation and blood stasis AS was 21.3%,and that in patients with phlegm turbidity and internal obstruction AS was 33.7%,with statistical difference among the three groups(P=0.023<0.05).These results indicated that the different syndromes of AS patients were correlated with iron metabolism.3.Experimental study on the regulation of AS lipid metabolism by Modified Erzhi Pills through iron autophagy and ferroptosis mediated by NCOA4(1)Modified Erzhi Pills could effectively regulate lipid metabolism in atherosclerotic mice①Changes of body weight of mice in each group:compared with mice in model group at 22 weeks,the body weight of mice in Fer-1 group,TCM low-dose group and highdose group decreased significantly(P<0.01).②Changes in serum lipid metabolism of mice in each group:compared with model group,the levels of TC and LDL-C in Fer-1 group and TCM high-dose group were significantly decreased(P<0.05),while the level of HDL-C was significantly increased(P<0.01).③Vascular lipid deposition and pathological structural changes in each group of mice:(A)Aorta gross oil red O staining of mice in each group:Compared with model group,Fer-1 group and TCM high-dose group had smaller aortic plaque area and significantly improved lipid deposition.(B)The results of aortic oil red O staining showed that mice in the Fer-1 group showed improved plaque area at the aorta,partial narrowing of the lumen,and reduced lipid deposition,compared with the model group.The mice in the low dose group showed a slight improvement in lipid deposition compared with the model group,with narrowing of the lumen and more plaques.The mice in the high dose group had less plaque area in the aorta and partial luminal stenosis.(C)Masson staining of the aorta showed that compared with the model group,more collagen fibrous hyperplasia and luminal stenosis in the Fer-1 group and the low-dose group of Chinese medicine.In the TCM high-dose group,the lumen was partially hyperplastic and the luminal stenosis was significantly improved.(D)The aortic HE staining results showed that there was a small amount of plaque at the root of the aortic sinus in the Fer-1 group of mice,and some foam cells were visible.Mice in the low dose group showed thickening of the vessel wall and a small amount of cholesterol crystals,and lumen narrowing was slightly reduced compared with the model group.A small amount of atheromatous plaque was seen in the mice in the high dose group,and the vessel wall was slightly thickened and the luminal stenosis was less severe.④Lipid deposition and pathological structural changes in the liver of mice in each group(A)The results of oil red O staining of mouse liver in all groups showed that the area of lipid droplets and the number of lipid droplets in Fer-1 group were reduced compared with the model group.The liver lipid deposition of mice in the low dose group and high dose group of Chinese medicine improved significantly compared with the model,and the area of lipid droplets decreased significantly.(B)Liver HE staining of mice in all groups showed that the liver of Fer-1 group mice had a more complete structure,a small amount of abnormal cell morphology,and a significant reduction of intracytoplasmic fat vacuoles compared with the model group.In the low dose group,some cells had abnormal morphology and a few fat vacuoles.In the high dose group,the hepatocyte structure of mice was clear and complete,and the hepatic sinusoids and hepatic cords were visible,without obvious fat-proof vacuoles.(2).Modified Erzhi Pills could effectively inhibit oxidative damage and ferroptosis in AS mice①Results of serum antioxidant indexes in mice of each group:Compared with model group,the expressions of MDA and Fe2+ in serum of Fer-1 group and TCM high-dose group were decreased(P<0.05).②Expression results of genes related to ferroptosis in liver tissues of mice in each group:Compared with model group,mRNA expressions of SLC7A11,GPX4 and Nrf2 in Fer-1 group and high-dose Chinese medicine group were increased(P<0.05).③Expression results of iron death related proteins in liver of mice in each group:compared with model group,SLC7A11 and GPX4 proteins in Fer-1 group and high-dose Chinese medicine group were increased(P<0.05);Compared with model group,SLC7A11 protein expression in low-dose group was increased(P<0.05).There was no significant difference in the expression of Nrf2 protein among the groups.④Immunohistochemical results of GPX4 protein in liver tissue of mice in each group:there were more brown particles in Fer-1 group and high-dose Chinese medicine group,and GPX4 protein expression was significantly increased.⑤ROS immunofluorescence staining results of liver tissues of mice in each group:there were more red ROS positive areas in the low dose group.The expression of red fluorescence in high-dose group was lower than that in model group.(3).Modified Erzhi Pills could inhibit the iron autophagy in regulating lipid metabolism in AS by AMPK/NCOA4/FTH1 pathway①Expression results of iron autophagy related genes in liver tissues of mice in each group:compared with model group,mRNA expression of FTH1 in Fer-1 group and highdose Chinese medicine group was increased(P<0.05),while mRNA expressions of LC3B,NCOA4 and AMPK were decreased(P<0.05).mRNA expressions of NCOA4 and AMPK were decreased in low-dose group compared with model group(P<0.01).②Iron autophagy protein expression:Compared with model group,LC3B,NCOA4,p-AMPKα protein expression decreased and FTH1 protein expression increased in highdose group(P<0.05).The expression of LC3B and NCOA4 protein decreased in Fer-1 group.LC3B protein expression in low-dose group was lower than that in model group(P<0.05).③Immunofluorescence staining of LC3B and NCOA4 in liver tissues of mice in each group:The Fer-1 group had significantly less yellow fluorescence.The yellow area of LC3B and NCOA4 was reduced in the high dose group of Chinese medicine compared with the model group,indicating that the high dose of Modified Erzhi Pills significantly inhibited the iron autophagy protein LC3B and NCOA4 co-expression.Conclusion1.Modified Erzhi Pills can prevent and treat atherosclerosis by working on multiple targets of NCOA4,GSH and ROS.Modified Erzhi Pills can regulate lipid metabolism of AS through iron autophagy-ferroptosis pathway.2.Serum iron deficiency is a common complication of AS,and risk factors for ID in patients with AS include anemia,rapid heart rate,and elevated LDL-C and CRP.There is a certain correlation between the ID and the TCM syndrome type of AS patients.3.Modified Erzhi Pills can inhibit NCOA4-mediated ferritin autophagy by regulating AMPK/NCOA4/FTH1 pathway,thus inhibiting the ferroptosis,effectively improving lipid deposition in the body,and thus effectively preventing atherosclerosis.