Study On The Mechanism Of Qingxin Jieyu Prescription Regulating Macrophage Ferroptosis To Stabilize Atherosclerotic Plaque

Atherosclerosis(AS)is a chronic,progressive vascular disease characterized by arterial wall dysfunction and atheromatous plaque formation,and is the pathological basis of atherosclerotic cardiovascular disease(ASCVD).Ferroptosis is a form of programmed cell death dependent on iron-mediated lipid peroxidation and plays an important role in the regulatory mechanisms of AS.It not only affects lipid metabolism,oxidative stress,and inflammatory responses,which are risk factors and pathophysiological processes of AS,but also iron metabolism disorder can lead to functional damage of macrophages and vascular endothelial cells,increasing plaque vulnerability and rupture risk.Studies have shown that imbalanced iron metabolism in macrophages accelerates the progression of AS,and inhibiting macrophage ferroptosis is a potential target for the prevention and treatment of ASCVD.The concept of "very high-risk ASCVD patients" has been proposed in recent years,and these patients have a higher risk of recurrent major adverse cardiovascular events and are recommended to achieve lower lipid-lowering goals.Currently,no studies are reporting the core pathogenesis and syndrome characteristics of traditional Chinese medicine(TCM)in very highrisk ASCVD patients.Investigating the syndromes features and syndrome types distribution of very high-risk ASCVD patients and further exploring the core pathogenesis of very high-risk ASCVD may provide references for TCM’s "stabilizing vulnerable plaques in the presence of disease" in AS.Qing-Xin-Jie-Yu Formula is an effective compound prescription based on the theory of"stasis and toxicity" in treating coronary heart disease.It is derived from the experience formula Yugeng Tongyu Decoction by National Medical Master Chen Keji.Preliminary multi-center randomized controlled trials have confirmed that Qing-Xin-Jie-Yu Formula can reduce cardiovascular endpoint events in patients with stable coronary heart disease.Proteomics studies suggest that Qing-Xin-Jie-Yu Formula can regulate programmed cell death in ASrelated cells.Network pharmacology studies show that Qing-Xin-Jie-Yu Formula can regulate macrophage lipid metabolism,ferroptosis,and other biological processes.Basic experiments have confirmed its therapeutic effects on AS from aspects such as macrophage pyroptosis and adjustment of intestinal flora.However,it is unclear whether Qing-Xin-Jie-Yu Formula stabilizes vulnerable plaques in AS by regulating ferroptosis.Therefore,this study conducted three parts:clinical research,in vivo,and in vitro experiments.Clinical trials explored the core pathogenesis of very high-risk ASCVD.In vivo and in vitro experiments proposed the following hypothesis,"Qing-Xin-Jie-Yu Formula can regulate the GPX4/xCT signaling pathway,inhibit macrophage ferroptosis,reduce iron accumulation and oxidative stress,thereby exerting a stabilizing effect on vulnerable plaques in AS." This hypothesis was validated at multiple levels,including animal,tissue,cellular,ultrastructural and molecular levels,to elucidate the mechanism of Qing-Xin-Jie-Yu Formula in regulating macrophage ferroptosis and stabilizes vulnerable plaques in AS.Part 1.Investigation of syndrome distribution patterns in very high-risk ASCVD patientsObjective:To investigate the distribution characteristics of TCM syndromes and syndrome types in very high-risk ASCVD patients,and to explore the core pathogenesis of very high-risk ASCVD.Methods:Patients diagnosed with very high-risk ASCVD who visited the outpatient clinic or ward of Xiyuan Hospital from March 1,2022,to December 31,2022,were included.Information on vital signs,personal history,past history,family history,TCM four diagnostic methods,and TCM diagnosis was collected.After data cleaning using Excel 2019,frequency analysis,factor analysis,and cluster analysis were conducted using IBM SPSS Statistics 25 software,and association rule analysis was conducted using IBM SPSS Modeler software based on the Apriori algorithm.Results:A total of 200 eligible patients with very-high-risk ASCVD were included,of whom 59.5%(119)were male,aged from 33 to 90 years old,with an average age of 63.87±10.19 years.The most common symptoms in very-high-risk ASCVD patients were bitter mouth,recurrent chest pain,progressive aggravation of chest pain,shortness of breath,dry mouth,tiredness and weakness,foul breath,impatience and irritability,yellow urine,and dark lips and mouth.The most frequent pulse signs were string pulse and slippery pulse,and string-slippery pulse were the most common concurrent pulse.The most common tongue manifestations were yellow coating,dark purple tongue,and old tongue.The syndrome distribution of very highrisk ASCVD patients was mainly characterized by blood stasis and toxins,qi deficiency,yang deficiency,phlegm and turbidity,and qi stagnation,with syndrome types mainly consisting of intermingled stasis and toxins,Qi deficiency/Yang deficiency,and phlegm-heat internal obstruction.Those with diabetes often had concurrent yin deficiency with hyperactive fire and qi deficiency,those with a history of ischemic stroke often had concurrent qi deficiency,those with multi-vessel coronary artery disease often had concurrent liver depression transforming into fire,and those with multi-vessel bed lesions often had concurrent yang deficiency.Conclusion:The core pathogenesis of very high-risk ASCVD patients is "deficiency of vital energy,and accumulation of phlegm and blood stasis with toxins." This study may provide references for TCM to stabilize vulnerable atherosclerotic plaques in AS through the concept of "preventing disease from exacerbating".Part 2 Effects and mechanism of Qing-Xin-Jie-Yu Granule on atherosclerotic vulnerable plaques in ApoE-/-miceObjective:To clarify the intervention effects of Qing-Xin-Jie-Yu Granule(QXJYG)on stabilizing vulnerable atherosclerotic plaques,inhibiting macrophage ferroptosis,and regulating the GPX4/xCT pathway in vivo.Methods:After establishing AS model with a high-fat diet for eight weeks,100 ApoE-/mice were randomly divided into five groups(20 mice per group),including the model group,atorvastatin group(5 mg/Kg/d),low-dose QXJYG group(L-QXJYG,1.28 g/Kg/d),mediumdose QXJYG group(M-QXJYG,2.56 g/Kg/d),and high-dose QXJYG group(H-QXJYG,5.12 g/Kg/d);and 20 C57BL/6J mice served as the control group with a normal diet.All mice were gavaged the corresponding intervention and continued the original diet for eight weeks.After that,the serum and aortic tissue of each group were separated and preserved.An automatic biochemical analyzer was used to detect the serum total cholesterol(TC),triglyceride(TG),low-density lipoprotein cholesterol(LDL-C),and high-density lipoprotein cholesterol(HDLC)levels.Oil Red O staining of the aorta,hematoxylin-eosin(HE)staining and Movat staining of the aortic root were used to assess the severity of aortic lesions and plaque stability in mice.Transmission electron microscopy(TEM)was used to observe the morphological characteristics of macrophages in aortic plaques.Prussian blue staining and the Iron assay kit were used to detect iron levels in aortic tissues.Enzyme-linked immunosorbent assay(ELISA)and biochemical reagent kits were used to detect oxidative stress and lipid peroxidation product levels in mouse serum:superoxide dismutase(SOD),malondialdehyde(MDA),4hydroxynonenal(4-HNE),8-isoprostane F2α(8-iso-PGF2α),and glutathione(GSH).ELISA was used to detect the levels of inflammatory factors:interleukin-1β(IL-1β),interleukin-6(IL6),and tumor necrosis factor-α(TNF-α).Immunofluorescence co-localization was used to detect the co-expression and distribution of GPX4 and CD68 in the aortic root.Immunohistochemistry and western blotting(WB)were used to locate and quantitatively detect the expression of GPX4 and xCT proteins in aortas.Results:After 16 weeks of a high-fat diet(including eight weeks of drug gavage),compared with the model group,TC,TG,and LDL-C levels were significantly lower(P<0.05),and HDL-C levels were significantly higher(P<0.05)in the M-QXJYG and the H-QXJYG group.Oil Red O staining of the aorta showed that the model group and the L-QXJYG group had multiple and dense plaques,while the M-QXJYG and the H-QXJYG group had relatively fewer plaques.HE staining showed that the percentage of plaques in each QXJYG group was significantly reduced compared with the model group(P<0.05).Movat staining of the aortic root showed that the proportion of macrophages in plaques was significantly reduced(P<0.05),and the proportion of collagen fibers in plaques was significantly increased(P<0.05)in each QXJYG group compared to the model group.TEM showed that the model group and the LQXJYG group exhibited typical ferroptosis changes,while ferroptosis was alleviated in the MQXJYG and H-QXJYG groups.Prussian blue staining of the aortic root showed no blue precipitates of iron in any group.The Iron assay kit results showed that total iron content in the aortic tissues of each QXJYG group was significantly reduced compared to the model group(P<0.05).ELISA and biochemical assays showed that the GSH and SOD levels were significantly increased(P<0.05),while the MDA.IL-1β,IL-6,and TNF-α levels were significantly decreased(P<0.05)in each QXJYG group compared to the model group,but there were no significant changes in 4-HNE and 8-iso-PGF2α levels(P>0.05).Immunofluorescence colocalization in the aortic root showed that the H-QXJYG group exhibited more expression of GPX4 and less expression of CD68.Immunohistochemistry and WB showed that GPX4 and xCT expression levels were significantly reduced(P<0.05)in each QXJYG group compared to the model group.Conclusion:QXJYG can regulate lipid metabolism,reduce plaque generation,alleviate iron accumulation,oxidative stress and inflammatory response,and inhibit macrophage ferroptosis,thereby stabilizing vulnerable atherosclerotic plaques.The effect may via the GPX4/xCT signaling pathway.Part 3:Effects and mechanism of Qing-Xin-Jie-Yu Granule on regulating ferroptosis in J774A.1 macrophageObjective:To investigate the effects of QXJYG on macrophage ferroptosis in RAS-selective lethal 3(RSL3)-induced J744A.1 cells.Methods:RSL3-induced ferroptosis in J744A.1 macrophage were established and divided into the model group.positive group(Fer-1),and QXJYG group(4%QXJYG-containing serum);J774A.1 macrophages under conventional culture were used as the control group.Cells were treated with QXJYG-containing serum for 24 hours.Alamar Blue assay was used to screen the optimal intervention conditions and detect cell viability.TEM was used to observe the effects of QXJYG on the ultrastructure of cells.FerroOrange fluorescent probe,Liperfluo fluorescent probe,and DFCH-DA fluorescent probe were used to observe the effects of QXJYG on the accumulation of ferrous iron(Fe2+).lipid peroxidation products(LPO)levels,and reactive oxygen species(ROS)levels in J774A.1 macrophage.WB was used to detect the effects of QXJYG on the expression of GPX4 and xCT proteins related to the GPX4/xCT signaling pathway in J774A.1 macrophage.Results:The Alamar Blue assay showed that QXJYG increased the survival rate of cells(P<0.05).TEM showed that the model group presented typical ferroptosis changes,while the Fer-1 group and QXJYG group had more abundant mitochondria,and the number of swollen mitochondria and ferroptotic cells significantly decreased.Compared with the model group,the Fe2+,LPO,and ROS levels of the Fer-1 group and QXJYG group were significantly reduced(P<0.05).WB showed that the QXJYG and the Fer-1 group had higher expression of the GPX4/xCT signal pathway(P<0.05).Conclusion:QXJYG alleviates RSL3-induced ferroptosis in J774A.1 macrophages.The effects partly via the GPX4/xCT signaling pathway.