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Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 6  |  Issue : 2  |  Page : 216-226

Effect of Yangxinshi tablets on the phenotype and function of monocytes in patients with unstable angina pectoris of coronary heart disease


1 Department of Cardiology, The Second Affiliated Hospital of Dalian Medical University; Graduate School, Dalian Medical University, Dalian 116044, China
2 Department of Cardiology, The Second Affiliated Hospital of Dalian Medical University, Dalian 116044, China

Date of Submission13-Oct-2019
Date of Acceptance02-Dec-2019
Date of Web Publication30-May-2020

Correspondence Address:
Prof. Yan-Chun Ding
5th Department of Cardiology, The Second Affiliated Hospital of Dalian Medical University, Dalian 116027
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/wjtcm.wjtcm_39_19

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  Abstract 


Objective: The objective of this study was to observe the effect of Yangxinshi tablets on the phenotype and function of peripheral blood monocytes in patients with unstable angina of coronary heart disease (CHD) and to explore the possible mechanism of Yangxinshi tablets in the treatment of CHD. Methods: A total of 100 patients with unstable angina of CHD were randomly divided into two groups: Group A – treatment group and Group B – control group. The phenotypic and functional changes in blood monocytes and the changes in serum inflammatory factors before and after treatment were compared in the two groups. Results: The expression of CD14+CD163+ interleukin (IL)-10+ was significantly higher in the control group than in the patients with unstable angina of CHD, whereas the expression of CD14+CD163IL-12+ was lower. The concentration of IL-10 was higher in the control group than in the patients with unstable angina, whereas the concentration of tumor necrosis factor-α (TNF-α) and IL-12 was significantly lower. In Group A, the expression of CD14+CD163+, CD14+CD206+, and CD14+CD163+CD206+ in peripheral blood monocytes increased after treatment than before treatment, whereas the expression of CD14+CD163CD206 decreased. The expression of CD14+CD163IL-12+ decreased after treatment than before, whereas CD14+CD163+IL-10+ expression increased. The serum concentration of IL-10 in Group A was higher after treatment than before, whereas that of IL-12 and TNF-α was lower. In Group B, the phenotype and function of the peripheral blood monocytes remained unchanged. Conclusion: Yangxinshi tablet therapy can change the phenotype of the peripheral blood monocytes in patients with unstable angina of CHD. Yangxinshi tablet therapy changes the inflammatory state in patients with an increase in the expression of anti-inflammatory factors and a decrease in the expression of inflammatory factors.

Keywords: Coronary heart disease, inflammatory factor, monocyte phenotype, unstable angina, Yangxinshi tablets


How to cite this article:
Yang LN, Wang MY, Tong YQ, Zhang SN, Wu WX, Ding YC. Effect of Yangxinshi tablets on the phenotype and function of monocytes in patients with unstable angina pectoris of coronary heart disease. World J Tradit Chin Med 2020;6:216-26

How to cite this URL:
Yang LN, Wang MY, Tong YQ, Zhang SN, Wu WX, Ding YC. Effect of Yangxinshi tablets on the phenotype and function of monocytes in patients with unstable angina pectoris of coronary heart disease. World J Tradit Chin Med [serial online] 2020 [cited 2020 Aug 14];6:216-26. Available from: http://www.wjtcm.net/text.asp?2020/6/2/216/276313




  Introduction Top


Coronary heart disease (CHD) is a leading cause of death and a serious threat to human health worldwide. The main pathological mechanism of CHD is the formation and development of atherosclerosis (AS), a chronic inflammatory response in the arterial vascular wall that is regulated by the body's innate and acquired immunity.[1] Monocyte/macrophages are a heterogeneous population of cells that adapt in response to a variety of micro-environmental signals; their phenotypes are very much a function of environmental cues. Macrophages are usually categorized as M1 (classically activated macrophages) or M2 (alternatively activated macrophages) based on their activation states and functions. In the 1980s, Mantovani reported that macrophages had continuous functional states and that M1 and M2 represented the two ends of this spectrum. The ability to switch phenotypes enables macrophages to perform different tasks. For example, M1-like macrophages display a cytotoxic and pro-inflammatory phenotype characterized by strong pathogen and tumor-cell clearance capabilities. In contrast, M2-like macrophages suppress immune and inflammatory responses but participate in tissue remodeling and tumor progression.[2] The relationship between the macrophage subtypes and the formation of AS has become a hot topic of research. The phenotypic polarization of macrophages may have a role in the fate of an atherosclerotic plaque. Pro- and anti-inflammatory macrophages participate in the development of AS. Therefore, understanding the mechanisms that influence the change of macrophage subtype, so as to increase anti-inflammatory polarization or inhibit pro-inflammatory macrophages, has become a new direction of research in AS prevention and control.[3]

Yangxinshi tablet is a traditional Chinese medicinal (TCM) preparation, made by combining ginseng, Ganoderma lucidum, Astragalus, Epimedium, Salvia miltiorrhiza, Angelica, The root of kudzu vine, hawthorn, and few other TCM compositions. The active ingredients are the total flavonoids of gegen, and Tanshinone, Epimedium Total Flavones, ginseng saponins, jaundice, Ferulic acid and Ganoderma lucidum polysaccharide, etc. Yangxinshi tablets are widely used in the treatment of CHD. They have the advantage of precise efficacy and high safety. These tablets are used in treating symptoms and etiology in combination with treatment and rehabilitation in CHD. In our initial studies, we found that Yangxinshi tablets inhibit the macrophages from swallowing lipids, interfere with the formation of foam cells, inhibit the secretion of active macrophages migration inhibitory factor and monocyte chemotactic protein-1 (MCP-1), and promote the transformation of macrophages from M1 to M2, all of which act to stabilize the plaques, inhibit inflammation, and are anti-atherosclerotic.[4] However, whether Yangxinshi tablets retain the above functions during clinical application has yet to be verified. In this study, we analyzed the differences in the phenotypes and functions of peripheral blood monocytes and the blood inflammatory status of healthy individuals and patients with unstable angina of CHD. Further, we explored the possible mechanisms of Yangxinshi tablets in anti-AS and treatment of CHD.


  Methods Top


Subjects

A total of 100 patients with unstable angina of CHD admitted to the Fifth Department of Cardiology, the Second Hospital of Dalian Medical University, between March 2016 and December 2017 were selected for the study. The cohort consisted of 47 males and 53 females, with an average age of 66.38 ± 10.72 years, and 15 healthy volunteers including 7 males and 8 females, with an average age of 54.45 ± 12.43 years. Admission criteria for the patients with unstable angina of CHD were in accordance with the 2007 Guidelines for the Diagnosis and Treatment of Unstable Angina and Non-ST-Segment Elevation Myocardial Infarction. The patients admitted had the following clinical manifestations: (1) resting angina: the angina pectoris occurs at rest and lasts for more than 20 min; (2) initial angina: The angina newly occurred within 1 month and may be characterized by spontaneous or exertional onset with pain scale at Level III or above; and (3) exacerbated exertional angina: The patient has a history of angina pectoris, the angina was exacerbated in a month and occurred frequently, the time was prolonged, or the pain threshold was reduced. The main exclusion criteria were the presence of (1) acute myocardial infarction, acute heart failure, poor management of severe hypertension, or severe arrhythmia; (2) severe diabetes and poor glucose control; (3) acute exacerbation of COPD; (4) severe skin diseases; (5) malignant tumors; (6) a history of autoimmune diseases or immunodeficiency; and (7) a variety of acute or chronic infectious diseases and severe liver and rental diseases. The patients (100 in total) with unstable angina of CHD were randomly divided into two groups: Group A – Yangxinshi tablet treatment group, with routine internal medicine, antiplatelet, and lipid-regulating treatment with Yangxinshi tablets (Shanghai Pharma Group Qingdao Growful Pharmaceutical Co. Ltd., GYZ Zi Z37021102, 0.6 g/tablet), three times a day, orally for 3 months and Group B – routine internal medicine, antiplatelet, and lipid-regulating treatment group for 3 months.

Materials

Human peripheral blood lymphocyte separation solution was purchased from Sigma. CD14-PerCP, CD163-FITC, CD206-PE, interleukin (IL)-10-PE, IL-12-APC, IgG-FITC, IgG-PE, and IgG-APC were purchased from BD. Human IL-10, IL-12, and tumor necrosis factor-α (TNF-α) ELISA kits were purchased from Abcam. The microplate reader (MD190), flow cytometer (BD FACS Calibur), 37°C carbon dioxide incubator, inverted phase-contrast microscope, and high-speed benchtop centrifuge were all provided by the Research Center of The Second Hospital of Dalian Medical University.

Basic clinical data

The gender, age, history of hypertension, history of diabetes, smoking history, height, and weight of all patients were recorded, and the body mass index (BMI) was calculated based on their weight and height (kg/m [2]). The total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), alanine aminotransferase (ALT), aspartate transaminase (AST), albumin (ALB), serum creatinine (SCr), urea nitrogen (UREA), cystatin C (Cys C), and uric acid (UA) were analyzed and recorded for all the patients.

Blood specimen collection

A total of 10 ml of peripheral venous blood was collected from all the patients and placed in ethylenediaminetetraacetic acid (EDTA) anticoagulation tubes in the morning in a fasting state for the extraction of monocytes, The collected blood samples were placed in a common serum tube at 3 ml, 2000 r / min, and centrifuged for 10 minutes at a reduced speed. The supernatant was collected in a 2 ml cryopreservation tube and stored in a -80 ° C refrigerator for testing.

Extraction of monocyte and determination of the phenotype and function

The peripheral blood monocytes were isolated using modified Ficoll-Hypaque density separation. Equal volumes of peripheral blood (anticoagulated with EDTA in two sterile 15 ml centrifuge tubes in the morning) and phosphate-buffered saline (PBS) solution were mixed. A total of 5 ml of lymphocyte separation solution was added to the two tubes, and the two-fold diluted peripheral blood was layered on the lymphocyte separation liquid slowly and uniformly along the tube walls and centrifuged at reduced speed 2000 rpm for 30 min at constant temperature. The buffy coat of the second layer was slowly sucked out with a pipette and washed twice, and the supernatant was discarded. Then, 200 μl of PBS was added, and the cells were counted and resuspended at a concentration of 1 × 10[7] cells/ml for further experiments.

To determine the monocyte phenotype, fluorescently labeled CD14 PerCP, CD163 FITC, CD206 PE, and isotype-matched control (IgG-FITC and IgG-PE) were added sequentially to 100 μl of the extracted monocyte suspension in a round bottom flow tube and incubated in the dark for 30 min. Then, 250 μl of PBS was added, and the cells were analyzed using flow cytometry.

To determine the monocyte function, the expression of CD14+ CD163 IL-12+ M1 and CD14+ CD163+ IL-10+ M2 monocytes was detected using flow cytometry. A total of 100 μl of monocyte suspension was added to each of the two wells in a 24-well plate and labeled as control and stimulation wells. Then, 50 μl of 0.1 mg/ml PMA (1-Methoxy-2-propyl acetate) and 50 μl of 0.5 mg/ml ionomycin were added into the stimulation well, whereas an equal volume of PBS was added to the control well to prepare 1000 μl of cell suspension, and the mixtures were incubated in an incubator for 1 h. The incubated plate was removed, and 0.5 mg of BFA (BrefeldinA) at 0.05 mg/ml was added to the stimulation well and the control well and replaced in the incubator for further 4 h. The cell suspension in the control and stimulation wells was transferred to flow tubes and centrifuged at 2000 rpm for 10 min. The supernatant was discarded, and 100 μl of cell suspension was prepared after the PBS washes. Fluorescently labeled CD14, CD163, and corresponding isotype controls were added to the two tubes and incubated for 20 min in the dark. Cell punching agent (250 μl) was added separately, and the mixture was incubated in a 4°C incubator for 20 min and then centrifuged at 2000 rpm for 10 min. The supernatant was discarded, and 100 μl of cell suspension was prepared after PBS washing. Fluorescently labeled IL-10, IL-12 and corresponding isotype controls were incubated for 30 minutes in the dark. After the incubation is completed, 200ul of PBS is added to the sample, and the fluorescence labeling result is detected by flow cytometry.

Inflammatory cytokine assay

The concentrations of IL-10, IL-12, and TNF-α in the serum were analyzed by ELISA following the protocol provided with the kit.

Statistical analysis

The SPSS statistical software for Windows (version 20.0 United States, IBM Corporation) was used for all statistical analyses. All measurements were expressed as mean ± standard deviation values of P < 0.05 were considered statistically significant. Normal distribution was tested for all continuous variables, and skewed data were normalized either by log or square root transformation. Comparisons between the groups for normally distributed data were performed with nonpaired t-tests and comparisons for nonparametric data were made with Mann–Whitney test. Analysis of variance was used for comparison among multiple groups, and least significant difference method was further used for pairwise comparison.


  Results Top


Basic clinical data of patients with unstable angina of coronary heart disease and healthy controls

The basic clinical data pertaining to the patients with unstable angina of CHD and the healthy controls are summarized in [Table 1]. There was no significant difference in age, BMI, SCr, UA, ALT, AST, ALB, UREA, Cys C, HDL-C, LDL-C, and TG between the two groups (P > 0.05). The level of TC in patients with unstable angina of CHD was significantly higher than that in the control group (P< 0.05).
Table 1: Comparison of basic clinical data between unstable angina group and control group

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Comparison of phenotype and function of peripheral blood monocytes between the unstable angina group and the control group

The morphology of peripheral blood monocytes is shown in [Figure 1]. For determination of the monocyte phenotype, the expressions of CD14+ CD163+, CD14+ CD206+, and CD14+ CD163+ CD206+ were analyzed. The expressions were significantly higher in the control group than in the unstable angina group [P< 0.05, [Table 2] and [Figure 2]. The expression of CD14+ CD163 CD206 in the control group was lower than that of patients with unstable angina [P< 0.05, [Table 2] and [Figure 2]. In terms of monocyte functions, the expression of CD14+ CD163+ IL-10+ was significantly higher in the control group than in the patients with unstable angina [P< 0.05, [Table 2] and [Figure 3]. The expression of CD14+ CD163 IL-12+ in the control group was lower than that of patients with unstable angina [P< 0.05, [Table 2] and [Figure 3]. Their results showed that compared to the control group, the phenotype of the peripheral blood monocytes in patients with unstable angina of CHD was M1 with the functional state of inflammatory activation.
Figure 1: (a) The morphology monocyte under of microscope (×40). (b) The morphology monocyte under of microscope (×200)

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Table 2: Comparison of phenotype and function of peripheral blood monocytes between unstable angina group and control group

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Figure 2: Expressions of CD163 and CD206 on monocyte of different groups

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Figure 3: Determination of monocyte function in different group (CD14+CD163+IL-10+, CD14+CD163-IL-12+ expression)

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Comparison of serum inflammatory factors between the control group and the unstable angina group

The concentration of IL-10 in the serum of the control group was higher than that of patients with unstable angina [P< 0.05, [Table 3]. The concentration of TNF-α and IL-12 in the serum of the control group was significantly lower than those of patients with unstable angina [P< 0.05, [Table 3]. These data indicated that the blood inflammation was in an activated state in patients with unstable angina, the pro-inflammatory cytokines TNF-α and IL-12 were elevated, and the anti-inflammatory factor IL-10 was decreased.
Table 3: Concentration of interleukin-10, interleukin-12, and tumor necrosis factor-a in serum of control group and unstable angina group

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Comparison of general conditions of patients with coronary heart disease and unstable angina pectoris with Yangxin's combined with conventional medical treatment group and conventional medical group

A total of 100 patients with unstable angina of CHD were randomly divided into two groups: Group A: Yangxinshi tablets treatment group, routine internal medicine, antiplatelet, and lipid-regulating treatment with Yangxinshi tablets, three times a day, orally for 3 months and Group B: routine internal medicine, antiplatelet, and lipid-regulating treatment group for 3 months. There were no significant differences between the two groups in terms of age, gender, BMI, and basic treatment with antihypertensive, lipid-regulating, and anticoagulant drugs [P > 0.05, [Table 4]. No significant differences were detected between the two groups in terms of SCr, UA, UREA, Cys C, ALT, AST, ALB, HDL-C, LDL-C, TG, and TC levels [P > 0.05, [Table 4].
Table 4: Comparison of basic clinical data between two groups

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Effect of Yangxinshi tablets on monocyte phenotype and function in patients with unstable angina

After treatment with Yangxinshi tablets and routine internal medicine for 3 months, the expression of CD14+ CD163+, CD14+ CD206+, and CD14+ CD163+ CD206+ significantly increased in Group A compared to the expression before treatment [P< 0.05, [Table 5] and [Figure 4]. However, the expression of CD14+ CD163 CD206 significantly decreased after treatment than before treatment [P< 0.05, Tables 5 and [Figure 4]. With regard to monocyte function, the expression of CD14+ CD163+ IL-10+ was higher after treatment than before treatment, whereas the expression of CD14+ CD163 IL-12+ was lower after treatment than before treatment in Group A. The differences were statistically significant [Table 5] and [Figure 5]. The IL-10 level in the serum of patients measured by ELISA was significantly higher after treatment than before treatment, whereas that of IL-12 and TNF-α decreased significantly following treatment in Group A [P< 0.05 and [Table 6]. In Group B, the monocyte phenotype and function, including the serum levels of IL-10, TNF-α, and IL-12, were not significantly different before and after treatment [P > 0.05, [Table 7], [Table 8] and [Figure 6], [Figure 7]. These results indicated that Yangxinshi tablets could change the phenotype and function of monocytes and the inflammatory status of patients with unstable angina, suggesting that Yangxinshi treatment-mediated change in the phenotype of monocytes could promote its anti-inflammatory transformation in patients with unstable angina. This change causes the upregulation of anti-inflammatory factors and downregulation of pro-inflammatory factors.
Table 5: Phenotypic and functional expression of monocytes before and after treatment of Group A

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Figure 4: Phenotypic expression of monocytes before and after treatment of Group A

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Figure 5: Functional expression of monocytes before and after treatment of group A

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Table 6: Changes of serum inflammatory factors before and after treatment of Group A

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Table 7: Phenotypic and functional expression of monocytes before and after treatment of Group B

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Figure 6: Phenotypic expression of monocytes before and after treatment of group B

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Figure 7: Functional expression of monocytes before and after treatment of group B

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Table 8: Changes in serum inflammatory factors before and after treatment of Group B

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  Discussion Top


CHD is a heart disease caused by myocardial ischemia, hypoxia, and necrosis resulting from coronary stenosis or even blockage due to coronary AS. Unstable angina is a type of acute coronary syndrome caused by acute or subacute cardiac oxygen supply reduction due to inner membrane bleeding of the coronary artery, plaque rupture, platelet and fibrinogen condensation, thrombosis, coronary spasms, and distal small vessel obstruction due to AS. The atherosclerotic lesions in patients with unstable angina of CHD are composed of various cell types that can produce cytokines to promote anti-inflammation and participate in the formation of plaques in the vascular wall.

Immunoinflammatory responses occur throughout the course of cardiovascular diseases and are closely related to the occurrence and development of coronary AS. The monocyte/macrophage system constitutes the main effector cells of innate immunity, which is involved in the pathophysiological processes of apoptosis, clotting, hypoxia, and vascular regeneration, and plays an important role in the formation of coronary AS. MCP-1 through binding to endothelial cell membrane glycoprotein, and under the action of human macrophage colony-stimulating factor, can promote monocytes to reach the endovascular membrane, so that it firmly adheres to the endothelial cells, and through signal transduction pathways promotes monocytes rolling along endothelial cells and transendothelial migration.[5] In the early stage of AS, low-density lipoproteins undergo numerous oxidative modifications to produce oxidized cholesterol, which increases the adhesion of monocytes to the arterial endothelium. The number of monocytes adhering to the endothelial cells is increased. They are attracted by chemotaxis to migrate between endothelial cells and converted into macrophages by cytokine stimulation.[6] The macrophages phagocytose the oxidized low-density lipoproteins into foam cells through scavenger receptors, activated by the action of the chemokine factor, then induce the recruitment of t-cells and mast cells, and promote smooth muscle cells to enter the atherosclerotic plaques and thereby promote the progress of AS. With the progression of AS, the apoptosis of macrophages and defects in their phagocytosis function can increase the inflammation of the atherosclerotic plaque, leading to the increase and instability of plaques.

As early as 1980, Russell Rose found macrophage infiltration in atherosclerotic plaques and proposed an inflammatory hypothesis of AS that has now been confirmed through extensive research. The activation of macrophages occurs throughout the formation and development of atherosclerotic plaques. Macrophages in atherosclerotic plaques are a group of highly heterogeneous cells that react differently to different signals in the microenvironment. Their phenotype is adapted to the functions of the microenvironment in which they are located. Macrophages were divided into classically activated (M1) and alternatively activated (M2) according to the classification of Th1 and Th2 cells. M1/M2 macrophages are involved throughout the development of AS.[7] The transformation of M1/M2 macrophages in the body and the conversion to either type affects the outcome of AS. The M1 macrophages secrete pro-inflammatory factors such as IL-12 and TNF-α, promoting inflammation occurrence which is associated with plaque development and rupture.[8] However, the M2 macrophages secrete anti-inflammatory cytokines, such as IL-10, and have an anti-AS effect. Studies have shown that M1 macrophages are mainly present in unstable atherosclerotic plaques. In the advanced stage of coronary AS, the cytokines produced in the patient's peripheral blood activate the M2 macrophages, promote fibrous production, repair the damage site, and maintain plaque stability. Many studies have demonstrated that the macrophages of types M1 and M2 have some differences in the expression of cytokines. The M1 macrophages are differentiated from Ly6C high monocytes, and their classical activation pathway which includes activation by lipopolysaccharide in the presence of interferon-γ (IFN-γ) that causes the increased expression of CD16, CD32, IL-12, IL-2, IL-23, IL-6, IL-1β, and TNF-α, while CD163, CD206, IL-10, etc., are highly expressed in the M2 macrophages.[9] In patients with CHD, the macrophage phenotype changes mainly through the following processes: (1) regulation of cellular metabolism: the M1 macrophages mainly rely on the glycolytic pathway, whereas the M2 macrophages mainly rely on fatty acid oxidative metabolism pathway. For the patients with CHD, the glycolytic activity of the M1 macrophages is enhanced. The rate-limiting enzyme M2 isoform of pyruvate kinase aggregation and translocation can promote the production and release of the pro-inflammatory cytokines. In the process of AS regression, fatty acid oxidative metabolism is more prominent and supplies energy in the long-term, resulting in a change in the balance of M1/M2 macrophages, and M2 macrophages become relatively increased, playing a role of anti-inflammation and stabilization of plaques.[10] (2) Regulation of lipids: The pro-inflammatory lipid regulator-oxidized phospholipid promotes the expression of pro-inflammatory genes in macrophages while activating inflammasomes by activating nuclear transcription factor 2 and thereby promoting the progression of AS. On the other hand, anti-inflammatory lipid regulators, polyunsaturated fatty acids, prevent the activation of pro-inflammatory genes in macrophages by counteracting saturated fatty acids, whereas high-density lipoproteins cause the increased expression of M2 macrophages in the atherosclerotic plaques by promoting reverse transcription of cholesterol.[11] (3) Antibacterial peptides: The only cathepsin statin class antibacterial peptide, LL-37, in human body is combined with vascular endothelial cells to activate the formazin receptor-2 of the M1 macrophages. Serum LL-37 levels are significantly increased in patients with unstable angina of CHD.[12] In this study, we found that compared to the healthy control group, the monocyte phenotype of patients with unstable angina of CHD was mainly of the pro-inflammatory M1 type, and the inflammatory factors IL-12 and TNF-α were elevated, and the monocytes were functionally in the state of inflammatory activation.

The IL-10 gene is located on chromosome 1 at 1q31-32. Three mutation-critical gene sitesIL-10 gene promoters -1082A / G, -819T / C and -592A / CA are present upstream of the transcription starting site of the IL-10 promoter and influence the expression of IL-10. It is reported that the −1082 AA IL-10 genotype is associated with a decrease in the level of IL-10 and consequently a decrease in the risk of developing CHD. In a meta-analysis of 16 recent case-control studies (involving 7779 cases and 7271 controls), the genotype AA at IL-10- 1082 was associated with the increased risk of AS and with coronary artery disease susceptibility.[13] IL-10 is produced by macrophages, Th2 cells, B-cells, and monocytes. IL-10 inhibits the adhesion and immersion of inflammatory cells during the inflammatory response and can inhibit the synthesis and secretion of cytokines caused by various inflammatory factors. It can induce the expression of anti-inflammatory cytokines, such as IL-4, and therefore act as an anti-inflammatory cytokine. Over the past few years, a series of studies have confirmed the role of endogenous IL-10 in animal models of AS. In mice with dual gene knockout of the ApoE and IL-10 genes, compared to a high-fat diet fed to mice with the ApoE gene knockout alone, the former enhanced the process of forming early atherosclerotic plaques. Mice with IL-10 gene defects were found to have more accumulation of immune cells, increased activation of T-cells, and increased pro-inflammatory factors in the early stages of atherosclerotic lesions, making them more likely to develop AS.[14] These results support the role of IL-10 in preventing the development of atherosclerotic lesions. Therefore, the molecules that regulate IL-10 expression can affect the formation and development of AS.[15]

IL-12, also known as NK cell stimulation factor, is produced by antigen-carrying cells and B-cells. IL-12 is a preinflammatory cytokine in the form of a heterogeneous dipolymer and is secreted out of the cell in this form. The plasma IL-12 level of patients with CHD is significantly higher than that of healthy individuals and has become one of the inflammatory markers of CHD. Studies have shown that IL-12-deficient mice have a slower progression of atherosclerotic plaque even on a high-fat diet. Injection of exogenous IL-12 can promote plaque progression.[16] IL-12 plays an important role in the development of instability angina mainly by affecting the polarization of the Th1 cells. IL-12 induces the activation of STAT4 transcription factor and T-box transcription factor T-bet, induces differentiation of CD4+ T-cells into Th1 cells, and promotes upregulation of IFN-γ, TNF-α, and IL-2 in Th1 cells. The IFN-γ produced can activate macrophages and dendritic cells, promoting their ability of present antigen, and cause the polarization of Th1 cells.[17]

TNF-α, secreted by endothelial cells, smooth muscle cells, and macrophages, is a cytokine with a variety of biological activities, including immunomodulation and enhancing monocyte activity, which plays an important role in regulating the immunoinflammatory response balance. It is expressed as a pro-inflammatory factor in early coronary lipid deposition and atherosclerotic plaques. Studies have shown that TNF-α levels are increased in the peripheral circulation in patients with CHD and those with acute coronary syndrome.

In this study, we found that the expression of M2 monocytes and its secreted cytokine, IL-10, was significantly higher in healthy individuals than in patients with unstable angina of CHD. Further, the expression of M1 monocytes and their secreted cytokines IL-12 and TNF-α was significantly lower in healthy individuals than in patients with unstable angina. Therefore, we infer that differences in monocyte/macrophage phenotype and function in patients with unstable angina may be the cause of the AS onset or progression. Currently, the relationship between monocyte/macrophage phenotype change and AS has become a hot topic of research. Pro- and anti-inflammatory macrophages jointly participate in the development of AS. Identifying ways to influence the change in the macrophages phenotypes, so that macrophages may be directed toward an anti-inflammatory polarization or ways to inhibit the production of pro-inflammatory macrophages, has become a new direction in AS prevention and treatment.

Our study found that compared to the conventional treatment, after 3-month treatment with Yangxinshi, the phenotype and function of peripheral blood monocytes changed in patients with unstable angina. The expression of CD14+ CD163+ CD206+ was upregulated in the monocytes, whereas the expression of CD14+ CD163 CD206 was downregulated. Concomitantly, the monocyte function also changed, CD14+ CD163+ IL-10+ expression was upregulated, and CD14+ CD163 IL-12+ expression was downregulated, confirming the transformation of monocytes from a pro-inflammatory type to an anti-inflammatory type. The level of IL-10 in the serum was higher, whereas that of IL-12 and TNF-α was lower after treatment than before treatment. However, there were no significant changes in the conventional treatment group. Our data suggest that Yangxinshi treatment can change the peripheral blood monocyte phenotype in patients with unstable angina, promote its transformation from pro-inflammatory M1 type to anti-inflammatory M2 type, adjust the inflammatory state of the patient, upregulate the anti-inflammatory cytokine IL-10 in serum, and downregulate the pro-inflammatory cytokines IL-12 and TNF-α.

Yangxinshi is a TCM preparation derived from the clinical experience of one of the famous veteran doctors of TCM, Ciqing Zhou, and includes a total of 13 Chinese herbal ingredients, such as ginseng, Astragalus, S. miltiorrhiza, Rhizoma corydalis, hawthorn, Codonopsis pilosula, Lucid ganoderma, Radix puerariae, Angelica sinensis, Epimedium, Rehmannia, Coptis chinensis, and honey-fried licorice root.[18] The active ingredients in Yangxinshi, such as Astragalus and R. puerariae, increase myocardial energy metabolism, increase myocardial blood flow, reverse myocardial remodeling, and have positive effects on ischemic cardiomyopathy.[19]G. lucidum has the functions of mind-tranquilizing, calming, and improving specific humoral immune.[20] To sum up, the active ingredients in Yangxinshi can improve myocardial blood supply, relieve clinical symptoms through improved circulation, disperse blood stasis, relieve pain, dispel melancholy, and instill mind-tranquilizing effect [21] through anti-inflammatory, antiplatelet aggregation, lipid regulation, and improved circulation. It can also interfere with the process of AS through multiple targets, thereby playing a role in the treatment of CHD. Therefore, it is widely used in the clinical treatment of CHD. In this study, through clinical treatment with Yangxinshi, it has been demonstrated that Yangxinshi can regulate the phenotypic and functional changes of monocyte/macrophage to reduce pro-inflammatory factors, increase the expression of anti-inflammatory factors, and change the inflammatory state in patients with CHD, thus stabilizing the plagues and relieving the symptoms of CHD.


  Conclusion Top


In patients with unstable angina of CHD, the peripheral blood monocytes present a pro-inflammatory phenotype. The inflammatory state is activated, the expression of pro-inflammatory factors in the blood is increased, and the expression of anti-inflammatory factors is decreased. Yangxinshi can change the phenotype of the peripheral blood monocyte in patients with unstable angina of CHD to promote its anti-inflammatory transformation and change the inflammatory state in the body, leading to the upregulated expression of anti-inflammatory factors and the downregulated expression of pro-inflammatory factors.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]



 

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