Nao-Xin-Qing tablet inhibits macrophage inflammatory response in atherosclerosis via AMPK-α/SIRT1/PPAR-γ pathway

Abstract

Background and aim

Nao-Xin-Qing (NXQ) tablets are standardized proprietary herbal products containing an extract of Chinese persimmon leaves (Diospyros kaki L.f., World Checklist) and other natural ingredients. NXQ has also been indicated for atherosclerosis (AS), but the mechanisms of its antiatherosclerotic activity are unclear. In this study, its mechanisms were investigated by using preclinical models, and provide evidence for its potential application against cardiovascular disorders.

Experimental procedure

In vivo, the apolipoprotein E-deficient (ApoE^−/−) mice were fed with a high-fat diet to induce AS. And the mice were treated with different concentrations of NXQ and Lipitor for 12 weeks. After the intervention, serum lipid levels and serum inflammatory factor levels were measured. The pathological changes in the aorta were observed by Oil-red-O staining and Hematoxylin and Eosin (HE) staining. Additionally, we investigated macrophage polarization both in vivo and in vitro. Using the NXQ fingerprint, we conducted network pharmacological analysis to predict and explore its antiatherosclerotic mechanism, which was validated in AS mice and LPS-induced macrophages.

Results

In our study, we found that NXQ significantly reduced atherosclerotic plaques in the aortic root and aorta and decreased serum lipid levels in HFD-fed ApoE^−/− mice. Meanwhile, NXQ promoted M2 macrophage polarization, which is regulated by the AMPK-α/SIRT1/PPAR-γ axis. Importantly, suppressing AMPK-α eliminated the effect of NXQ on macrophages.

Conclusion

NXQ exerted a preventive effect on the development and progression of AS by promoting M2 macrophage polarization through modulation of the AMPK-α/SIRT1/PPAR-γ axis.

Graphical abstract

Highlights of the findings and novelties

• •Naoxinqing is an antiatherosclerotic, which can regulate macrophage polarization.
• •Naoxinqing can regulate the expression level of AMP-activated protein kinase-α.
• •Naoxinqing offers an alternative for patients experiencing adverse reactions to Lipitor.

List of abbreviations

ACC

Acetyl-CoA carboxylase

AMPK-α

AMP-activated protein kinase-α

Arg-1

Arginase-1

AS

Atherosclerosis

ApoE ^−/−

Apolipoprotein E ^−/−

BCA

Bicinchoninic acid

BPs

Biological processes

CC

Compound C

CCK-8

Cell Counting Kit-8

DEGs

Differential expression genes

DMSO

Dimethyl sulfoxide

ECL

Enhanced chemiluminescence

ELISA

Enzyme-linked immunosorbent assay

ETCM

Encyclopedia of Traditional Chinese Medicine

FBS

Fetal bovine serum

Fizz 1

Found in inflammatory zone 1

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

HDL-C

High-density lipoprotein cholesterol

HFD

High-fat diet

H&E

Hematoxylin and eosin

HPLC

High performance liquid chromatography

IL-1β

Interleukin-1β

IL-10

Interleukin-10

IL-6

Interleukin-6

iNOS

inducible nitric oxide synthase

JAK

Janus kinase

LDL-C

Low-density lipoprotein cholesterol

LPS

Lipopolysaccharide

NF-κB

Nuclear factor-kappa B

NOS

Nitric oxide synthetase

NXQ

Naoxinqing

MyD88

Myeloid differentiation factor 88

MFs

Molecular functions

OMIM

Online Mendelian Inheritance in Man

ORO

Oil red O

PBS

Phosphate buffer saline

PGC1α

Peroxisome proliferator-activated receptor gamma coactivator-1 alpha

PPAR-γ

Peroxisome proliferator-activated receptor-γ

PPI

Protein-Protein Interaction

PS

Penicillin–streptomycin

QAMS

Quantitative analysis of multi-components by single-marker

qRT‒PCR

Quantitative reverse transcription-polymerase chain reaction

RAW264.7

mouse leukemia cells of monocyte macrophage

SIRT1

Sirtuin 1

STAT6

Signal transducer and activator of transcription 6

TBS-T

Tris-buffered saline with 0.1 % tween 20

TC

Total cholesterol

TCM

Traditional Chinese medicine

TCMSP

Traditional Chinese Medicine Systems Pharmacology

TG

Triacylglycerol

TLR4

Toll-like receptor 4

TNF-α

Tumor necrosis factor-α

3D

Three-dimensional

1. Introduction

Atherosclerosis (AS) is a chronic inflammatory disease involving the arterial wall, characterized by progressive lipid accumulation and inflammatory reaction, in which the build-up of vessel-occluding plaques can lead to stenosis in medium- and large-size arteries.¹ AS plaques are mainly composed of necrotic cores, accumulated lipids, infiltrating macrophages, and fibrous caps formed by proliferative smooth muscle cells. The small necrotic core and thick fibrous membrane are the keys to plaque stability. A pronounced inflammatory response can cause a large number of inflammatory cells in plaques to infiltrate and secrete metalloproteinases, degrade the extracellular matrix, greatly increase plaque instability, and thus more easily induce plaque rupture, leading to the occurrence of acute ischemic events. Clinically, current conventional therapies for AS include statins, aspirin, nitroglycerin, nonsteroidal anti-inflammatory drugs, and so on. For example, Lipitor (atorvastatin) has been proven to have observable effects on promoting the polarization of macrophages toward the M2 phenotype, which has protective effects in the process of AS.² However, some side effects limit their clinical application, such as the side effects observed with long-term use of statins, including liver damage, rhabdomyolysis, constipation, flatulence, and so on.^3,4 Consequently, developing more effective and safer strategies for AS treatment is urgently needed.

As commonly understood, effective treatment of AS hinges on plaque stability, with macrophage infiltration and its polarization direction being pivotal factors influencing plaque stability. Macrophages are key cells in tissue homeostasis and inflammatory processes, playing indispensable roles in various stages of AS. Their main functions include responding to pathogens and regulating adaptive immune responses, inducing and dissipating inflammation, tissue repair, and homeostasis.⁵ During the early stages, a significant migration and infiltration of macrophages into the blood vessel wall occur, where they uptake ox-LDL to form foam cells, thereby facilitating the expansion of the plaque lipid core. As the disease advances, the imbalance between pro-inflammatory and anti-inflammatory macrophages progressively worsens. In the later stages, there's a substantial release of pro-inflammatory factors from macrophages infiltrating the plaque shoulder, further aggravating plaque instability.^6,7 Based on the distinct polarization phenotypes of macrophages, they are categorized into M1 and M2 types. M1 macrophages primarily secrete pro-inflammatory factors, whereas M2 macrophages facilitate inflammation regression and tissue repair. Macrophages are dynamic and adaptable, and their differentiation is influenced by a variety of factors, such as the specific microenvironment and the metabolic state of the macrophage.⁸ Promoting the regression of vascular plaque inflammation by modulating macrophage polarization represents a novel strategy for atherosclerosis treatment.

According to the Pharmacopoeia of the People's Republic of China 2020 edition, The Nao-Xin-Qing (NXQ) tablet is a pure formulation of traditional Chinese medicine refined by modern extraction methods.⁹ A variety of chemical compounds can be found in the fresh or dried leaves of Chinese persimmons (Diospyros kaki L.f., World Checklist), chiefly protocatechuic acid, hyperoside, isoquercitrin, quercetin, and kaempferol, and they can regulate blood lipids, inhibit arteriosclerosis, scavenge oxygen free radicals, and inhibit excessive lipid oxidation.10, 11, 12 Besides, NXQ has been shown to have a favorable effect on the treatment of cerebral apoplexy and atherosclerosis, as well as the protection of nerve and myocardial cell activity in past studies.^13,14 However, the specific mechanism is still unknown.

In this study, our aim was to investigate NXQ's potential in ameliorating AS and modulating macrophage polarization. Furthermore, a combination of fingerprinting and network pharmacology was employed to predict and explore the anti-atherosclerosis mechanism of NXQ, which was subsequently validated both in vivo and in vitro.

2. Materials and methods

2.1. Chemicals and reagents

The following chemicals and reagents were used in this study: HFD (TP26305, Trophic Animal Feed High-Tech Co., Ltd, China), DMEM (PM150210, Procell, Wuhan, China), FBS (164,210–50, Procell, Wuhan, China), PrimeScript™ RT Reagent Kit (Takara, Osaka, Japan), LPS (Lot No. 5164948, Lianke Biotechnology, Hangzhou, China), CCK-8 kit (GK10001, GLPBIO, CA, USA), TrIzol™ Reagent (15596018CN, Invitrogen, USA), JC-1 Kit (C2006, Beyotime, Nantong, Jiangsu, China), Total cholesterol assay kit, Triglyceride assay kit, High-density lipoprotein cholesterol assay kit, and Low-density lipoprotein cholesterol assay kit (A111-1-1, A110-1-1, A112-1-1, A113-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), IL-10, IL-6 and TNF-α ELISA kits (MM-0176M1, MM-1011M2, MM-0312M1, Jiangsu Meimian Industrial Co., Ltd), RIPA Lysis Buffer (P0013B, Beyotime Biotechnology Co., Ltd., Shanghai, China). BCA protein assay kit (P0010, Beyotime Biotechnology Co., Ltd., Shanghai, China). Oil red O staining kit (G1261, Service, Wuhan, China). Dorsomorphin dihydrochloride (HY-13418, MedChemExpress, USA). APC anti-mouse CD86 Antibody (105,011, Biolegend, CA, USA). CD206 Antibody (60143-1-Ig, Wuhan Sanying Biotechnology Co., LTD). FITC anti-mouse F4/80 Antibody (123,107, Biolegend, CA, USA). Antibodies against PPAR-γ (2443, 1:1000), NF-κB P65 (4764, 1:1000), GAPDH (2118, 1:1000), and phosphor–NF–κB P65 (3033, 1:1000) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against AMPK-α (ab176323, 1:1000), SIRT1 (ab52617, 1:1000), and p-AMPK-α (ab33915, 1:1000) were purchased from Abcam (Cambridge, UK). The secondary antibodies were purchased from Abcam Co., Ltd. (ab288151, Shanghai, China).

2.2. Drug preparation

Preparation of Nao-Xin-Qing tablet (Hutchison Whampoa Guangzhou Baiyunshan Chinese Medicine Co., Ltd): took 50 g persimmon leaf extract and added appropriate amount of starch, sucrose powder, magnesium stearate, microcrystalline cellulose as well as other auxiliary materials; then mixed them well, made the mixture into granules, dried them, pressed them into 1000 or 500 tablets and then coated the tablets with film.

Preparation method of persimmon leaf extract: took dried persimmon leaves, and added water to boil twice (the first time for 2 h, the second time for 1 h); then combined the decoction, filtered it, and concentrated the filtrate to a relative density of 1.12–1.15 (60 °C); added ethanol to reach 85 % alcohol content, let it stand overnight, filtered the supernatant, and set aside; subsequently washed the sediment twice with 65 % ethanol, combined the washing solution, let it sit overnight, filtered out the supernatant, and merged it with the backup supernatant; next, recovered ethanol, added an appropriate amount of water and mixed well; after mixing, filtered the mixture, extracted the filtrate with ethyl acetate four times, combined the ethyl acetate solution, recovered the ethyl acetate and concentrate it into a thick paste, and finally dried it at low temperature to obtain the extract.

2.3. Animals

Guangzhou University of Chinese Medicine's Ethics Committee approved the in vivo study procedures (No. SCXK 20, 201, 229,009). Beijing Vital River Laboratory Animal Technology Co., Ltd. (License no.: SCXK (Beijing, China) 2021–0006) provided the ApoE^−/− male mice on C57BL6 backgrounds at five weeks of age. All animal studies were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and the China Physiological Society's Guiding Principles in the Care and Use of Animals. For one week, mice were maintained in a typical laboratory environment (12/12 h light/dark cycle, 45–55 % humidity, 22 ± 1 °C temperature) with free access to standard chow.

After one week of acclimatization to the diet, Apoe^−/− mice were randomly assigned to six groups. The normal control group was fed a standard diet, while the other five groups, including the model group, NXQ-L group, NXQ-M group, NXQ-H group, and Lipitor group, were fed a high-fat diet for 12 weeks. HE staining was used to determine whether the model was successful by measuring plaque formation in the mouse aorta. After establishing the model, NXQ groups received oral doses of 40, 80, and 160 mg/kg NXQ daily for 12 weeks. The Lipitor group received 10 mg/kg Lipitor orally, while the model and blank control groups were given normal saline.

2.4. Blood lipid detection and enzyme-linked immunosorbent assay (ELISA)

To measure blood lipids, fasting blood samples were taken from the orbital at the end of the study. The serum was centrifuged at 12,000 g and 4 °C for 15 min and analyzed immediately after centrifugation. Serum lipid level were determined by enzymatic colorimetric methods using commercial assay kits, including TC, HDL-C, LDL-C, TG four indexes.

In vivo, anti-inflammatory effects were evaluated by ELISA. Serum from ApoE^−/− mice were used as the test sample after centrifugation. IL-6, TNF-α, IL-10, and Arg-1 cytokines were detected by ELISA kits according to the manufacturer's instructions. A Multiskan GO microplate reader (Thermo Fisher Scientific, USA) was used to measure the absorbance (reference length at 570 nm).

2.5. Oil-red-O staining and HE staining

Through the left ventricle, anesthesia was administered to the animals and PBS was perfused. We carefully dissected entire aortas, and gently removed the connective tissue and fixed it with 4 % paraformaldehyde. Oil-red-O staining was employed to evaluate lipid deposition in the entire aorta and the aortic root, while HE staining was conducted to analyze morphological lesions in the abdominal aorta. We captured images using a Leica microscope and analyzed the data using Image-Pro Plus software (Image-Pro Plus, RRID: SCR_007369).

2.6. High performance liquid chromatography

Using Agilent Eclipse Plus C₁₈ chromatographic column (250mm × 4.6 mm, 5 μm), the mobile phase was acetonitrile −0.1 % phosphoric acid aqueous solution with gradient eluting. The flow rate was 1.0 ml/min, the detection wavelength was 360 nm, the column temperature was 30 °C, and the injection volume was 10 μL. With Quercetin as the internal reference, the relative correction factors between five flavonoids were determined by QAMS, and the contents of six index components in persimmon leaf extracts were determined by QAMS.

2.7. Network pharmacological analysis

We obtained the drug target corresponding to each chemical component of NXQ through the TCMSP and ETCM platforms based on NXQ fingerprint analysis provided by Guangzhou Baiyunshan Hutchison Huangpu Traditional Chinese Medicine Co., Ltd. Potential genes associated with AS were collected from GeneCards and OMIM. Genecards is a comprehensive database of searchable genes, where we can obtain information on almost all known human genes. PPI networks were constructed with Cytoscape v-3.7.1. The Metascape database was used to explore BP, CC, and MF through GO enrichment analysis. We performed pathway enrichment analysis using the KEGG data obtained from the DAVID 6.8 platform and OmicShare tools.

2.8. Cell culture and treatment

The mouse macrophage line (RAW264.7) was acquired from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). At 37 °C in a 5 % CO₂ atmosphere, and RAW264.7 cells were maintained in DMEM supplemented with 10 % FBS as well as 1 % penicillin/streptomycin. BMDMs had been obtained from the bone marrow of mouse tibias and femurs, and then had been maintained in 1 % nonessential amino acids, 10 % FBS, 1 % penicillin/streptomycin, and 40 ng/mL M-CSF for seven days. RAW264.7 cells were divided into the control group, model group, NXQ-L group, NXQ-M group, NXQ-H group, and Compound C group; BMDMs were divided into the control group, model group, and NXQ group. In vitro models were established by incubating with 100 ng/ml LPS for 24 h. Before LPS treatment, Compound C and NXQ (10, 20, and 40 μg/ml) were added to the culture medium 24 h in advance as needed.

2.9. Cell viability assays

Under the manufacturer's instructions, we conducted cell viability assays with the CCK-8 kit. 10⁵ cells per well were seeded in 96-well plants and incubated overnight to allow cells to adhere. Then we treated cells with NXQ (0, 5, 10, 20, 40, 60, 80, and 160 μg/ml) for 24 h and incubated them with CCK-8 solution at 37 °C for 2 h. We measured absorbance at 450 nm using a microplate reader.

2.10. qRT‒PCR

From mouse aorta tissue and experimental cells, we extracted the total RNA using TRIzol reagent. PrimeScriptTM RT Reagent Kit was used to reverse-transcribe the RNA into cDNA according to the manufacturer's instructions. With SYBR Green as the detection fluorophore, we performed qRT-PCR using an Applied Biosystems 7900HT Fast Real-Time PCR System. Objective to normalize gene expression for GAPDH, we used the 2^−ΔΔCt method to determine the relative RNA expression. (The primer sequence is shown in Supplementary materials Table 1).

2.11. Western blotting

With the BCA protein assay kit, we quantified total protein concentrations from the homogenized aorta, and experimental cells were treated with a lysis buffer. Electrophoresis of 10 % sodium dodecyl sulfate-polyacrylamide gels was used to separate equal amounts of protein extracts. A polyvinylidene difluoride membrane preactivated with methanol was then used to transfer the proteins. Next, the membranes had been blocked using 5 % skim milk at room temperature for 1 h and subsequently incubated with primary antibodies overnight at 4 °C. We had incubated secondary antibodies bound to horseradish peroxidase for 1 h at room temperature after washing with TBST. Finally, chemical-enhanced luminescence was used to visualize the proteins, and the bands were imaged with a chemiluminescence imaging system.

2.12. Flow cytometry

The cell marker F4/80 was utilized for identifying macrophages, while the cell markers CD86 and CD206 were employed to distinguish between M1 and M2 macrophages, respectively. Following this, the cells were incubated with PE-labeled anti-CD206 antibody and APC-labeled anti-CD86 antibody. The cells were acquired on a Beckman Gallios, and analysis was done using Flow Jo software (Tree Star, Inc., Ashland, OR).

2.13. JC-1 procedure

We evaluated mitochondrial membrane potentials using a JC-1 Kit (Beyotime, Nantong, Jiangsu, China). As previously described, BMDM cells were cultured for 24 h in serum-containing culture media in 48-well plates for JC-1 staining. The culture medium was discarded and the cells were incubated with JC-1 (20 min; 37 °C; dark). During incubation, an appropriate amount of JC-1 staining buffer (1X) was prepared by adding 4 ml of distilled water to every 1 ml of JC-1 staining buffer (5X) and placed in an ice bath. After incubation at 37 °C, the supernatant was removed and washed twice with JC-1 staining buffer (1X), and the cell morphology was observed under a fluorescence microscope (Invitrogen EVOS M5000, USA).

2.14. Statistical analysis

Analysis of the data was carried out using GraphPad Prism V.7 software. All data were expressed as mean ± standard deviation (SD) values. We conducted a one-way analysis of variance (ANOVA) to determine the statistical significance of the disparities. P values < 0.05 were considered to be statistically significant.

3. Results

3.1. NXQ has been shown to have a protective effect on atherosclerosis induced by a high-fat diet (HFD) in ApoE^−/− mice

The experimental scheme for mice can be seen in Fig. 1A. Following that, we detected TNF-α, IL-6, IL-10, and Arg-1 levels in the serum of mice by ELISA. NXQ and Lipitor treatment significantly decreased the above inflammatory indicators in ApoE^−/− mice (Fig. 1B). At the same time, we measured the levels of lipid-related indexes including TC, TG, LDL-C, and HDL-C in the serum of mice. Compared with the model group, H-NXQ, and Lipitor groups showed significant reductions in serum TG, TC, and LDL-C levels, while HDL-C levels increased significantly (Fig. 1C). To evaluate tissue structure, hearts and aortas from mice was stained after treatment (Fig. 2A). In ApoE^−/− mice, significant histological changes were occurring in the aortic root as a result of HFD, including the formation of atherosclerosis plaques and lipid accumulation under the intima. Treatment with NXQ and Lipitor effectively alleviated these pathological changes induced by HFD (Fig. 2B and C).

Fig. 1.

Animal experiment process and ApoE^−/− mouse serological testing.

(A) Roadmap of Animal Experiment Technology. (B) The levels of TNF-α, IL-10, IL-6, and Arg-1 were measured in the serum of ApoE^−/− mice by ELISA. (C) Levels of blood lipids HDL-C, LDL-C, TG, and TG in each group (n = 5). Data are expressed as the mean ± SD.**p < 0.01. *p < 0.05, ***p < 0.001, the same below.

Fig. 2.

Pathological section staining of ApoE^−/− mouse aorta.

(A) Whole aorta Oil-red-O staining representative image. (B) Positive area statistics of Oil-red-O staining in whole aorta. (C) Representative images of H&E staining and Oil-red-O staining of the aortic root and abdominal aorta sections of ApoE^−/− mice. (H&E: magnification, 20 × and scale, 50 μm; Oil-red-O: magnification, 10 × and scale, 100 μm). (D) Statistics of positive area of HE staining on cross-sections of the abdominal aorta (n = 5). (E) Quantified lipid content was stained with Oil-red-O in the aortic root zone (n = 5).

3.2. NXQ can decrease the levels of inflammatory cytokines in ApoE^−/−mice

Evaluated by using qPCR and WB to determine both the transcript and the protein levels. We utilized qRT-PCR and Western blotting to evaluated both the transcript and the protein levels of IL-6, IL-10, and TNF-α in the aortas of ApoE^−/−mice. Compared to the control group, the model group showed a significant decrease in IL-10 mRNA expression. However, after treatment with NXQ, the expression increased significantly. The IL-6 and TNF-α mRNA expression levels in the model group were significantly higher than those in the control group and decreased significantly after NXQ treatment (Fig. 3A). This result corroborates the qRT-PCR results, showing that the model group's IL-10 protein expression was significantly lower than the control group's, and its expression was significantly increased after NXQ treatment. The protein expression levels of IL-6 and TNF-α in the model group were significantly higher than those of the control group and significantly decreased after NXQ treatment (Fig. 3B and C). These results indicate that NXQ could reverse the increased inflammatory factors' expressions induced by the HFD diet.

Fig. 3.

The levels of inflammatory cytokines in ApoE^−/− mice can be decreased by NXQ.

(A) Expression of inflammation-related genes (IL-10, iNOS, TNF-α, Arg-1, IL-6, and YM1) in aortic tissue (n = 5). (B) Levels of inflammation-related proteins (TNF-α, IL-6, and IL-10) in aortic tissue. (C) Densitometric analysis of IL-10, IL-6, and TNF-α in aortic tissue (n = 3). (D) Flow analysis of peritoneal macrophage of APOE^−/− mice in each group. (E) The proportion of M1 and M2 type macrophages in peritoneal macrophages of APOE^−/− mice in each group.

3.3. NXQ can regulate the polarization direction of peritoneal macrophages in ApoE^−/−mice

We harvested peritoneal macrophages from mice in each group and analyzed their polarization using flow cytometry. The findings revealed that in the model group, there was a high proportion of M1 polarization among the macrophages. However, after NXQ intervention, the number of M1-polarized macrophages decreased significantly. Conversely, there was a notable increase in M2-polarized macrophages following NXQ intervention, suggesting that NXQ can facilitate the transition of macrophages to M2 polarization (Fig. 3D and E).

3.4. NXQ can inhibit LPS-induced inflammation in RAW264.7 macrophages

Our assessment of NXQ's viability against RAW264.7 macrophages was carried out using the CCK-8 assay. NXQ significantly decreased RAW264.7 macrophage viability at 60 μg/ml, while cell viability was unaffected by NXQ at concentrations≤40 μg/ml. After LPS stimulation, the effects of NXQ at different drug concentrations on the viability of macrophages remained the same as before (Fig. 4A). In subsequent in vitro experiments, we used 10, 20, and 40 μg/ml NXQ, with 40 μg/ml NXQ selected as the highest concentration.

Fig. 4.

NXQ can decrease inflammation in LPS-induced RAW264.7 cells.

(A) Effects of NXQ on RAW264.7 macrophage viability. Cells were treated with 0, 1, 5, 10, 20, 40, 60, 80, and 160 μg/ml NXQ for 24 h. Cell viability was then measured using the CCK-8 assay. Cells were treated with LPS and 0, 10, 20, 40, 60, and 80 μg/ml NXQ for 24 h. Cell viability was then measured using the CCK-8 assay. (B) Expression of inflammation-related genes (IL-10, iNOS, TNF-α, Arg-1, IL-6 and YM1) in RAW264.7 cells. (C) The levels of inflammation-related proteins (TNF-α, IL-6, and IL-10) in RAW264.7 cells. (D) Densitometric analysis of IL-10, IL-6, and TNF-α in RAW264.7 (n = 3). (E) Representative images of M1-type macrophage surface markers (CD86) and M2-type macrophage surface markers (CD206) in BMDMs were analyzed by flow cytometry. (F) Percentage of CD206+ or CD86⁺ macrophages (n = 5).

To detect the mRNA and protein expression levels of IL-6, IL-10, and TNF-α, respectively, in macrophages, we performed qRT‒PCR and Western blot analysis. We found that the expression of IL-10 mRNA in the model group was significantly lower than that in the control group and that its expression was significantly increased following NXQ treatment. The IL-6 and TNF-α mRNA expression levels in the model group were significantly higher than those in the control group and decreased significantly after NXQ treatment (Fig. 4B). Under the qRT-PCR findings, there was a notable decrease in the expression of IL-10 protein in the macrophages of the model group compared to the control group. Nevertheless, following the administration of NXQ, there was a significant increase in the expression of IL-10 protein. The protein expression levels of IL-6 and TNF-α in the model group were significantly higher than those of the control group and significantly decreased after NXQ treatment (Fig. 4C and D). The results imply that NXQ has the potential to decrease the expression levels of pro-inflammatory cytokines present in macrophages. NXQ was observed to reduce the proportion of CD86⁺ (M1) and increase the proportion of CD206+ (M2) in BMDMs as determined by flow cytometry. (Fig. 4E and F).

3.5. Network pharmacological analysis predicted the potential targets and signaling pathways of NXQ in treating atherosclerosis

The chemical fingerprint of NXQ were measured by HPLC analysis as previously described.^7,8 We identified six compounds: protocatechuic acid, hyperoside, isoquercitrin, quercitrin, quercetin, and kaempferol (Fig. 5A). To further elucidate the potential molecular mechanism of NXQ's antiatherosclerotic effect, the pharmacology of the network was used to predict NXQ targets and signaling pathways. In particular, we screened for targets related to the chemical composition of NXQ (Supplementary materials Table 2) and displayed their intersecting and key targets with atherosclerosis (Supplementary materials Tables 3–4). Furthermore, based on cross goals, an overall network of component target paths was built (Fig. 5B). Diagram of the obtained network shows the six major components of NXQ across 185 action targets with atherosclerosis. To understand these chemical components and targets more clearly and intuitively, we generated networks of target paths and composite targets (Fig. 5C). To conduct the GO enrichment analysis, we utilized the Metascape database to detect overrepresented BPs, cellular components, and MFs (Fig. 5D). The study found that "lipid and atherosclerosis" and "AMPK signaling pathway" have an obvious correlation with NXQ (Fig. 5E). Based on the above information and the published literature, it was proposed that the AMPK-α/SIRT1/PPAR-γ signaling axis might be the key mechanism of NXQ's antiatherosclerotic effect.

Fig. 5.

Chemical fingerprint and network pharmacology analysis

(A) Chemical fingerprints of six major NXQ compounds. (B) The drug-component-target-disease network. The green hexagon represents NXQ, the blue rectangle represents the active ingredient in NXQ, the yellow sphere represents a common atherosclerosis-related NXQ target gene identified through text mining, and the pink rectangle represents atherosclerosis disease. (C) PPI network for common targets. The yellow square represents the core targets. The blue squares represent other targets. (D) GO enrichment analysis of the BPs, CCs, and MFs of target proteins identified using the Metascape database. (E) Kyoto Encyclopedia of Genes and Genomes (KEGG) used the DAVID 6.8 platform and OmicShare tools to analyze the top 20 target protein enrichment pathways associated with NXQ and atherosclerosis.

In conclusion, the cross targets and signaling pathways of NXQ and atherosclerosis are related to lipid metabolism and inflammation, providing reliable clues for the molecular mechanism of the antiatherosclerotic effect of NXQ.

3.6. NXQ can regulate the AMPK-α/SIRT1/PPARγ signaling axis

To determine the influence of NXQ on the AMPK-α/SIRT1/PPAR-γ signaling axis, Western blot analysis was used to measure the levels of p-AMPK-α/AMPK-α, SIRT1, PPAR-γ, and p-P65/P65 in ApoE^−/− mouse aortas and LPS-stimulated RAW264.7 macrophages. HFD enhanced NF-κB protein expression in the mouse aorta, and NXQ and Lipitor treatment reversed this effect and enhanced p-AMPK-α, SIRT1, and PPAR-γ isoprotein expression levels (Fig. 6A and B). Similarly, Stimulation with LPS increased the level of p-P65 in RAW264.7 macrophages, whereas treatment with NXQ significantly decreased the level of p-P65 and enhanced p-AMPK-α, SIRT1, and PPAR-γ isoprotein expression levels (Fig. 6C and D).

Fig. 6.

NXQ can regulate the AMPK-α/SIRT1/PPARγ signaling axis in vitro and in vivo. (A) Levels of AMPK-α/SIRT1/PPARγ signaling axis-related proteins, including p-AMPK-α, AMPK-α, SIRT1, PPAR-γ, P65, and p-P65, in atherosclerotic mice. (B) Densitometric analysis of p-AMPK-α/AMPK-α, SIRT1, PPAR-γ, and p-P65/P65 in atherosclerotic mice (n = 3). (C) Levels of AMPK-α/SIRT1/PPAR-γ signaling axis-related proteins, including p-AMPK-α, AMPK-α, SIRT1, PPAR-γ, P65, and p-P65, in RAW264.7 cells. (D) Densitometric analysis of p-AMPK-α/AMPK-α, SIRT1, PPAR-γ, and p-P65/P65 in RAW264.7 cells (n = 3).

3.7. Effect of combined treatment with NXQ and compound C in LPS-induced BMDMs

After intervention with Compound C, the mRNA expression levels of iNOS and TNF-α were higher compared with those of the NXQ group, and the mRNA expression levels of YM1 and Arg-1 were lower compared with those of the NXQ group (Fig. 7A). After Compound C intervention, the p-AMPK-α/AMPK-α ratio and SIRT1, PPAR protein expression levels were significantly lower than those in the NXQ group (Fig. 7B and C), indicating that AMPK-α inhibitors can reduce the therapeutic effect of NXQ on macrophages. In our study, the changes in cell membrane potential (MMP, ΔΨm) were measured by JC-1 staining red-green method. When the concentration of MMPs is high, JC-1 will polymerize and produce red light, while when the concentration of MMPs is low, JC-1 will produce green light. The red-to-green fluorescence ratio decreased in cells treated with LPS for 24 h, suggesting reduced mitochondrial aggregation and MMP. However, supplementation with NXQ increased the ratio (Fig. 7D and E).

Fig. 7.

AMPK-α inhibitors can reduce the therapeutic effect of NXQ on macrophages.

(A) Inflammation-related genes expressed in BMDMs (TNF-α, iNOS, Arg-1, and YM1). (B) Levels of AMPK-α/SIRT1/PPARγ signaling axis-related proteins, including p-AMPK-α/AMPK-α, SIRT1, and PPAR-γ in BMDMs. (C) Densitometric analysis of p-AMPK-α/AMPK-α, SIRT1, and PPAR-γ in BMDMs (n = 3). (D) Assessments of mitochondrial membrane potential in BMDMs using the JC-1 procedure. (E) Decreases in the red/green fluorescence intensity ratio reflect mitochondrial membrane depolarization during the apoptosis process (n = 3).

4. Discussion

AS is a chronically progressive inflammatory disease that results in high morbidity and mortality worldwide. Anti-inflammatory treatment is recognized as an effective approach to ameliorate AS. However, the development of drugs that can effectively prevent and halt the progression of AS with minimal side effects remains an ongoing challenge. Recently, there has been increasing interest in the use of natural medicine. Previous studies reported that the primary effective constituents of NXQ are flavonoids, organic acids, and coumarins, which exhibit antioxidative and anti-inflammatory activities and improve blood supply.^15,16 In this study, ApoE^−/− mice were fed a high-fat diet for 12 weeks to establish an atherosclerosis mouse model, and varying doses of NXQ and Lipitor were administered as interventions. NXQ was observed to dose-dependently reduce circulating inflammation and lipid levels, with high doses of NXQ showing comparable efficacy to Lipitor.

The direction of macrophage polarization influences the extent of inflammation in AS mice. M1 and M2 are two macrophage phenotypes. M1 macrophages are typically activated by LPS, IFN-γ, or M-CSF. M1 macrophages can secrete abundant pro-inflammatory factors, including TNF-α, IL-6, and iNOS, which results in inflammatory cell recruitment and accelerated plaque development.^5,17 Conversely, M2 macrophages produce anti-inflammatory cytokines, such as Arg-1, IL-10, and YM1, inhibiting cell recruitment and tissue remodeling and consequently increasing plaque stability.^5,18 Therefore, the abundance of M1 and M2 macrophages serves as an indicator of plaque progression or regression. To explore this further, we analyzed the transcription levels of M1 and M2 macrophage markers in plaques. Our findings demonstrated that NXQ intervention reduced levels of TNF-α, IL-6, and iNOS in M1-type macrophages, while simultaneously increasing levels of Arg-1, IL-10, and YM1 in M2-type macrophages. Flow cytometry analysis of macrophages labeled with CD86 and CD206 antibodies provides a more intuitive visualization of macrophage polarization.

Analysis of abdominal macrophages in AS mice before and after NXQ intervention reveals a decrease in the number of CD86⁺ macrophages and an increase in the number of CD206+ macrophages following NXQ treatment.These results are consistent with the observed effects of NXQ on BMDM in vitro, suggesting that NXQ may influence the direction of macrophage polarization. However, it's important to note that during flow analysis of peritoneal macrophages, distinct macrophage clusters were observed, while primary BMDM exhibited a unimodal distribution. This difference may be due to the greater heterogeneity and maturity of peritoneal cavity-isolated macrophages, with various polarization states of M1 and M2 macrophages tending to concentrate within specific subgroups. In contrast, BMDM cells demonstrate high homogeneity, with uniform activation of polarization markers across cells. Nonetheless, both in vivo peritoneal macrophages and in vitro BMDM showed plasticity in their polarization phenotype.¹⁹

Regulating the balance of macrophage polarization presents a novel strategy for treating AS. Previous research indicates that various pathways, including the NLRP3 inflammasome, TLR, HIF-α, NF-κB signaling pathways, GM–CSF–related pathways, JAK-STAT pathways, and the TBK/IRF3 pathway, play roles in regulating macrophage polarization and influencing the progression of AS.²⁰ To further investigate how NXQ regulates macrophage polarization phenotype to improve AS, this study predicted potential targets and signaling pathways through network pharmacological analysis. The findings suggest that the AMPK signaling axis might be a crucial mechanism underlying the anti-atherosclerotic effects of NXQ. The AMPK signaling axis exert its effects in the response to inflammation, regulating the inflammatory reaction and immune response.²¹ AMPK, known as a cellular energy sensor, is an important regulator of cellular metabolism. Mitochondria are the mainly suppliers of ATP in most organisms, and AMPK is activated to play multiple roles in mitochondrial homeostasis when mitochondria are damaged.^22,23 Activated AMPK enhances the transcription and activity of NAM phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in NAD + synthesis, consequently increasing the [NAD+]/[NADPH] ratio. This increase contributes to the activation of SIRT1, thereby exerting an anti-inflammatory effect.²⁴ SIRT1, short for Silent Information Regulator Transcription 1, is an NAD + -dependent histone deacetylase involved in chromatin packaging. Current research suggests that the SIRT1 pathway serves as the primary anti-inflammatory pathway downstream of AMPK. On one hand, SIRT1 directly inhibits the transcriptional activity of the NF-κB. NF-κB, when activated, triggers the expression of various pro-inflammatory factors like IL-6, TNF-α, and iNOS in immune cells during inflammation.^25,26 On the other hand, SIRT1 activates downstream genes associated with energy metabolism, such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), which enhances the expression of PPAR-γ. Increased PPAR-γ negatively regulates NF-κB expression, preventing its binding to corresponding sites in the promoter region of pro-inflammatory genes. As a result, this regulates the transcriptional activity of pro-inflammatory genes.^27,28

To validate this hypothesis, we conducted a series of in vitro and in vivo experiments. In our studies, following treatment with NXQ, we observed an increase in the expression of p-AMPK-α and PPAR-γ in both tissues and cells. Conversely, the expression of phosphorylated P65 (p-P65) decreased. P65 (also known as RelA) is a subunit of NF-κB family. Phosphorylation of p65 promotes the translocation of the NF-κB transcription factor subunit to the nucleus, thereby inducing the expression of target genes. These results suggest that NXQ may facilitate the activation of the AMPK/SIRT1/PPAR-γ pathway. This led to a reduction in the release of inflammatory factors and the suppression of the inflammatory response in AS. Furthermore, we confirmed the effect of NXQ in activating AMPK and enhancing its mitochondrial function through the detection of mitochondrial membrane potential. In addition, after intervention with compound C (an effective, reversible, and selective AMPK inhibitor), there was no significant change in the expression of p-AMPK-α, SIRT1, and PPAR-γ compared to the model group. This further suggested that the mechanism by which NXQ improves AS may be through the AMPK-α/SIRT1/PPAR-γ signaling axis. Based on the aforementioned results, we posit that NXQ regulates macrophage polarization to suppress inflammation by enhancing the expression of AMPK/SIRT1/PPAR-γpathway, thereby impeding the progression of AS. The potential mechanism is illustrated in Fig. 8.

Fig. 8.

Mechanism of NXQ in treating atherosclerosis. AMPK-α, adenosine monophosphate-activated protein kinase-alpha; IL-10, interleukin-10; SIRT1, sirtuin 1; PGC1α, peroxisome proliferator-activated receptor gamma coactivator-1 alpha; TNF-α, tumor necrosis factor-alpha; PPAR-γ, peroxisome proliferator-activated receptor-γ; IL-6, interleukin-6; NF-κB, nuclear factor kappa B; iNOS, inducible nitric oxide synthase; Arg-1, arginase-1.

However, there were several limitations in this study. However, previous studies have shown that as a master nuclear receptor regulating lipid metabolism, the activation of PPAR-γ reduces the uptake of ox-LDLs by macrophages, thereby attenuating foam cell formation.²⁹ Based on the AMPK-α/SIRT1/PPAR-γ pathway, while it is known to regulate macrophage polarization for its anti-inflammatory effects, it remains unclear whether NXQ also influences glucolipid regulation, promotes fatty acid oxidation in macrophages, and facilitates lipid efflux.Furthermore, AMPK, functioning as an energy sensor, plays a crucial role in maintaining mitochondrial homeostasis. While this study primarily examines the impact of NXQ on mitochondrial membrane potential, further investigation is required to determine whether NXQ can regulate mitochondrial oxidative stress and mitochondrial autophagy.

Overall, this study demonstrates the considerable potential of NXQ in treating AS. With its low side effects and notable efficacy, NXQ is poised to emerge as a natural remedy for effectively managing AS. Additionally, exploring its synergistic effects with current treatments could offer valuable insights for clinical application. However, given the complex nature of AS pathology, further investigation is warranted to elucidate the multi-faceted mechanisms through which NXQ may ameliorate AS.

Authors' contributions

Conceptualization of the study: CL, ML, and HG; Performing the experiments and data analysis: GZ,CW, ZL, RZ, LL, SW, JX, YX, YZ, DC, CZ, JL, and HG; Resources: GZ, ML, JL, HG, and CL; Writing and editing the manuscript: all authors.

Funding

This study was supported by the Technology project of Guangzhou Baiyun Mountain Hutchison Whampoa Chinese Medicine Co., LTD [grant numbers 202012]; The Guangdong Province Key Area Research Program [grant numbers 2022A0446]; Guangzhou Science and Technology Plan Project: Service Platform for the Deep Integration of Production, Learning, and Research of Famous and Excellent Chinese Patent Drugs [grant numbers 20212210006]; Guangdong University student science and technology innovation cultivation special fund [PDJH2022b0118].

Ethics approval and consent to participate

Guangzhou University of Chinese Medicine's Ethics Committee approved the in vivo study procedures (No. SCXK 20, 201, 229,009).

Consent for publication

The manuscript has been approved by all authors for publication.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Availability of data and material

All relevant data and material of this study are available from the corresponding author upon request.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article: