Resibufogenin protects against atherosclerosis in ApoE^-/- mice through blocking NLRP3 inflammasome assembly

Graphical abstract

Highlights

• •Therapeutic Potential in Atherosclerosis: Through various experiments, including animal models and cell cultures, Resibufogenin (RBG) showed significant efficacy in alleviating atherosclerosis-related characteristics in ApoE^-/- mice. The compound reduced inflammatory infiltration, lipid accumulation, and fibrosis, highlighting its potential as a novel therapeutic strategy for cardiovascular diseases.
• •RBG as a Potent NLRP3 Inflammasome Inhibitor: The study identifies RBG as a potent inhibitor of the NLRP3 inflammasome, demonstrating its ability to form a non-covalent bond with the CYS-279 residue of the NLRP3 protein. This binding effectively hinders inflammasome assembly, thereby reducing pro-inflammatory cytokine release and macrophage foam cell formation, which are crucial in the progression of atherosclerosis.
• •Impact on Macrophage Polarization and Inflammatory Response: RBG was observed to inhibit the activation of M1 macrophages while promoting M2 macrophage polarization, which is vital for reducing inflammatory responses and enhancing tissue repair. This dual action underscores the compound’s broader potential in managing inflammatory diseases beyond atherosclerosis.

Abstract

Introduction

Atherosclerosis (AS), a major cause of cardiovascular diseases, is characterized by lipid accumulation and chronic inflammation within arterial walls. Traditional treatments, such as statins, are often ineffective for many patients, highlighting the need for novel therapeutic strategies.

Objective

This study explores the potential of Resibufogenin (RBG) as an NLRP3 inflammasome inhibitor for treating AS in ApoE^-/- mice.

Methods

We performed experiments encompassing cellular studies, animal model assessments, molecular simulations, and binding assays to assess RBG’s impact on the NLRP3 inflammasome, inflammatory cytokine release, and foam cell formation.

Results

RBG treatment alleviated AS in ApoE^-/- mice, evidenced by reduced body weight, smaller atherosclerotic plaques, and improved serum lipid profiles. Transcriptomics and molecular biology demonstrated that RBG suppressed the expression of key inflammatory markers such as NLRP3. RBG also reduced macrophage infiltration and promoted polarization toward the anti-inflammatory M2 phenotype. Molecular docking, SPR, Pull-down studies identified a non-covalent interaction between RBG and the CYS-279 residue of NLRP3, confirming its role as a potent NLRP3 inhibitor.

Conclusion

RBG effectively inhibits NLRP3 inflammasome activation, reduces pro-inflammatory cytokine release, and decreases formation of foamy macrophages, thereby slowing the progression of AS. Although these findings highlight RBG as a promising therapeutic approach for cardiovascular diseases, further research is necessary to assess its safety and effectiveness in humans and to investigate possible synergistic effects with other treatments.

Introduction

Atherosclerosis (AS), a primary cause of global cardiovascular diseases, involves the progressive buildup of lipids and fibrous tissue in the vessel wall, resulting in vascular narrowing and hardening [[1], [2], [3]]. Although traditional treatment strategies, such as statins, have shown some efficacy in lowering cholesterol levels and slowing disease progression, a significant number of patients do not respond well to existing therapies and experience considerable side effects [[4], [5], [6], [7], [8], [9]]. Thus, investigating novel pathological mechanisms and drug discovery is crucial for AS prevention and treatment [4,10].

Current research highlights the critical role of inflammatory responses, particularly involving the NLRP3 inflammasome, in the development of AS [3,6,[11], [12], [13], [14], [15], [16], [17]]. Elevated NLRP3 activity has been observed in patients, while animal studies show that NLRP3 deficiency reduces plaque formation and inflammation [6,[12], [13], [14], [15], [16], [17], [18], [19], [20], [21]]. Cellular studies suggest a strong association between NLRP3 gene regulation and the onset and development of AS [20,22]. Together, these findings emphasize the significance of NLRP3 in mediating inflammatory responses throughout the disease process. Macrophages are pivotal effector cells in the inflammatory response, exhibiting a dual role in advancing AS [4,13,23]. They can ingest oxidized low-density lipoproteins (ox-LDL) and transform them into foam cells, aiding in the initial development of plaques [[24], [25], [26], [27]]. Activated macrophages secrete pro-inflammatory factors interleukin-1 beta (IL1β), which intensify inflammation and contribute to plaque instability and rupture [13,22,[28], [29], [30]]. Inhibiting macrophage-mediated inflammatory responses is crucial for preventing and treating AS.

The NOD-like receptor protein 3 (NLRP3) inflammasome, a cytoplasmic multiprotein complex, is activated by diverse pathogen-associated and damage-associated molecular patterns. It is crucial in the inflammatory response linked to AS [15,16,18,[31], [32], [33], [34]]. Activation of the NLRP3 inflammasome triggers caspase-1, leading to the maturation and release of pro-inflammatory cytokines IL1β and IL18, which enhance the inflammatory response and contribute to AS progression. Therefore, based on previous literature and results from clinical, animal, and cellular studies, there is a close relationship between NLRP3 and AS, which underscores the importance of NLRP3 inhibitors. Therefore, targeting the inhibition of NLRP3 inflammasome activation is regarded as a promising strategy for preventing and treating AS [6,13,16].

Resibufogenin (RBG), a natural compound derived from Venenum Bufonis (Chansu, 蟾酥 in Chinese), a traditional medicinal substance obtained from the secretions of Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider [[35], [36], [37]]. RBG exhibits diverse biological activities such as anti-inflammatory, anti-tumor, and cardiovascular protective effects [[38], [39], [40], [41], [42], [43], [44]]. Preliminary studies suggest that RBG may inhibit inflammatory responses through multiple mechanisms, particularly in macrophages [35,45]. However, its specific role and mechanisms in AS have not been comprehensively elucidated. Research suggests that RBG may directly or indirectly inhibit NLRP3 inflammasome assembly and activation in macrophages, contributing to its anti-inflammatory and vascular protective effects [35,46,47].

This study examines the effectiveness of RBG in AS and its mechanisms by regulating the NLRP3 inflammasome in macrophages. We employed animal models, cell experiments, molecular simulations, in vitro binding assays, and gene manipulation to investigate the impact of RGB on the NLRP3 inflammasome and its protective effects against atherosclerotic lesions. Our study aims to demonstrate the component's potential in AS treatment, offering theoretical and experimental foundations for novel therapeutic approaches.

Materials and methods

Animal experiments

ApoE^-/- mice on a C57BL/6 background were obtained from Guangzhou Ruige Biotechnology Co., Ltd, Guangzhou, China. The control group (NCD group) was fed a normal chow diet, while the remaining groups were given high fat diet (HFD) for 12 weeks. To assess the impact of RGB, mice received daily oral doses for 8 weeks, starting from the fifth week, of either 0.9 % normal saline (NCD and HFD groups), rosuvastatin (2.1 mg/kg, RVS group), varying concentrations of RGB (5 mg/kg for high dose, 3 mg/kg for medium dose, and 1 mg/kg for low dose in RBG-H, RBG-M, and RBG-L groups, respectively), or MCC950 (20 mg/kg, MCC950 group). Aorta-specific NLRP3 knock-down mice were created using AAV9-U6-mNLRP3-shRNA, while wild-type mice received an intravenous injection of AAV9-CMV-GFP-CON three days prior to MCC950 or RBG administration. Western blot and immunofluorescence analyses were used to assess knock-down efficiency. The Animal Ethics Committee of Guangzhou University of Chinese Medicine approved all animal care and experimental procedures, following their guidelines (approval No. 20201013002).

Chemicals and Reagents

Primary antibodies for NLRP3 (ab15101), NF-κB (ab4764), NEK7 (ab10054), ASC (ab67824), Caspase-1 (ab24232), IL18 (ab57058), IL1β (ab31202), CD68 (ab97778), GAPDH (ab2118), and β-ACTIN (ab4970) were obtained from Cell Signaling Technology, Denvers, MA, USA.The antibodies PE Anti-Mouse CD86 (GL-1), APC Anti-Mouse CD206/MMR (C068C2), and PE/Cyanine7 Anti-Mouse F4/80 (CI A3-1) were procured from Elabscience Biotechnology Co., Ltd. (Wuhan, China). AAV9-U6-mNLRP3-shRNA (5.02 × 10^13 vg/mL, ShRNA sequence: GGATGAACGTGTTCC-AGAA), AAV9-CMV-GFP-CON (4.24 × 10^13 vg/mL), SiRNA-NLRP3 and SiRNA-Con were purchased from Kidan Bio Co. Ltd (Guangzhou, China). The plasmids pcADV-EF1-mNeonGreen-CMV-Nlrp3 and the control pcADV-EF1-mNeoGreen-CMV-MCS were obtained from Heyuan Biotechnology Co., Ltd, Shanghai, China. MCC950 was purchased from Selleck (Houston, USA). ox-LDL (YB-002) were purchased from Zhuosheng Biotechnology Co., Ltd (Guangzhou, China). LPS (S11060) and ATP (b25057) were sourced from Yuanye Biotechnology Co., Ltd, Shanghai, China. IFN-γ (P00028) was purchased from Solaibao Technology Co., Ltd (Beijing, China). Human NLRP3 protein (24013068P-1) was obtained from Pujian Biotechnology Co., Ltd (Wuhan, China). TSA Fluorescence System Kits (K1050, K1051, and K1052) were purchased from APExBIO (Houston, USA). High-fat diet (D12492) was purchased from Guangdong Medical Laboratory Animal Center. ELISA kits for ICAM-1, VCAM-1, IL18, IL1β, TNF-α, IL10, TG, TC, LDL-C, HDL-C, and Caspase-1 were sourced from Elabscience Biotechnology Co., Ltd. (Wuhan, China). All other commercially available chemicals were of the highest quality. Primers (Table S1), including those for mouse-specific NLRP3, ASC, NEK7, Caspase-1, IL18, IL1β, PPAR-α, ACC, CPT1A, SREBP1were acquired from Shenggong Bioengineering Co., Ltd (Shanghai, China). All other commercially available chemicals were of the highest quality. EZ-Link™ Biotinylation Reagent was purchased from Thermo Fisher Scientific Co., Ltd. The purified protein NLRP3 was purchased from Huamei Biotechnology Co., Ltd.

Culturing and treating cells

RAW264.7 cells purchased from ATCC were cultured in DMEM medium with 5 % CO₂ at 37 °C. Extraction and cultivation of Bone marrow-derived macrophages (BMDMs) follow the method established [48]. Foam cell formation was induced by incubating cells in serum-free DMEM with 50 μg/mL ox-LDL for 24 h. Cells were incubated in DMEM with LPS (1 μg/mL for 24 h) and ATP (5 mM for 30 min) to establish an inflammatory activation model. Cells underwent M1 polarization with DMEM supplemented with LPS (100 ng/mL) and IFN-γ (50 ng/mL) for 24 h, whereas M2 polarization was induced using DMEM with IL4 (10 ng/mL) for the same duration.

Western blot analysis

Proteins were extracted from tissues or cells using RIPA buffer (Beyotime, Hangzhou) with protease inhibitors and quantified via BCA assay (Dingguo Biology, Guangzhou). Equal concentrations of samples were mixed with LDS buffer, resolved by 8–15 % SDS-PAGE, and transferred to PVDF membranes. Membranes were blocked with 5 % non-fat milk in TBS-T for 1.5 h at room temperature. After overnight incubation with primary antibodies at 4 °C, membranes were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. Proteins were visualized using ECL kits (Millipore, Billerica, USA) at last. Protein band intensities were analyzed using ImageJ software, normalized to β-ACTIN or GAPDH levels.

Quantitative real-time polymerase chain reaction analysis (RT-qPCR)

Utilize an RNA extraction kit for RNA isolation. RNA should be transcribed into complementary DNA (cDNA) through reverse transcription. The qPCR reaction system requires mixing cDNA, primers for target RNA sequence amplification, and SYBR Green for detecting PCR products. Primer sequences refer to supplemental Table 1. A thermal cycling PCR machine is required for multiple qPCR cycles, and fluorescence data should be analyzed using the 2^-ΔΔCT method to assess target RNA expression levels.

ELISA assay

Cytokines or metabolites concentrations, including IL18, IL1β, TNF-α, IL10, TG, TC, LDL-C, HDL-C, and caspase-1, were quantified in serum, tissue supernatants, and cell culture media via ELISA. The procedure was meticulously followed as per the manufacturer's guidelines.

CCK-8 assay

RAW or BMDMs were seeded into 96-well plates at suitable densities and incubated at 37 °C with 5 % CO₂ for 24 h to facilitate attachment. Different concentrations of RBG were added to the culture medium, along with a control group. Cells were further cultured for varying durations (3, 6, 12, or 24 h). A 10 % volume of CCK-8 reagent was added to each well, followed by a 1–4 h incubation to ensure complete reaction. A microplate reader measured the optical density at 450 nm. Cell viability and proliferation were evaluated by measuring changes in OD values.

Pathological staining

Aorta, aortic sinus sections, and cell slides were fixed in tissue fixation solution and washed thrice with PBS. HE, Masson, and Oil-Red O staining were conducted following the kits' instructions. Aorta tissues underwent washing with normal saline, dehydration, paraffin embedding, and fixation in 10 % buffered formalin. They were then sectioned into 5 μm slices, deparaffinized, and stained using HE, Masson, and Oil Red-O for histological analysis.Samples were visualized under a panoramic scanning system (Olympus SLIDEVIEW VS200, Tokyo, Japan) or a stereoscope (Olympus X70, Tokyo, Japan).

Immunofluorescence analysis

Cells or tissue slices were fixed with paraformaldehyde and subjected to antigen retrieval by microwave heating for 15 min. Cells or tissue slices were washed thrice with PBS, permeabilized using 1 % Triton X-100 in PBS for 15 min, and blocked with 5 % BSA for 1 h at room temperature. Endogenous peroxidase was quenched using 3 % hydrogen peroxide for 1 h. Primary antibodies for NF-κB, NLRP3, ASC, and Caspase-1, each at a 1:200 dilution (CST), along with HRP-linked secondary antibodies (1:200, Affinity), were diluted in BSA solution and incubated at room temperature for 1 h. Following washing, nuclei were stained with DAPI solution for 10 min at room temperature. After an additional PBS wash, samples were either mounted or photographed directly. Images were examined using a fluorescence microscope (Olympus IXplore SpinSR, Tokyo, Japan).

Transcriptome sequencing

RNA extraction from the samples was performed using TRIZOL reagent following the manufacturer's guidelines. The quality and quantity of RNA were assessed using a Nanodrop spectrophotometer and an Agilent Bioanalyzer. RNA libraries were constructed with the NEBNext Ultra RNA Library Prep Kit and sequenced using an Illumina platform. Low-quality reads were filtered from the raw sequencing data, and the resulting clean reads were aligned to the reference genome using STAR. Gene expression was quantified via feature counts, and differential expression analysis was performed using DESeq2, applying a significance threshold of p-value < 0.05.

Flow cytometry assay

Cells (6 × 10^5) were resuspended in 100 μL PBS and incubated with fluorescent antibodies F4/80, CD11b (Invitrogen, CA, USA), CD86, and CD206.The mixture was kept in the dark at room temperature for 25 min. Following incubation, 1.5 mL of PBS was added, and the sample was centrifuged at 1000 g for 5 min at room temperature. After washing to remove residual antibodies, the resultant pellet was resuspended and analyzed by BD flow cytometry. Agilent NovoCyte Flow Cytometer Systems were used for on-board testing.

Molecular docking

We used Maestro from Schrödinger 2021–3 for molecular docking, with compounds from PubChem and a protein structure from PDB (ID: 7vtq). LigPrep prepared the compounds by determining their charge state and energy-minimized conformation. Glide (SP precision) was used for docking, and we selected the best non-covalent binding mode. Covalent docking simulations of Michael addition reactions were then performed. PyMOL visualized these covalent complexes for further dynamics simulations. Docking results suggested an R147A mutation in the small molecule-protein complex, which served as the starting point for AMBER 18 simulations. Charges for non-standard amino acids, including small molecules and CYS275, were calculated using antechamber and Gaussian 09 (HF SCF/6-31G* method). We followed tutorials to develop force field parameters for these amino acids and used the ff14SB force field. LEaP added hydrogen atoms, and a TIP3P solvent box with Na+/Cl- ions was placed 10 Å away to balance charges. Final files were prepared for simulation.

Molecular dynamics simulation

AMBER 18 software was used for molecular dynamics simulations. Before starting, the system underwent energy optimization with 2500 steps of steepest descent and conjugate gradient methods. The system was then heated to 298.15 K over 200 ps while maintaining a fixed volume. A 500 ps NVT simulation at 298.15 K ensured uniform solvent distribution. This was followed by a 500 ps NPT equilibrium simulation. Both complex systems were then simulated for 500 ns under NPT conditions with periodic boundary conditions. During the simulation, non-bonded interactions were cutoff at 10 Å, and electrostatics were handled by the PME method. Hydrogen bonds were constrained using SHAKE, temperature was controlled by Langevin dynamics, and pressure was maintained at 1 atm. Trajectories were recorded every 10 ps for analysis.

Surface plasmon resonance analysis

The COOH chip was set up according to OpenSPR TM instrument protocols. PBST (pH 7.4) was used as the detection buffer at a max flow rate of 150 µL/min. After establishing a baseline, 200 µL of isopropanol was introduced with a 10-second bubble purge. The system was then washed and air-purged. The flow rate was reduced to 20 µL/min, and the chip was activated with a 1:1 EDC/NHS solution. A diluted ligand was injected for 4 min, followed by washing and purging. A blocking solution was added, and the system was washed again. The analyte buffer was changed, and stability was confirmed over 5 min. The analyte, diluted as per experimental results, was injected at 20 µL/min for 240 s of binding and 360 s of dissociation. Data was analyzed using TraceDrawer software with the One To One binding model.

Pull-down assays

To investigate the specific interaction between RBG and NLRP3, we first biotinylated RBG using a commercial biotinylation reagent following the manufacturer's instructions. Briefly, RBG was dissolved in a suitable buffer at an optimal concentration, and the biotinylation reagent was added in a molar ratio that ensured efficient labeling of RBG molecules. The reaction mixture was incubated at the recommended temperature and time to allow the formation of a stable biotin-RBG conjugate. After biotinylation, excess reagent was removed by dialysis or gel filtration to obtain pure biotinylated RBG. Concurrently, we constructed full-length NLRP3 protein, as well as its various fragments and mutant variants, using standard molecular cloning techniques. These constructs were then expressed in a suitable expression system (e.g., bacterial or mammalian cells) and purified to homogeneity using affinity chromatography or other purification methods. For the Pull-down assay, the biotinylated RBG was immobilized on streptavidin-coated beads, which were then incubated with the purified NLRP3 proteins (full-length, fragments, or mutants) under gentle agitation at 4 °C for several hours to allow the formation of RBG-NLRP3 complexes. Simultaneously, perform competitive binding validation experiments by incubating with free RBG. After incubation, the beads were washed extensively to remove unbound proteins, and the bound proteins were eluted using a suitable elution buffer. The eluted proteins were then analyzed by SDS-PAGE followed by Western blotting using specific antibodies against NLRP3 to detect the interaction between RBG and NLRP3. This experimental approach allowed us to determine the specific binding of RBG to different regions of NLRP3 and to assess the impact of mutations on this interaction.

Statistical analyses

Figures present values as MEAN ± SEM. Data analysis utilized GraphPad Prism Version 6.0 (GraphPad Software, La Jolla, CA, USA) with one-way ANOVA, followed by LSD or Dunnett’s multiple comparisons test. Statistical significance was defined as p-values less than 0.05.

Results

RBG mitigates HFD-Induced AS in ApoE^-/- mice

Based on previous literature studies, we administered RBG or rosuvastatin via gavage to ApoE^-/- mice starting from the fifth week (Fig. 1A). The study found that RBG treatment significantly decreased the body weight of ApoE^-/- mice on a high-fat diet (Fig. 1B). Macroscopic Oil Red O staining of the aorta indicated that RBG treatment may protect against AS in ApoE^-/- mice (Fig. 1C, 1E). Histological analyses, including HE, Masson, and Oil Red O staining of the aortic root, demonstrated reduced plaque size in RBG-treated mice (Fig. 1D, 1F-H). Serum analysis indicated that RBG treatment partially enhanced the levels of TG, TC, LDL-C, and HDL-C in ApoE^-/- mice (Fig. 1I-L). Our analysis of aortic lipid metabolism-related genes revealed that RBG treatment significantly increased the mRNA expression levels of PPAR-α, ACC, CPT1A, and SREBP1 (Fig. S1A-D). Immunofluorescence staining at the aortic root demonstrated that RBG decreased α-SMA expression, suggesting reduced fibrosis (Fig. S1E, F). These results suggest that RBG treatment can effectively alleviate AS in ApoE^-/- mice and has regulatory effects on lipid levels, body weight, fibrosis, and lipid metabolism. In addition, we also found that RBG can improve serum adhesion factors in APOE^-/- mice, indicating an improvement in endothelial cell function (Fig. S8).

Fig. 1.

RBG mitigates HFD-induced AS in ApoE^-/- mice. (A) Establishment of the model and treatment protocol. (B) Weight-time curve of mice (n = 10). (C and E) Representative images and quantification of en face Oil Red O staining of the aorta (n = 3). (D, F, G, H) Representative images and quantitation of HE, Masson, and Oil Red O staining in the aortic sinus (n = 3). (I, J, K, L) TG, TC, LDL-C, and HDL-C were measured (n = 10). *P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001; ns indicates not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

RBG treatment inhibits NLRP3 inflammasome and inflammation in ApoE^-/- mice aorta

To investigate the precise mechanisms by which RBG treatment alleviates AS, we conducted transcriptomic sequencing of the aortas from ApoE^-/- mice. The results revealed the presence of differentially expressed genes such as NLRP3, ASC, and CASPASE-1 (Fig. 2A). Gene Ontology (GO) enrichment analysis revealed that the anti-atherosclerotic effects of RBG were linked to multiple biological processes (Fig. 2B). KEGG enrichment analysis identified the NOD-like receptor and NF-κB signaling Additionally, by taking the intersection of differentially expressed genes between the NCD and HFD groups, as well as between the HFD and RBG-H groups, we identified 92 differentially expressed genes (Fig. S9A–C). Through Gene Set Enrichment Analysis (GSEA), we found enrichment in the NOD-like receptor signaling pathway (Fig. S9D). Furthermore, we validated the transcriptomic results using PCR, which demonstrated that RBG exerts regulatory effects on inflammation-related signaling pathways beyond the NF-κB/NLRP3 signaling pathway (Fig. S10). We reasonably speculated that the anti atherosclerotic effect of RBG might be related to NLRP3 signaling pathway, and then we conducted further experiments. ELISA results indicated that RBG treatment enhanced serum levels of IL18, IL1β, TNF-α, and IL10 in HFD-induced ApoE^-/- mice (Fig. 2E-H). Western blot analysis indicated that RBG treatment decreased the levels of NLRP3, ASC, ASC oligomerization, NEK7, caspase-1, cleaved caspase-1, pro-IL18, and IL18 proteins in the aorta of ApoE^-/- mice on a high-fat diet (Fig. 2D, 2I-P). PCR results demonstrated that RBG treatment reduced mRNA levels of NLRP3, ASC, ASC oligomerization, NEK7, caspase-1, IL18, and IL1β in the aorta of HFD-induced ApoE^-/- mice (Fig. S2A-F). Immunofluorescence analysis revealed that RBG treatment decreased the expression and co-localization of NLRP3, ASC, and caspase-1 in the aortic sinus of HFD-induced ApoE^-/- mice (Fig. 2Q-U). Additionally, we found through immunofluorescence that RBG treatment could decrease macrophage infiltration in the aortic sinus of ApoE^-/- mice induced by HFD (Fig. S2G, H). The findings indicate that RBG significantly lowers inflammation in HFD-induced ApoE^-/- mice, correlating with reduced macrophage infiltration and suppression of the NLRP3 signaling pathway and inflammasome assembly.

Fig. 2.

RBG Inhibits NLRP3 Inflammasome Assembly and Inflammation in Atherosclerotic Plaques of ApoE^-/- Mice. (A) Volcano plot of differentially expressed genes between the RBG-H group and HFD group (n = 3). (B) GO enrichment analysis of genes with differential expression. (C) KEGG enrichment analysis of genes with differential expression. (E, F, G, H) Serum levels of IL18, IL1β, TNF-α, and IL10 were measured (n = 10). (D, I, J, K, L, M, N, O, P) Western blot analysis and Statistical analysis was conducted for NLRP3, ASC total protein, ASC oligomerization, NEK7, Caspase-1, Cleaved Caspase-1, Pro IL18, and IL18 (n = 6). (Q, R, S, T, U) Representative images and statistical analysis of immunofluorescence staining for NLRP3, ASC, and Caspase-1 in the aortic sinus (n = 3). *P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001; ns indicates not significant.

RBG inhibits Macrophage infiltration and reduces ox-LDL induced foam cell formation and NLRP3 inflammasome expression and assembly

Through CCK-8 assays, we found that RBG exhibited no cytotoxic effects on BMDM and Raws at concentrations below 1 μM (Fig. S3A, B). Considering the pharmacokinetics from previous envidences, we established three doses of RBG: 100 nM, 50 nM, and 25 nM. By treating BMDM with ox-LDL, we replicated the foam cell model. Oil Red O staining demonstrated that RBG significantly decreased lipid accumulation and foam cell formation in BMDM (Fig. 3A, 3B). Treatment with ox-LDL and LPS + IFN-γ showed that RBG reduces M1 macrophage infiltration and enhances M2 macrophage infiltration (Fig. 3C-G). Similarly, we observed comparable results in Raws (Fig. S3C-G). ELISA results demonstrated that RBG enhanced the levels of IL18, IL1β, TNF-α, IL10, TG, and TC in the supernatant of BMDM cultures stimulated by ox-LDL (Fig. 3H-M). Similarly, analogous findings were observed in Raws (Fig. S3N-S). Western blot analysis demonstrated that RBG decreased the levels of NLRP3, ASC, ASC oligomerization, NEK7, caspase-1, cleaved caspase-1, pro-IL18, and IL18 in ox-LDL-induced BMDM (Fig. 3N-V). PCR results indicated that RBG treatment reduced mRNA levels of NLRP3, ASC, NEK7, caspase-1, IL18, and IL1β in both ox-LDL-induced BMDM (Fig. S7A-F) and Raws (Fig. S3H-N). Immunofluorescence analysis demonstrated that RBG decreased the expression and co-localization of NLRP3, ASC, and caspase-1 in ox-LDL induced BMDM (Fig. 3W, 3a-d) and similar effects were observed in Raws (Fig. S3V-Z). Immunofluorescence analysis revealed that RBG reduces NF-κB nuclear translocation (Fig. 3T, 3U).

Fig. 3.

RBG enhances foam cell formation in BMDMs induced by ox-LDL. (A and B) Representative images and Statistical analysis of Oil Red O staining in BMDMs are shown for both A and B (n = 4). (C, D, E, F, G) RBG reduces the ratio of M1 macrophages while increases the ratio of M2 macrophages in BMDM polarization induced by ox-LDL or LPS + IFN-γ (n = 4). (H, I, J, K, L, M) Levels of IL18, IL1β, TNF-α, IL10, TG, and TC in the supernatants of BMDM cultures (n = 8). (N, O, P, Q, R, S, T, U, V) Western blot and statistical analysis was conducted for NLRP3, total ASC protein, ASC oligomerization, NEK7, Caspase-1, Cleaved Caspase-1, Pro IL18, and IL18 (n = 3). (W, a, b, c, d) Representative images and Statistical analysis of Immunofluorescence staining of NLRP3, ASC, and Caspase-1 in BMDMs (n = 3). (X and Y) Representative images of NF-κB immunofluorescence in BMDMs. Ratio of NF-κB fluorescence intensity in the nucleus to the cytoplasm (n = 3). *P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001; ns indicates not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

CYS-279 residue of NLRP3 is the binding site for RBG pharmacological inhibition

As shown in Fig. 4A, PyMOL 2.5.5 identified five exposed cysteine residues (CYS-279, CYS-514, CYS-598, CYS-319, CYS-673) on NLRP3′s NACHT domain potentially interacting with RBG. Molecular docking revealed RBG binds mainly to CYS-279 and CYS-598, with CYS-279 showing a more favorable interaction (−5.4 kcal/mol). Fig. 4B depicts RBG in a central position on NLRP3, interacting with CYS-279 and forming hydrogen bonds with ARG-147, stabilizing the molecular recognition pocket and facilitating a stable association. In contrast, Fig. 4C shows RBG docked with the R147A mutant positioned far from CYS-279, making strong interactions unlikely. Weak hydrogen bonding with R237 indicates instability and reduced efficacy. Molecular dynamics simulations support this stability. Fig. 4D shows stable RMSD fluctuations around 3 Å during three 500 ns simulations, indicating RBG and NLRP3′s NACHT domain stably associate, with RBG suppressing the protein's dynamic motion. Fig. 4E monitors the Coulomb and Lennard-Jones potential energies, revealing stable fluctuations, which confirm the simulation's correctness and data reliability. Mutating ARG-147 disrupted RBG's stable binding trend, as shown in Fig. 4F, highlighting ARG-147′s role in the interaction's stability. Extended simulations showed low, stable RMSD fluctuations, demonstrating RBG's inhibitory effect on NLRP3 over time. Finally, SPR results (Fig. 4G, Table S2) confirmed RBG's direct binding to NLRP3 with good affinity (KD = 62.1uM), supporting the interaction mechanism.

Fig. 4.

CYS-279 residue of NLRP3 is the binding site for RBG. (A) Sitemap predicted cavity of human NLRP3 protein, along with a covalent site map where spheres represent the CYS covalent reaction site, and the cyan block indicates the pocket cavity. (B) Binding mode of RBG with wild-type human NLRP3 protein. (C) Binding mode of RBG with the R147A mutant of human NLRP3 protein. (D) Variations in the root mean square deviation (RMSD) of the system throughout the molecular dynamics simulation. (E) Temporal variations in energy. (F) RMSD changes over time after 500 ns of extended simulation and for the R147A mutant simulation. (G) SPR response of RBG with purified human NLRP3 protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

To further confirm the specific binding site of RBG on NLRP3, we constructed a series of virtual mutations of the NLRP3 protein for molecular dynamics simulations. Compared to other mutants, the C279A mutant exhibited significantly higher dynamic parameters such as Ligand RMSD, Complex RMSD, and Radius of Gyration (RoG), and displayed larger fluctuations, indicating that its complex underwent greater structural expansion and instability during the simulation (Fig. S11A). Additionally, the ligand conformation of the C279A mutant differed significantly between the 0 ns and 100 ns time points, with large molecular shifts, suggesting that the C279A mutation led to the loss of ligand binding, preventing the molecule from binding effectively within the pocket (Fig. S11B, C). Finally, through Pull-down experiments, we found that biotinylated RBG could pull down the NLRP3 protein, while free RBG competitively bound to NLRP3, resulting in reduced pull-down (Fig. S11D–G). However, mutation of CYS-279 led to the failure of biotinylated RBG pull-down (Fig. S11H, I).

RBG inhibits NLRP3 inflammasome assembly induced by LPS + ATP in Macrophages

ELISA results demonstrated that RBG could inhibit the levels of IL18 and IL1β in the supernatant of BMDM cultures induced by LPS + ATP (Fig. 5A, 5B). Interestingly, similar results were obtained in Raws (Fig. S4A, B). PCR analysis demonstrated that RBG decreased mRNA levels of NLRP3, ASC, NEK7, Caspase-1, IL18, and IL1β in LPS + ATP induced BMDM (Fig. 5C–H), with comparable findings in Raws (Fig. S4C–H). Western blot analysis revealed that RBG reduced the levels of NLRP3, ASC, ASC oligomerization, NEK7, caspase-1, cleaved caspase-1, pro-IL18, and IL18 in BMDM stimulated with LPS + ATP (Fig. 5I-Q). Immunofluorescence analysis demonstrated that RBG decreased the expression and co-localization of NLRP3, ASC, and caspase in BMDM cells stimulated with LPS + ATP (Fig. 5R, 5U-X), with comparable findings in Raw cells (Fig. S4I-M). Immunofluorescence analysis revealed that RBG reduce NF-κB nuclear translocation (Fig. 5S-T, S4N-O).

Fig. 5.

RBG Inhibits Inflammation and NLRP3 Inflammasome Assembly Induced by LPS + ATP in BMDMs. (A and B) Concentrations of IL18 and IL1β in BMDM culture supernatants (n = 8). (C–H) Relative mRNA expression levels of NLRP3, NEK7, ASC, Caspase-1, IL18, and IL1β were measured in BMDMs (n = 4). (I-Q) Western blot analysis and statistical analysis of NLRP3, ASC total protein, ASC oligomerization, NEK7, Caspase-1, Cleaved Caspase-1, Pro IL18, and IL18 (n = 3). (R, U, V, W, X) Representative immunofluorescence images and statistical analysis of NLRP3, ASC, and Caspase-1 in BMDM (n = 3). (S and T) Representative images of NF-κB immunofluorescence in BMDMs. Ratio of NF-κB relative fluorescence intensity between the nucleus and cytoplasm (n = 3). *P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001; ns indicates not significant.

The inhibitory effect of RBG on LPS + ATP-Induced inflammatory responses in Macrophages Depends on NLRP3

ELISA results indicated that NLRP3 overexpression counteracted the RBG-induced reduction of Caspase-1, IL18, and IL1β levels in the supernatant of BMDM cultures stimulated with LPS + ATP (Fig. 6A-C). Conversely, knockdown of NLRP3 reversed the release of Caspase-1, IL18, and IL1β in the supernatant of BMDM cultures induced by LPS + ATP, and RBG did not exhibit any additive improvement (Fig. 6N-P). We replicated these results in Raws as well (Fig. S5A-F). Western blot analysis indicated that neither overexpression nor knockdown of NLRP3 affected the total protein expression of ASC in BMDM induced by LPS + ATP. However, NLRP3 overexpression reversed the effect of RBG in reducing ASC oligomerization induced by LPS + ATP (Fig. 6D-F), while NLRP3 knockdown reversed the increase in ASC oligomerization in BMDM induced by LPS + ATP, and RBG did not show any additive improvement (Fig. 6Q-S). Immunofluorescence analysis revealed that NLRP3 overexpression counteracted RBG's impact on the expression and co-localization of NLRP3, ASC, and caspase-1 in BMDM triggered by LPS + ATP (Fig. 6G-K). NLRP3 knockdown counteracted the expression and co-localization of NLRP3, ASC, and caspase-1 in BMDM stimulated with LPS + ATP, with RBG showing no additional enhancement (Fig. 6T-X). This phenomenon was also replicated in Raws (Fig. S5G-P). NLRP3 overexpression counteracted RBG's impact on NF-κB nuclear translocation in BMDM stimulated by LPS + ATP (Fig. 6L, 6 M), whereas NLRP3 knockdown reversed the NF-κB nuclear translocation level induced by LPS + ATP without additional enhancement from RBG (Fig. 6Y, 6Z). This phenomenon was also replicated in Raws (Fig. S5Q-T).

Fig. 6.

The Inhibitory Effect of RBG on LPS + ATP Induced Inflammatory Responses in BMDMs Depends on NLRP3. (A, B, C) Concentrations of IL18, IL1β, and Caspase-1 in the culture supernatants of NLRP3-overexpressing BMDMs (n = 8). (D, E, F) Analysis of total protein and ASC oligomerization in NLRP3-overexpressing BMDMs (n = 3). (G, H, J, K) Representative immunofluorescence images and statistical analysis of NLRP3, ASC, and Caspase-1 in NLRP3-overexpressing BMDMs (n = 3). (L and M) Representative images of NF-κB immunofluorescence in NLRP3-overexpressing BMDMs. Ratio of fluorescence intensity of NF-κB in the nucleus to that in the cytoplasm (n = 3). Concentrations of IL18, IL1β, and Caspase-1 in the supernatants of NLRP3-knockdown BMDMs (n = 8). (Q, R, S) Total protein and oligomerization levels of ASC in NLRP3-knockdown BMDMs (n = 3). Representative immunofluorescence images and statistical analysis of NLRP3, ASC, and Caspase-1 staining in NLRP3-knockdown BMDMs (n = 3). (Y and Z) Representative images of NF-κB immunofluorescence in NLRP3-knockdown BMDMs. Ratio of NF-κB relative fluorescence intensity between the nucleus and cytoplasm (n = 3). *P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001; ns indicates not significant.

The enhancement of RBG in reducing foam cell formation induced by ox-LDL in macrophages is contingent upon NLRP3

Oil Red O staining demonstrated that NLRP3 overexpression counteracted the reduction in foam cell formation and lipid droplet accumulation in BMDM induced by ox-LDL, as achieved by RBG (Fig. 7A). NLRP3 knockdown counteracted ox-LDL induced foam cell formation and lipid droplet accumulation in BMDM, with no additional enhancement observed from RBG (Fig. 7O). ELISA results showed that NLRP3 overexpression reversed the effect of RBG on the levels of IL18, IL1β, TNF-α, IL10, TG, and TC in the supernatant of BMDM cultures induced by ox-LDL (Fig. 7B-G). NLRP3 counteracted the effects of ox-LDL on IL18, IL1β, TNF-α, IL10, TG, and TC levels in BMDM culture supernatants, with no additional enhancement observed from RBG (Fig. 7P-U). We also observed this phenomenon in Raws, as illustrated in Fig. S6A-L. Immunofluorescence analysis indicated that NLRP3 overexpression counteracted the impact of RBG on the expression and co-localization of NLRP3, ASC, and caspase-1 in BMDM stimulated by ox-LDL (Fig. 7H-L). NLRP3 knockdown counteracted the ox-LDL-induced expression and co-localization of NLRP3, ASC, and caspase-1 in BMDM, with RBG showing no additional enhancement (Fig. 7V-7Z). This phenomenon was similarly observed in Raws (Fig. S6M-V). NLRP3 overexpression counteracted RBG's impact on NF-κB translocation in ox-LDL induced BMDM (Fig. 6M, 6 N), whereas NLRP3 knockdown reversed ox-LDL induced NF-κB nuclear translocation without additional enhancement from RBG (Fig. 7a, 7b). This phenomenon was also replicated in Raws (Fig. S6W-Z).

Fig. 7.

The Inhibitory Effect of RBG on Ox-LDL Induced Inflammatory Responses and Foam Cell Formation in BMDMs Depends on NLRP3. (A) Representative images of Oil Red O staining in NLRP3-overexpressing BMDMs (n = 6). (B, C, D, E, F, G) Concentrations of IL18, IL1β, TNF-α, IL10, TG, and TC in the culture supernatants of NLRP3-overexpressing BMDMs (n = 8). (H, I, J, K, L) Representative immunofluorescence images and statistical analysis of NLRP3, ASC, and Caspase-1 in NLRP3-overexpressing BMDMs (n = 3). (M and N) Representative images of NF-κB immunofluorescence in NLRP3-overexpressing BMDMs. Ratio of relative fluorescence intensity of NF-κB in the nucleus to that in the cytoplasm (n = 3). (O) Representative images of Oil Red O staining in NLRP3-knockdown BMDMs (n = 6). (P, Q, R, S, T, U) Concentrations of IL18, IL1β, TNF-α, IL10, TG, and TC in the culture supernatants of NLRP3-knockdown BMDMs (n = 8). (V, W, X, Y Z) Representative immunofluorescence images and statistical analysis of NLRP3, ASC, and Caspase1 in NLRP3-knockdown BMDMs (n = 3). (a and b) Representative images of NF-κB immunofluorescence in NLRP3-knockdown BMDMs. Ratio of relative fluorescence intensity of NF-κB in the nucleus to that in the cytoplasm (n = 3). *P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001 and ns: Not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

The ameliorative impact of RBG on HFD-induced AS in ApoE^-/- mice is contingent upon NLRP3

After administering HFD to ApoE^-/- mice for 4 weeks, we performed tail vein injections of AAV- shRNA-NLRP3 or control empty vector as part of an intervention scheme (Fig. 8A). Our study demonstrated that the Knockdown of NLRP3 did not alter the weight-reducing impact of RBG in ApoE^-/- mice subjected to a high-fat diet (Fig. 8B, 8Q). Macroscopic Oil Red O staining of the aorta indicated that RBG treatment offered protection against AS in ApoE^-/- mice; however, this protective effect was diminished by NLRP3 knockdown (Fig. 8C, 8D). HE, Masson, and Oil Red O staining of the aortic root revealed smaller plaques in RBG-treated mice. Although NLRP3 knockdown did not fully eliminate these effects, as pathological changes persisted at the aortic root, this was not consistent with the overall aortic changes (Fig. 8M-P). Serum analysis demonstrated that RBG treatment partially improved TG, TC, LDL-C, and HDL-C levels in ApoE^-/- mice injected with the empty virus. Although the injection of AAV-NLRP3 virus had some impact, the improvement effect of RBG was still observed (Fig. 8E-H). Knockdown of NLRP3 counteracted the HFD-induced alterations in serum levels of IL18, IL1β, TNF-α, and IL10 in ApoE^-/- mice, diminishing the ameliorative effects of RBG on these parameters (Fig. 8I-L).

Fig. 8.

The beneficial impact of RBG on AS caused by HFD in ApoE^-/- mice relies on NLRP3. (A) Construction of vascular-specific NLRP3 knockdown in ApoE^-/- mice, model replication, and treatment protocol. (B) Body weight-time curve of mice (n = 10). (C, D) Representative images and quantification of lesion area of Oil Red O staining of the aorta (n = 3). (E-L) Serum levels of TG, TC, LDL-C, HDL-C, IL18, IL1β, TNF-α, and IL10 were measured (n = 10). (M−P) Representative images and quantification of lesion area of HE, MASSON, and Oil Red O staining (n = 3). (Q) Comparison of mouse body weight at the end of the experiment. *P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001; ns indicates not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Discussion

AS significantly contributes to cardiovascular disease, marked by chronic inflammation and lipid accumulation in arterial walls [49,50]. In this research, we explored the potential therapeutic effects of RBG on AS using ApoE^-/- mice (Fig. 9). Our results indicate that RBG significantly reduces AS-related characteristics, including inflammatory infiltration, lipid accumulation, and fibrosis. Cellular studies revealed that RBG improved the resolution of macrophage foam cell formation, inflammatory profiles, and lipid metabolism dysregulation induced by ox-LDL. RNA-seq analysis revealed that the protective effects of RBG are linked to anti-inflammatory signaling pathways. We found that RBG suppresses inflammation in mice by inhibiting NLRP3 expression and activity. Surface plasmon resonance analysis revealed that RBG directly binds to the CYS-279 residue in the NATCH domain of NLRP3, inhibiting NLRP3 inflammasome assembly and reducing IL18 and IL1β secretion induced by LPS + ATP. Moreover, molecular docking and dynamic simulations further corroborated this interaction. However, mutating this residue significantly diminished the binding affinity between RBG and NLRP3, uncovering a new site for inhibiting NLRP3. Importantly, vessel-specific knockdown of NLRP3 significantly weakened the protective effects of RBG on AS-related characteristics. This study highlights RBG's therapeutic potential in cardiovascular diseases, focusing on its effectiveness as a potent NLRP3-targeted inhibitor for AS prevention and treatment.

Fig. 9.

Schematic Diagram of the Mechanism of RBG in Treating AS.

AS, as a major cardiovascular disease, has a complex and diverse pathogenesis, with inflammatory responses identified as one of the key driving factors [1,2,[51], [52], [53]]. Clinical research has linked NLRP3 activation with increased inflammatory markers in patients with AS [12,13,53]. Animal models demonstrate that NLRP3 knockout mice exhibit reduced plaque formation and inflammation [14,17,19,20,30]. In vitro, cholesterol crystals activate NLRP3 in macrophages, promoting IL1β secretion and foam cell transformation. Gene manipulation studies demonstrate that NLRP3 overexpression increases cytokine release, whereas its knockdown diminishes inflammation and foam cell formation, underscoring NLRP3′s pivotal role in atherosclerotic progression. The NLRP3 inflammasome is an intracellular multiprotein complex that detects pathogen-associated and damage-associated molecular patterns, resulting in its activation. Once activated, the NLRP3 inflammasome engages caspase-1, facilitating the maturation and release of pro-inflammatory cytokines like IL1β and IL18 [[32], [33], [34]]. The cytokine release intensifies local inflammation and promotes macrophage transformation into foam cells, thereby advancing AS progression [3,10,14,17,19,23,28]. The dual role of macrophages in AS makes them a focal point of research. Macrophages ingest ox-LDL to form foam cells, which contribute to the initial development of plaques [26,27,54]. Conversely, macrophage-released pro-inflammatory cytokines exacerbate inflammation, causing plaque instability and rupture [3,4,12,54]. The assembly and activation of NLRP3, along with its role in mediating macrophage foam cell formation, function as a double-edged sword. In the early stages or under low lipid load, this process can have a protective effect on blood vessels, but under prolonged lipid overload, the rupture of foam cells and the resulting release of their contents can trigger a negative feedback loop of inflammation, causing continuous progression of AS [3,20,24,26,27,54]. This was confirmed in experiments involving NLRP3 knockdown in APOE^-/- mice, where, although no lipid plaques appeared in the aorta, pathological damage was observed in the aortic sinus, a region with complex hemodynamics. Furthermore, RBG or MCC950 could not alleviate these effects owing to NLRP3 knockdown. Many evidences indicates that NLRP3 inhibitors show significant potential in the treatment of AS. Inhibiting NLRP3 inflammasome activation mitigates the inflammatory response and prevents foam cell formation. Our research offers experimental and theoretical evidence supporting RBG as a novel NLRP3 inhibitor for AS prevention and treatment. RBG inhibits M1 macrophage activation and promotes M2 macrophage polarization. This transition is significant for reducing inflammatory responses and promoting tissue repair. Future research should investigate the clinical potential of RBG, especially in combination with other therapies for AS. Understanding the mechanisms of NLRP3 and its inhibitors can lead to more effective treatment strategies, while also assessing RBG’s safety and efficacy in diverse patient populations. RBG targets the NLRP3 inflammasome in macrophages, presenting a promising therapeutic strategy for AS. The study highlights the crucial role of the NLRP3 inflammasome in macrophage foam cell formation and suggests RBG's potential as a treatment option. Continued research and clinical trials may position RBG as a valuable anti-inflammatory option for cardiovascular patients.

Our study thoroughly explored the mechanism by which RBG inhibits NLRP3, identifying the CYS-279 residue in the NACHT domain as crucial for this inhibition. The core region responsible for the ATPase activity of the NLRP3 protein is the NACHT domain, with the CYS-279 residue playing a critical role in maintaining its functional integrity [15,18,33]. Our experimental results, through molecular docking, dynamic simulations, and surface plasmon resonance (SPR) experiments, clarified the binding mode of RBG with the NLRP3 protein. The SPR results indicated that RBG binds to NLRP3 with a fast dissociation rate, suggesting a non-covalent binding interaction. This non-covalent binding mode offers several advantages. Firstly, non-covalent interactions are generally more reversible than covalent bonds, allowing for dynamic regulation of the binding process in response to cellular signals or changes in the microenvironment. This reversibility is crucial for maintaining the balance of biological processes and ensuring that the inhibition of NLRP3 by RBG can be finely tuned as needed. Secondly, the fast dissociation rate observed in the SPR experiments implies that RBG can rapidly bind to and dissociate from NLRP3. This rapid kinetics not only enhances RBG’s binding affinity for NLRP3 by allowing it to sample multiple binding sites and conformations more efficiently but also provides sustained inhibition of NLRP3 activity without causing prolonged or irreversible effects. This is particularly beneficial in the context of therapeutic applications, where minimizing off-target effects and maintaining physiological homeostasis are paramount. Compared to other NLRP3 inhibitors reported in the literature, RBG exhibits unique advantages. Many inhibitors rely on non-covalent binding but may require higher concentrations or exhibit different binding kinetics to be effective [55]. In contrast, RBG achieves effective inhibition at relatively low concentrations through its specific non-covalent binding mode, which is characterized by the SPR results showing fast dissociation. This means that RBG can exert its inhibitory effect more efficiently, reducing the potential for side effects associated with high drug concentrations. The binding of RBG to NLRP3 effectively inhibits its activity, significantly preventing macrophage foam cell formation and decreasing inflammatory factor release. In experiments with high-fat diet (HFD)-induced APOE-/- mice, RBG treatment significantly reduced the arterial expression of NLRP3, ASC, and caspase-1, and effectively controlled serum levels of IL1β and IL18. The findings suggest that RBG significantly mitigates macrophage-mediated inflammation and foam cell formation by inhibiting NLRP3 activation, thus playing a protective role in the progression of atherosclerosis (AS). The NLRP3 inflammasome is crucial in AS, expediting disease progression through its role in inflammatory responses and foam cell formation. We conducted a series of mutations followed by molecular dynamics simulations and Pull-down experiments to confirm that RBG specifically targets the CYS-279 residue in the NACHT domain of NLRP3. These experiments demonstrated that the interaction between RBG and the CYS-279 residue is essential for the inhibitory effect on NLRP3. Unlike some traditional inhibitors, RBG offers more durable and efficient inhibition through its specific non-covalent binding to CYS-279, thereby minimizing potential off-target effects. The non-covalent nature of the binding allows RBG to selectively target NLRP3 without affecting other proteins with similar structures or functions, reducing the risk of unwanted side effects. Furthermore, the fast dissociation rate of RBG from NLRP3 contributes to its safety and efficacy. By rapidly dissociating from the target, RBG can avoid prolonged inhibition of NLRP3, which could potentially lead to undesirable physiological effects. This rapid dissociation also allows for better control of the therapeutic window, enabling precise dosing and timing of RBG administration to achieve optimal therapeutic outcomes. The mechanisms of RBG offer a robust theoretical basis for its application in AS prevention and treatment. Our research not only reveals the molecular-level mechanism of RBG but also verifies its practical effects under physiological conditions through animal models. The potential of RBG in AS prevention and treatment is reflected not only in its inhibition of NLRP3 but also in its multiple roles in regulating macrophage function and inflammatory responses.

The NLRP3 inflammasome is essential for inflammatory responses, foam cell formation, and the regulation of macrophage polarization into M1 and M2 phenotypes [56]. Macrophage polarization directly affects their immune response functions [22,26,29,30,56]. M1 macrophages are recognized as pro-inflammatory phenotypes that release significant amounts of pro-inflammatory factors, including IL1β, TNF-α, and IL6, thereby intensifying inflammation and advancing AS progression. M2 macrophages are considered anti-inflammatory, playing a role in tissue repair and resolving inflammation. M1 macrophage polarization is strongly linked to NLRP3 inflammasome activation. Its activation promotes the maintenance and enhancement of M1 macrophages, thereby intensifying local inflammatory responses. This pro-inflammatory environment not only accelerates the formation and instability of atherosclerotic plaques but also hinders the polarization and function of M2 macrophages. Modulating NLRP3 inflammasome activity can alter macrophage polarization, thereby achieving balanced inflammatory response regulation. Our experimental results further confirm this perspective. In HFD-induced APOE^-/- mouse model, treatment with RBG significantly reduced inflammation levels associated with AS while promoting M2 macrophage polarization. RBG modulates macrophage activity by inhibiting the NLRP3 inflammasome, thereby diminishing the pro-inflammatory response of M1 macrophages and promoting the anti-inflammatory effects of M2 macrophages. The transition from M1 to M2 is crucial for alleviating the inflammatory burden of atherosclerotic plaques and promoting plaque stability. Our findings indicate that the NLRP3-mediated macrophage transition between M1 and M2 phenotypes serves a dual function in both preventing and treating AS. Inhibiting NLRP3 activity reduces M1 macrophage-related inflammation, alleviating AS's inflammatory burden, while promoting M2 macrophage polarization aids tissue repair and plaque stability, lowering cardiovascular event risk. RBG's involvement in NLRP3-mediated polarization transitions offers a novel perspective for AS treatment and a scientific foundation for new anti-inflammatory drug development. RBG shows significant potential in treating inflammatory diseases by modulating macrophage polarization.

This article explores RBG's inhibitory effects on the NLRP3 inflammasome and its potential applications in AS. However, there are several shortcomings in the study. While animal models offer important insights, clinical trials validating the safety and efficacy of RBG in humans are lacking [57,58]. Secondly, the paper does not comprehensively evaluate the potential side effects and toxicity associated with prolonged RBG usage. Additionally, the effects of RBG when combined with other therapeutic approaches have not been systematically studied, which limits its application prospects as part of a comprehensive treatment strategy. Finally, while the molecular mechanisms of NLRP3 inhibition have been explored, other potential molecular targets have not been adequately investigated.

Conclusion

This research explores RBG's potential as an NLRP3 inflammasome inhibitor for treating AS. Through cell experiments, animal models, and molecular simulations, RBG is shown to effectively inhibit NLRP3 inflammasome activation, reduce pro-inflammatory cytokine release, and decrease macrophage foam cell formation, thereby slowing AS progression. However, further research is necessary to assess RBG’s safety and efficacy in humans and its potential synergistic effects with other therapies. Overall, RBG offers a promising therapeutic strategy for cardiovascular diseases, laying the groundwork for future research and clinical applications.

Ethics statement

All experiments involving mice were performed in accordance with the ethical policies and procedures approved by the Animal Ethics Committee of Guangzhou University of Chinese Medicine (approval No. 20201013002).

CRediT authorship contribution statement

Chen Xiaoyang: Writing – review & editing. Chen Yijun: Conceptualization, Methodology, Software. Zhai Chenguang: Data curation, Writing – original draft. Du Wanying: Data curation. Chen Zijun: Software, Validation. Wang Jun: Visualization, Investigation. Xu Xuegong: Funding acquisition, Conceptualization. Wang Wei: Project administration, Conceptualization, Methodology. Li Chun: Project administration, Conceptualization, Methodology.

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 material

The following are the Supplementary data to this article: