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

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.

Keywords: Atherosclerosis; Inflammation; Macrophage; NLRP3; Resibufogenin.

Graphical abstract

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.)

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.

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.)

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.)

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.

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.

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.)

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.)

Fig. 9

Schematic Diagram of the Mechanism of RBG in Treating AS.