Sparstolonin B and Curcumin alleviate atherogenesis by modulating T helper 17-stromal cell interactions associated with interleukin-17 receptor A (IL-17RA) - transforming growth factor-β-activated kinase 1 (TAK1) - nuclear factor-kappa B (NF-κB) signaling

Background: Sparstolonin B (SSNB) and Curcumin (Cur), from a pair of compatible herbs, were previously identified as anti-inflammation and T helper 17 (Th17) modulation reagents. However, their compatible roles in atherosclerosis (AS) and underlying mechanisms remain uninvestigated.

Methods: In vivo, the apoE^-/- mice fed with high fat diet were used as mice model of AS. In vitro, macrophages, vascular smooth muscle cells (VSMC) were used in biological tests for vascular atherogenesis. For the Th17 differentiation, bone marrow-derived dendritic cells (DC) from wild-type mice were stimulated and co-cultured with sorted naïve CD4⁺T cells from mice spleen, and. Functional assessments included flow cytometry, Oil Red O staining, ELISA, Western blotting (WB), and label-free proteomics of mouse peripheral blood. Molecular docking and Surface plasmon resonance (SPR) was used for evaluating binding affinity of ligands to target proteins.

Results: The gene-disease interaction and hub gene network reveals Th17-associated genes in the pathogenesis of atherosclerosis. In vitro, SSNB and Cur reduced oxLDL-induced BMDC activation by downregulating CD36. SSNB showed stronger inhibition to inflammatory activation of DC, while Cur more intensively suppressed co-stimulatory molecules. For the Th17/Treg bias in co-culture of BMDC and CD4⁺T cells, SSNB exerted a more significant inhibitory effect on Th17 differentiation, whereas SSNB and Cur exerted stronger effect on promoting Treg expansion. Cur exerts a more significant inhibitory effect on macrophages inflammation, and SSNB showed better inhibition of oxLDL uptake in VSMC foam cell formation. In vivo, SSNB and Cur reduced aortic atherosclerotic lesions and IL-17A in aortic valve, inflammatory cells infiltration and related Th17 immune response in apoE^-/- mice. Proteomics identified proteolysis, oxidation-reduction, protein phosphorylation and metabolic process as top biological processes in the effects of SSNB and Cur. WB indicated IL-17RA-TAK1-NF-κB interaction as the related underlying molecular pathways. SSNB mainly inhibited TAK1 phosphorylation, while Cur more directly inhibited NF-κB P65 phosphorylation. Molecular docking indicated SSNB was superior binding to TLR4 and CD36 than Cur, and the binding result was further confirmed by molecular dynamic simulations and SPR verification.

Conclusion: Our findings reveal, for the first time, that SSNB and Cur alleviate AS by modulating Th17-stromal cell interactions, with the IL-17RA-TAK1-NF-κB pathway as a related mediator. Notably, SSNB and Cur exhibit distinct anti-atherogenic roles. SSNB primarily targets TLR4/CD36 to inhibit DC activation, Th17 differentiation, VSMC inflammation and mainly inhibited TAK1 phosphorylation, while Cur more significant inhibited macrophage inflammation, and more directly inhibited NF-κB P65 phosphorylation. This study will be valuable for developing novel and precise Adjuvant therapies for AS.

Atherosclerosis; Macrophages and VSMC; Proteomic analysis; SSNB and Cur; Th17/Treg bias.