Costunolide alleviates atherosclerosis in high-fat diet-fed ApoE^−/− mice through covalently binding to IKKβ and inhibiting NF-κB-mediated inflammation

Costunolide (CTD) is a sesquiterpene lactone isolated from costus root and exhibits various biological activities including anti- inflammation. Since atherosclerosis is a chronic inflammatory disease, we herein investigated the anti-atherosclerotic effects of CTD and the underlying mechanism. Atherosclerosis was induced in ApoE^−/− mice by feeding them with a high-fat diet (HFD) for 8 weeks, followed by administration of CTD (10, 20 mg ·kg⁻¹·d⁻¹, i.g.) for 8 weeks. We showed that CTD administration dose- dependently alleviated atherosclerosis in HFD-fed ApoE^−/− mice. Furthermore, we found that CTD dose-dependently reduced inflammatory responses in aortas of the mice, as CTD prevented infiltration of inflammatory cells in aortas and attenuated oxLDL uptake in macrophages, leading to reduced expression of pro-inflammatory and pro-fibrotic molecules in aortas. Similar results were observed in oxLDL-stimulated mouse primary peritoneal macrophages (MPMs) in vitro. We showed that pretreatment with CTD (2.5, 5. 10 μM) restrained oxLDL-induced inflammatory responses in MPMs by blocking pro-inflammatory NF-κB/p65 signaling pathway. We further demonstrated that CTD inactivated NF-κB via covalent binding to cysteine 179 on IKKβ, a canonical upstream regulator of NF-κB, reducing its phosphorylation and leading to conformational change in the active loop of IKKβ. Our results discover IKKβ as the target of CTD for its anti-inflammatory activity and elucidate a molecular mechanism underlying the anti-atherosclerosis effect of CTD. CTD is a potentially therapeutic candidate for retarding inflammatory atherosclerotic diseases.