ICAM-1 overexpression and subsequent adhesion of leukocytes to endothelial cells play critical roles in the early stage of atherosclerosis. Danshenol A (DA) is an abietane-type diterpenoid isolated from traditional Chinese herb Salvia miltiorrhiza Bunge. The mechanisms under its regulation of adhesion of molecular expression are explored. Here, the effect of DA on TNF-α-induced ICAM-1 expression was investigated in endothelial cells. TNF-α-induced ICAM-1 expression and subsequent adhesion of monocytes, as well as elevated reactive oxygen species (ROS) generation and NOX4 expression were all significantly reversed by DA, siNOX4 and NOX4 inhibitor GKT137831. Furthermore, TNF-α-induced ICAM-1 expression, which was increased via IKKβ/IκBα-mediated activation of NF-κB p65, was also inhibited by DA. Interestingly, NOX4 overexpression suppressed the ICAM-1 expression, and this finding may be ascribed to the activation of Nrf-2. Additionally, NF-κB inhibitor PDTC, siNOX4, or DA can decrease the TNF-α-induced ICAM-1 expression and suppress the adhesion of monocytes. In all, DA inhibited TNF-α-induced ICAM-1 expression and subsequent monocyte adhesion to endothelial cells through the NOX4-dependent IKKβ/NF-κB pathway. Besides, NOX4 played dual role in regulating ICAM-1 expression via diverse signal pathway. This novel bioactivity will make DA a good candidate to be further explored for therapeutic or preventive application for atherosclerosis.

Hispidulin, a polyphenolic flavonoid extracted from the traditional Chinese medicinal plant S involucrata, exhibits anti-tumor effects in a wide array of human cancer cells, mainly through growth inhibition, apoptosis induction and cell cycle arrest. However, its precise anticancer mechanisms remain unclear. In this study, we investigated the molecular mechanisms that contribute to hispidulin-induced apoptosis of human clear-cell renal cell carcinoma (ccRCC) lines Caki-2 and ACHN. Hispidulin (10, 20 μmol/L) decreased the viability of ccRCC cells in dose- and time-dependent manners without affecting that of normal tubular epithelial cells. Moreover, hispidulin treatment dose-dependently increased the levels of cleaved caspase-8 and caspase-9, but the inhibitors of caspase-8 and caspase-9 only partly abrogated hispidulin-induced apoptosis, suggesting that hispidulin triggered apoptosis via both extrinsic and intrinsic pathways. Moreover, hispidulin treatment significantly inhibited the activity of sphingosine kinase 1 (SphK1) and consequently promoted ceramide accumulation, thus leading to apoptosis of the cancer cells, whereas pretreatment with K6PC-5, an activator of SphK1, or overexpression of SphK1 significantly attenuated the anti-proliferative and pro-apoptotic effects of hispidulin. In addition, hispidulin treatment dose-dependently activated ROS/JNK signaling and led to cell apoptosis. We further demonstrated in Caki-2 xenograft nude mice that injection of hispidulin (20, 40 mg·kg⁻¹·d⁻¹, ip) dose-dependently suppressed tumor growth accompanied by decreased SphK1 activity and increased ceramide accumulation in tumor tissues. Our findings reveal a new explanation for the anti-tumor mechanisms of hispidulin, and suggest that SphK1 and ceramide may serve as potential therapeutic targets for the treatment of ccRCC.