Hydroxysafflor yellow A regulates lymphangiogenesis and inflammation via the inhibition of PI3K on regulating AKT/mTOR and NF-κB pathway in macrophages to reduce atherosclerosis in ApoE-/- mice

Background: Macrophage-mediated inflammatory infiltration and pathological lymphangiogenesis around atherosclerotic plaques are newly highlighted treatment targets of atherosclerosis. Although the effect of Hydroxysafflor yellow A(HSYA) on atherosclerosis was clear, few studies focus on the regulation of HSYA on such mechanisms.

Purpose: This study aimed to uncover the key site of HSYA on improving atherosclerosis by regulating macrophage-induced inflammation and lymphangiogenesis.

Study design: This study was designed to explore the new mechanism of HSYA on alleviating atherosclerosis in vitro and in vivo.

Methods: We determined the expression of vascular endothelial growth factor C(VEGF-C) in Raw264.7 cells and high-fat diet fed ApoE knockout (ApoE^-/-) mice. Raw264.7 cells were treated with HSYA under the stimulation of LPS and ox-LDL. HFD induced ApoE^-/- mice were given different concentrations of HSYA-saline solution by tail vein injection and ATV-saline suspension by gavage. C57/B6j mice fed with chow diet were used for the control group. H&E, oil red O and immunofluorescence staining analysis were used for visualizing the pathological changes. The biological impact of HSYA was evaluated by body weight, lipid metabolism, inflammation levels, and corresponding function indexes of kidney and liver. RT-qPCR and western blot methods were conducted to determine the expression of the inflammation and lymphangiogenesis factors. Molecular docking and microscale thermophoresis analysis were used to verify the combination of HSYA and PI3K.

Results: In vivo, HSYA reduced the plaque formation, hepatic steatosis and inflammation-related lymphangiogenesis (IAL). It also changed the serum levels of inflammation (VEGF-C, TNF-α, IL-6, VCAM1, MCP1), lipid indexes (LDL, CHOL, TRIG) and relevant lymphangiogenesis (VEGF-C and LYVE-1) and inflammation (VCAM-1 and IL-6) signals in the aorta. In vitro, HSYA regulated Akt/mTOR and NF-κB activation by the inhibition of PI3K in macrophages.

Conclusion: HSYA affects inflammation and inflammation-associated lymphangiogenesis via suppressing PI3K to affect Akt/mTOR and NF-B pathway activation in macrophages, showing a comprehensive protective effect on atherosclerosis.

Atherosclerosis; Hyperlipidemia; Inflammation; Lymphangiogenesis; hepatic steatosis; hydroxysafflor yellow A.