Aspalathin-rich unfermented Aspalathus linearsis extract mitigates vascular endothelial inflammation by inhibiting the TNF-α/Hexokinase 2/NF-κB signaling pathway

Inflammation of vascular endothelium initiates cardiovascular diseases. We investigated if unfermented green Aspalathus linearis (rooibos) extract (GRT) and its main flavonoid aspalathin may suppress inflammation in cardiovascular endothelial cells. Human coronary artery endothelial cells (HCAEC) and human umbilical vein endothelial cells (HUVEC) were used as cell models, while β-aminopropionitrile (BAPN)-induced inflammation in mice was used as animal models. Tube formation assay, transwell migration assay, wound healing assay and monolayer permeability assay were used to examine the inflammatory effects on vascular endothelial cells, while cytokine array, qRT-PCR, ELISA, reporter gene assay, fluorescent microscopy, western blot, IHC staining and Seahorse mitochondrial analyzer were used to dissect the molecular mechanisms lying underneath. TNF-α decreased angiogenesis and VE-cadherin expression but increased cellular migration, monolayer permeability, secretion of inflammation cytokines IL-6 and IL-8, as well as NF-κB activity in HCAEC and HUVEC cells. Treatment with GRT or aspalathin reversed these TNF-α-induced inflammatory effects. We discovered that hexokinase 2 (HK2) was essential in regulation of TNF-α-induced inflammation in vascular endothelial cells, but was inhibited by GRT or aspalathin. Overexpression of HK2 induced NF-κB phosphorylation, activity and inflammatory effects. However, these phenomenon was repressed by GRT, aspalathin or HK2 knockdown. GRT or aspalathin reversed the TNF-α-enhanced interaction between HK2 and YB-1, vimentin or drebrin. Gavage of GRT repressed the expression of IL-6 and HK-2 in aortic tissues in BAPN-induced inflammation murine model. Our study demonstrated that GRT or aspalathin can suppress the TNF-α-induced inflammation in human vascular endothelial cells via inhibition of TNF-α/HK2/NF-κB signaling axis.