Lonicerin targets ADRA1D and RSPO3 to ameliorate diabetes-induced vascular injury through Ca2+/Calcineurin/NFAT1-dependent anti-EndMT pathway.

Cardiovascular complications are the primary cause of mortality and disability among diabetic patients. Lonicerin, a major bioactive compound in Lonicera japonica Thunb., has unclear effects and mechanisms on diabetic vascular injury. The research aims to investigate the therapeutic effects of lonicerin on diabetic vascular injury and elucidate its underlying molecular mechanisms. Streptozotocin (STZ)-induced mice and high glucose-treated human aortic endothelial cells (HAECs) were employed as animal and cellular models of diabetes. Endothelial-to-mesenchymal transition (EndMT) was validated by examining EndMT-related markers and endothelial/mesenchymal functions. RNA sequencing was used to identify potential mechanisms through which lonicerin alleviates EndMT. The relationship between lonicerin and its targets was investigated using RNAi, plasmid overexpression, western blot, qRT-PCR, immunofluorescence, flow cytometry, and calcineurin activity assays. Lonicerin dose-dependently alleviates diabetes-induced vascular injury (intimal damage, inflammation, calcification, and fibrosis) and EndMT. Lonicerin produces anti-EndMT effect by inhibiting cytoplasmic Ca²⁺ levels. Further analysis reveals that adrenoceptor alpha 1D (ADRA1D) and R-spondin 3 (RSPO3) are targets of lonicerin. Lonicerin reduces high glucose-induced upregulation of ADRA1D and RSPO3, leading to decreased cytoplasmic Ca²⁺ levels. This reduction inhibits calcineurin activity, promotes nuclear factor of activated T cells 1 (NFAT1) phosphorylation, and prevents its nuclear translocation, ultimately exerting an anti-EndMT effect. EC-specific overexpression of ADRA1D or RSPO3 negates the inhibitory effects of lonicerin on EndMT and its therapeutic impact on diabetic vascular injury. Lonicerin targets ADRA1D and RSPO3 to ameliorate diabetes-induced vascular injury through the Ca2+/Calcineurin/NFAT1-dependent anti-EndMT pathway. Thus, this study provides the first evidence that lonicerin is a potential novel therapeutic agent for diabetic vasculopathy.