Effects of Rhaponticum carthamoides (Willd.) Iljin on endothelial dysfunction and the inflammatory response in type 2 diabetes mellitus mice

Background: Diabetes mellitus (DM) and its complications seriously threaten human life and health. Rhaponticum carthamoides (Willd.) Iljin (RC) is widely used to treat cardiovascular diseases. Previous studies reported that RC reduces blood glucose levels in rats with type 1 DM. However, the effects of RC on type 2 diabetes and vascular complications, as well as its related active components and underlying mechanisms, remain unclear. Purpose: This study aimed to investigate the effects of RC on endothelial dysfunction and the inflammatory response in type 2 DM mice and to explore its underlying mechanism and active ingredients. Study Design/Methods: Male C57BL/6J mice were used to establish a type 2 DM mouse model. After 12 weeks of oral administration of RC extract (60, 120, and 240 mg/kg) to mice, blood glucose and lipid levels were assessed. The morphological structures of the liver and kidney tissues were observed using hematoxylin and eosin (HE) staining, and their functions were evaluated by detecting relevant biochemical indicators in the serum. Then, aorta morphology was observed via HE staining. In addition, serum levels of markers of endothelial function and inflammatory factors were detected, and the expression of inflammatory factors and the phosphorylation levels of key proteins in the aorta were examined. Furthermore, prediction and enrichment analyses of potential targets of RC acting on diabetic vascular lesions were performed on the basis of pharmacophore matching using various databases. Then, the expression, localization and phosphorylation levels of potential targets in the aortas of DM mice treated with RC were assessed using Western blotting, immunofluorescence, and RT‒PCR. Finally, the active components of RC were identified through virtual screening, and their ability to improve endothelial cell dysfunction was verified. Results: RC reduced blood glucose levels and serum lipid levels of total triglyceride (TG), total cholesterol (TC), and low density lipoprotein cholesterol (LDL-c), increased high density lipoprotein cholesterol (HDL-c) levels, and improved liver and kidney function in type 2 DM mice. RC decreased endothelial cell shedding in the aortas of type 2 DM mice, increased serum nitric oxide (NO) and nitric oxide synthase (NOS) levels, and reduced soluble cluster of differentiation 40 ligand (sCD40L), tumor necrosis factor α (TNF-α), and interleukin-1β (IL-1β) levels. Further findings indicated that RC reduced the expression of aortic inflammatory factors, namely, CD40, CD40L, IL-1β, and interleukin-6 (IL-6), and increased endothelial NOS (eNOS) phosphorylation levels. Sirtuin 6 (SIRT6), protein kinase B (AKT), and eNOS were predicted to be key node targets of RC acting on DM vascular lesions, and it was confirmed that RC increased SIRT6 expression and AKT phosphorylation levels in aortic endothelial cells. 20-Hydroxyecdysone (20E), daucosterol (Dau), euscaphic acid (Eus), and syringin (Syr) were identified as active components of RC. These components protect against TNF-α-induced human umbilical vein endothelial cell (HUVEC) damage and decrease the release of lactate dehydrogenase (LDH) and IL-1β and increased the release of NO in TNF-α-induced HUVECs in a dose-dependent manner. Conclusion: RC reduced blood glucose and lipid levels in mice with type 2 DM and protected liver and kidney function. RC promotes SIRT6 expression in endothelial cells; upregulates the NO/NOS system by increasing AKT/eNOS phosphorylation levels to regulate vascular tone factors; and reduces the levels of inflammatory factors such as CD40, TNF-α, and IL-1β to inhibit endothelial inflammatory responses. Based on these mechanisms, RC improves endothelial dysfunction.