Orientin Mitigates High Glucose/Ox‐LDL–Triggered Endothelial Cell Injury and Atherosclerosis by Regulating MARCH8‐Mediated NLRP3 Inflammasome Activation

Endothelial cells under oxidative stress and inflammation are vital contributors to the progression of atherosclerosis. Although Orientin possesses antioxidant and anti-inflammatory activities, the effects of Orientin on oxidized low-density lipoprotein and high glucose (ox-LDL/HG)-triggered endothelial cell injury and diabetes-accelerated atherosclerosis remain unclear. ApoE^-/- mice were administered streptozotocin (STZ), fed with high-fat diet (HFD), and then treated with Orientin to test the efficacy of Orientin on ameliorating atherosclerosis through pathological and biochemical assays. Human aortic endothelial cells (HAECs) were stimulated by ox-LDL/HG followed by Orientin treatment, and the effects of Orientin on regulating HAEC viability, oxidative stress, inflammation, and endothelial-mesenchymal transition (EndMT) were assessed using cell counting kit-8 (CCK-8), fluorescein diacetate (FDA) staining, quantitative real-time PCR, immunofluorescence (IF), and western blot assays. The results showed that Orientin treatment decreased atherosclerotic plaque burden, lipid lesion, and collagen content in aortic and femoral arteries in diabetic mice. Meanwhile, Orientin alleviated hypercholesterolemia, as evidenced by decreased levels of total cholesterol, LDL-cholesterol, and triglyceride. In HAECs, Orientin treatment increased cell viability and decreased inflammation, oxidative stress, and EndMT induced by ox-LDL/HG. Furthermore, Orientin significantly inhibited reactive oxygen species (ROS)-triggered NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and pyroptosis, as suggested by cleavage of caspase-1 and gasdermin-D (GSDMD), generation of interleukin (IL)-18 and IL-1β, and lactate dehydrogenase (LDH) release. Mechanistically, Orientin increased E3 ubiquitin ligase membrane-associated RING-CH 8 (MARCH8) expression in HAECs and resulted in subsequent MARCH8-mediated ubiquitination and proteasomal degradation of the NLRP3 protein. Taken together, these data demonstrate that Orientin, which alleviates HAEC inflammation and pyroptosis through regulating the MARCH8/NLRP3 axis, might be a potential candidate for treating diabetes-accelerated atherosclerosis.