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

Orientin treatment decreased atherosclerotic plaque burden, lipid lesion, and collagen content in aortic and femoral arteries in diabetic ApoE-/- mice.

• ApoE-/- mice were administered streptozotocin (STZ) and fed a high-fat diet (HFD) to model diabetes-accelerated atherosclerosis.
• Orientin was administered as a treatment following induction of the diabetic atherosclerosis model.
• Pathological and biochemical assays were used to assess plaque burden, lipid lesion, and collagen content.
• Reductions were observed in both aortic and femoral arteries.

Orientin alleviated hypercholesterolemia in diabetic atherosclerotic mice, evidenced by decreased levels of total cholesterol, LDL-cholesterol, and triglyceride.

• Biochemical assays were used to measure circulating lipid levels.
• Decreased total cholesterol, LDL-cholesterol, and triglyceride levels were observed following Orientin treatment.
• This was assessed in the ApoE-/- mouse model treated with STZ and HFD.

Orientin increased cell viability and decreased inflammation, oxidative stress, and endothelial-mesenchymal transition (EndMT) in human aortic endothelial cells (HAECs) stimulated by ox-LDL/HG.

• HAECs were stimulated with oxidized low-density lipoprotein and high glucose (ox-LDL/HG) to model endothelial injury.
• Cell viability was assessed using cell counting kit-8 (CCK-8) and fluorescein diacetate (FDA) staining.
• Inflammation, oxidative stress, and EndMT markers were assessed using quantitative real-time PCR, immunofluorescence (IF), and western blot assays.

Orientin significantly inhibited ROS-triggered NLRP3 inflammasome activation and pyroptosis in ox-LDL/HG-stimulated HAECs.

• Inhibition of NLRP3 inflammasome activation was evidenced by reduced cleavage of caspase-1 and gasdermin-D (GSDMD).
• Reduced generation of interleukin (IL)-18 and IL-1β was observed following Orientin treatment.
• Pyroptosis was also assessed by lactate dehydrogenase (LDH) release, which was decreased with Orientin treatment.
• Reactive oxygen species (ROS) were implicated as upstream triggers of NLRP3 inflammasome activation in this context.

Orientin increased E3 ubiquitin ligase MARCH8 expression in HAECs, leading to MARCH8-mediated ubiquitination and proteasomal degradation of the NLRP3 protein.

• MARCH8 (membrane-associated RING-CH 8) is an E3 ubiquitin ligase whose expression was upregulated by Orientin treatment.
• The mechanism involves MARCH8-mediated ubiquitination of NLRP3 followed by its proteasomal degradation.
• This MARCH8/NLRP3 axis was identified as the mechanistic basis for Orientin's anti-inflammatory and anti-pyroptotic effects.
• Western blot assays were among the methods used to assess protein expression and modification.