Fig. 8.

Impact of Fundc1 regulation on the intestinal endothelial ferroptosis level and microvascular function. A. Endothelial cells isolated from intestinal tissue were used to evaluate MDA, GSH, GSSH and Fe²⁺ levels by ELISAs. B–C. Intracellular Fe²⁺ in the intestinal endothelium was evaluated using the FerroOrange probe. D-E. Western blot analysis was used to detect ferroptosis-related proteins in endothelial cells isolated from the intestine. F-G. Intestinal blood perfusion in WT and Fundc1^ΔEC mice was measured using a laser speckle blood flow analysis system. H. Intestinal and serum syndecan-1 levels were assessed using an ELISA kit. I-J. Pathological staining and immunohistochemistry were performed to evaluate intestinal microvascular function. The intestinal microvascular morphology was observed by HE and Masson’s trichrome staining and evaluated and graded according to the Chiu score system. TUNEL staining was used to evaluate microvascular cells undergoing apoptosis. Intestinal microvascular damage and permeability were assessed using immunohistochemistry for VCAM-1 and albumin. K-L. Western immunoblotting was used to examine VCAM-1 and VE-cadherin protein expression. M-N. To evaluate microvascular damage, immunofluorescence colocalization of CD31 and VE-cadherin was conducted in the intestine. Data correspond to the means ± SD, ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.