Impairment of the intestinal epithelium barrier through alteration of proliferation and differentiation balance in alcohol use disorder

Figure 9 Generation and alcohol stimulation of intestinal enteroids from healthy subjects and alcohol use disorder patients. A: Alcohol use disorder (AUD)-derived enteroids retained the morphological impairments observed in their host. A subset of AUD enteroids lacked budding structures, whereas another subset showed excessive budding. Images were acquired by optical microscopy at 10 × magnification; B: Gene expression analysis showing unchanged levels of LGR5 (stem cells) and lysozyme (Paneth cells) in AUD-derived enteroids, with a significant upregulation of MUC2, indicating increased goblet cell differentiation. Healthy (n = 3); AUD (n = 8); C: Experimental design of three-dimensional enteroids cultured in Matrigel and treated with 40 mmol/L or 70 mmol/L ethanol from day 2 after passage until full differentiation at day 13. Ethanol-containing medium was refreshed daily; D: Confocal microscopy of fixed and stained three-dimensional enteroids shows no significant differences in Ki67⁺ proliferating cells (green) across ethanol treatments (black: 70 mmol/L; gray: 40 mmol/L) as well as in the expression of cyclinB1 and Wnt target genes CD44 and Axin2; E: Experimental design of two-dimensional enteroids treated for three days after full monolayer confluence two-dimensional enteroids exposed to 70 mmol/L ethanol exhibited significantly increased proliferation; F: Further supported by upregulation of cyclin B1 mRNA. Expression of Wnt target genes (CD44, Axin2) was not changed upon ethanol exposure. Scale bar (50 μm). Healthy (n = 3). AUD: Alcohol use disorder; 3D: Three-dimensional; 2D: Two-dimensional.