Fig. 2: ALA inhibits ISC aging and promotes epithelial regeneration in old mice.

a–g Young and old mice were treated with vehicle (veh) or ALA (100 mg/kg/day) in drinking water for 3 months. Experimental design (a), EdU (red) and SOX9(green) staining (b, scale bars, 25μm), yellow arrow: ISCs, and quantification of crypts (c–e, n = 16 crypts/group from 7 mice;). TEM analysis of ISC mitochondria (f, scale bars: 1μm and 500 nm; (g), n = 15 ISCs/group from 3 mice). Yellow dashed lines: ISCs; M (red), ISC mitochondria; N (blue), nucleus. h–p Crypts from young and old mice were cultured with vehicle or ALA (100 μM) for 6 days. Experimental design (h), representative images of intestinal organoids (i, scale bars, 250 μm), organoid number(j, n = 7 mice/group, each biological replicate represents an independent organoid culture from one mouse), bud morphology (k, scale bars, 50μm), and quantification (l, n = 26 organoids/group from 7 mice), RT-qPCR of Lgr5, Olfm4, and Ascl2 (n = 6 mice/group) (m), staining of DAPI, Olfm4, and EdU (n, scale bars, 50μm); quantification of EdU⁺ and Olfm4⁺ cells (o, p, n = 14 organoids/group from 7 mice) q–u Young and old mice were injected with indomethacin (10 mg/kg) for 1 day followed by 7 days vehicle or ALA. Experimental design (q). H&E staining and histology scores (r, s, scale bars, 75μm; n = 5 mice/group), TEM images of crypts (t, 1 μm and 500 nm) and mitochondrial integrity quantification (u, n = 15 cells from 3 mice/group). Yellow lines: ISCs; N (blue), nucleus; M (red), mitochondria. Data are mean ± SD. One-way ANOVA with Tukey’s test (c–e, j, l, o, p): (c), p  =  4e–9 (young vs old), 8e–9 (old vs old+ALA); (l), p  =  2e–9 (young vs old); (p), p  =  6e–10 (young vs old), 3e–7 (old vs old+ALA). Two-way ANOVA with Tukey’s (g, m) or Sidak’s test (s, u): (g) within 0–50% mitochondrial integrity, p = 8e–9 (young vs old), 1e–10 (young vs old+ALA, old vs old+ALA); (m) p = 8e–6 for Olfm4 (old vs old+ALA); (u) at 0–50% integrity, p = 2e–7; at 0%, p = 1e–15. Source data are provided as a Source Data file.