Fig. 3: ALA supplementation restores abnormal Paneth cell accumulation in human and mouse intestines upon aging.

a, b Representative images of Paneth cells (a, scale bars, 50 μm) and quantification in young and old human crypts (b, n = 27 crypts/group from 6 independent donors). c–e Diagram of lysozyme distribution patterns: normal (D0), disordered (D1), diminished (D2), diffuse (D3), excluded (D4), enlarged (D5) (c). Representative staining showing lysozyme granules (d, scale bars, 25 μm); white/yellow arrowheads indicate normal/abnormal granules. Quantification of distribution patterns (e, n = 15 crypts/group from independent 6 donors). f–j Young and old mice were administered drinking water with vehicle or ALA (100 mg/kg/day) for 3 months. Representative images of Paneth cells (f, scale bars, 75 μm), and quantification (g, n = 33 crypts/group from 7 biologically independent mice), Representative images showing lysozyme granules (h, scale bars, 10 μm; white/yellow arrowheads indicate normal/abnormal granules in Paneth cells). Distribution of lysozyme patterns (i, n = 14 crypts/group from 7 mice). Representative TEM images of Paneth cells (j, scale bars, 1μm; yellow/red stars indicate normal/abnormal granules). k, l Intestinal crypts from young and old humans were cultured with vehicle or ALA (100 μM) for 7 days. Representative lysozyme staining in organoids (k, scale bars, 75μm) and Paneth cell quantification (l, n = 7 organoids/group from 3 independent human donors). m–p Mouse intestinal crypts were cultured with vehicle or ALA (100 μM) for 6 days. Representative lysozyme staining (m, scale bars, 75μm) and Paneth cell quantification (n, n = 18 organoids/group from 7 mice). ELISA of Reg3g and Defa6 in organoid supernatants (o, p, n = 3 biological replicates/group). All data are mean ± SD. P values were calculated by unpaired two-tailed t test (b), one-way ANOVA with Tukey’s test (g, l, n, o, p); for (n), p = 6e–9 (young vs old), 8e–5 (old vs old+ALA). Two-way ANOVA with Sidak’s test (e); for (e), p = 2e–8 (young vs old within D0 and D1–D5). Two-way ANOVA with Tukey’s test (i). Source data are provided as a Source Data file.