Fig 2: L-arginine increased expansion of ISCs in SI crypts and accelerates ISC-mediated intestinal epithelial regeneration.By constructing a co-culture model, we found that CD90+ stromal cells, a key constituent of the mammalian ISC niches, augmented stem-cell function in response to L-arginine. Moreover, increased expression of Wnt2b in CD90+ stromal cells mediated the effects of L-arginine on ISC function. We found that L-arginine stimulated Wnt2b secretion of CD90+ stromal cells through mTORC1 signals.

Fig 3: L-arginine supplementation protects the gut from 5-FU and TNF-a induced intestinal epithelial damage in a Wnt2b-dependent manner.a–c Mice were treated with normal water or 7 mg/ml L-arginine in their drinking water for 14 days and then injected with 5-FU (300 mg/kg) or PBS as a control once a day for 5 days. In Wnt2b antibody neutralization experiment, every mouse were given 300 µg Wnt2b neutralizing antibody once on the first day of 5-FU injection. a Pathology of the small intestine by H&E staining. Scale bar, 200 µm. b FCM analysis of live Lgr5hi ISC frequency in SI crypts, n = 3. c SI organoid frequency of crypts from mice in different groups. Representative images of crypt culture from each group are shown, n = 6. Scale bar, 50 µm. d–e SI organoids-CD90+ stromal cells co-culture model was cultured with ENR-medium or ENR-medium supplemented L-arginine (1 mM), TNF-a (100 ng/ml), WNT2B protein (100 ng/ml), Wnt2b neutralizing antibody (1 µg/ml) and their combinations for 3 days. d The light microscope observation of the co-culture model treated with L-arginine, TNF-a, and their combinations. Scale bar, 200 µm. The number of total organoids and disrupted organoids with altered morphology per well were counted, n = 6. e The light microscope observation of co-culture model treated with L-arginine, TNF-a, WNT2B protein, Wnt2b neutralizing antibody and their combinations. Scale bar, 200 µm. The number of total organoids and disrupted organoids with altered morphology per well were counted, n = 6. Data are the mean ± SD; comparisons performed with t-tests (two groups) or analysis of variance (ANOVA) (multiple groups). *P < 0.05, **P < 0.01, ***P < 0.001. Results are representative of two or three independent experiments.

Fig 4: L-arginine supplementation stimulates Wnt2b synthesis in CD90+ stromal cells through the mTORC1 signalling pathway.CD90+ stromal cells were cultured in medium supplemented with L-arginine (1 mM) for 3 days. a qPCR of relative mRNA expression of Slc7a1 and Slc7a4 genes in CD90+ stromal cells treated with L-arginine or not. Expression show is relative to GAPDH gene, n = 3. b Immunostaining of Slc7a1 (red) and DAPI (blue) in CD90+ stromal cells treated with L-arginine or not. Scale bar, 10 μm. Quantitation for Slc7a1 was measured by MFI, n = 6. c, d Protein levels of GAPDH, mTOR, P-mTOR, P70S6K, P-P70S6K, 4EBP1, and P-4EBP1 were measured by western blotting assay in CD90+ stromal cells treated with L-arginine or not, n = 3. e Immunostaining of Wnt2b (red), Vimentin (green), and DAPI (blue) in CD90+ stromal cells treated with L-arginine (1 mM), MHY1485 (5 μM), Rapamycin (5 μM) and their combinations for 3 days. Scale bar, 10 μm. f The concentration of Wnt2b in CD90+ stromal cells was detected by ELISA, n = 3. Data are the mean ± SD; comparisons performed with t-tests (two groups) or analysis of variance (ANOVA) (multiple groups). *P < 0.05, **P < 0.01, ***P < 0.001. Results are representative of two or three independent experiments.