Figures & data
Figure 1. Gut microbiota is involved in host melatonin production.
(a) Diagram for the description of antibiotic-treated mouse model. (b) The level of tryptophan, 5-HTP, 5-HT, NAS, and melatonin in the serum of control and antibiotic-treated mice (n = 8-12). (c) The level of tryptophan, 5-HTP, NAS, and melatonin in the colon of control and antibiotic-treated mice (n = 10-12). Melatonin levels were detected in batches and normalized by external standard. (d) Diagram for the description of treatment in SPF mice and GF mice. (e) The level of tryptophan, 5-HTP, 5-HT, NAS, and melatonin in the serum of SPF and GF mice (n = 15-20). (f) The level of tryptophan, 5-HTP, 5-HT, NAS, and melatonin in the colon of SPF and GF mice (n = 10-20). Data were analyzed by one-way ANOVA (b and c: Tryptophan, 5-HTP), or Kruskal-Wallis (c: NAS, Melatonin) or unpaired t test (e and f: 5-HTP, Melatonin), or Mann-Whitney U test (f: Tryptophan, 5-HT, NAS) and represented as mean ± SD. *p < .05, **p < .01, ***p < .001 and ****p < .0001.
Figure 2. Melatonin production is regulated by fecal microbiota transplantation.
(a) Diagram for the description of co-housed mouse model. (b) The level of melatonin in the serum and colon of control (CON) and metronidazole-treated (MNZ) mice (n = 9-12). (c) The level of melatonin in the serum and colon of CON and MNZ mice after co-housing (n = 8). (d) Diagram for the description of CON FMT in CON and MNZ mice. (e) The level of melatonin in the serum and colon of CON and MNZ mice with CON FMT (n = 9-10). (f) Diagram for the description of MNZ FMT in CON and MNZ mice. (g) The level of melatonin in the serum and colon of CON and MNZ mice with MNZ FMT (n = 10). (h) Diagram for the description of FMT in Abx cocktail pretreated mice. (i) The level of melatonin in the serum and colon of Abx cocktail pretreated mice with FMT from CON or MNZ mice (n = 12). Data were analyzed by unpaired t test (b-c, e, g, i) and represented as mean ± SD. *p <.05. FMT, fecal microbiota transplantation; Abx, antibiotics.
Figure 3. L. R, L. I, L. J or E. coli colonization affects melatonin production in vivo.
(a) Diagram for the description of L. R colonization in Abx cocktail pretreated mice. (b and c) The level of melatonin in the serum (b) and colon (c) of Abx cocktail pretreated micecolonized with L. R or not (n = 6-10). Melatonin levels were detected in batches and normalized by external standard. (d) Diagram for the description of L. I colonization in SPF and Abx cocktail pretreated mice. (e) The level of melatonin in the serum of SPF and Abx cocktail pretreated mice colonized with L. I or not (n = 7-8). (f) Diagram for the description of L. J colonization in Abx cocktail pretreated mice. (g) The level of melatonin in the serum of Abx cocktail pretreated mice colonized with L. J or not (n = 9-10). (h) Diagram for the description of L. R colonization in GF mice. (i) Relative abundance of Lactobacillus in the intestinal contents of GF mice colonized with L. R or not (n = 4). (j) The level of melatonin in the serum of GF mice colonized with L. R or not (n = 11-12). (k) Diagram for the description of E. coli colonization in Abx cocktail pretreated mice. (l and m) The level of melatonin in the serum (l) and colon (m) of Abx cocktail pretreated mice colonized with E. coli or not (n = 10-12). (n) Diagram for the description of E. coli culture supernatant gavage in mice. (o and p) The level of melatonin in the serum (o) and colon (p) of mice with LB broth or E. coli culture supernatant gavage (n = 9-10). Data were analyzed by unpaired t test (e, g, j, l-m) or Mann-Whitney U test (b-c, o-p) and represented as mean ± SD. *p < .05, **p < .01.
Figure 4. Gut microbiota regulates AANAT expression through NF-κB signaling.
(a) The protein abundance of AANAT, p-IκB-α, IκB-α, p-IκB-β, IκB-β, p-p65 and p65 in the colon of mice treated with metronidazole or not (n = 5). (b) Immunofluorescence analysis of AANAT (green) in the colon tissue of mice treated with metronidazole or not. Scale bars, 50 μm (n = 6). (c) The protein abundance of AANAT, p-IκB-α, IκB-α, p-p65 and p65 in the colon of Abx cocktail pretreated mice colonized with L. R or not (n = 7). (d) The protein abundance of AANAT, p-IκB-α, IκB-α, p-p65 and p65 in the colon of Abx cocktail pretreated mice colonized with E. coli or not (n = 4). (e) Diagram for the description of Caco-2 cell co-culture system with L. R and PDTC. (f) The protein abundance of AANAT, p-p65 and p65 inL. R co-cultured Caco-2 cells treated with PDTC or not (n = 4). (g) Diagram for the description of Caco-2 cell co-culture system with E. coli and PDTC. (h) The protein abundance of AANAT, p-p65 and p65 in E. coli co-cultured Caco-2 cells treated with PDTC or not (n = 4). (i) Diagram for the description of Caco-2 cell co-culture system with L. R/E. coli and PDTC. (j) Immunofluorescence analysis of AANAT (purple) in L. R or E. coli co-cultured Caco-2 cells treated with PDTC or not. Scale bars, 5 μm (n = 3-5). Data were analyzed by unpaired t test (a-d, f, h, j) and represented as mean ± SD. *p < .05, **p < .01, ***p < .001 and ****p < .0001.
Figure 5. MyD88 deficiency eliminates the effects of metronidazole treatment on melatonin production.
(a) Diagram for the description of metronidazole treatment in Myd88flox/flox and Myd88ΔIEC mice. (b and c) The level of melatonin in the serum (b) and colon (c) of Myd88flox/flox and Myd88ΔIEC mice treated with metronidazole or not (n = 4-10). (d) The protein abundance of MyD88, AANAT, p-p65 and p65 in the colon of Myd88flox/flox and Myd88ΔIEC mice treated with metronidazole or not (n = 3). (e) Confocal microscopy analysis of MyD88 (purple), AANAT (green), and Villin (red) in the colon tissue of Myd88flox/flox and Myd88ΔIEC mice treated with metronidazole or not. Scale bars, 5 μm (n = 10-12). Data were analyzed by Kruskal-Wallis (b) or one-way ANOVA (c-e) and represented as mean ± SD. *p < .05, **p < .01, ***p < .001.
Figure 6. MyD88 deficiency eliminates the effects of L. R or E. coli colonization on melatonin production.
(a) Diagram for the description of L. R orE. coli colonization in Myd88ΔIEC mice. (b and c) The level of melatonin in the serum (b) and colon (c) of Myd88ΔIEC mice colonized with L. R/E. coli or not (n = 5). (d) Confocal microscopy analysis of MyD88 (purple), AANAT (green) and Villin (red) in the colon tissue of Myd88ΔIEC mice colonized with L. R/E. coli (Scale bars, 20 μm, n = 6). Data were analyzed by one-way ANOVA (b-d) and represented as mean ± SD.
Figure 7. Gut microbiota activates TLR2 and TLR4 signaling in the colon.
(a) The protein abundance of TLR2 and TLR4 in the colon of mice treated with metronidazole or not (n = 5). (b) The protein abundance of TLR2 and TLR4 in the colon of Abx cocktail pretreated mice colonized with L. R or not (n = 7). (c) The protein abundance of TLR2 and TLR4 in the colon of Abx cocktail pretreated mice colonized with E. coli or not (n = 4). (d) Diagram depicting the mechanisms of gut microbiota regulating melatonin production in colonic epithelial cells. The diagram was created with BioRender.com. Data were analyzed by unpaired t test (a-c) and represented as mean ± SD. **p < .01 and ****p < .0001.
Supplemental material
Supplemental Material
Download Zip (3 MB)Data availability statement
The raw 16S rDNA sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra) under the accession number PRJNA1047195.