The Role of Aeromonas-Goblet Cell Interactions in Melatonin-Mediated Improvements in Sleep Deprivation-Induced Colitis

Abstract

Background: Our previous studies demonstrated that melatonin could effectively ameliorate sleep deprivation- (SD-) caused oxidative stress-mediated gut microbiota disorder and colitis. The research further clarified the mechanism of melatonin in improving colitis from the perspective of the interaction between Aeromonas and goblet cells.

Methods: A seventy-two hours SD mouse model with or without melatonin intervention and fecal microbiota transplantation (FMT) to explore the vital position of Aeromonas-goblet cell interactions in melatonin improving SD-induced colitis. Moreover, Aeromonas or LPS-supplied mice were assessed, and the influence of melatonin on Aeromonas-goblet cell interactions-mediated oxidative stress caused colitis. Furthermore, in vitro experiment investigated the regulation mechanism of melatonin.

Results: Our study showed that SD induced colitis, with upregulation of Aeromonas and LPS levels and reductions in goblet cells number and MUC2 protein. Similarly, FMT from SD mice, Aeromonas veronii colonization, and LPS treatment restored the SD-like goblet cells number and MUC2 protein decrease and colitis. Moreover, LPS treatment downregulated the colonic antioxidant capacity. Yet, melatonin intervention reversed all consequence in SD, A.veronii colonization, and LPS-treated mice. In vitro, melatonin reversed A. veronii- or LPS-induced MUC2 depletion in mucus-secreting human HT-29 cells via increasing the expression level of Villin, Tff3, p-GSK-3β, β-catenin, and melatonin receptor 2 (MT2) and decreasing the level of p-IκB, p-P65, ROS, TLR4, and MyD88 proteins, while the improvement effect was blocked with pretreatment with a MT2 antagonist but were mimicked by TLR4 and GSK-3β antagonists and ROS scavengers.

Conclusions: Our results demonstrated that melatonin-mediated MT2 inhibits Aeromonas-goblet cell interactions to restore the level of MUC2 production via LPS/TLR4/MyD88/GSK-3β/ROS/NF-κB loop, further improving colitis in SD mice.

Figure 1

Melatonin improved SD-induced Aeromonas and LPS level increase and MUC2 deficiency in mice. (a) DAI score; relative abundance of colonic Aeromonas (b) and LPS (c); (d) PAS staining of colon tissue sections (scale: 50 μm); (e) immunohistochemical staining of MUC2 in colon sections (scale: 50 μm); (f) IOD of MUC2 protein; (g) Villin mRNA; (h) Tff3 mRNA; (i) TLR4 protein; (j) immunohistochemical staining of TLR4 in colon sections (scale: 50 μm); (k) IOD of TLR4 protein; (l) MyD88 mRNA; (m) p-GSK-3β and (n) β-catenin proteins in CON, SD, and SD+MT groups. Values are presented as mean ± SE. Differences were assessed using ANOVA and are denoted as follows: different lowercase letters: P < 0.05; different uppercase letters: P < 0.01; same letter: P > 0.05. The bottom is the same.

Figure 2

FMT reestablished the intestinal microecology similar to CON, SD, and SD+MT mice. (a) PAS staining of colon tissue sections (scale: 50 μm); (b) the number of goblet cells per um² in the colon; (c) MUC2 protein; (d) the ratio of F:B; Relative abundance of Aeromonas (e) and LPS (f) in the colonic content; colonic TLR4 (g), MyD88 (h), GSK-3β (i), β-cateinin (j), p-P65 (k), and p-IκB (l) mRNA and proteins of the F-CON, F-SD, F-SM, and F-MT groups.

Figure 3

Aeromonas veronii colonization promoted the occurrence of colitis in mice. (a): fecal occult blood; (b) relative luciferase activity for colonic permeability; (c) H&E staining photographs (scale: 50 μm); (d) histopathological score; colonic TNF-α (e), IL-1β (f), and IL-10 (g), and IFN-γ (h) concentrations were measured by ELISA; (i) PAS staining of colon tissue sections (scale: 50 μm); (j) the number of goblet cells per um² in the colon; (k) MUC2 protein; (l) Villin mRNA; (m) Tff3 mRNA in C-CON, C-A, and C-AM groups.

Figure 4

Melatonin improved LPS induced colitis in mice. (a) body weight; (b) and (c) colonic length; (d) H&E staining photographs (scale: 50 μm); (e) histopathological score; (f) relative luciferase activity for colonic permeability; (g–j) TNF-α (g); IL-1β (h); IL-10 (i), and IFN-γ (j) concentrations were measured by ELISA in the colon of the CON, LPS, LPS+MT, and LPS+TAK-242 groups.

Figure 5

Melatonin improved LPS-induced MUC2 deficiency and the changes of expression levels in signalling proteins in mice. (a–d) PAS staining of colon tissue sections (scale: 50 μm); (e) the number of goblet cells per um² in the colon; (f) colonic MUC2 protein; (g) Villin, and (h) Tff3 mRNA; (a) colonic TLR4, (b) MyD88, (c) p-GSK-3β, (d) β-catenin, (e) p-IκB, and (f) p-P65 proteins and mRNA production in the CON, LPS, LPS+MT, and LPS+TAK-242 groups.

Figure 6

Melatonin suppressed the changes in expression levels of signalling proteins in Aeromonas-treated HT-29 cells. MUC2 (a), Villin (b), Tff3 (c), TLR4 (d), MyD88 (e), p-IκB (f), p-P65 (g), p-GSK-3β (h) and β-catenin (i) proteins, and mRNA in various treatment groups.

Figure 7

Melatonin suppressed the changes in expression levels of signalling proteins (MUC2, Tff3, Villin, TLR4, and MyD88) in LPS-treated HT-29 cells. MUC2 (a), Tff3 (b), Villin (v), TLR4 (d), MyD88 (e) proteins and mRNA content and ROS (f) in various treatment groups. NAC: ROS scavenger; TAK-242: an antagonist of TLR4; TWS119: an antagonist of GSK-3β; 4P-PDOT: an antagonist of MT2. The bottom is the same.

Figure 8

Melatonin suppressed the changes in expression levels of signalling proteins (p-P65, p-IκB, p-GSK-3β, β-catenin, MT1, and MT2) in LPS-treated HT-29 cells. p-P65 (a), p-IκB (b), p-GSK-3β (c), β-catenin (d), MT1 (e), and MT2 (f) proteins in various treatment groups.