Long-term exposure to constant light disrupts intestinal stem cells through sympathoexcitation-induced Wnt5a signaling inhibition

Abstract

Background: Long-term exposure to constant light is becoming a prevalent lifestyle that is associated with irritable bowel syndrome (IBS), a chronic functional gastrointestinal disorder. Intestinal stem cells (ISCs) are an important population of cells that maintain homeostasis and function of intestinal tissues. The purpose of this study was to identify the effects of long-term constant light exposure on gastrointestinal function and the potential mechanisms of sympathetic activity on ISC.

Methods: Rats housed in a 24 h constant light chamber for 4 weeks were used as the constant light exposure animal model. Hematoxylin-eosin staining and immunohistochemical examination were used to determine the pathological changes of the intestine. Propranolol (ARs inhibitor; 40 mg/kg/day), metoprolol (ADRβ1 inhibitor; 50 mg/kg/day), and Box5 (Wnt5a inhibitor; 2 μg/day) were used to examine the effect of sympathoexcitation and Wnt signaling pathway on constant light-induced gastrointestinal disorders.

Results: We found that 4 weeks of constant light exposure in rats resulted in a decrease in the number of ISC and an increase in sympathetic activity. Intestinal β1-adrenoceptor expression and reactive oxygen species (ROS) were significantly increased, but Wnt5a expression decreased in the continuous light-exposed rats. Similarly, we found that administration of the β1-adrenoceptor antagonist metoprolol for 4 weeks attenuated the effects of continuous light exposure on the intestine, which was rescued by the reintroduction of Wnt5a.

Conclusion: Taken together, these data indicate that sympathoexcitation is critical for disruption of ISC under constant light exposure, suggesting that targeting β1-adrenoceptor/oxidative stress/Wnt5a axis may be a potential strategy for ISC disruption induced by prolonged sustained light exposure, providing a new direction for IBS treatment.

Keywords: IBS; ROS; Wnt5a; constant light; intestinal stem cells; sympathoexcitation.

Figure 1.

Constant light disrupts gastrointestinal function. (A, B) Statistical graphs of gastric emptying rate, intestinal propulsion rate in LD and LL groups. (C) AWR scores in LD and LL groups. (D) Representative images of abdominal cavity of LD and LL groups. (E, F) Representative images of intestine length and statistical graphs in LD and LL groups. (G, H) Representative HE-stained images of the intestine showing thickness of the muscle layer, number of goblet cells, length of villi, and length of crypts, with corresponding statistical graphs (scale bar = 400 μm). (I) Statistical graphs of relative ZO-1 gene expression in LD and LL groups. n = 5–6 per group. LD = 12 h light: 12 h dark, LL = 24 h light, AWR = abdominal withdrawal score, HE = hematoxylin-eosin.

Figure 2.

Sympathoexcitation is identified to be involved in ISC injury by constant light. (A, B) Representative IHC staining images of Olfm4 positive cells (ISC) in each crypt and statistical graphs in the LD and LL groups (scale bar = 100 μm). (C) Statistical graphs of relative Olfm4 gene expression in the two groups. (D) The concentration of NE and EPI in plasma of two groups. (E, F) Representative images of TH IOD in intestine submucosa and muscular layer in two groups with corresponding statistical graphs (scale bar = 200 μm). (G, H) Representative IHC staining images of Olfm4 positive cells (ISC) in each crypt (scale bar = 100 μm) and statistical graphs. (I) Relative changes in Olfm4 gene expression. n = 5–6 per group. LD = 12 h light: 12 h dark, LL = 24 h light, IHC = immunohistochemical, ISC = intestinal stem cells, NE = norepinephrine, EPI = epinephrine, TH = tyrosine hydroxylase, IOD = integrated optical density.

Figure 3.

Constant light causes ISC injury through β1-adrenoreceptor. (A) Representative IHC staining images of ADRβ1, ADRβ2, and ADRβ3 in LD and LL groups (scale bar = 200 μm). (B, C) Representative IHC staining images of Olfm4 positive cells in each crypt and statistical graphs in LD_NS, LD_Met, LL_NS, and LL_Met groups (scale bar = 100 μm). (D) Relative Olfm4 gene expression in the four groups. (E) Expression of NOX2 was examined by Western blot and quantitated. n = 5–6 per group. LD = 12 h light: 12 h dark, LL = 24 h light, IHC = immunohistochemical, Met = metoprolol, NS = 0.9% NaCl solution.

Figure 4.

Sympathoexcitation inhibits Wnt5a signaling pathway by constant light. (A) Relative changes in the expression of 13 genes in the Wnt signaling pathway. (B) Relative Wnt5a, Wnt8b, and Wnt10a gene expression in LD_NS, LD_Met, LL_NS, and LL_Met groups. (C) AWR scores in LD_Met, LD_Met+Box5, LL_Met, and LL_Met+Box5 groups. (D, E) Statistical graphs of gastric emptying rate and intestinal propulsion rate in the four groups. (F, G) Representative HE-stained images of the intestine showing thickness of the muscle layer, number of goblet cells, length of villi, and length of crypts, with corresponding statistical plots (scale bar = 400 μm.). (H) Statistical graphs of relative changes in ZO-1 gene expression in the four groups. (I, J) Representative IHC staining images of Olfm4 positive cells in each crypt and statistical graphs in four groups and statistical graph (scale bar = 100 μm.). (K) Relative changes in Olfm4 gene expression in the four groups. Here rats in metoprolol + saline group were abbreviated as Met. n = 5–6 per group. LD = 12 h light: 12 h dark, LL = 24 h light, IHC = immunohistochemical, Met = metoprolol, NS = 0.9% NaCl solution, AWR = abdominal withdrawal score.

Figure 5.

Restoration of light rhythm protected gastrointestinal function by constant light. (A, B) Representative HE-stained images of the intestine showing thickness of the muscle layer, number of goblet cells, length of villi, and length of crypts, with corresponding statistical plots (scale bar = 400 μm.). (C) Statistical graphs of relative ZO-1 gene expression in LL, LL_6W, and LL_R groups. (D, E) Representative IHC staining images of Olfm4 positive cells in each crypt and statistical graphs in the three groups and statistical graph (scale bar = 100 μm.). (F) Relative changes in Wnt5a gene expression in three groups. Rats with 2 weeks of light rhythm restoration after 4 weeks of constant light exposure were abbreviated as LL_R. n = 5–6 per group. AWR = abdominal withdrawal score, HE = hematoxylin-eosin, LL = 4 weeks of continuous light exposure, LL_6W = 6 weeks of continuous light exposure, LL_R = 2 weeks of light rhythm recovery after 4 weeks of continuous light exposure.

Figure 6.

The schematic model of constant light on intestinal stem cells via sympathoexcitation-induced inhibition of Wnt5a signaling.