Positive Correlation between nNOS and Stress-Activated Bowel Motility Is Confirmed by In Vivo HiBiT System

Abstract

Neuronal nitric oxide synthase (nNOS) has various roles as a neurotransmitter. However, studies to date have produced insufficient data to fully support the correlation between nNOS and bowel motility. This study aimed to investigate the correlation between nNOS expression and gastrointestinal (GI) tract motility using a stress-induced neonatal maternal separation (NMS) mouse model. In this study, we generated a genetically modified mouse with the HiBiT sequence knock-in into the nNOS gene using CRISPR/Cas9 for analyzing accurate nNOS expression. nNOS expression was measured in the stomach, small intestine, large intestine, adrenal gland, and hypothalamus tissues after establishing the NMS model. The NMS model exhibited a significant increase in nNOS expression in large intestine, adrenal gland, and hypothalamus. Moreover, a significant positive correlation was observed between whole gastrointestinal transit time and the expression level of nNOS. We reasoned that NMS induced chronic stress and consequent nNOS activation in the hypothalamic-pituitary-adrenal (HPA) axis, and led to an excessive increase in intestinal motility in the lower GI tract. These results demonstrated that HiBiT is a sensitive and valuable tool for analyzing in vivo gene activation, and nNOS could be a biomarker of the HPA axis-linked lower intestinal tract dysfunction.

Keywords: HiBiT; IBS; NMS; bowel motility; nNOS; stress.

Figure 1

Generation of an nNOS-HiBiT knock-in (KI) mouse and evaluation of HiBiT analysis. (A) Brief schematic of nNOS-HiBiT fusion protein for luminescence detection. (B) Single guide RNA was designed for 33-base pair of HiBiT sequence KI upstream of the stop codon in the nNOS gene. Blue alphabets: sgRNA binding sequence, Red alphabets: protospacer adjacent motif sequence. (C) PCR and Sanger-based genotyping was conducted for obtained pups. Red symbol: KI expected pups, Red alphabet: an expected correct sequence of HiBiT. (D) neuronal nitric oxide synthase (nNOS) protein was detected by HiBiT blotting using the nNOS-HiBiT KI and wild type. Western blotting for α-tubulin was conducted to confirm protein integrity. (E) nNOS expression was measured by HiBiT lytic analysis with 18 different organs from an nNOS-HiBiT KI mouse. Luminescence value was subtracted by background control.

Figure 2

NMS-mediated IBS mouse model and analysis nitric oxide concentration. (A) Schematic for inducing NMS-mediated IBS mouse model. Briefly, NMS was conducted for 3 h per day for 21 consecutive days (3–24 days old). Mice were weaned at 28 days old, and sacrificed and sampled at 40 days old. (B) nNOS-HiBiT expression was analyzed by HiBiT lytic analysis, and each dot indicates data from the individual animals (control, n = 16, NMS, n = 16). nNOS-HiBiT expression was displayed as a luminescence intensity and represented as a mean ± SEM. (C) Nitric oxide (NO) concentration was analyzed with the large intestine, adrenal, and hypothalamus tissues (control: n = 5, NMS: n = 5). Statistical analysis was performed using Student’s t-test. **: p < 0.01 and ****: p < 0.0001. ST: Stomach, SI: Small intestine, LI: Large intestine, Ad: Adrenal gland, HT: Hypothalamus. (D) NMS was conducted using wild-type mouse, and mice from each group were sacrificed and sampled at 40 days old. Tissue sample proteins from 5 mice with the same group (control vs. NMS) were quantified and merged, and subjected to western blotting to detect iNOS, eNOS, and nNOS. iNOS: inducible NOS, eNOS: endothelial NOS.

Figure 3

Positive correlation between nNOS expression and whole gastrointestinal transit time. (A) Whole gastrointestinal transit time was measured in NMS and control mice. The time of the first observation of carmine-stained feces was designated transit time. Transit time was analyzed by grouping for 30 min (left graph) and compared using individual transit times (right graph) (control, n = 16, NMS, n = 16). (B) Transit time from male and female mice was analyzed (male and female: control, n = 8, NMS, n = 8). Each dot indicates data from the individual animals, and data were represented as a mean ± SEM. Statistical analysis was performed using Student’s t-test. n.s.: not significant, *: p < 0.05, and ***: p < 0.001. (C) Correlation analysis for nNOS expression and whole gastrointestinal transit time on several organs was conducted. Each dot indicates data from individual mice. Black dot: data from control mice, Red dot: data from NMS mice. Statistical analysis was performed using Pearson R and Student’s two-tailed t-test.