Shu-Xie decoction alleviates oxidative stress and colon injury in acute sleep-deprived mice by suppressing p62/KEAP1/NRF2/HO1/NQO1 signaling

Abstract

Introduction: Sleep disorders are common clinical psychosomatic disorders that can co-exist with a variety of conditions. In humans and animal models, sleep deprivation (SD) is closely related with gastrointestinal diseases. Shu-Xie Decoction (SX) is a traditional Chinese medicine (TCM) with anti-nociceptive, anti-inflammatory, and antidepressant properties. SX is effective in the clinic for treating patients with abnormal sleep and/or gastrointestinal disorders, but the underlying mechanisms are not known. This study investigated the mechanisms by which SX alleviates SD-induced colon injury in vivo. Methods: C57BL/6 mice were placed on an automated sleep deprivation system for 72 h to generate an acute sleep deprivation (ASD) model, and low-dose SX (SXL), high-dose SX (SXH), or S-zopiclone (S-z) as a positive control using the oral gavage were given during the whole ASD-induced period for one time each day. The colon length was measured and the colon morphology was visualized using hematoxylin and eosin (H&E) staining. ROS and the redox biomarkers include reduced glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD) were detected. Quantitative real-time PCR (qRT-PCR), molecular docking, immunofluorescence and western blotting assays were performed to detect the antioxidant signaling pathways. Results: ASD significantly increased FBG levels, decreased colon length, moderately increased the infiltration of inflammatory cells in the colon mucosa, altered the colon mucosal structure, increased the levels of ROS, GSH, MDA, and SOD activity compared with the controls. These adverse effects were significantly alleviated by SX treatment. ASD induced nuclear translocation of NRF2 in the colon mucosal cells and increased the expression levels of p62, NQO1, and HO1 transcripts and proteins, but these effects were reversed by SX treatment. Conclusion: SX decoction ameliorated ASD-induced oxidative stress and colon injury by suppressing the p62/KEAP1/NRF2/HO1/NQO1 signaling pathway. In conclusion, combined clinical experience, SX may be a promising drug for sleep disorder combined with colitis.

Keywords: NRF2; ROS; oxidative stress; sleep deprivation; traditional Chinese medicine.

FIGURE 1

SX alleviates ASD-induced clinical symptoms associated with gastrointestinal dysfunction in mice. (A) Schematic representation of the experimental design. After 14 days of habituation, mice were sleep deprived for 3 days and orally administered with distilled water, SX, or S-z, once a day. Fasting blood glucose levels, body weight, and food and water consumption were recorded at all points. (B) Grouping of mice. CON = control; ASD = acute sleep deprived; Veh = vehicle; SXL = low-dose SX; SXH = high-dose SX; S-z = S-zopiclone. (C) Schematic diagram shows acute sleep deprivation modeling. SX alleviates ASD-induced weight loss. (D, E) Body weights of the CON, ASD, ASD + SXL, ASD + SXH, and ASD + S-z group mice during habituation and ASD modeling. p < 0.05, CON vs. ASD; (F, G) Fasting blood glucose levels of the CON, ASD, ASD + SXL, ASD + SXH, and ASD + S-z group mice during habituation and ASD modeling. p < 0.05, ASD vs. SXL, SXH and S-z. Data were expressed as means ± SEM. n = 7 per group. Two-way ANOVA for D and F; one-way ANOVA for E and G; ^* p < 0.05, ^** p < 0.01 vs. control group; ^# p < 0.05 vs. ASD group; ns, no significant.

FIGURE 2

SX alleviates acute sleep deprivation-induced histopathological injury in the colon. (A) Representative images show the lengths of colons harvested from the CON, ASD, ASD + SXL, ASD + SXH, and ASD + S-z groups of mice. (B) Quantitative data shows the colon lengths of mice belonging to the CON, ASD, ASD + SXL, ASD + SXH, and ASD + S-z groups. (C) Body weights of the CON, ASD, ASD + SXL, ASD + SXH, and ASD + S-z group mice after modeling. (D) Colon mass indices (%) (colonic weight/body weight) of the CON, ASD, ASD + SXL, ASD + SXH, and ASD + S-z groups of mice. (E) The colon histopathological scores of the CON, ASD, ASD + SXL, ASD + SXH, and ASD + S-z group mice. p < 0.05, ASD vs. SXL, SXH and S-z. (F) Representative H&E-stained images show the morphological changes in the colon mucosa of mice belonging to the CON, ASD, ASD + SXL, ASD + SXH, and ASD + S-z groups (×1, ×20, and ×40 magnification). M: mucosa; SM: submucosa; red arrow: multiple mucinous glands; yellow arrow: inflammatory cells. Data were expressed as means ± SEM. n = 6–7 per group. Statistical significance was determined by one-way ANOVA; ^* p < 0.05 vs. control group; ^# p < 0.05 vs. ASD group; ns, no significant.

FIGURE 3

SX alleviates ASD-induced oxidative stress in the mouse serum. (A) (A). ROS fluorescence intensity of blood cells after 72 h of ASD, waveforms are images obtained in “multi-point scanning” mode for each well, with the top and bottom wells being multiple wells. The average of each well was taken for statistical analysis. Positive control Rosup concentration was 100 μM. (B). Fluorescence intensity before and after ASD was measured at 488 nm excitation wavelength and 535 nm emission wavelength, time point by time point (0, 24, 48, 72 h). (B) ROS levels in the blood cells of ASD group mice at 0, 24, 48, and 72 h. (C) Serum GSH levels in the CON, ASD, ASD + SXL, ASD + SXH, and ASD + S-z groups of mice. p < 0.0001, ASD vs. SXL, SXH, S-z. (D) Serum MDA levels in the CON, ASD, ASD + SXL, ASD + SXH, and ASD + S-z groups of mice. p < 0.0001, ASD vs. SXL, SXH, S-z. (E) Total SOD activity in the serum of CON, ASD, ASD + SXL, ASD + SXH, and ASD + S-z groups of mice. p < 0.05, ASD vs. SXH; p < 0.001 ASD vs. S-z. The data are represented as means ± SEM. n = 7 each group. Statistical significance was determined based on one-way ANOVA; ^**** p < 0.0001, ^*** p < 0.001, ^** p < 0.01 vs. CON group; ^#### p < 0.0001, ^### p < 0.001, ^## p < 0.01, ^# p < 0.05 vs. ASD group.

FIGURE 4

Molecular docking diagrams of BAI and PAE with p62, KEAP1, NRF2, HO1, and NQO1. (A) Molecular docking diagrams of BAI with p62, KEAP1, NRF2, HO1, and NQO1 proteins. (B) Binding affinity (kcal/mol) of BAI with p62, KEAP1, NRF2, HO1, and NQO1 proteins. (C) Molecular docking diagrams of PAE with p62, KEAP1, NRF2, HO1, and NQO1 proteins. (D) Binding affinity (kcal/mol) of PAE with p62, KEAP1, NRF2, HO1, and NQO1 proteins. BAI, Baicalin; PAE, Paeoniflorin.

FIGURE 5

SX modulates ASD-induced oxidative stress in the colon through the p62/KEAP1/NRF2/HO1/NQO1 signaling pathway. (A) Representative immunofluorescence images show the localization of NRF2 protein in the colon sections based on staining with the fluorophore-labeled anti-NRF2 antibody (red fluorescence). The nuclei are stained with DAPI (blue fluorescence). (B) Representative western blot shows the expression levels of p62, KEAP1, NRF2, NQO1, HO1, and β-actin proteins in the colons of mice belonging to the CON, ASD, ASD + SXL, ASD + SXH, and ASD + S-z groups. β-actin was used as the internal loading control. The experiment was repeated three times. (C) The dot plot shows the numbers of NRF2 positive cells (based on immunofluorescence staining in A) in the colonic mucosa of mice belonging to the CON, ASD, ASD + SXL, ASD + SXH, and ASD + S-z groups. p < 0.05, ASD vs. SXL, SXH, and S-z. (D–H) The relative expression levels of p62, KEAP1, NRF2, NQO1, and HO1 proteins compared to the β-actin protein levels in the colons of mice belonging to the CON, ASD, ASD + SXL, ASD + SXH, and ASD + S-z groups. Note: for p62 in (D), p < 0.05, ASD vs. SXL, SXH; p = 0.4302, ASD vs. S-z); for KEAP1 in (E), p < 0.05 ASD vs. SXH; p < 0.01 vs. S-z); for NRF2 in (F), p < 0.01, ASD vs. SXL, SXH, and S-z); for NQO1 in (G), p < 0.05, ASD vs. SXL, SXH, and S-z; for HO1 in (H) p < 0.05, ASD vs. SXH); (I–K) qRT-PCR results show the transcript levels of KEAP1, NRF2, and NQO1 compared with the β-actin transcript levels. For KEAP1 in (I), p < 0.05, ASD vs. SXH; for NRF2 in (J), p < 0.01 ASD vs. SXL; p < 0.05, ASD vs. S-z; for NQO1 in (K), p < 0.001 ASD vs. SXL and S-z; p < 0.01, ASD vs. SXH; KEAP1, Kelch-like ECH-associated protein 1; NRF2, nuclear factor erythroid 2-related factor 2; NQO1, NAD(P)H: quinone oxidoreductase 1; HO1, heme oxygenase 1; Data are expressed as means ± SEM. n = 4 per group. Statistical significance was determined by one-way ANOVA; ^* p < 0.05, ^** p < 0.01 vs. CON; ^# p < 0.05, ^## p < 0.01 vs. ASD. ns, no significant. Scale bar: 20 μm.

FIGURE 6

Immunofluorescence analysis of NRF2 (red), HO1 and NQO1 (green) in colon mucosal layers (×400 magnification). (A) Representative fluorescence confocal images show NRF2 and HO1 staining in the colon sections of mice belonging to the CON, ASD, ASD + SXL, ASD + SXH, and ASD + S-z groups. The nuclei were stained with DAPI (blue). (B) Representative fluorescence confocal images show NRF2 and NQO1 staining in the colon sections of mice belonging to the CON, ASD, ASD + SXL, ASD + SXH, and ASD + S-z groups. The nuclei were stained with DAPI (blue). (C) Quantitative analysis of NRF2 fluorescence. (D) Quantitative analysis of HO1 fluorescence. (E) Quantitative analysis of NQO1 fluorescence. The immunofluorescence signal intensity in the images was quantified using the ImageJ software. n = 3 per group. The experiment was repeated three times. The data are shown as mean ± S.E.M. ^** p < 0.01 vs. CON group; ^# p < 0.05, ^## p < 0.01 vs. ASD group. Scale bar: 50 μm.

FIGURE 7

Diagrammatic representation shows the mechanism by which SX alleviates ASD-induced oxidative stress and colon injury.