Naringenin attenuates slow-transit constipation by regulating the AMPK/mTOR/ULK1 signalling pathway: in vivo and in vitro studies

Abstract

Background: Slow-transit constipation (STC) is a widespread functional gastrointestinal condition distinguished by decreased colonic motility as an essential clinical characteristic. The excessive autophagy of interstitial cells of Cajal (ICCs) causes phenotypic changes and functional abnormalities, which are important in colonic dysmotility. Naringenin (NAR) has been shown to regulate gastrointestinal motility disorders. The present study aimed to elucidate the regulatory role of naringenin in autophagy in STC and its underlying mechanism.

Methods: In vitro, ICCs were stimulated with L-glutamic acid (GA) to induce autophagy and treated with NAR. A CCK8 assay was performed to evaluate the cytotoxic effect of NAR. Annexin V-FITC/PI staining was used to examine NAR apoptosis. The expression of the autophagy markers Beclin1 and LC3B, as well as proteins related to the AMPK/mTOR/ULK1 pathway was investigated through quantitative PCR, Western blot analysis and immunofluorescence staining. The small interfering RNA (siRNA) technique was used to knockdown selective autophagy receptors (NDP52, OPTN, NBR1, and p62) in ICCs. Coimmunoprecipitation (co-IP) was used to evaluate the binding of pS757-ULK1 to the autophagy receptors NDP52 and OPTN in ICCs. Immunofluorescence (IF) staining was performed to observe the colocalization of pS757-ULK1 with exogenous NDP52 and OPTN in ICCs. In vivo, male C57BL/6 mice were administered loperamide (10 mg/kg) to establish a constipation model and then treated with NAR (75/150/300 mg/kg) for 2 weeks. Finally, colonic tissues were collected for a histological analysis and immunohistochemical for cell growth factor receptor kit (c-Kit) and anoctamin-1 (ANO1).

Results: Our results indicated that NAR improved the survival and apoptosis of ICCs after GA by inhibiting autophagy through the partial suppression of the AMPK/mTOR/ULK1 signalling pathway. Moreover, NAR inhibited the autophagic degradation of pS757-ULK1 by weakening the interactions between pS757-ULK1 and the selective autophagy receptor genes NDP52 and OPTN. Further research revealed that NAR could increase the moisture content of faeces; increase the rate of small intestinal propulsion in mice; increase the serum concentrations of excitatory neurotransmitters such as GAS, 5-HT, MTL, and SP; and increase the expression levels of ANO1 and c-Kit in the colon, and the molecular mechanism was consistent with the in vitro results.

Conclusion: NAR attenuates the AMPK/mTOR/ULK1 pathway in ICCs, thereby improving STC colonic dysmotility and underscoring its promise as a therapeutic option for STC.

Keywords: AMPK/mTOR/ULK1 signalling pathway; autophagy; interstitial cells of Cajal; naringenin; slow transit constipation.

Naringenin attenuates slow-transit constipation by regulating the AMPK/mTOR/ULK1 signalling pathway, both in vitro and in vivo, improving colonic function.

FIGURE 1

NAR suppressed autophagy in ICCs stimulated by GA. (A) Chemical structure of NAR. (B) Cytotoxicity of NAR in ICCs (n = 3). (C,D) The expression levels of LC3, P62, and Beclin1 were detected by Western blot (n = 3). (E) The expression level of LC3 was detected by Western blot (n = 3). Data are presented as the means ± SD. ^*** P < 0.001 vs Control group. ^# P < 0.05, ^## P < 0.01, and ^### P < 0.001 vs GA group. NAR: Naringenin; GA: L- Glutamic acid; BafA1: Bafilomycin A1.

FIGURE 2

NAR improved the survival and apoptosis of ICCs after GA by suppressing autophagy. (A) CCK8 assay was used to determine the survival of ICCs in each treatment group (n = 3). (B,C) Flow cytometry (FCM) was used to assess the apoptosis of ICCs in each treatment group (n = 3). Data are presented as the means ± SD. ^** P < 0.01, ^*** P < 0.001 vs. Control group. ^### P < 0.001 vs. GA group. ^&&& P < 0.001 vs Control + BafA1 group. ^△△△ P < 0.001 vs GA + BafA1 group. NAR: Naringenin; GA: L- Glutamic acid; BafA1: Bafilomycin A1.

FIGURE 3

NAR influenced autophagy by inhibiting the activation of the AMPK/mTOR/ULK1 signaling pathway in ICCs after GA. (A–L) Western blot was used to assess the expression of AMPK, mTOR, ULK1 and their phosphorylated proteins (n = 3). Data are presented as the means ± SD. ^** P < 0.01, ^*** P < 0.001 vs. Control group. ^# P < 0.05, ^## P < 0.01 vs. GA group. ^& P < 0.05 vs. GA + NAR group. NAR: Naringenin; GA: L- Glutamic acid.

FIGURE 4

NAR inhibited the autophagic degradation of pS757-ULK1 in ICCs after GA. (A) Before and after treatment with the autophagy inhibitor BafA1, the expression level of pS757-ULK1 was detected by Western blotting (n = 3). (B) After knockdown of the autophagy-related gene Atg5 in murine ICCs, the expression level of pS757-ULK1 was detected by Western blotting (n = 3). Data are presented as the means ± SD. ^** P < 0.01 vs. Control group. ^# P < 0.05, ^## P < 0.01 vs. GA group. ^& P < 0.05 vs. GA + NAR group. NAR: Naringenin; GA: L- Glutamic acid; BafA1: Bafilomycin A1.

FIGURE 5

NAR inhibited autophagic activity in ICCs after GA by suppressing the interaction between pS757-ULK1 and the selective autophagy receptor genes NDP52 and OPTN. (A,B) After knockdown of the selective autophagy receptor gene NDP52, OPTN, NBR1 and p62, Western blot was used to assess the expression levels of pS757-ULK1 protein (n = 3). (C) Co-immunoprecipitation was performed to evaluate the interactions between pS757-ULK1 and NDP52, as well as between pS757-ULK1 and OPTN. (D) The expression levels of NDP52 and OPTN were detected by Western blot. (E,F) Immunofluorescence staining was performed to observe the co-localization of pS757-ULK1 with NDP52 and OPTN, and representative images are displayed (n = 3). Data are presented as the means ± SD. ^* P < 0.05, ^** P < 0.01, and ^*** P < 0.001 vs. Control group. ^### P < 0.001 vs. GA + NAR group. ^&& P < 0.01 vs. GA + KD group. NAR: Naringenin; GA: L- Glutamic acid; NDP52: Nuclear dot protein 52 kDa; OPTN: Optineurin; NBR1: Neighbor of BRCA1 gene one protein.

FIGURE 6

NAR alleviates loperamide-induced constipation in mice. Constipation was induced by administering Lop intragastrically, followed by treatment with or without NAR. (A) Flow chart of the establishment of the mouse model and drug administration. During the experiment, the following parameters were measured simultaneously: (B) fecal quantity, (C) fecal water content, and (D,E) small intestinal propulsion rate (n = 8). The length of charcoal from stomach in the intestine was measured after the administration of charcoal. Charcoal transit ratio (%) = (distance travelled by the charocal)/(total length of small intestine) × 100%. (F) Detection of neurotransmitter concentrations in the serum of mice (n = 3). (G) HE staining and AB-PAS staining were used to observe morphological changes in the colonic tissue of mice (magnification = ×100 , 400 ×) (H) Colonic pathology score of mice in each group (n = 8). (I) Changes of mucosal layer thickness in colon tissue of mice in each group (n = 6). (J,K) Changes of the thickness of Internal circular muscle layer and lateral longitudinal muscle layer in mice of each group (n = 6). (L) Mean optical density of mucin expression/MOD (n = 8). (M) Changes of mucus layer thickness in colon tissue of mice in each group (n = 8). Data are presented as the means ± SD. ^* P < 0.05, ^** P < 0.01 vs. Control group. ^# P < 0.05, ^## P < 0.01 vs. Lop group. NAR: Naringenin; Lop: loperamide; Gas: gastrin; MTL: motilin; 5-HT: 5-hydroxy tryptamine; SP: substance P; VIP: vasoactive intestinal peptide; SS: somatostatin.

FIGURE 7

Effect of NAR on AMPK/mTOR/ULK1 signaling pathway in colon tissues of Lop-induced constipated mice. (A) Evaluation of cellular microstructures in the colonic mucosa by transmission electron microscopy. (B) Immunohistochemical analysis was performed to detect the expression of ANO1 and c-Kit in the colonic tissue of Lop-induced constipation mice (magnification = × 400). (C,D) The IOD value of immunohistochemical positive signals (n = 3). (E,F) The expression levels of AMPK, pT172-AMPK, mTOR, pS2448-mTOR, ULK1, pS757-ULK1, P62, LC3II/I, Beclin1, and ChAT were detected by Western blot (n = 3). Data are presented as the means ± SD. ^*** P < 0.001 vs. Control group. ^# P < 0.05, ^## P < 0.01, and ^### P < 0.001 vs Lop group. TEM: transmission electron microscopy; NAR: Naringenin; Lop: loperamide.