Wang Yahui, Wang Xiaopeng, Qian Yifei, Sun Mingming, Yang Huiju, Su Lianlin, Yan Shuai
Department of Anorectal Surgery, Suzhou TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Suzhou, Jiangsu, China.
Department of Chemistry, University of Wisconsin-Madison, Madison, WI, United States.
Front Pharmacol. 2025 Jun 17;16:1550458. doi: 10.3389/fphar.2025.1550458. eCollection 2025.
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.
, 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. , 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).
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 results.
NAR attenuates the AMPK/mTOR/ULK1 pathway in ICCs, thereby improving STC colonic dysmotility and underscoring its promise as a therapeutic option for STC.
慢传输型便秘（STC）是一种广泛存在的功能性胃肠疾病，其主要临床特征为结肠动力下降。 Cajal间质细胞（ICCs）的过度自噬会导致表型变化和功能异常，这在结肠动力障碍中起重要作用。柚皮素（NAR）已被证明可调节胃肠动力障碍。本研究旨在阐明柚皮素在STC自噬中的调节作用及其潜在机制。
用L-谷氨酸（GA）刺激ICCs诱导自噬，并用NAR处理。进行CCK8试验以评估NAR的细胞毒性作用。采用Annexin V-FITC/PI染色检测NAR诱导的细胞凋亡。通过定量PCR、蛋白质免疫印迹分析和免疫荧光染色研究自噬标志物Beclin1和LC3B以及与AMPK/mTOR/ULK1通路相关蛋白的表达。使用小干扰RNA（siRNA）技术敲低ICCs中的选择性自噬受体（NDP52、OPTN、NBR1和p62）。采用免疫共沉淀（co-IP）法评估pS757-ULK1与ICCs中自噬受体NDP52和OPTN的结合。进行免疫荧光（IF）染色以观察pS757-ULK1与外源性NDP52和OPTN在ICCs中的共定位。将雄性C57BL/6小鼠给予洛哌丁胺（10mg/kg）建立便秘模型，然后用NAR（75/150/300mg/kg）治疗2周。最后，收集结肠组织进行组织学分析以及细胞生长因子受体试剂盒（c-Kit）和anoctamin-1（ANO1）的免疫组织化学检测。
我们的结果表明，NAR通过部分抑制AMPK/mTOR/ULK1信号通路抑制自噬，从而改善GA处理后ICCs的存活和凋亡。此外，NAR通过减弱pS757-ULK1与选择性自噬受体基因NDP52和OPTN之间的相互作用，抑制pS757-ULK1的自噬降解。进一步研究发现，NAR可增加粪便含水量；提高小鼠小肠推进率；增加血清中兴奋性神经递质如GAS、5-HT、MTL和SP的浓度；增加结肠中ANO1和c-Kit的表达水平，其分子机制与上述结果一致。
NAR减弱ICCs中的AMPK/mTOR/ULK1通路，从而改善STC的结肠动力障碍，并突出其作为STC治疗选择的前景。
