The First School of Clinical Medicine, Lanzhou University, Lanzhou, 730000, PR China.
The First School of Clinical Medicine, Lanzhou University, Lanzhou, 730000, PR China; Department of Anesthesiology, The First Hospital of Lanzhou University, Lanzhou, 730000, PR China.
Phytomedicine. 2024 Dec;135:156095. doi: 10.1016/j.phymed.2024.156095. Epub 2024 Sep 27.
Intestinal ischemia/reperfusion injury (IRI) is a significant clinical emergency, and investigating novel therapeutic approaches and understanding their underlying mechanisms is essential for improving patient outcomes. Naringenin (Nar), a flavanone present in tomatoes and citrus fruits, is frequently consumed in the human diet and recognized for having immunomodulatory, anti-inflammatory, and antioxidant properties. Despite Nar being able to alleviate intestinal IRI, the exact molecular mechanisms remain elusive.
To investigate Nar's protective properties on intestinal IRI and elucidate the mechanisms, a comprehensive approach that combines network pharmacology analysis with experimental verification in vitro and in vivo was adopted.
The oxygen-glucose deprivation/reoxygenation (OGD/R) model in IEC-6 cells and a murine model of intestinal IRI were used. Nar's effects on intestinal IRI were assessed through histological analysis using H&E staining and tight junction (TJ) protein expression. Ferroptosis-related parameters, including iron levels, superoxide dismutase (SOD), glutathione (GSH), reactive oxygen species (ROS), malondialdehyde (MDA), and mitochondrial morphology, were analyzed. Network pharmacology was utilized to predict the pathways through which Nar exerts its anti-ferroptosis effects. Further mechanistic insights were obtained through si-RNA transfection, YAP inhibitor (verteporfin, VP) treatment, ferroptosis inhibitor (Ferrostatin-1) and ferroptosis inducer (Erastin) application, co-immunoprecipitation (Co-IP) and Western blotting.
Our results revealed that pretreatment with Nar significantly mitigated intestinal tissue damage and improved gut barrier function, as evidenced by increased TJ proteins (ZO-1 and Occludin). Nar reduced iron, MDA, and ROS, while it increased GSH and SOD levels. Additionally, Nar alleviated mitochondrial damage in mice. Nar treatment increased GPX4 and SLC7A11, while decreasing ACSL4 levels both in vivo and in vitro. Network pharmacology analysis suggested that Nar may target the Hippo signaling pathway. Notably, YAP, a key transcriptional co-activator within the Hippo pathway, was downregulated in intestinal IRI mice and OGD/R-induced IEC-6 cells. Nar pretreatment activated YAP, thereby augmenting anti-ferroptosis effects. The inhibition of YAP activation by VP or YAP knockdown increased p-STAT3 expression, thereby diminishing Nar's efficacy. Co-IP and immunofluorescence studies confirmed the interaction between YAP and STAT3.
This study shows that Nar can inhibit ferroptosis in intestinal IRI via activating YAP, which in turn suppresses STAT3 phosphorylation, thereby unveiling a novel mechanism and supporting Nar's potential to be a promising therapeutic agent for intestinal IRI.
肠缺血/再灌注损伤（IRI）是一种严重的临床急症，研究新的治疗方法并了解其潜在机制对于改善患者预后至关重要。柚皮素（Nar）是一种存在于番茄和柑橘类水果中的类黄酮，在人类饮食中经常摄入，具有免疫调节、抗炎和抗氧化特性。尽管 Nar 能够减轻肠 IRI，但确切的分子机制仍不清楚。
采用网络药理学分析结合体外和体内实验验证的综合方法，研究 Nar 对肠 IRI 的保护作用，并阐明其机制。
采用 IEC-6 细胞的氧葡萄糖剥夺/再氧合（OGD/R）模型和肠 IRI 小鼠模型。通过 H&E 染色和紧密连接（TJ）蛋白表达评估 Nar 对肠 IRI 的影响。分析铁水平、超氧化物歧化酶（SOD）、谷胱甘肽（GSH）、活性氧（ROS）、丙二醛（MDA）和线粒体形态等铁死亡相关参数。利用网络药理学预测 Nar 发挥抗铁死亡作用的途径。通过 si-RNA 转染、YAP 抑制剂（verteporfin，VP）处理、铁死亡抑制剂（Ferrostatin-1）和铁死亡诱导剂（Erastin）应用、免疫共沉淀（Co-IP）和 Western blot 获得进一步的机制见解。
我们的结果表明，预处理 Nar 可显著减轻肠组织损伤并改善肠道屏障功能，表现为 TJ 蛋白（ZO-1 和 Occludin）增加。Nar 降低了铁、MDA 和 ROS，同时增加了 GSH 和 SOD 水平。此外，Nar 减轻了小鼠的线粒体损伤。Nar 处理增加了体内和体外的 GPX4 和 SLC7A11，同时降低了 ACSL4 水平。网络药理学分析表明，Nar 可能靶向 Hippo 信号通路。值得注意的是，YAP，Hippo 通路中的关键转录共激活因子，在肠 IRI 小鼠和 OGD/R 诱导的 IEC-6 细胞中下调。Nar 预处理激活了 YAP，从而增强了抗铁死亡作用。VP 或 YAP 敲低抑制 YAP 激活会增加 p-STAT3 的表达，从而降低 Nar 的疗效。Co-IP 和免疫荧光研究证实了 YAP 和 STAT3 之间的相互作用。
本研究表明，Nar 通过激活 YAP 抑制肠 IRI 中的铁死亡，从而抑制 STAT3 磷酸化，揭示了一种新的机制，并支持 Nar 成为治疗肠 IRI 的有前途的治疗药物。
