Dai Wen, Zhou Lei, Hao Hao, Wang Diankui, Zhang Feihu, Wang Peng, Wang Lin, Kong Li
The First Clinical Medical College, Shandong University of Traditional Chinese Medicine, Jinan, Shandong, 250014, China.
Department of Emergency, Yantai Affiliated Hospital of Binzhou Medical University, Yantai, Shandong, 264100, China.
Comb Chem High Throughput Screen. 2025 Apr 29. doi: 10.2174/0113862073349597250410111941.
This study aims to investigate therapeutic effects and involved molecular mechanisms of Banxia Xiexin Decoction (BXD) in reducing gastrointestinal complications associated with sepsis.
Sepsis is a common critical illness that threatens patient survival and costs society a lot. This syndrome is a prominent cause of death in ICUs due to its high mortality rate, which exceeds 30% after 28 days and 35.5% after 90 days. Sepsis remains a major medical challenge despite a 20%-30% drop in fatality rates due to a better understanding of its physiological and pathological features and better therapeutic techniques. There is no pharmacological treatment for sepsis, highlighting the need for more study.
We explored the protecting effects of BXD on intestinal functionality in sepsis by investigating its roles in the regulation of mitochondrial autophagy and mitochondrial functioning in small intestinal epithelial cells, primarily via the PINK1/Parkin signaling pathway.
We established a cell model of Human Intestinal Epithelial Cell (HIEC) injury induced by lipopolysaccharide (LPS) and a cecal ligation and perforation (CLP) sepsis model in Sprague Dawley (SD) rats. The cell model and animal model of sepsis were divided into control groups and different treatment groups that received different doses of BXD. We utilized HIECs with PINK1 knockdown to assess BXD's protective effects on the sepsis intestinal barrier and its regulatory mechanism both on the PINK1/Parkin signaling pathway, exploring both its facilitative and inhibitory effects. ELISA method was used to measure inflammatory markers IL-6, IL-1β, and intestinal injury-related molecules IFABP and DAO. Pathological assessments were performed with H&E staining, and tight junction proteins ZO-1 and Occludin were detected using immunohistochemical staining. Mitochondrial membrane protein TOM20 was detected through immunofluorescence staining. Mitochondrial membrane potential and autophagy were assessed via flow cytometry. The expression levels of PINK1, Park, LC3, and p62 proteins and mRNA, integral to the PINK1/Parkin autophagy pathway, were evaluated using Western Blot and RT-PCR.
Compared to the control group, BXD therapy significantly lowered serum DAO, IFABP, and DA. The BXD therapy group showed a more significant and sustained drop in IL-6 and IL-1β levels than the control group. The BXD therapy reduced intestinal mucosa damage by lowering DAO and IFABP. BXD also restored tight junction proteins ZO-1 and Occludin, improving intestinal mucosal barrier function. In septic rats, BXD therapy lowered serum IL-6 and IL-1β levels, avoiding inflammation and reducing intestinal damage. BXD enhanced TOM20, which protected intestinal epithelial cell mitochondria against decreasing mitochondrial membrane potential. BXD increased the PINK1/Parkin mitochondrial autophagy pathway at the molecular level. Mitochondrial autophagy can repair mitochondria, reduce oxidative stress, maintain mitochondrial homeostasis, and help intestinal epithelial cells survive and function.
BXD could improve intestinal mucosal damage and systemic inflammation caused by sepsis. BXD mainly promotes the PINK1/Parkin mitophagy pathway by upregulating PINK1 protein.
本研究旨在探讨半夏泻心汤（BXD）在减轻脓毒症相关胃肠道并发症方面的治疗作用及相关分子机制。
脓毒症是一种常见的危重病，威胁患者生命，给社会带来巨大负担。该综合征是重症监护病房（ICU）死亡的主要原因之一，死亡率很高，28天后超过30%，90天后超过35.5%。尽管由于对其生理和病理特征有了更好的了解以及治疗技术的改进，脓毒症的死亡率下降了20%-30%，但其仍然是一个重大的医学挑战。目前尚无针对脓毒症的药物治疗方法，这凸显了进一步研究的必要性。
我们通过研究半夏泻心汤在调节小肠上皮细胞线粒体自噬和线粒体功能中的作用，主要通过PINK1/Parkin信号通路，探讨其对脓毒症肠道功能的保护作用。
我们建立了脂多糖（LPS）诱导的人肠上皮细胞（HIEC）损伤细胞模型以及Sprague Dawley（SD）大鼠的盲肠结扎穿孔（CLP）脓毒症模型。将脓毒症细胞模型和动物模型分为对照组和接受不同剂量半夏泻心汤的不同治疗组。我们利用敲低PINK1的HIEC来评估半夏泻心汤对脓毒症肠屏障的保护作用及其对PINK1/Parkin信号通路的调节机制，探讨其促进和抑制作用。采用酶联免疫吸附测定（ELISA）法检测炎症标志物白细胞介素-6（IL-6）、白细胞介素-1β（IL-1β）以及肠道损伤相关分子肠脂肪酸结合蛋白（IFABP）和二胺氧化酶（DAO）。用苏木精-伊红（H&E）染色进行病理评估，采用免疫组织化学染色检测紧密连接蛋白闭合蛋白（ZO-1）和闭锁蛋白（Occludin）。通过免疫荧光染色检测线粒体膜蛋白转位酶亚基20（TOM20）。通过流式细胞术评估线粒体膜电位和自噬。使用蛋白质免疫印迹法（Western Blot）和逆转录-聚合酶链反应（RT-PCR）评估PINK1/Parkin自噬途径中重要的PINK1、Park、微管相关蛋白轻链3（LC3）和p62蛋白及mRNA的表达水平。
与对照组相比，半夏泻心汤治疗显著降低了血清DAO、IFABP和二胺（DA）水平。半夏泻心汤治疗组的IL-6和IL-1β水平较对照组有更显著且持续的下降。半夏泻心汤通过降低DAO和IFABP减轻了肠黏膜损伤。半夏泻心汤还恢复了紧密连接蛋白ZO-1和Occludin，改善了肠黏膜屏障功能。在脓毒症大鼠中，半夏泻心汤治疗降低了血清IL-6和IL-1β水平，避免了炎症反应并减轻了肠道损伤。半夏泻心汤增强了TOM20，保护肠上皮细胞线粒体免受线粒体膜电位降低的影响。半夏泻心汤在分子水平上增强了PINK1/Parkin线粒体自噬途径。线粒体自噬可以修复线粒体、减少氧化应激、维持线粒体稳态，并有助于肠上皮细胞存活和发挥功能。
半夏泻心汤可改善脓毒症引起的肠黏膜损伤和全身炎症反应。半夏泻心汤主要通过上调PINK1蛋白促进PINK1/Parkin线粒体自噬途径。
