Huang Na, Wei Yu, Wang Maxizi, Liu Meng, Kao Xingyu, Yang Zhen, He Mingfeng, Chen Jingli
The Eighth School of Clinical Medicine (Foshan Hospital of Traditional Chinese Medicine), Guangzhou University of Chinese Medicine, Foshan, 528000, China.
The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, 510000, China.
J Ethnopharmacol. 2025 Jan 30;337(Pt 3):118937. doi: 10.1016/j.jep.2024.118937. Epub 2024 Oct 16.
Dachaihu decoction (DCH) is a famous and ancient TCM formula, extensively utilized for over 1800 years in treating gastrointestinal and inflammatory conditions. Our previous study showed that DCH ameliorated intestinal damage and modulated the gut microflora in septic rats. However, the material basis for these effects and the underlying mechanism of action remains ill-defined. We aimed to explore the pharmaceutical ingredients of DCH and its mechanism in mitigating sepsis-induced intestinal epithelial barrier disruption (IEBD).
Ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) was used to identify DCH composition. A septic rat model and Caco-2 cells were employed to investigate DCH's effects on IEBD. Transcriptomics and network pharmacology were used to predict potential mechanisms, which were further validated by molecular docking and dynamics simulations. The Modified Murine Sepsis Score (mMSS) and histological assessments were performed. Serum fluorescence intensity of FD4 and the expression of Occludin were evaluated to assess intestinal barrier integrity. And p-PI3K P85, PI3K P85, p-AKT, AKT, Bax and Bcl-2 were determined by Western blot. Cell viability was determined using CCK-8 assay, IL-6 and TNF-α by ELISA and quantitative Real-time PCR (RT-qPCR). The integrity and permeability of single layer of Caco-2 cells were assessed via transepithelial resistance (TEER), alkaline phosphatase (ALP) activity and FD4 permeability.
UHPLC-HRMS identified 180 compounds in DCH. DCH significantly reduced mMSS, improved pathological conditions in the ileum, decreased FD4 serum fluorescence, and enhanced Occludin expression. Transcriptomic and network pharmacology analyses identified the PI3K/AKT pathway as a critical mechanism of action. Molecular docking and dynamics simulations confirmed strong binding of DCH components to PIK3R1. DCH upregulated p-PI3K and p-AKT in ileum tissue of septic rats. DCH improved cell viability, decreased IL-6 and TNF-α, promoted cell survival and Occludin level, and upregulated p-PI3K and p-AKT in LPS-stimulated Caco-2 cells. DCH also maintained TEER, ALP activity and decreased FD4 permeability and these effects were reversed by PI3K inhibitor, LY294002. DCH also downregulated Bax expression and increased Bcl-2 levels in both septic rats and LPS-stimulated Caco-2 cells.
DCH ameliorates sepsis-induced IEBD via PI3K/AKT pathway activation, offering a novel therapeutic perspective for sepsis-related intestinal dysfunction.
大柴胡汤（DCH）是一个著名的古老中药方剂，在治疗胃肠道疾病和炎症性疾病方面已广泛应用了1800多年。我们之前的研究表明，DCH可改善脓毒症大鼠的肠道损伤并调节肠道微生物群。然而，这些作用的物质基础和潜在作用机制仍不明确。我们旨在探索DCH的药物成分及其减轻脓毒症诱导的肠上皮屏障破坏（IEBD）的机制。
采用超高效液相色谱-高分辨率质谱（UHPLC-HRMS）鉴定DCH的成分。采用脓毒症大鼠模型和Caco-2细胞研究DCH对IEBD的影响。利用转录组学和网络药理学预测潜在机制，并通过分子对接和动力学模拟进一步验证。进行改良小鼠脓毒症评分（mMSS）和组织学评估。评估FD4的血清荧光强度和闭合蛋白的表达以评估肠道屏障完整性。通过蛋白质免疫印迹法测定p-PI3K P85、PI3K P85、p-AKT、AKT、Bax和Bcl-2。使用CCK-8法测定细胞活力，通过酶联免疫吸附测定法（ELISA）和定量实时聚合酶链反应（RT-qPCR）测定白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）。通过跨上皮电阻（TEER）、碱性磷酸酶（ALP）活性和FD4通透性评估单层Caco-2细胞的完整性和通透性。
UHPLC-HRMS鉴定出DCH中的180种化合物。DCH显著降低mMSS，改善回肠病理状况，降低FD4血清荧光，并增强闭合蛋白表达。转录组学和网络药理学分析确定PI3K/AKT信号通路是关键作用机制。分子对接和动力学模拟证实DCH成分与PIK3R1有强烈结合。DCH上调脓毒症大鼠回肠组织中的p-PI3K和p-AKT。DCH提高脂多糖（LPS）刺激的Caco-2细胞的活力，降低IL-6和TNF-α水平，促进细胞存活和闭合蛋白水平，并上调p-PI3K和p-AKT。DCH还维持TEER、ALP活性，降低FD4通透性，而PI3K抑制剂LY294002可逆转这些作用。DCH还下调脓毒症大鼠和LPS刺激的Caco-2细胞中Bax的表达并增加Bcl-2水平。
DCH通过激活PI3K/AKT信号通路改善脓毒症诱导的IEBD，为脓毒症相关肠道功能障碍提供了新的治疗前景。
