目的 阐明衢陈皮提取物（Quzhou Citri Reticulatae Pericarpium extract，CPE）的物质基础，并探讨其通过修复肠屏障改善功能性消化不良（functional dyspepsia，FD）的作用机制。方法 采用水煎提取联合冷冻干燥制备CPE，利用超高效液相色谱-四级杆飞行时间质谱联用技术与高效液相色谱技术进行成分定性与定量分析，通过网络药理学预测潜在作用靶点与通路。体内实验采用碘乙酰胺联合不规律进食法建立FD大鼠模型，给予药物干预后，检测胃排空率、小肠推进率；采用苏木素-伊红染色分析胃肠组织病理学变化；采用ELISA检测血清中脂多糖（lipopolysaccharide，LPS）、二胺氧化酶（diamine oxidase，DAO）水平及十二指肠组织肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）水平；采用免疫组化法检测十二指肠组织闭锁小带蛋白-1（zonula occludens-1，ZO-1）和闭合蛋白（Occludin）的表达。体外实验构建TNF-α诱导的Caco-2肠道细胞屏障损伤模型，测定跨上皮电阻（transepithelial electrical resistance，TEER）值和相对分子质量为4×10⁴的异硫氰酸荧光素-葡聚糖（fluorescein isothiocyanate-dextran with relative molecular mass 4×10⁴，FD-4）通透性；采用免疫荧光染色观察ZO-1和Occludin蛋白分布；采用qRT-PCR检测ZO-1、Occludin、白细胞介素-6（interleukin-6，IL-6）和TNF-α的mRNA表达；采用ELISA检测IL-6分泌；采用Western blotting检测c-Src原癌基因（cellular Src，c-Src）和蛋白激酶B（protein kinase B，Akt）的磷酸化水平。结果 从CPE中鉴定出84个化学成分，其中8个为首次在陈皮中报道；4种主要黄酮类成分的含量由高到低为橙皮苷＞芸香柚皮苷＞川陈皮素＞桔皮素。网络药理学预测显示，CPE改善FD的潜在核心靶点涉及SRC、AKT1等，相关通路富集于磷脂酰肌醇3-激酶（phosphoinositide 3-kinase，PI3K）/Akt信号通路及紧密连接调控。体内实验表明，CPE可显著改善FD大鼠的胃肠动力障碍（P＜0.05、0.01、0.001），修复十二指肠黏膜损伤，显著降低血清LPS和DAO水平（P＜0.01、0.001），抑制十二指肠组织TNF-α蛋白及基因表达（P＜0.01、0.001），上调Occludin蛋白表达（P＜0.05、0.001）。体外实验证实，CPE中橙皮苷的实际含量仅为橙皮苷单体组的10.48%，但在稳定TEER、降低FD-4通透性方面与橙皮苷单体等效（P＜0.01、0.001）；CPE可显著上调ZO-1和Occludin的mRNA表达（P＜0.001），并维持其连续网状分布，显著抑制IL-6的转录与分泌（P＜0.01、0.001），并显著降低p-c-Src/c-Src和p-Akt/Akt的值（P＜0.05）。结论 CPE可显著改善FD大鼠的胃肠动力障碍与十二指肠黏膜屏障损伤，并改善TNF-α诱导的Caco-2细胞屏障功能与炎症状态，其作用依赖于对c-Src/Akt信号通路过度激活的抑制，且其整体效应体现了多组分协同增效的特点，为衢陈皮在胃肠疾病中的应用提供了实验依据。

Objective To elucidate the material basis of Quzhou Chenpi (Citri Reticulatae Pericarpium) extract (CPE) and to investigate its mechanism in improving functional dyspepsia (FD) by repairing the intestinal barrier. Methods CPE was prepared by water decoction combined with freeze-drying. Qualitative and quantitative analyses of its chemical components were performed using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and high-performance liquid chromatography (HPLC), followed by network pharmacology to predict potential targets and pathways. In vivo, an FD rat model was established by iodoacetamide combined with irregular feeding. After drug intervention, gastric emptying rate and small intestinal propulsion rate were measured. Histopathological changes in the stomach and duodenum were examined by hematoxylin-eosin staining. Levels of lipopolysaccharide (LPS), diamine oxidase (DAO) in serum as well as tumor necrosis factor-α (TNF-α) in duodenal tissue were determined by ELISA. The expressions of zonula occludens-1 (ZO-1) and Occludin in duodenal tissue was assessed by immunohistochemistry. In vitro, a TNF-α-induced Caco-2 intestinal barrier injury model was used. Transepithelial electrical resistance (TEER) value and fluorescein isothiocyanate-dextran with relative molecular mass 4 × 10⁴ (FD-4) permeability were measured. The distribution of ZO-1 and Occludin proteins was observed by immunofluorescence staining. The mRNA expressions of ZO-1, Occludin, interleukin-6 (IL-6) and TNF-α were detected by qRT-PCR. IL-6 secretion was measured by ELISA. The phosphorylation levels of cellular Src (c-Src) and protein kinase B (Akt) were detected by Western blotting. Results A total of 84 chemical components were identified from CPE, eight of which were reported in Citri Reticulatae Pericarpium for the first time. The contents of the four major flavonoids were ranked from high to low as hesperidin > narirutin > nobiletin > tangeretin. Network pharmacology predicted that the potential core targets of CPE for improving FD involved SRC, AKT1, etc., with enrichment in phosphoinositide 3-kinase (PI3K)/Akt signaling pathway and tight junction regulation. In vivo, CPE significantly ameliorated gastrointestinal motility disorders in FD rats (P < 0.05, 0.01, 0.001), alleviated duodenal mucosal injury, reduced levels of LPS and DAO in serum (P < 0.01, 0.001), suppressed TNF-α protein and mRNA expressions in duodenal tissue (P < 0.01, 0.001), and up-regulated Occludin protein expression (P < 0.05, 0.001). In vitro, the actual hesperidin content in CPE was only 10.48% of that in the hesperidin monomer group, yet CPE was equivalent to hesperidin monomer in stabilizing TEER and reducing FD-4 permeability (P < 0.01, 0.001). CPE significantly up-regulated the mRNA expressions of ZO-1 and Occludin (P < 0.001), maintained their continuous network-like distribution, significantly inhibited IL-6 transcription and secretion (P < 0.01, 0.001), and significantly reduced the values of p-c-Src/c-Src and p-Akt/Akt (P < 0.05). Conclusion CPE significantly ameliorates gastrointestinal motility disorders and duodenal mucosal barrier injury in FD rats, and alleviates barrier dysfunction and inflammation in TNF-α-induced Caco-2 cells. Its effect depends on the inhibition of over-activation of c-Src/Akt signaling pathway. The overall efficacy demonstrates the multi-component synergistic characteristics of traditional Chinese medicine, providing an experimental basis for the application of Quzhou Citri Reticulatae Pericarpium in gastrointestinal diseases.

R285.5

浙江省中医药科技计划项目（762213V00102）