丹参注射液改善粘连性肠梗阻大鼠肠黏膜屏障功能的作用及机制研究

Effect and mechanism of Danshen injection in improving intestinal mucosal barrier function in rats with adhesive intestinal obstruction

摘要:

背景与目的 粘连性肠梗阻（AIO）是由腹腔或肠道粘连引起的机械性肠道梗阻，其发生和进展与肠黏膜屏障功能受损密切相关。丹参注射液作为一种常用的活血化瘀类中药，具有改善胃肠道微循环、扩张血管等作用，临床上在多种胃肠疾病中已表现出一定疗效。但其在AIO中的作用机制尚不明确。本研究旨在探讨丹参注射液对AIO大鼠肠黏膜屏障功能的影响及其潜在作用机制。方法 将40只大鼠建立AIO动物模型随机均分为3组，分别腹腔注射生理盐水（模型组）与不同剂量（1、2、4 mL/kg）丹参注射液（低、中、高剂量丹参注射液干预组），1次/d，连续7 d。另取10只正常大鼠，在同样的时间段，以同样的方式注射生理盐水作为正常对照组。末次干预完成后，麻醉处死大鼠，用HE染色观察大鼠小肠组织病理形态，通过ELISA法检测各组大鼠外周血D-乳酸、内毒素水平，并通过qRT-PCR法检测小肠组织中黏液层主要成分黏蛋白2（MUC2）、黏蛋白3（MUC3）以及肠道免疫因子人防御素5（HD5）表达，比色法检测小肠组织氧化应激指标NO合酶（NOS）、丙二醛（MDA）、超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GSH-Px）水平，Western blot检测小肠组织中内源性抗氧化信号通路蛋白核因子E2相关因子2（Nrf2）、血红素氧合酶-1（HO-1）和氧化还原酶1（NQO1）的表达。结果 HE染色结果显示，正常对照组肠组织无明显病理学变化，肠黏膜损伤评分为0，模型组与低、中、高剂量丹参注射液干预组均呈现不同程度的小肠绒毛排列紊乱与组织水肿，肠黏膜损伤评分分别为4.69±0.62、3.36±0.41、2.29±0.22、1.53±0.14，低、中、高剂量丹参注射液干预组的评分均明显低于模型组（均P<0.05）；与正常对照组比较，其余各组外周血D-乳酸、内毒素水平均明显升高（均P<0.05）；小肠组织中MUC2和MUC3基因表达明显升高，HD5基因表达明显降低（均P<0.05）；小肠组织中NOS、MDA水平明显升高，SOD、GSH-Px水平明显降低（均P<0.05）；小肠组织中Nrf2、HO-1、NQO1蛋白表达明显下调（均P<0.05）。与模型组比较，低、中、高剂量丹参注射液干预组的以上所有指标的变化程度均明显减弱（均P<0.05），且呈一定的剂量依赖趋势。结论 丹参注射液可改善AIO大鼠的肠黏膜损伤，其作用机制可能与活化Nrf2/HO-1/NQO1信号通路，抑制氧化应激反应，从而保护肠黏膜屏障有关。

Abstract:

Background and Aims Adhesive intestinal obstruction (AIO) is a type of mechanical bowel obstruction caused by abdominal or intestinal adhesions, and its onset and progression are closely associated with impaired intestinal mucosal barrier function. Danshen injection, a commonly used traditional Chinese medicine preparation with properties of promoting blood circulation and removing blood stasis, has shown therapeutic potential in various gastrointestinal diseases by improving microcirculation and promoting vasodilation. However, its specific mechanism of action in AIO remains unclear. This study was conducted to investigate the effects and potential mechanisms of Danshen injection on intestinal mucosal barrier function in a rat model of AIO.Methods Forty rats with experimentally induced AIO were equally randomized into four groups: the model group (receiving intraperitoneal saline) and three Danshen-treated groups administered low, medium, and high doses of Danshen injection (1, 2, and 4 mL/kg, respectively), once daily for 7 consecutive d. An additional 10 healthy rats received saline injections in the same manner and served as the normal control group. After the final intervention, all rats were euthanized under anesthesia. Hematoxylin and eosin (HE) staining was performed to evaluate the histopathological morphology of small intestinal tissues. Levels of D-lactic acid and endotoxin in peripheral blood were measured using enzyme-linked immunosorbent assay (ELISA). The expression levels of mucin 2 (MUC2), mucin 3 (MUC3), and human defensin 5 (HD5)—key components of the intestinal mucus layer and innate immune response—were analyzed using quantitative real-time PCR (qRT-PCR). Colorimetric assays were conducted to assess oxidative stress markers in intestinal tissue, including nitric oxide synthase (NOS), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Western blot was used to determine the protein expression levels of endogenous antioxidant pathway components: nuclear factor erythroid 2–related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and NAD(P)H:quinone oxidoreductase 1 (NQO1).Results HE staining showed no significant histological changes in the intestinal tissues of the normal control group, with a mucosal injury score of 0. The model and treatment groups exhibited varying degrees of villous disorganization and tissue edema, with injury scores of 4.69±0.62, 3.36±0.41, 2.29±0.22, and 1.53±0.14 in the model, low-, medium-, and high-dose groups, respectively (all P<0.05 vs. model group). Compared with the normal control group, the other groups showed significantly increased levels of D-lactic acid and endotoxin in the blood (all P<0.05); elevated expression of MUC2 and MUC3, reduced HD5 expression (all P<0.05); increased NOS and MDA levels, decreased SOD and GSH-Px levels (all P<0.05); downregulated expression of Nrf2, HO-1, and NQO1 proteins in intestinal tissues (all P<0.05). These changes were significantly attenuated in the Danshen-treated groups in a dose-dependent manner (all P<0.05).Conclusion Danshen injection can alleviate intestinal mucosal injury in AIO rats, possibly by activating the Nrf2/HO-1/NQO1 signaling pathway and reducing oxidative stress, thereby enhancing the intestinal mucosal barrier function.