Cen Shui-Mei, Zou Ying, Zeng Jia-Yang, Li Rou-de, Zhao Jing-Mei, Liao Xiao-Xia, Cheng Jin-Peng, Chi Hong-Gang
the First Dongguan Affiliated Hospital of Guangdong Medical University Dongguan 523710, China the Second Clinical Medical College, Guangdong Medical University Dongguan 523808, China.
the First Dongguan Affiliated Hospital of Guangdong Medical University Dongguan 523710, China Liaobu Hospital, Guangdong Medical University Dongguan 523400, China.
Zhongguo Zhong Yao Za Zhi. 2024 Oct;49(20):5555-5565. doi: 10.19540/j.cnki.cjcmm.20240611.701.
This study aims to elucidate the mechanism of Huangqin Decoction(HQD) in treating ulcerative colitis(UC) by investigating the relationship between tryptophan metabolism and intestinal barriers. In the in vivo experiments, 3% dextran sulfate sodium(DSS) was used to induce a mouse model of acute colitis, with mesalazine as a positive control. The therapeutic effect of HQD on mice with UC was evaluated according to body weight, disease activity index(DAI), colon length, and pathological changes. Targeted metabolomics was used to detect the concentration of tryptophan and its metabolites in mouse feces. Western blot and RT-qPCR techniques were used to assess the expression levels of colonic aryl hydrocarbon receptor(AhR), myosin light chain kinase(MLCK), myo-sin light chain(MLC), and p-MLC. Serum FITC-dextran concentration, bacterial culture of mesenteric lymph nodes and spleen, as well as fluorescence probe in situ hybridization technique were used to evaluate intestinal epithelial permeability. Alcian blue and nuclear fast red staining, Western blot, and RT-qPCR techniques were used to detect the expression of mucin secreted by the mouse's intestinal epithelial goblet cells. Transmission electron microscopy was utilized to observe the connections of the mouse's intestinal epithelial cells. Immunofluorescence, Western blot, and RT-qPCR techniques were used to assess the expression of tight junction proteins in the mouse's intestinal epithelium. In the in vitro experiments, lipopolysaccharide(LPS) was used to induce intestinal epithelial barrier injury model in Caco2 cells, and AhR siRNA was used to further clarify the mechanism of HQD in activating AhR to improve intestinal barrier function. The results demonstrated that HQD effectively alleviated symptoms and pathological changes in the colon of DSS-induced mice with colitis. Treatment with HQD could regulate tryptophan metabolism in the feces of mice with colitis, activate AhR, and improve the intestinal epithelial barrier. Additionally, the results of the in vitro experiments confirmed that HQD could restore the expression of tight junction proteins in the intestinal epithelium of colitis cells by activating AhR to regulate the MLCK/p-MLC signaling pathway.
本研究旨在通过探究色氨酸代谢与肠道屏障之间的关系，阐明黄芩汤（HQD）治疗溃疡性结肠炎（UC）的机制。在体内实验中，使用3%葡聚糖硫酸钠（DSS）诱导小鼠急性结肠炎模型，以美沙拉嗪作为阳性对照。根据体重、疾病活动指数（DAI）、结肠长度和病理变化评估HQD对UC小鼠的治疗效果。采用靶向代谢组学检测小鼠粪便中色氨酸及其代谢产物的浓度。运用蛋白质免疫印迹法（Western blot）和逆转录定量聚合酶链反应（RT-qPCR）技术评估结肠芳烃受体（AhR）、肌球蛋白轻链激酶（MLCK）、肌球蛋白轻链（MLC）和磷酸化肌球蛋白轻链（p-MLC）的表达水平。采用血清异硫氰酸荧光素-葡聚糖（FITC-葡聚糖）浓度、肠系膜淋巴结和脾脏细菌培养以及荧光探针原位杂交技术评估肠道上皮通透性。运用阿尔新蓝和核固红染色、蛋白质免疫印迹法和RT-qPCR技术检测小鼠肠道上皮杯状细胞分泌的黏蛋白的表达。利用透射电子显微镜观察小鼠肠道上皮细胞的连接情况。运用免疫荧光、蛋白质免疫印迹法和RT-qPCR技术评估小鼠肠道上皮中紧密连接蛋白的表达。在体外实验中，使用脂多糖（LPS）诱导Caco2细胞肠道上皮屏障损伤模型，并使用AhR小干扰RNA（siRNA）进一步阐明HQD激活AhR改善肠道屏障功能的机制。结果表明，HQD有效减轻了DSS诱导的结肠炎小鼠结肠的症状和病理变化。HQD治疗可调节结肠炎小鼠粪便中的色氨酸代谢，激活AhR，并改善肠道上皮屏障。此外，体外实验结果证实，HQD可通过激活AhR调节MLCK/p-MLC信号通路，恢复结肠炎细胞肠道上皮中紧密连接蛋白的表达。
