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伍冲梅, 潘坤, 谢果珍, 郑淘, 惠华英.葛根芩连汤对肠道湿热证泄泻小鼠肠道机械屏障的影响[J].湖南中医药大学学报英文版,2024,44(8):1351-1359.[Click to copy
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| 葛根芩连汤对肠道湿热证泄泻小鼠肠道机械屏障的影响 |
| 伍冲梅,潘坤,谢果珍,郑淘,惠华英 |
| (湖南中医药大学药学院, 湖南 长沙 410208;湖南中医药大学医学院, 湖南 长沙 410208) |
| 摘要: |
| 目的 探讨葛根芩连汤对肠道湿热证泄泻模型小鼠肠道机械屏障的作用机制。方法 选取15只SPF级雄性昆明小鼠，随机分为正常组5只和模型组10只。采取“高糖高脂+高温高湿+白酒稀释液+冰水法”复制肠道湿热证泄泻小鼠模型，造模成功的10只小鼠随机分为自愈组和治疗组，每组5只。治疗组灌胃5.07 g/（kg·d）葛根芩连汤水煎液，正常组和自愈组给予等容积无菌水，连续灌胃4 d后无菌提取各组小鼠十二指肠、空肠和回肠肠段。观察各组小鼠一般状态；HE染色观察各组肠段形态；采用Image J图像处理软件测量各组绒毛长度（villus height, VH）、隐窝深度（crypt depth, CD），计算VH与CD的比值（绒隐比，V/C）、杯状细胞数目和紧密连接蛋白平均光密度值（average optical density value, AOD）；采用二步法免疫组织化学法分析各肠段密封蛋白1（Claudin 1）、闭合蛋白（Occludin）和紧密连接蛋白-1（zonula occluden-1, ZO-1）的表达。结果 正常组小鼠毛发有光泽，精神状态佳，饮水量、肛温和二便均正常；治疗组小鼠毛发洁白、精神状态良好、大便颗粒饱满、肛温恢复正常、泄泻症状消失；自愈组小鼠毛发暗淡、肛温高、大便含水量较多、挤压不成型。与正常组相比，自愈组十二指肠、空肠和回肠肠黏膜结构部分受损，小肠腺出现萎缩，出现炎性症状。与自愈组相比，治疗组肠黏膜结构较清晰，肠上皮细胞和固有层细胞排列紧密，炎症情况明显改善。与正常组相比，自愈组十二指肠CD升高（P＜0.01），十二指肠V/C和杯状细胞数降低（P＜0.05，P＜0.01）；治疗组十二指肠V/C降低（P＜0.05）。与正常组相比，自愈组空肠、回肠VH和V/C均明显降低（P＜0.05，P＜0.01）。与自愈组相比，治疗组十二指肠CD显著降低（P＜0.01），十二指肠、回肠V/C升高（P＜0.05，P＜0.01）。十二指肠肠段，与正常组相比，自愈组和治疗组Claudin 1、Occludin、ZO-1 AOD值降低（P＜0.05，P＜0.01）；与自愈组相比，治疗组Claudin 1、Occludin、ZO-1 AOD值显著升高（P＜0.01）。与正常组比较，自愈组空肠、回肠肠段Occludin AOD值明显降低（P＜0.01）。与自愈组比较，治疗组空肠肠段Claudin 1 AOD值升高（P＜0.05），回肠肠段Occludin AOD值明显升高（P＜0.01）。结论 葛根芩连汤可改善肠道湿热证泄泻小鼠肠道炎症水平、促进紧密连接蛋白Claudin1、Occludin和ZO-1的表达，其机制可能与修复肠道机械屏障功能有关。 |
| 关键词: 泄泻 肠道湿热证 葛根芩连汤 机械屏障 肠道形态 紧密连接蛋白 |
| DOI：10.3969/j.issn.1674-070X.2024.08.001 |
| Received:May 15, 2024 |
| 基金项目:国家自然科学基金项目（81703985）；湖南省卫生健康委员会科研计划项目（D202303038538）。 |
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| Effects of Gegen Qinlian Decoction on the intestinal mechanical barrier in diarrhea mice with intestinal dampness-heat pattern |
| WU Chongmei, PAN Kun, XIE Guozhen, ZHENG Tao, HUI Huaying |
| (School of Pharmacy, Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;School of Medicine, Hunan University of Chinese Medicine, Changsha, Hunan 410208, China) |
| Abstract: |
| Objective To explore the effects of Gegen Qinlian Decoction on the intestinal mechanical barrier in diarrhea mice with intestinal dampness-heat pattern. Methods Fifteen SPF male Kunming mice were randomized into normal group (n=5) and model group (n=10). The diarrhea model with intestinal dampness-heat pattern was replicated by the method of "high sugar and high fat + high temperature and high humidity + liquor dilution + ice water", and 10 successfully modeled mice were randomized into self-healing group and treatment group, with five mice in each group. The treatment group was administered with 5.07 g/(kg·d) Gegen Qinlian Decoction, and the normal group and self-healing group were given equal volumes of sterile water. The duodenum, jejunum, and ileum segments of mice in each group were aseptically extracted after 4 d of gavage. The general condition of the mice was observed; HE staining was used to observe the morphology of each intestinal segment. Image J processing software was used to measure the villus height (VH) and crypt depth (CD) in each group, and the ratio of VH to CD (V/C), the number of goblet cells, and the average optical density (AOD) value of tight junction protein were calculated. Two-step immunohistochemistry was used to analyze the expressions of Claudin 1, Occludin, and zonula occluden-1 (ZO-1). Results The normal group of mice had shiny hair, good mental state, normal water intake and rectal temperature, and normal stool and urine. The treatment group of mice had white hair, good mental state, full stool particles, normally recovered rectal temperature, and disappearance of diarrhea symptoms. The self-healing group of mice had dull hair, high rectal temperature, stool with high water content, and the stool did not hold shapes when squeezed. Compared with the normal group, the mucosal structure of the duodenum, jejunum, and ileum in the self-healing group was partially damaged, with atrophy of the intestinal glands and inflammatory symptoms. Compared with the self-healing group, the mucosal structure in the treatment group was clearer, with tightly arranged intestinal epithelial cells and lamina propria cells, and the inflammatory condition was significantly improved. In comparison to the normal group, the CD of the duodenum in the self-healing group was higher (P＜0.01), and the V/C and the number of goblet cells were lower (P＜0.05, P＜0.01) in the duodenum; the V/C in the treatment group was lower (P＜0.05). Compared with the normal group, the VH and V/C of the jejunum and ileum in the self-healing group were significantly reduced (P＜0.05, P＜0.01). Compared with the self-healing group, the CD of the duodenum in the treatment group was significantly lower (P＜0.01), while the V/C of the duodenum and ileum were significantly higher (P＜0.05, P＜0.01). In the duodenal segment, compared with the normal group, the AOD values of Claudin 1, Occludin, and ZO-1 proteins were lower in both the self-healing and treatment groups (P＜0.05, P＜0.01); compared with the self-healing group, the AOD value of Claudin 1, Occludin, and ZO-1 proteins in the treatment group were significantly higher (P＜0.01). Compared with the normal group, the AOD value of Occludin protein in the jejunum and ileum was significantly lower in the self-healing group (P＜0.01). Compared with the self-healing group, the AOD value of Claudin 1 protein in the jejunum was higher in the treatment group (P＜0.05), and the AOD value of Occludin protein in the ileum was significantly elevated (P＜0.01). Conclusion Gegen Qinlian Decoction can improve the level of intestinal inflammation in diarrhea mice with intestinal dampness-heat pattern and promote the expression of tight junction proteins Claudin 1, Occludin, and ZO-1. Its mechanism may be related to its repairing function on intestinal mechanical barrier. |
| Key words: diarrhea intestinal dampness-heat pattern Gegen Qinlian Decoction mechanical barrier intestinal morphology tight junction protein |
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