【摘要】 目的 基于网络药理学、分子对接和动物实验探讨枳术散调节肝郁脾虚型腹泻型肠易激综合征（IBS-D）的潜在作用机制。方法 检索TCMSP、Uniprot数据库筛选枳术散活性成分及作用靶点；采用Gene Gards、OMIM、TTD数据库筛选IBS-D的关联靶点；利用Venny 2.1获取的交集靶点构建蛋白质-蛋白质相互作用（PPI）网络，拓扑分析筛选关键靶点及核心活性成分；利用David数据库进行基因本体（GO）功能分析和京都基因与基因组百科全书（KEGG）通路富集分析；选择度值前4位的核心活性成分和PPI网络中有高度中心性的前4位关键靶点进行分子对接。选取8周龄SPF级雄性KM小鼠40只，按照随机数字表法将其分为正常组、模型组、阳性对照组和枳术散组，每组10只。除正常组外，其余组小鼠进行肝郁脾虚型IBS-D造模。阳性对照组给予匹维溴铵片药液15 mg/kg，枳术散组给予枳术散药液（2 g生药/kg）0.2 ml/只；正常组和模型组给予等体积蒸馏水，连续给药21 d。比较各组行为表现、粪便污迹直径与含水量，HE染色观察结肠组织病理变化，Western blot法检测通路相关蛋白。结果 经过筛选共得到枳术散16个活性成分、124个相关联靶点，与1 745个疾病相关靶点取交集，获得66个药物-疾病交集靶点。“药物-成分-靶点-疾病”网络中排名前4位的活性成分是木犀草素、异橙黄酮、柚皮素、3β-乙酰氧基苍术酮。度值排名前10位的靶蛋白为Akt1、IL-6、TNF、TP53、VEGFA、IL-1B、CASP3、PTGS2、EGFR和MMP9。GO功能富集分析得到生物过程4 047项、细胞组分357项、分子功能517项。KEGG通路富集分析得到233条信号通路，主要涉及PI3K/Akt信号通路、HIF-1信号通路等。分子对接结果显示，核心成分与关键靶点均有较强的潜在结合能力。与正常组比较，模型组总路程缩短、平均速度降低、墨汁推进比例减小、粪便污迹直径及含水量增加（P<0.05或P<0.01）；与模型组比较，枳术散组总路程增加、墨汁推进比例增大、粪便污迹直径及含水量降低（P<0.05或P<0.01）。正常组小鼠整体组织结构完整，无断裂或明显损伤；模型组小鼠结肠黏膜层细胞存在坏死脱落，黏膜下层可见炎症细胞浸润与水肿；枳术散组小鼠结肠组织炎症有一定程度的减轻，黏膜脱落损伤减少。与正常组比较，模型组p-Akt/Akt、p-PI3K/PI3K、e NOS蛋白表达降低（P<0.05或P<0.01）；与模型组比较，枳术散组p-Akt/Akt、p-PI3K/PI3K、e NOS蛋白表达升高（P<0.05或P<0.01）。结论枳术散可能通过调控PI3K/Akt/e NOS通路发挥治疗肝郁脾虚型IBS-D的作用。更多还原

【Abstract】 Objective To explore the potential mechanism of Zhizhu Powders in the treatment of diarrhea-predominant irritable bowel syndrome(IBS-D) with liver depression and spleen deficiency type based on network pharmacology,molecular docking, and animal experiment. Methods The active ingredients and target points of Zhizhu Powders were screened by searching TCMSP and Uniprot databases; the associated targets of IBS-D were screened by using Gene Gards,OMIM, and TTD databases; the proteinprotein interaction(PPI) network was constructed using the intersection targets obtained by Venny 2.1, and topological analysis was conducted to screen the key target points and core active components;gene ontology(GO) function analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis were analyzed using David database; and the top four core active ingredients with the highest degree of selection and the top four with high centrality in the PPI network were selected as key target points for molecular docking. Forty 8-week-old SPF-grade male KM mice were selected and they were divided into the normal group, the model group, the positive control group, and the Zhishu Powders group according to the random number table method, with 10 mice in each group. Except for the normal group, the IBS-D model of liver depression and spleen deficiency type was established in the mice of the other groups. The positive control group was given Piveronium Bromide Tablets solution at a dose of 15 mg/kg, while the Zhishu Powders group was given Zhishu Powders solution(2 g crude drug/kg) at a dose of 0.2 ml per mouse; the normal group and the model group were given equal volumes of distilled water for continuous administration for 21 days. The behavioral manifestations, fecal stain diameters, and water content of each group were compared; the pathological changes of colon tissue were observed by HE staining, and the pathway-related proteins were detected by Western blot. Results After screening, a total of 16 active ingredients and 124 associated targets of Zhizhu Powders were obtained, and they were intersections with 1 745 disease-related targets, resulting in 66 drug-disease intersection targets. The top four active ingredients in the“drug-component-target-disease”network were luteolin, isohesperidone, naringin, and 3β-acetoxyloxone.The top 10 target proteins ranked by degree values were Akt1, IL-6, TNF, TP53, VEGFA, IL-1B, CASP3, PTGS2, EGFR,and MMP9. GO functional enrichment analysis yielded 4 047 biological processes, 357 cellular components, and 517molecular functions. KEGG pathway enrichment analysis yielded 233 signaling pathways, mainly involving the PI3K/Akt signaling pathway, HIF-1 signaling pathway, etc. The results of molecular docking showed that both the core components and the key targets have strong potential binding capabilities. Compared with the normal group, the total distance of the model group was shortened, the average speed was reduced, the proportion of ink propulsion was decreased, and fecal stain diameters and water content were increased(P<0.05 or P<0.01); compared with the model group, the total distance of the Zhishu Powders group increased, the proportion of ink propulsion increased, and the fecal stain diameters and water content decreased(P<0.05 or P<0.01). The overall tissue structure of mice in the normal group was intact, with no fractures or obvious injuries; in the model group of mice, there was necrosis and shedding of cells in the colonic mucosal layer, and inflammatory cell infiltration and edema could be seen in the submucosa; and the colonic tissue inflammation in the Zhishu Powders group of mice was alleviated to a certain extent, and the mucosal shedding injury was reduced. Compared with the normal group, the expressions of p-Akt/Akt, p-PI3K/PI3K, and e NOS protein in the model group decreased(P<0.05 or P<0.01); compared with the model group, the expressions of p-Akt/Akt, p-PI3K/PI3K, and e NOS protein in the Zhishu Powders group increased(P<0.05 or P<0.01). Conclusion Zhizhu Powders may exert its therapeutic effect on IBS-D of liver depression and spleen deficiency type by regulating the PI3K/Akt/e NOS pathway.更多还原