中药药理与临床

目的:本研究旨在通过整合化学分析、网络药理学、分子对接及动物实验验证，系统探讨枳术丸治疗溃疡性结肠炎(UC)的作用机制，初步明确其药效物质基础及作用靶点。方法:采用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱(UPLC-Q-Exactive MS2)技术对生药材枳术丸、炮制品及其含药血清进行化学成分定性分析，鉴定入血成分。基于SwissTargetPrediction和Genecards数据库筛选枳术丸治疗UC的潜在作用靶点，构建“药物-成分-靶点-疾病”网络及“通路-靶点-入血成分”网络。通过STRING数据库绘制蛋白互作(PPI)网络，结合David数据库进行GO功能分析和KEGG通路富集分析，并采用分子对接技术验证。将56只Balb/c小鼠随机分为正常对照组、模型对照组、生药材枳术丸9、18 g/kg组、炮制饮片枳术丸9、18 g/kg组和柳氮磺吡啶0.25 g/kg组。造模小鼠自由饮用3.5%DSS溶液。造模同时，正常对照组和模型对照组灌胃生理盐水，各给药组灌胃给予相应药物，连续给药7 d。观察枳术丸对结肠长度、疾病活动指数(DAI)评分及肠黏膜损伤的影响;通过HE染色、阿利新蓝-过碘酸雪夫(AB-PAS)染色评估结肠病理变化及杯状细胞数量;采用TUNEL染色检测结肠上皮细胞凋亡情况;通过Western blot法分析凋亡相关蛋白裂解的半胱天冬酶-3(Cleaved Caspase-3)、B细胞淋巴瘤-2(BCL-2)及Bcl-2相关X蛋白(BAX)的表达。结果:从枳术丸中鉴定出38个化合物，其中18个为原型吸收成分，包括9个黄酮类、3个萜类、2个有机酸类及4个其他类化合物。网络药理学分析显示，异橙皮内酯、柠檬苦素、橘皮素、白术内酯Ⅰ、白术内酯Ⅱ及白术内酯Ⅲ等为枳术丸改善UC的潜在活性成分，BCL2、半胱天冬酶-3(CASPASE-3)等为关键靶点。分子对接结果表明，6个关键活性成分均与BCL2、CASPASE-3具有较高的结合力。动物实验结果显示，与正常对照组比较，模型对照组UC小鼠体质量，结肠长度及DAI评分显著下降(P <0.01)，结肠组织BCL-2蛋白表达显著下调(P <0.01),BAX、Cleaved Caspase-3蛋白表达明显上调(P <0.05或P <0.01);与模型对照组比较，生药材及炮制品枳术丸各组UC小鼠体质量、结肠长度及DAI评分明显降低(P <0.05或P <0.01)，肠黏膜损伤减轻，杯状细胞数量增加，炮制饮片枳术丸18 g/kg组肠上皮细胞凋亡率降低(P <0.05)，结肠组织BCL-2蛋白表达上调(P <0.05),BAX及Cleaved Caspase-3表达下调(P <0.01)。结论:炮制饮片枳术丸和生药材枳术丸化学成分变化不大。对小鼠UC模型具有显著的改善作用，炮制品略优于生品。结合网络药理学分析及动物实验验证，枳术丸可能通过调控细胞凋亡途径，减轻肠黏膜损伤，从而发挥治疗UC的作用。

Objective:To elucidate the therapeutic mechanism of Zhizhu Pill(枳术丸) in treating ulcerative colitis(UC) through a combination of chemical analysis, network pharmacology, molecular docking, and animal experimentation and to preliminarily explore its therapeutic material basis and action targets. Methods:The chemical constituents and serum components of both raw and processed Zhizhu Pill were identified using UPLC-Q-Exactive MS² technology to identify the blood components. Potential therapeutic targets were predicted using the SwissTargetPrediction and Genecards databases, and the “drug-component-target-disease” and “pathway-target-blood component” networks were constructed. Protein-protein interaction(PPI) networks were generated via the STRING database. Functional enrichment analyses, including Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG),were conducted using the David database, with molecular docking employed for validation. For animal experimentation, 56 Balb/c mice were randomly divided into a normal control group, a model control group, 9 g/kg and 18 g/kg raw Zhizhu Pill groups, 9 g/kg and 18 g/kg processed Zhizhu Pill groups, and a 0.25 g/kg sulfasalazine(SASP) group. The mice were fed for 3 days for acclimation. The normal control group was fed with normal diet and water, and the other groups were free to drink 3.5% DSS solution. During modeling, the normal control group and the model control group were given normal saline by gavage, and each medication group was given the corresponding drug by gavage for 7 days. The effects of Zhizhu Pill on colon length, disease activity index(DAI) score, and intestinal mucosal injury were observed. The pathological changes in the colon, as well as the number of goblet cells, were assessed using hematoxylin and eosin(HE) staining and Alcian blue-periodic acid Schiff(AB-PAS) staining. Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) staining was used to detect apoptosis in colon epithelial cells, and Western blot analysis was performed to analyze the expression of apoptosis-related proteins, including cleaved caspase-3,B-cell lymphoma-2(Bcl-2),and Bcl-2-associated X protein(Bax). Results:38 compounds were identified in Zhizhu Pill, of which 18 were prototypical absorbing components, including 9 flavonoids, 3 terpenoids, 2 organic acids, and 4 other types of compounds. Network pharmacology analysis showed that isomeranzin, limonin, tangeretin, atractylenolide I,atractylenolide II,and atractylenolide III were potential active ingredients of Zhizhu Pill that could improve UC,and BCL2 and CASPASE-3 were key targets. Molecular docking results showed high affinity of the six key active ingredients to BCL2 and CASPASE-3. The results of animal experiments showed that compared with those in the normal control group, the body weight, colon length, and DAI score of UC mice in the model control group were decreased significantly(P<0.01). In addition, in the model control group, the expression of Bcl-2 protein in colon was significantly downregulated(P<0.01),while the expression of Bax and cleaved caspase-3 was significantly upregulated(P<0.05 or P<0.01). Compared with the model control group, both the raw and processed forms of Zhizhu Pill significantly improved the body weight, colon length, and DAI score of UC mice(P<0.05 or P<0.01),reduced intestinal mucosal injury, and increased goblet cell number. The 18 g/kg processed Zhizhu Pill group exhibited significantly inhibited apoptosis of intestinal epithelial cells(P<0.05),upregulated expression of Bcl-2(P<0.05),and downregulated expression of Bax and cleaved caspase-3(P<0.01). Conclusion:The chemical components of Zhizhu Pill have no obvious change before and after processing. It can significantly improve UC injury, with the processed product slightly outperforming the raw form. The results of network pharmacology analysis and animal experiment verification show that it plays its therapeutic role against UC probably by regulating the cell apoptosis pathway and reducing mucosal injury.