枳术丸中饮片炮制前后成分变化及抗溃疡性结肠炎损伤的作用机制OA

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 se-rum 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 data-bases,and the"drug-component-target-disease"and"pathway-target-blood component"networks were constructed.Pro-tein-protein interaction(PPI)networks were generated via the STRING database.Functional enrichment analyses,inclu-ding 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 di-vided 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 ga-vage,and each medication group was given the corresponding drug by gavage for 7 days.The effects of Zhizhu Pill on co-lon 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 ex-pression 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 compo-nents,including 9 flavonoids,3 terpenoids,2 organic acids,and 4 other types of compounds.Network pharmacology anal-ysis 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 pro-cessed 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 reduc-ing mucosal injury.