基于动物实验和网络药理学研究白竭散保留灌肠治疗溃疡性结肠炎的作用机制

Mechanism of Baijie Powder retention enema for ulcerative colitis:based on animal experiments and network pharmacology mining

目的基于动物实验和网络药理学研究白竭散保留灌肠治疗溃疡性结肠炎(UC)的作用机制。方法将42只SD大鼠随机抽出10只为空白组,剩余32只大鼠通过5%2,4,6-三硝基苯磺酸溶液(TNBS)灌肠法构建UC大鼠模型。在32只大鼠中随机抽出2只,麻醉处死,剪取距肛门约8 cm肠道,显微镜下观察结肠组织,见黏膜层炎症改变,溃疡形成,造模成功。余鼠30只按照随机数字表法分为3组,模型组、美沙拉秦组、白竭散组,每组10只。造模成功后次日,白竭散组予白竭散混悬液2.3 g/(kg•d)灌肠,美沙拉秦组予美沙拉秦灌肠液0.7 g/(kg•d)灌肠,模型组、空白组予0.9%氯化钠注射液2 mL灌肠。观察各组大鼠治疗后症状表现及结肠组织损伤情况;比较各组大鼠疾病活动指数(DIA)、结肠黏膜损伤指数(CMDI)评分变化;观察各组大鼠结肠组织苏木精-伊红(HE)染色结果。通过相关数据库提取白竭散有效成分和靶点、UC疾病靶点基因,构建“药物-有效成分-疾病靶点”网络图、蛋白质-蛋白质相互作用(PPI)网络,进行基因本体生物学过程(GOBP)及京都基因与基因组百科全书(KEGG)通路富集分析,挖掘其作用机制。结果治疗后空白组大鼠体质量无明显变化,活动量正常,精神反应灵敏,大便质软成形,无黏液脓血便;模型组大鼠活动量减少,体质量减轻,大便不成形,见脓血便;美沙拉秦组及白竭散组大鼠精神良好,反应尚灵敏,体质量基本正常,大便轻度松散,无黏液脓血便。治疗后空白组大鼠结肠壁结构完整,无损伤;模型组大鼠结肠壁可见多处溃疡,大小不等,最大约1.1 cm,炎症较重;美沙拉秦组及白竭散组大鼠结肠壁溃疡面较少,面积较小,炎性反应轻。模型组大鼠DAI及CMDI评分均高于空白组(P<0.05);美沙拉秦组、白竭散组大鼠DAI及CMDI评分均低于模型组(P<0.05);美沙拉秦组与白竭散组大鼠DAI及CMDI评分比较差异无统计学意义(P>0.05)。空白组大鼠肠壁结构完整,未见明显炎症细胞;模型组大鼠肠壁结构不完整,固有腺体及腺上皮杯状细胞明显减少,固有间质及肠壁全层可见弥漫大量炎症细胞浸润,血管扩张充血,黏膜下层水肿明显,结构紊乱。美沙拉秦组及白竭散组大鼠肠壁结构基本完整,可见部分修复,黏膜固有层水肿不明显,可见少量炎症细胞浸润,固有腺体及其杯状细胞数量较模型组增多,排列稍疏松,黏膜下层及固有肌层未见明显炎症细胞浸润。通过各数据库筛选出白竭散有效成分106个、靶点500个,UC疾病靶点801个,“药物-疾病”交集靶点105个,获得蛋白激酶B1(Akt1)、血管内皮生长因子A(VEGFA)、白细胞介素2(IL-2)等27个核心靶点,炎性反应、活性氧代谢等20类生物过程,癌症信号通路、磷脂酰肌醇3-激酶-蛋白激酶B(PI3K-Akt)等25条相关通路。结论白竭散灌肠可有效缓解UC大鼠肠黏膜损伤,其机制可能是通过白及多糖、黄酮类及酚类等成分调控相关基因和通路,抑制炎性反应和氧化应激反应,提高活性氧代谢,从而缓解UC肠黏膜损伤。

Objective Mechanism of Baijie Powder retention enema for ulcerative colitis(UC)based on animal experiments and network pharmacology mining.Methods Ten of 42 SD rats were randomly selected as blank group,and the remaining 32 rats were given enema intervention of 2,4,6-trinitrobenzenesulfonic acid(TNBS,concentration 5%)to establish UC rat model.Two of the 32 rats were randomly sacrificed by anesthesia,and then the intestinal tract about 8 cm away from the anus was obtained observing the colon tissue under the microscope;when mucosal inflammation changes and ulcer formation were observed,here,the model was successfully established.The next day,a random number table method make 30 rats into 3 groups:model group,mesalazine group[0.7 g/(kg•d),once daily(QD)]and Baijie Powder group[2.3 g/(kg•d),QD],with 10 rats in each group,model group and blank group were given 2 mL of 0.9%sodium chloride injection to enema.Posttreatment symptoms and colic injury of rats in each group were observed.The key observation was disease activity index(DIA)and colonic mucosal injury index(CMDI)and HE staining results of colon tissue in groups.The active ingredients and targets of Baijie Powder and UC disease targets were extracted in relevant databases,a"drug-active ingredient-disease target"network diagram and a"protein-protein interaction(PPI)"network were built,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis was performed,exploring their mechanisms of action.Results After treatment,the rats in the blank group had no significant changes in body weight,normal activity levels,sensitive responses,soft-formed stools,and no mucus,pus and blood in the stools;the rats in the model group had reduced activity,decreased body weight,unformed stools,and pus and blood stools in the stools;posttreatment symptoms similar to blank group were found in Rats of the mesalazine group and Baijie Powder group.After treatment,the structure of intestine wall of the rats in the blank group was complete,and with no damage;the colon wall of the rats in the model group had multiple ulcers,ranging in size,the maximum was about 1.1 cm,and the inflammation was severe;the rats in mesalazine group and Baijie Powder group had less ulcer surface,smaller area and lighter inflammatory reaction.The DAI and CMDI scores of the rats in the model group were higher than those in the blank group(P<0.05);the DAI and CMDI scores of the rats in the mesalazine group and Baijie Powder group were lower than those in the model group(P<0.05).There was no significant difference in DAI and CMDI scores between mesalazine group and Baijie Powder group(P>0.05).The structure of intestinal wall of the rats in the blank group was complete,and with no obvious inflammatory cells;the structure of intestinal wall of the rats in the model group was incomplete,the goblet cells of the intrinsic glands and glandular epithelium were significantly reduced,and a large number of diffuse inflammatory cells infiltration were seen in the interstitium proper and the full thickness of the intestinal wall,with vascular dilatation and congestion,obvious submucosal edema,and architecture distortion.In the mesalazine group and Baijie Powder group,the structure of intestinal wall was basically intact,with partial repair.The lamina propria edema was not obvious,and a small amount of inflammatory cell infiltration was seen.Compared with the model group,the number of intrinsic glands and goblet cells increased.Slightly loose,no obvious inflammatory cell infiltration was found in the submucosa and muscularis propria.A total of 106 effective components of Baijie Powder,500 targets,801 UC disease targets,105"drugs-diseases"intersection targets were screened out in various databases;totally 27 core targets such as protein kinase B1(Akt1),vascular endothelial growth factor A(VEGFA),and interleukin 2(IL-2)were obtained,and including 20 biological processes such as inflammatory response and reactive oxygen species metabolism,and 25 related pathways such as cancer signaling pathway and phosphatidylinositol 3 kinase(PI3K)-protein kinase B(PI3K-Akt).Conclusion For UC rats,Baijie Powder retention enema can effectively alleviate the intestinal mucosal injury,and its mechanism may be related to the regulation related genes and pathways by polysaccharide,flavonoids and phenols of Bletilla striata,inhibiting inflammatory response,improving active oxygen metabolism and inhibiting oxidative stress response to alleviate the intestinal mucosal injury of UC.