基于斑马鱼模型的复方苦参注射液安全性及治疗炎症性肠病的有效性评价  

Evaluation of Safety of Compound Kushen Injection and Efficacy in Treatment of Inflammatory Bowel Disease Based on Zebrafish Model

作　　者：陈小橹 李嘉琪 陈林珍 陈祺 马志强 赵崇军 CHEN Xiaolu;LI Jiaqi;CHEN Linzhen;CHEN Qi;MA Zhiqiang;ZHAO Chongjun(Beijing Key Laboratory for Quality Evaluation of Chinese Materia Medical/Traditional Chinese Medicine(TCM)Processing Technology Inheritance Base of National Administration of TCM,School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 102488,China)

机构地区：[1]北京中医药大学中药学院中药品质评价北京市重点实验室/国家中医药管理局中药炮制技术传承基地,北京102488

出　　处：《中国实验方剂学杂志》2025年第4期71-78,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家科技重大专项(2018ZX01031-301-005)。

摘　　要：目的:采用斑马鱼模型对复方苦参注射液(CKI)安全性/有效性进行整体评价,并探讨可能的作用机制。方法:将斑马鱼暴露于不同浓度CKI溶液中,统计24 h后的死亡率;在亚致死浓度(<LC_(10))和安全剂量浓度(<LC_(0))下暴露24 h,基于斑马鱼肝脏吖啶橙染色、肝脏面积变化、肝脏组织病理切片等指标评价CKI安全性。通过2,4,6-三硝基苯磺酸(TNBS)建立斑马鱼肠炎模型,以斑马鱼肠道中性红染色面积变化、中性粒细胞数量变化、阿尔新蓝染色面积变化,结合体内紧密连接蛋白(Occludin)、闭锁小带蛋白-1(ZO-1)、肿瘤坏死因子(TNF)-α与前列腺素E_(2)(PGE_(2))含量等指标,综合评价CKI对炎症性肠病(IBD)的治疗有效性;使用网络药理学预测CKI治疗IBD的作用机制,并通过实时荧光定量聚合酶链式反应(Real-time PCR)技术验证。结果:在安全性评价中,与空白组比较,亚致死浓度CKI能够引起斑马鱼肝脏细胞凋亡、肝脏面积减小、肝脏组织结构排列松散紊乱及明显空泡化等病理特征,而安全剂量暴露处理不会对上述指标造成明显变化。在有效性评价中,与模型组比较,安全剂量的CKI能呈剂量依赖性增加肠道中性红染色面积,改善肠道吞噬功能;减少肠道中性粒细胞聚集情况;增加阿尔新蓝染色面积,增强肠道杯状细胞分泌情况;显著增加Occludin及ZO-1含量;减少TNF-α及PGE_(2)含量;网络药理学分析得到CKI治疗IBD潜在靶点288个,蛋白质-蛋白质相互作用(PPI)网络分析度值排名前5的靶点分别为表皮生长因子受体A(EGFRA)、蛋白激酶B1(Akt1)、热休克蛋白90(HSP90AA1)、禽肉瘤病毒致癌基因同源物(SRC)、原癌基因(JUN);京都基因与基因组百科全书(KEGG)结果显示,CKI治疗IBD可能与Wnt信号通路、胆碱能突触通路有关,Real-time PCR结果验证了以上通路。结论:超剂量使用CKI能诱导斑马鱼出现明显肝损伤;而合理使用CKI不会造成明显毒性反应,�Objective:To evaluate the safety/efficacy of compound Kushen injection(CKI)by zebrafish model and explore the possible mechanism.Methods:Zebrafish were exposed to different concentrations of CKI solution,and the mortality rate after 24 h was calculated.After exposure to sublethal concentration(<LC_(10))and safe dose concentration(<LC_(0))for 24 h,the safety of CKI was evaluated based on acridine orange staining for the liver,liver area changes,and liver histological sections.The inflammatory bowel disease(IBD)model of zebrafish was established with 2,4,6-trinitrobenzenesulfonic acid sol(TNBS).The efficacy of CKI in the treatment of IBD was evaluated by the changes in the area of neutral red staining,the number of neutrophils,the area of alcian blue staining,and the contents of tight junction protein(Occludin),zonula occludens-1(ZO-1),tumor necrosis facto(r TNF)-α,and prostaglandin E_(2)(PGE_(2))in the intestine of zebrafish.The mechanism of CKI in the treatment of IBD was predicted by network pharmacology and verified by real-time polymerase chain reaction(Real-time PCR).Results:In the safety evaluation,compared with the blank group,the sublethal concentration of CKI could cause liver cell apoptosis,liver area reduction,loose and disordered arrangement of liver cell structure,obvious vacuolation,and other pathological characteristics of zebrafish,while these indicators showed no significant changes under safe dose conditions.In the effectiveness evaluation,compared with the model group,the safe dose of CKI could increase the neutral red staining area of the intestine in a dosedependent manner and improve the intestinal phagocytosis function.It could reduce the accumulation of intestinal neutrophils,increase the alcian blue staining area,enhance intestinal goblet cell secretion,improve the contents of Occludin and ZO-1,and decrease the contents of TNF-αand PGE_(2).Network pharmacology analysis identified 288 potential targets for CKI treatment of IBD.The top five targets obtained by protein-protein interaction(P

关 键 词：斑马鱼 复方苦参注射液 炎症性肠病 安全性 有效性 作用机制 

分 类 号：R285[医药卫生—中药学] R516.1[医药卫生—中医学] R287