摘要
基于网络药理学预测,结合动物实验验证以阐明牛黄治疗溃疡性结肠炎(ulcerative colitis,UC)的潜在作用及机制。运用BATMAN-TCM等数据库挖掘牛黄抗UC的潜在靶点,并进行通路富集分析。70只C57BL/6J小鼠随机分为空白组,模型组,溶剂模型(2%聚山梨酯80)组,柳氮磺胺吡啶(SASP,0.40 g·kg^(-1))组,体外培育牛黄(Bovis Calculus Sativus,BCS)0.20、0.10、0.05 g·kg^(-1)组共7组。3%葡聚糖硫酸钠(dextran sulfate sodium,DSS)水溶液自由饮用7 d建立UC小鼠模型,各给药组采用灌胃预给药3 d,边造模边给药7 d(连续给药10 d)。观察小鼠体质量,记录疾病活动指数(disease activity index,DAI)评分分值。造模7 d后,测量结肠长度,苏木素-伊红(HE)染色观察结肠组织病理变化,酶联免疫吸附法(ELISA)检测小鼠结肠组织中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)和白细胞介素-17(IL-17)水平,实时荧光定量聚合酶链式反应(RT-PCR)检测IL-17、IL-17RA、Act1、TRAF2、TRAF5、TNF-α、IL-6、IL-1β、CXCL1、CXCL2及CXCL10 mRNA的表达水平,Western blot法检测IL-17、IL-17RA、Act1、p-p38 MAPK、p-ERK1/2蛋白的表达。网络药理学预测结果显示,牛黄可能通过IL-17信号通路、TNF信号通路等发挥治疗作用。动物实验结果显示,给药第10天,与溶剂模型组比较,BCS 3个剂量组均能显著增加体质量,降低DAI评分,增加结肠长度,改善结肠黏膜病理损伤,并能显著抑制结肠组织TNF-α、IL-6、IL-1β和IL-17表达量。BCS 0.20 g·kg^(-1)组可显著降低UC模型小鼠结肠组织中IL-17、Act1、TRAF2、TRAF5、TNF-α、IL-6、IL-1β、CXCL1及CXCL2 mRNA的表达水平,有下调IL-17RA及CXCL10 mRNA表达水平的趋势;还能显著抑制IL-17RA、Act1、p-ERK1/2蛋白表达,有降低IL-17、p-p38 MAPK蛋白表达的趋势。首次从整体-器官-组织-分子水平,揭示了体外培育牛黄可能通过抑制IL-17/IL-17RA/Act1信号通路,降低促炎细胞因子和趋化因子的表达,从而改善DSS诱导的UC小鼠结肠组织炎症损伤,发挥“清热解毒”功效。
This study aimed to elucidate the effect and underlying mechanism of Bovis Calculus in the treatment of ulcerative colitis(UC)through network pharmacological prediction and animal experimental verification.Databases such as BATMAN-TCM were used to mine the potential targets of Bovis Calculus against UC,and the pathway enrichment analysis was conducted.Seventy healthy C57BL/6J mice were randomly divided into a blank group,a model group,a solvent model(2%polysorbate 80)group,a salazosulfapyridine(SASP,0.40 g·kg^(-1))group,and high-,medium-,and low-dose Bovis Calculus Sativus(BCS,0.20,0.10,and 0.05 g·kg^(-1))groups according to the body weight.The UC model was established in mice by drinking 3%dextran sulfate sodium(DSS)solution for 7 days.The mice in the groups with drug intervention received corresponding drugs for 3 days before modeling by gavage,and continued to take drugs for 7 days while modeling(continuous administration for 10 days).During the experiment,the body weight of mice was observed,and the disease activity index(DAI)score was recorded.After 7 days of modeling,the colon length was measured,and the pathological changes in colon tissues were observed by hematoxylin-eosin(HE)staining.The levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6),and interleukin-17(IL-17)in colon tissues of mice were detected by enzyme-linked immunosorbent assay(ELISA).The mRNA expression of IL-17,IL-17RA,Act1,TRAF2,TRAF5,TNF-α,IL-6,IL-1β,CXCL1,CXCL2,and CXCL10 was evaluated by real-time polymerase chain reaction(RT-PCR).The protein expression of IL-17,IL-17RA,Act1,p-p38 MAPK,and p-ERK1/2 was investigated by Western blot.The results of network pharmacological prediction showed that Bovis Calculus might play a therapeutic role through the IL-17 signaling pathway and the TNF signaling pathway.As revealed by the results of animal experiments,on the 10th day of drug administration,compared with the solvent model group,all the BCS groups showed significantly increased body weight,decreased DAI score,increased colon length,improved pathological damage of colon mucosa,and significantly inhibited expression of TNF-α,IL-6,IL-1β,and IL-17 in colon tissues.The high-dose BCS(0.20 g·kg^(-1))could significantly reduce the mRNA expression levels of IL-17,Act1,TRAF2,TRAF5,TNF-α,IL-6,IL-1β,CXCL1,and CXCL2 in colon tissues of UC model mice,tend to down-regulate mRNA expression levels of IL-17RA and CXCL10,significantly inhibit the protein expression of IL-17RA,Act1,and p-ERK1/2,and tend to decrease the protein expression of IL-17 and p-p38 MAPK.This study,for the first time from the whole-organ-tissue-molecular level,reveals that BCS may reduce the expression of pro-inflammatory cytokines and chemokines by inhibiting the IL-17/IL-17RA/Act1 signaling pathway,thereby improving the inflammatory injury of colon tissues in DSS-induced UC mice and exerting the effect of clearing heat and removing toxins.
作者
袁健梅
芦丹妮
王佳俊
徐卓
李勇
任敉宏
李金秀
龚道银
王建
YUAN Jian-mei;LU Dan-ni;WANG Jia-jun;XU Zhuo;LI Yong;REN Mi-hong;LI Jin-xiu;GONG Dao-yin;WANG Jian(School of Pharmacy,Chengdu University of Traditional Chinese Medicine,Chengdu 611137,China;Key Laboratory of Systematic Research of Distinctive Chinese Medicine Resources in Southwest China,Chengdu University of Traditional Chinese Medicine,Chengdu 611137,China;Affiliated Hospital of Chengdu University of Traditional Chinese Medicine,Chengdu 610075,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2023年第9期2500-2511,共12页
China Journal of Chinese Materia Medica
基金
国家自然科学基金面上项目(81873023)
成都中医药大学西南特色中药资源重点实验室开放研究基金项目(2020XSGG025)。
作者简介
袁健梅,硕士研究生,主要从事中药药性理论与应用研究,E-mail:1937171404@qq.com;通信作者:王建,博士生导师,教授,主要从事中药药性理论与应用研究,E-mail:jianwang08@163.com。
