连翘叶总黄酮对斑马鱼溃疡性结肠炎的作用研究  

Effect of total flavonoids from Forsythia suspensa leaf on zebrafish ulcerative colitis

作　　者：吕允珂 郑萱 张菊 崔施展 王明雪 周春伶 贾东升[2] LYU Yun-ke;ZHENG Xuan;ZHANG Ju;CUI Shi-zhan;WANG Ming-xue;ZHOU Chun-ling;JIA Dong-sheng(College of Biology Science and Engineering,Hebei University of Economics and Business,Shijiazhuang 050061,China;Institute of Cash Crop,Institute of Cash Crops,Hebei Academy of Agriculture and Forestry Sciences,Shijiazhuang 050051,China)

机构地区：[1]河北经贸大学生物科学与工程学院,石家庄050061 [2]河北省农林科学院经济作物研究所,石家庄050051

出　　处：《天然产物研究与开发》2024年第8期1279-1287,共9页Natural Product Research and Development

基　　金：国家中药材产业体系-全草类药材岗位(CARS-21);河北省重点研发计划(23372509D);河北省现代农业产业技术体系项目(HBCT2023080204);河北省农林科学院科技创新专项(2022KJCXZX-JZS-4)。

摘　　要：研究连翘叶总黄酮(Forsythia suspensa leaf total flavonoid,FSLF)对2,4,6-三硝基苯磺酸(2,4,6-trinitrobenzene sulfonicacid,TNBS)诱导斑马鱼溃疡性结肠炎的疗效作用。采用D101大孔树脂对FSLF分离纯化并使用超高效液相色谱-质谱联用仪(UPLC-MS)分析成分;将36条野生斑马鱼分为空白组、模型组、FSLF低、中、高剂量给药组(10、20、40μg/mL)、地塞米松组(50μg/mL),除空白组外,其余各组均用160 mmol/L TNBS肛门注射诱导斑马鱼溃疡性结肠炎模型,使用苏木素-伊红、阿利新蓝染色法观察斑马鱼结肠组织病理和杯状细胞数量变化,通过试剂盒检测斑马鱼结肠组织活性氧(reactive oxygen species,ROS)、超氧化物歧化酶(superoxide dismutase,SOD)含量和活性变化,经荧光定量PCR法检测斑马鱼结肠组织中髓样分化因子88(mycloid differentiation factor 88,MyD88)、肿瘤坏死因子相关分子6(TNF receptor associated factor 6,TRAF6)、核因子-κB(nuclear factor-κB,NF-κB)p65、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-10(interleukin-10,IL-10)mRNA表达量。结果显示,FSLF 30%乙醇洗脱部位总黄酮含量最高,UPLC-MS共鉴定出芦丁、异槲皮苷、山柰酚-3-葡萄糖鼠李糖苷、金丝桃苷、山柰酚-3-O-芸香糖苷、槲皮素、橙皮苷、山柰酚、紫云英苷9种黄酮类化学成分。与模型组相比FSLF可改善斑马鱼结肠组织结构,提高杯状细胞数量(P<0.001),降低ROS含量(P<0.05)和MyD88/TRAF6/NF-κBp65、TNF-α、IL-1βmRNA表达量,升高SOD活性(P<0.01)和IL-10 mRNA表达量(P<0.05)。本研究表明FSLF对斑马鱼溃疡性结肠炎有明显治疗作用,其作用机制可能与通过调节信号通路NF-κB,下调炎性因子TNF-α、IL-1β和上调IL-10 mRNA表达量有关。This study aims to investigate the impact of total flavonoids extracted from Forsythia suspensa leaf on zebrafish ulcerative colitis induced by 2,4,6-trinitrobenzene sulfonic acid(TNBS).The FSLF was purified using D101 macroporous resin and its components were analyzed via UPLC-MS.Thirty-six wild zebrafish were divided into a control group,model group,low-dose FSLF group(10μg/mL),medium-dose FSLF group(20μg/mL),high-dose FSLF group(40μg/mL),and dexamethasone group(50μg/mL).ulcerative colitis was induced in all groups except for the blank group by anal injection of 160 mmol/L TNBS.The colonic histopathology and goblet cell number of zebrafish were observed using hematoxylin-eosin and alcian blue staining.The levels of reactive oxygen species(ROS)and superoxide dismutase(SOD)in zebrafish colon tissue were assessed using a detection kit.Fluorescence quantitative PCR detected the presence of MyD88 and TNF receptor associated factor 6(TRAF6)in the colon tissue of zebrafish.Additionally,mRNA expression of nuclear factor-κB(NF-κB)p65,tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and interleukin-10(IL-10)was also observed.The highest concentration of total flavonoids was found in the 30%ethanol-eluting fraction of FSLF,UPLC-MS analysis identified nine flavonoids,including rutin,isoquercetin,kaempferol-3-glucorhamnoside,hypericin,kaempferin-3-O-rutinoside,quercetin,Hesperidin,kaempferol and porphyrin.Compared to the model group,FSLF demonstrates an improvement in the colonic structure of zebrafish,as evidenced by an increase in goblet cell number(P<0.001),a decrease in ROS content(P<0.05),downregulation of MyD88/TRAF6/NF-κBp65,TNF-α,IL-1βmRNA expression levels,and an increase in SOD activity(P<0.01)and IL-10 mRNA expression levels(P<0.05).This study indicates that FSLF exerts a significant therapeutic impact on zebrafish ulcerative colitis,potentially through the modulation of inflammatory factors TNF-αand IL-1β,as well as the enhancement of IL-10 mRNA expression via regulation of the NF-κB signalin

关 键 词：连翘叶 总黄酮 斑马鱼 溃疡性结肠炎 

分 类 号：R285.5[医药卫生—中药学]