西维来司钠抑制中性粒细胞弹性蛋白酶调控肝内胆管MUC5AC表达  

Sivelestat sodium inhibits neutrophil elastase to regulate intrahepatic biliary mucin 5AC expression

作　　者：辜习卯 叶海军 徐承雷 林杼颖 李江[1] Gu Ximao;Ye Haijun;Xu Chenglei;Lin Zhuying;Li Jiang(Department of Hepatobiliary Surgery,First Affiliated Hospital of Kunming Medical University,Kunming 650032,Yunnan,China)

机构地区：[1]昆明医科大学第一附属医院肝胆外科,云南昆明650032

出　　处：《中华危重病急救医学》2024年第6期609-615,共7页Chinese Critical Care Medicine

基　　金：云南省科技厅科技计划项目(202101AY070001-129)。

摘　　要：目的探讨西维来司钠是否能够通过抑制中性粒细胞弹性蛋白酶(NE)进而减少黏蛋白5AC(MUC5AC)在肝内胆管上皮细胞中的表达,为治疗肝内胆管结石(IBDS)提供新的潜在治疗思路。方法①生信分析:基于基因表达数据库(GEO)对胆结石及胆囊炎的测序数据进行差异基因分析,筛选中性粒细胞及黏蛋白相关的显著差异基因,使用基因与蛋白质相互作用检索数据库(STRING)进行蛋白互作分析,以预测NE基因与MUC5AC是否存在互作关系。②动物实验:将18只雄性SD大鼠按随机数字表法分为假手术组、胆管炎模型组和西维来司钠治疗组,每组6只。结合预实验于大鼠右前叶肝脏一次性注射1.25 mg/kg脂多糖(LPS)建立胆管炎大鼠模型;假手术组肝脏注射等体积生理盐水。建模后,西维来司钠治疗组尾静脉注射西维来司钠100 mg/kg;假手术组和胆管炎模型组尾静脉注射等体积生理盐水;每日1次,连续给药5 d。2周后处死大鼠取肝胆管组织,光镜下观察肝胆管组织病理学改变;免疫组织化学染色检测肝胆管组织NE和MUC5AC表达;蛋白质免疫印迹试验(Western blotting)检测肝胆管组织NE、MUC5AC、Toll样受体4(TLR4)的蛋白表达。③细胞实验:将原代人肝内胆管上皮细胞株(HiBEpiC)传代后分为空白对照组、NE组(10 nmol/L NE)、NE+西维来司钠低剂量组(10 nmol/L NE+1×10-8 g/L西维来司钠1 mL)、NE+西维来司钠中剂量组(10 nmol/L NE+1×10-7 g/L西维来司钠1 mL)、NE+西维来司钠高剂量组(10 nmol/L NE+1×10-6 g/L西维来司钠1 mL)。培养48 h后收集细胞,行5-乙炔基-2''-脱氧尿嘧啶核苷(EdU)检测细胞增殖活性;酶联免疫吸附试验(ELISA)和Western blotting检测细胞MUC5AC的表达。结果①生信分析:NE基因(ELANE)与MUC5AC存在互作关系。②动物实验:光镜下显示,胆管炎模型组肝细胞水肿,肝细胞呈弥漫性点、灶状坏死,汇管区纤维组织及肝内胆管增生与炎症细胞浸润;西维来司Objective To explore whether sivelestat sodium could reduce the expression of mucin 5AC(MUC5AC)in intrahepatic bile duct epithelial cells by inhibiting neutrophil elastase(NE)and thus provide new potential therapeutic ideas for the treatment of intrahepatic bile duct stone(IBDS).Methods①Bioinformatics analysis:differential gene analysis was performed on gallbladder stone cholecystitis sequencing data based on the gene expression omnibus(GEO)to screen for significantly different genes related to neutrophils and mucins.The search tool for the retrieval of interacting genes database(STRING)was used for protein interaction analysis to predict whether there was an interaction between NE and MUC5AC genes.②Animal experiment:a total of 18 male SD rats were divided into the sham-operated group,cholangitis model group and sivelestat sodium treatment group according to the random number table method,with 6 rats in each group.The cholangitis rat model was established by a one-time injection of 1.25 mg/kg lipopolysaccharide(LPS)into the right anterior lobe of the liver of rats in combination with the pre-experiment;the liver of the sham-operated group was injected with an equal volume of saline.After the modelling,100 mg/kg of sivelestat sodium was injected into the tail vein of the cevalexin treatment group once a day for 5 days,and an equal volume of saline was injected into the tail vein of the sham-operated group and the cholangitis model group.Two weeks later,the rats were euthanized and their liver and bile duct tissues were taken.The pathological changes in the liver and bile duct tissues were observed under the light microscope.Immunohistochemical staining was used to detect the expressions of NE and MUC5AC in liver and bile duct tissues.The protein expressions of NE,MUC5AC and Toll-like receptor 4(TLR4)were detected by Western blotting.③Cell experiment:primary human intrahepatic biliary epithelial cell line(HiBEpiC)was divided into blank control group,NE group(10 nmol/L NE),NE+sivelestat sodium low dose group(1

关 键 词：西维来司钠 中性粒细胞弹性蛋白酶 黏蛋白5AC 肝内胆管结石 

分 类 号：R575[医药卫生—消化系统]