清解扶正颗粒调控Cathepsin B/NLRP3通路减轻大肠癌小鼠5-FU化疗性肠黏膜炎的机制研究  

Mechanism of Qingjie Fuzheng Granules on Alleviating 5-FU Chemotherapy-Induced Intes⁃tinal Mucositis in Mouse Model of Colorectal Cancer By Regulating Cathepsin B/NLRP3 Pathway

作　　者：袁颖绯 林久茂[1,2,3] 林明和 赵锦燕 陈旭征[1,2] 刘锦洪 朱晓勤 YUAN Yingfei;LIN Jiumao;LIN Minghe;ZHAO Jinyan;CHEN Xuzheng;LIU Jinhong;ZHU Xiaoqin(Academy of Integrative Medicine(College of Integrative Medicine),Fujian University of Traditional Chinese Medicine,Fuzhou,Fujian 350122,China;Fujian Key Laboratory of Integrative Medicine on Geriatrics,Fujian University of Traditional Chinese Medicine,Fuzhou,Fujian 350122,China;Key Laboratory of Integrative Medicine of Fujian Province University,Fuzhou,Fujian 350122,China)

机构地区：[1]福建中医药大学中西医结合学院、中西医结合研究院,福建福州350122 [2]福建省中西医结合老年性疾病重点实验室,福建福州350122 [3]中西医结合基础福建省高校重点实验室,福建福州350122

出　　处：《康复学报》2024年第5期473-482,489,共11页Rehabilitation Medicine

基　　金：国家自然科学基金面上项目(81774121);福建省自然科学基金项目(2021J01941,2021J01939);福建省教育厅A类科技项目(JAT200230);福建中医药大学校管项目(X2020015)。

摘　　要：目的基于Cathepsin B/NLRP3通路探讨清解扶正颗粒对大肠癌小鼠经5-氟尿嘧啶(5-FU)化疗后所致的肠黏膜炎的治疗作用及作用机制。方法24只雄性BALB/c小鼠采取右前肢腋部皮下接种CT26细胞的方法构建大肠癌皮下移植瘤小鼠模型,待瘤体生长至100~300 mm3时,按照瘤体体积大小将小鼠随机分为对照组、5-FU组、5-FU+清解扶正颗粒组(5-FU+QFG组),每组8只。对照组给予生理盐水灌胃和腹腔注射,其他2组小鼠腹腔注射5-FU[50 mg/(kg·d^(-1))],每日1次,连续5 d;5-FU+QFG组在化疗的同时灌胃给予清解扶正颗粒(1.0 g/kg·d^(-1)),每日1次,连续7 d。每天观察小鼠的腹泻情况,记录体质量及瘤体积等情况。第7天末次给药后禁食12 h,动物取材,进行实验检测。采用苏木精-伊红染色(HE)法观察小鼠空肠组织病理变化;采用酶联免疫吸附试验(ELISA)法检测3组小鼠血清中二胺氧化酶(DAO)、D-乳酸(DLA)、内毒素(ET)、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)表达水平;采用免疫组织化学法检测空肠组织中组织蛋白酶B(Cathepsin B)的阳性表达;采用实时定量聚合酶链式反应(qPCR)检测空肠组织中紧密连接蛋白ZO-1、Occludin mRNA的相对表达水平,采用Western blot法检测空肠组织中ZO-1、Occludin、Cathepsin B、NLRP3、ASC、cleaved Caspase-1蛋白的相对表达量。结果①小鼠一般情况:在实验周期内,清解扶正颗粒能够显著减轻5-FU化疗引起的腹泻症状(P<0.05),对5-FU化疗引起的体质量下降无显著改善(P>0.05),对5-FU抑制瘤体生长没有显著的协同效应(P>0.05)。②空肠组织病理变化:清解扶正颗粒可显著改善5-FU化疗引起的肠黏膜绒毛变短、隐窝变深、炎性细胞浸润等肠黏膜损伤症状。③血清DAO、D-LA、ET以及TNF-α、IL-1β、IL-6含量:与5-FU组比较,清解扶正颗粒可以显著降低大肠癌小鼠血清中DAO、D-LA、ET以及促炎细胞因子TNF-α、IL-1β�Objective To explore the therapeutic effect and underlying mechanism of Qingjie Fuzheng Granules(QFG)on intestinal mucositis caused by 5-fluorouracil(5-FU)chemotherapy in mice with colorectal carcinoma based on the Cathepsin B/NLRP3 pathway.Methods A total of twenty-four male BALB/c mice were selected for subcutaneous inoculation of CT26 cells in the axilla of the right forelimb to construct a subcutaneous graft mouse model of colorectal carcinoma.When the tumor volume grew to 100-300 mm3,the mice were randomly divided into control group,5-FU group,and 5-FU+Qingjie Fuzheng Granule group(5-FU+QFG group),with 8 mice in each group.The control group was given saline gavage and intraperitoneal injection,and mice in the other groups received intraperitoneal injection of 5-FU(50 mg/kg·d^(-1)),once daily for 5 days;the 5-FU+QFG group was given Qingjie Fuzheng Granules(1.0 g/kg·d^(-1))by gavage during chemotherapy,once daily for 7 days.Mice were observed daily for diarrhea,and their body mass and tumor volume were recorded.After the last administration on the 7th day,the mice were fasted for 12 hours,and then sampled and tested.The histopathological changes in the jejunum were observed by hematoxylin-eosin staining(HE);the expression levels of diamine oxidase(DAO),D-lactic acid(D-LA),endotoxin(ET),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6)were measured by enzyme-linked immunosorbent assay(ELISA);the positive expression of Cathepsin B in the jejunal tissue was detected by immunohistochemistry;the relative expression level of tight junction protein ZO-1 and Occludin mRNA in jejunal tissue were determined by real-time quantitative polymerase chain reaction(RT-qPCR),and the relative expression level of ZO-1,Occludin,Cathepsin B,NLRP3,ASC,and cleaved Caspase-1 protein in the jejunal tissue were determined by the Western blot method.Results(1)General condition of mice:During the experimental period,QFG significantly reduced the diarrhea symptoms caused by 5-FU chemotherapy(P<0.05),had no sign

关 键 词：大肠癌 Cathepsin B/NLRP3通路 肠黏膜炎 5-氟尿嘧啶 清解扶正颗粒 

分 类 号：R285.5[医药卫生—中药学]