基于NLRP3相关炎症反应和肠道黏膜屏障功能探究汉黄芩素治疗溃疡性结肠炎小鼠的作用机制  

Mechanism of wogonin in the treatment of ulcerative colitis mice based on NLRP3-related inflammatory response and intestinal mucosal barrier function

作　　者：陆璐 刘宇婧 李瑗 徐汉辰[1] 季光[1] LU Lu;LIU Yu-jing;LI Yuan;XU Han-chen;JI Guang(Longhua Hospital Shanghai University of Traditional Chinese Medicine,Institute of Digestive Diseases,Shanghai University of TCM,Shanghai 200032,China)

机构地区：[1]上海中医药大学附属龙华医院,上海中医药大学脾胃病研究所,上海200032

出　　处：《中华中医药杂志》2022年第10期5992-5999,共8页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：国家自然科学基金青年科学基金项目(No.81874206);上海市炎癌转化病证生物学前沿研究基地(No.2021KJ03-12);上海市科委青年科技启明星计划(No.20QA1409300);上海市高校青年东方学者(No.QD2019034)。

摘　　要：目的:基于肠道黏膜屏障功能和含NLR家族Pyrin域蛋白3(NLRP3)相关炎症反应,探讨汉黄芩素对小鼠溃疡性结肠炎的保护作用及分子机制。方法:将32只C57BL/6小鼠随机分为正常对照组、模型组、汉黄芩素组(100 mg/kg)和美沙拉嗪组(50 mg/kg),每组8只。实验第1~7天,正常对照组小鼠自由饮水饮食,模型组、汉黄芩素组和美沙拉嗪组小鼠饮用3%DSS水溶液,同时汉黄芩素组和美沙拉嗪组小鼠分别腹腔注射和灌胃相应的药物,正常对照组和模型组小鼠腹腔注射等量的0.9%氯化钠溶液;每日记录小鼠体质量、饮食饮水、大便性状和活动状态等一般情况。第7天实验结束后12 h,动物取材进行实验检测。同时,分别采用THP-1来源的巨噬细胞和Caco-2细胞进行肠道黏膜屏障模型体外实验,评价汉黄芩素的作用机制。绘制不同组别小鼠体质量变化曲线,记录结肠长度,统计疾病活跃指数(DAI)评分;进行小鼠结肠组织HE染色,F4/80和Occludin免疫组化染色;免疫荧光检测小鼠结肠组织ZO-1和NLRP3表达,Caco-2体外模型ZO-1和Occludin表达;qPCR检测小鼠结肠组织肿瘤坏死因子α(TNF-α)、白细胞介素-1β(IL-1β)、NLRP3、凋亡相关斑点(ASC)、紧密连接组分闭合蛋白(Occludin)mRNA水平,体外巨噬细胞TNF-α、IL-1β、NLRP3、ASC mRNA水平,体外Caco-2模型TNF-α、IL-1β、Occludin mRNA水平;ELISA检测巨噬细胞体外实验上清液中TNF-α和IL-1β水平。结果:汉黄芩素可显著缓解UC小鼠体质量下降(P<0.01),增加结肠长度(P<0.05),降低DAI评分(P<0.05);模型组小鼠结肠组织中上皮黏膜结构部分缺失,腺体的排列紊乱,可见炎症细胞浸润和巨噬细胞浸润,汉黄芩素可明显改善上述情况;汉黄芩素可以抑制小鼠结肠组织中促炎细胞因子TNF-αmRNA的表达(P<0.01),减少NLRP3和接头蛋白ASC及其下游促炎因子IL-1βmRNA水平(P<0.01);抑制小鼠结肠组织中NLRP3表达,增加紧密连接蛋白ZO-1和Occludin的Objective:To explore the protective effect and molecular mechanism of wogonin on ulcerative colitis in mice based on intestinal mucosal barrier function and NLRP3-related inflammation response.Methods:Thirty-two C57BL/6 mice were randomly divided into normal control group,model group,wogonin group(100 mg/kg)and mesalazine group(50 mg/kg),8 mice in each group.On day 1-7 of the experiment,the mice in the normal control group drank water and diet freely.The mice in the model group,the wogonin group and the mesalazine group drank 3%DSS aqueous solution.At the same time,the wogonin group injected the corresponding drugs while the mesalazine group gavaged.The mice in the normal control group and the model group were injected with the same amount of normal saline in the abdominal cavity;the mice’s body weight,diet and drinking water,stool characteristics and activity status were recorded daily.On the 7th day,12 hours after the end of the experiment,the animals were collected for experimental detection.At the same time,macrophages-derived from THP1 and intestinal mucosal barrier model by Caco-2 were used to evaluate the mechanism of wogonin.The weight change curve of mice in different groups was drawn,the colon length was recorded,and the DAI score was calculated.HE staining,F4/80 and Occludin immunohistochemical staining were performed on mouse colon tissue.The expression of ZO-1 and NLRP3 in mouse colon tissue and the expression of ZO-1 and Occludin in Caco-2 in vitro model were detected by immunofluorescence.qPCR was used to detect the mRNA levels of TNF-α,IL-1β,NLRP3,ASC and Occludin in mouse colon tissue,the mRNA levels of TNF-α,IL-1β,NLRP3 and ASC in macrophages in vitro,and the mRNA levels of TNF-α,IL-1βand Occludin in the Caco-2 model in vitro.The levels of TNF-αand IL-1βin the supernatant of macrophage in vitro experiments were detected by ELISA.Results:Wogonin can significantly alleviate the weight loss of ulcerative colitis mice(P<0.01),increase the length of the colon(P<0.05),and reduce the DAI scor

关 键 词：汉黄芩素 溃疡性结肠炎 肠道黏膜屏障 含NLR家族Pyrin域蛋白3 炎症 

分 类 号：R285.5[医药卫生—中药学]