急性敌草快中毒大鼠肠道损伤机制的研究  

Mechanism of intestinal injury induced by acute diquat poisoning in rats

作　　者：张建爽 孙艺青 高恒波[1] 苑霖 姚冬奇[1] 刘亮[1] 吕宝谱 田英平[1] Zhang Jianshuang;Sun Yiqing;Gao Hengbo;Yuan Lin;Yao Dongqi;Liu Liang;Lyu Baopu;Tian Yingping(Department of Emergency,the Second Hospital of Hebei Medical University,Shijiazhuang 050000,Hebei,China)

机构地区：[1]河北医科大学第二医院急诊科,石家庄050000

出　　处：《中华危重病急救医学》2024年第3期293-297,共5页Chinese Critical Care Medicine

基　　金：河北省自然科学基金(H2019206317);河北省医学科学研究课题计划项目(20230499)。

摘　　要：目的探讨敌草快(DQ)对大鼠肠道焦亡相关蛋白及紧密连接蛋白表达的影响,分析细胞焦亡在急性DQ中毒大鼠肠道损伤中的作用。方法将36只Wistar雄性大鼠随机分为对照组及染毒3 h、12 h、36 h、3 d组,每组6只。各染毒组均给予1/2半数致死量(LD50)115.5 mg/kg DQ一次性灌胃;对照组给予等量生理盐水灌胃。对照组于DQ灌胃后3 h麻醉取空肠组织;各染毒组分别于DQ灌胃后3 h、12 h、36 h、3 d麻醉取空肠组织。记录大鼠的一般情况;采用苏木素-伊红(HE)染色观察各组大鼠空肠组织病理学改变;采用免疫组织化学染色观察空肠组织焦亡相关蛋白〔NOD样受体蛋白3(NLRP3)、天冬氨酸特异性半胱氨酸蛋白酶1(caspase-1)、Gasdemin D(GSDMD)〕的表达;采用蛋白质免疫印迹试验(Western blotting)检测空肠组织焦亡相关蛋白和紧密连接蛋白〔闭合蛋白(Occludin)、封闭蛋白-1(Claudin-1)〕的表达。结果光镜下显示,大鼠空肠组织在染毒早期(3 h)即出现病理学改变,染毒12 h组损伤最重,可见大量炎症细胞浸润,染毒3 d组损伤明显减轻。免疫组织化学染色结果显示,NLRP3、caspase-1和GSDMD在对照组和各染毒组大鼠空肠黏膜组织中均有表达,其中对照组阳性细胞表达较少,着色浅;各染毒组大鼠上述蛋白表达均明显增加,着色深。Western blotting结果显示,各染毒组大鼠空肠组织NLRP3蛋白表达均较对照组升高,以染毒36 h组升高最显著(NLRP3/β-actin:1.47±0.06比0.43±0.14,P<0.01);染毒3 h、12 h、36 h组GSDMD蛋白表达均较对照组升高,其中染毒3 h和12 h组GSDMD蛋白表达明显升高(GSDMD/β-actin:1.04±0.40、1.25±0.15比0.65±0.25,均P<0.05);染毒36 h组caspase-1蛋白表达较对照组有所升高(caspase-1/β-actin:1.44±0.34比0.98±0.19,P>0.05)。与对照组比较,各染毒组Occludin、Claudin-1蛋白表达均降低,其中染毒3 h、12 h、36 h组Occludin蛋白表达明显降低(Occludin/β-actin:0.74±0.17、0.91±0.20、Objective To investigate the effects of diquat(DQ)on the expression of intestinal pyroptosis-related proteins and tight junction proteins in rats,and to analyze the role of pyroptosis in the intestinal injury of rats with acute DQ poisoning.Methods A total of 36 Wistar male rats were randomly divided into control group,and 3 hours,12 hours,36 hours and 3 days exposure groups,with 6 rats in each group.Each exposure group was given 1/2 median lethal dose(LD50)of 115.5 mg/kg DQ by one-time gavage.The control group was given the same amount of normal saline by gavage.The control group was anesthetized at 3 hours after DQ gavage to take jejunal tissues;each exposure group was anesthetized at 3 hours,12 hours,36 hours,and 3 days after DQ gavage to take jejunal tissues,respectively.The general conditions of the rats were recorded.The pathological changes of jejunum tissue were observed by hematoxylin-eosin(HE)staining.The expression of intestinal pyroptosis-related proteins[NOD-like receptor protein 3(NLRP3),cysteine aspartate-specific protease 1(caspase-1),Gasdemin D(GSDMD)]in the intestinal tissues was observed by immunohistochemical staining.Western blotting was used to detect the expression of intestinal pyroptosis-related proteins and intestinal tight junction proteins(Occludin and Claudin-1).Results Light microscopy showed that pathological changes occurred in jejunum tissue at the early stage of exposure(3 hours),and the injury was the most serious in the 12 hours exposure group,with a large number of inflammatory cells infiltrating in the tissue,and the damage was significantly reduced after 3 days exposure.Immunohistochemical results showed that NLRP3,caspase-1 and GSDMD were expressed in the jejunal mucosa of the control group and the exposure groups,and the positive cells in the control group were less expressed with light staining.The expression of the above proteins in the exposed group was increased significantly and the staining was deep.Western blotting results showed that compared with the control group

关 键 词：敌草快 肠道 焦亡相关蛋白 紧密连接蛋白 

分 类 号：R595.4[医药卫生—内科学]