清热化痰解毒方对脑缺血再灌注大鼠肺组织TXNIP/NLRP3炎性通路的影响  

Effect of Qingre Huatan Jiedu Formula on TXNIP/NLRP3 Inflammatory Pathway in Lung Tissue of Rats with Cerebral Ischemia-reperfusion

作　　者：王涛[1] 刘宏祥[1] 王颖[1] 齐姣[1] 陈小静[1] 闫丽静 史小盼[1] 罗金花[1] WANG Tao;LIU Hong-xiang;WANG Ying;QI Jiao;CHEN Xiao-jing;YAN Li-jing;SHI Xiao-pan;LUO Jin-hua(Affiliated Hospital of Hebei University, Baoding 071000, China)

机构地区：[1]河北大学附属医院

出　　处：《中国实验方剂学杂志》2018年第12期114-122,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：河北省科技计划项目(132777203)

摘　　要：目的：探究清热化痰解毒方对脑缺血再灌注大鼠肺组织的影响,并初步探讨相关分子机制。方法：将SD大鼠分为假手术组、模型组、阳性药组（灌胃尼莫地平200 mg·kg^-1）、清热化痰解毒方低、高剂量组（100,200 mg·kg^-1）,每组10只,除假手术组外,采用改良4动脉阻断法制备大鼠全脑缺血再灌注损伤模型,造模后,给药3 d。肺湿/干重（W/D）分析,采用苏木素-伊红（HE）染色进行组织病理学分析;酶联免疫吸附法检测支气管肺泡灌洗液（BALF）中白细胞介素（interleukin,IL）-1β和IL-18水平;肺髓过氧化物酶试剂盒检测髓过氧化物酶（myeloperoxidase,MPO）活性;免疫组化分析核苷酸结合域样受体蛋白3（nucleotide-binding domain-like receptor 3,NLRP3）阳性细胞数;半胱氨酸天冬氨酸酶-1（Caspase-1）比色测定试剂盒检测Caspase-1活性;实时荧光定量聚合酶链式反应（Real-time PCR）和蛋白免疫印迹法（Western blot）检测NLRP3,ASC,Caspase-1mRNA和蛋白表达,免疫共沉淀检测硫氧还蛋白相互作用蛋白（TXNIP）-NLRP3蛋白表达。结果：与假手术组比较,模型组肺损伤评分,肺W/D,BALF中IL-1β和IL-18水平、总细胞和多形核白细胞数量,MPO活性均明显增加（P〈0.05）;与模型组比较,尼莫地平组、清热化痰解毒方低、高剂量组肺损伤评分,肺W/D,BALF中IL-1β和IL-18水平、总细胞和多形核白细胞数量及MPO活性均明显降低（P〈0.05）。与假手术组比较,模型组肺组织中NLRP3阳性细胞数,Caspase-1活性,NLRP3,ASC,Caspase-1蛋白和mRNA相对表达明显增加（P〈0.05）;与模型组比较,尼莫地平组、清热化痰解毒方低、高剂量组肺组织中NLRP3阳性细胞数,Caspase-1活性,NLRP3,ASC,Caspase-1蛋白和mRNA相对表达均明显降低（P〈0.05）。与假手术组比较,模型组肺组织中ROS产生,TXNIP蛋白表达均明显增加（P〈0.05）;与模型组比较,尼莫地平组、清热化痰解毒方低、高�Objective： To investigate the effect of Qingre Huatan Jiedu formula on lung tissue in rats with cerebral ischemia-reperfusion injury and explore the underlying molecular mechanisms. Method： SD rats were divided into shame group,model group,positive control group（ PC group,nimodipine 200 mg·kg^-1）,low-dose Qingre Huatan Jiedu formula group（ LT group,100 mg·kg^-1） and high-dose Qingre Huatan Jiedu formula group（ HT group,200 mg·kg^-1）,with 10 rats in each group. Except for sham group,modified 4 artery occlusion method was used to prepare the global cerebral ischemia reperfusion injury model in rats. After modeling,the drug was given for 3 d. Lung wet/dry weight（ W/D） analysis was made. htoxylin easin（ HE） staining was performed for histopathological analysis; the levels of interleukin（ IL）-1β and IL-18 in bronchoalveolar lavage fluid（ BALF） were detected by enzyme linked immunosorbent assay（ ELISA）; pulmonary myeloperoxidase kit assay was used to detect myeloperoxidase（ MPO） activity; immunohistochemical analysis was performed for nucleotidebinding domain-like receptor 3（ NLRP3） positive cells; Caspase-1 colorimetric assay kit was applied in detecting Caspase-1 activity; Real-time PCR and Western blot were used to detect the mRNA and protein expressions of NLRP3, ASC and Caspase-1. Co-immunoprecipitation was used to detect thioredoxon interaction protein（ TXNIP）-NLRP3 protein expression. Result： Compared with shame group,lung injury score,W/D ratio of lung,IL-1β and IL-18 levels in BALF,total number of cells and polymorphonuclear leukocytes and MPO activity in model group were significantly increased（ P〈0. 05）. Compared with model group,lung injury score,W/D ratio of lung,IL-1β and IL-18 levels in BALF,total cell and polymorphonuclear leukocyte and MPO activity in PC group,LT group and HT group were significantly decreased（ P〈0. 05）. Compared with shame group,the relative expression of NLRP3 positive cells,Caspase-1 activity and NLRP3/Asc/Caspase-1

关 键 词：脑缺血再灌注 清热化痰解毒方 肺损伤 核苷酸结合域样受体蛋白3(NLRP3) 硫氧还蛋白相互作用蛋白(TXNIP) 

分 类 号：R22[医药卫生—中医基础理论] R24[医药卫生—中医学]