汤鲁明; 柯迦勒; 徐晨希; 王林霞; 潘小东; 孙来芳; 龚裕强

doi:10.3877/cma.j.issn.1674-6880.2019.01.001

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萝卜硫素对脓毒症急性肺损伤大鼠肺组织Toll样受体4/核因子κB信号通路的影响

中华危重症医学杂志（电子版）, 2019,12(1) : 3-8. DOI: 10.3877/cma.j.issn.1674-6880.2019.01.001

摘要

目的

评价萝卜硫素对于脓毒症急性肺损伤（ALI）大鼠肺组织炎症反应以及Toll样受体4（TLR4）/核因子κB（NF-κB）信号通路的影响。

方法

将30只雄性Sprague Dawley大鼠按随机数字表法分成假手术组、模型组和治疗组，每组各10只。模型组和治疗组大鼠采用盲肠结扎穿孔（CLP）法制备大鼠脓毒症模型，治疗组大鼠在术后立即腹腔注射50 mg/kg的萝卜硫素注射液，其余两组注入等量等渗NaCl溶液；于术后24 h大鼠右侧颈总动脉采集动脉血标本，然后处死大鼠取肺组织测定肺组织湿/干比。采用酶联免疫吸附测定（ELISA）法检测三组大鼠肺组织匀浆标本肿瘤坏死因子α（TNF-α）、白细胞介素1β（IL-1β）、NF-κB p65表达水平；荧光实时定量PCR检测三组大鼠肺组织TLR4 mRNA的表达。

结果

假手术组、模型组及治疗组大鼠肺组织湿/干比[（4.00 ± 0.13）、（6.10 ± 0.05）、（5.80 ± 0.08）]、氧合指数[（315 ± 11）、（177 ± 7）、（200 ± 12）mmHg]、TNF-α[（6.05 ± 0.29）、（45.06 ± 0.52）、（27.09 ± 0.85）ng/L]、IL-1β[（8.02 ± 0.21）、（38.23 ± 0.81）、（32.73 ± 1.12）ng/L]及NF-κB p65[（0.375 ± 0.013）、（1.230 ± 0.045）、（0.988 ± 0.043）ng/L]表达水平比较，差异均有统计学意义（F = 480.891、255.309、4 245.262、1 918.168、564.842，P均< 0.001）。进一步两两比较发现，模型组和治疗组大鼠肺组织湿/干比、TNF-α、IL-1β、NF-κB p65表达水平均较假手术组大鼠显著升高（P均< 0.05），治疗组大鼠肺组织湿/干比、TNF-α、IL-1β、NF-κB p65表达水平均较模型组大鼠显著降低（P均< 0.05）；而模型组和治疗组大鼠氧合指数均较假手术组大鼠显著降低（P均< 0.05），治疗组大鼠氧合指数较模型组大鼠显著升高（P < 0.05）。荧光实时定量PCR结果显示，三组大鼠肺组织TLR4 mRNA表达水平比较，差异具有统计学意义（F= 224.538，P < 0.001）。进一步两两比较发现，与假手术组比较，模型组及治疗组大鼠肺组织TLR4 mRNA表达均显著升高（P均< 0.05）；而治疗组大鼠肺组织TLR4 mRNA表达较模型组显著降低（P < 0.05）。

结论

萝卜硫素可减轻脓毒症急性肺损伤大鼠肺组织炎症反应、肺水肿程度，改善缺氧状态，对于肺组织具有一定保护作用，且其作用机制可能与干预TLR4/NF-κB信号途径相关。

引用本文: 汤鲁明, 柯迦勒, 徐晨希, 等. 萝卜硫素对脓毒症急性肺损伤大鼠肺组织Toll样受体4/核因子κB信号通路的影响 [J/OL] . 中华危重症医学杂志（电子版）, 2019, 12(1) : 3-8. DOI: 10.3877/cma.j.issn.1674-6880.2019.01.001.

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脓毒症所致的急性肺损伤（acute lung injury，ALI）/急性呼吸窘迫综合征（acute respiratory distress syndrome，ARDS）是引起危重症患者急性呼吸衰竭的一个较为常见的病因，常常导致患者死亡。据统计，仅在美国15岁以上人群中ALI/ARDS每年发病人数为58.7 ~ 78.9/10万人，病死率为38.5% ~ 41.1%，且临床上缺乏有效的预防和治疗措施^[1]。业已明确，过度炎症反应导致促炎/抗炎递质失衡是造成脓毒症诱发ALI/ARDS发生发展的一个主要机制^[2]。目前认为，炎症反应主要是指宿主对外界炎症因子刺激的适应性免疫应答过程，而外界病原微生物致炎组分主要由存在细胞表面的Toll样受体（Toll like receptors，TLR）来识别，通过胞内一系列信号转导，最终引起核因子κB（nuclear factor-kappa B，NF-κB）活化以及炎症因子的表达，其中TLR4主要识别细菌内毒素-脂多糖（lipopolysaccharide，LPS）^[3,4]。研究显示，采用选择性的TLR4阻断剂可以降低NF-κB的表达，降低炎症反应，对脓毒症及器官损伤具有一定的保护作用^[5,6]。萝卜硫素作为一种从自然界十字花科植物中提取的有机硫复合物，已被证实具有多种生物学活性，如抗癌、抗氧化、抗血小板活化、抗炎症以及去毒性等作用^[7]。本课题组前期研究证实，萝卜硫素对脓毒症大鼠急性肺损伤具有一定的保护作用，但其作用机制尚有待研究^[8]。本研究拟通过采用盲肠结扎穿孔（cecal ligation and puncture，CLP）法构建脓毒症ALI/ARDS大鼠模型，观察萝卜硫素治疗对大鼠肺组织炎症反应以及TLR4/NF-κB信号通路的作用。

贡献者信息

汤鲁明

325000　浙江温州，温州医科大学附属第二医院急诊医学科

柯迦勒

325000　浙江温州，温州医科大学附属第二医院急诊医学科

徐晨希

325000　浙江温州，温州医科大学附属第二医院急诊医学科

王林霞

325000　浙江温州，温州医科大学附属第二医院重症医学科

潘小东

325000　浙江温州，温州医科大学附属第二医院急诊医学科

孙来芳

325000　浙江温州，温州医科大学附属第二医院急诊医学科

龚裕强

325000　浙江温州，温州医科大学附属第二医院重症医学科

通信作者

龚裕强

325000　浙江温州，温州医科大学附属第二医院重症医学科

Email：gyq12120@163.com

关键词

脓毒症; 急性肺损伤; Toll样受体4; 萝卜硫素; 炎症反应; 核因子κB;

基金项目

温州市公益性科技计划项目（Y20160100）

作者声明

汤鲁明，柯迦勒，徐晨希，等.萝卜硫素对脓毒症急性肺损伤大鼠肺组织Toll样受体4/核因子κB信号通路的影响[J/CD].中华危重症医学杂志（电子版），2019，12（1）：3-8.

历史

出版日期：2019-02-01

收稿日期：2019-01-26

本文编辑

于莉

Original Article

Effect of sulforaphane on Toll like receptor 4/nuclear factor-kappa B signaling pathway in rats with sepsis-induced acute lung injury

Luming Tang, Jiale Ke, Chenxi Xu, Linxia Wang, Xiaodong Pan, Laifang Sun, Yuqiang Gong

Published 2019-02-01

Cite as Chin J Crit Care Med(Electronic Edition), 2019, 12(1): 3-8. DOI: 10.3877/cma.j.issn.1674-6880.2019.01.001

Abstract

Objective

To evaluate the effects of sulforaphane on inflammatory reaction and Toll like receptor 4 (TLR4)/ nuclear factor-kappa B (NF-κB) signaling pathway in rats with sepsis-induced acute lung injury (ALI).

Methods

Thirty male Sprague Dawley rats were randomly divided into the sham operation group, model group and treatment group, with 10 rats in each group. Rats in the model group and treatment group were prepared by cecal ligation and puncture (CLP). Rats in the treatment group were intraperitoneally injected with 50 mg/kg sulforaphane immediately after operation, while the other two groups were injected with isosmotic NaCl solution. Arterial blood samples were collected from the right common carotid artery 24 hours after operation, and then lung tissues were taken from rats to determine their wet/dry ratio. The expression levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and NF-κB p65 in the lung tissue homogenate of rats in three groups were detected by enzyme-linked immunosorbent assay (ELISA), and TLR4 mRNA expressions were measured by real-time fluorescence quantitative PCR.

Results

The expression levels of lung tissue wet/dry ratio [(4.00 ± 0.13), (6.10 ± 0.05), (5.80 ± 0.08)], oxygenation index [(315 ± 11), (177 ± 7), (200 ± 12) mmHg], TNF-α [(6.05 ± 0.29), (45.06 ± 0.52), (27.09 ± 0.85) ng/L], IL-1β [(8.02 ± 0.21), (38.23 ± 0.81), (32.73 ± 1.12) ng/L] and NF-κB p65 [(0.375 ± 0.013), (1.230 ± 0.045), (0.988 ± 0.043) ng/L] were statistically significantly different in three groups (F = 480.891, 255.309, 4 245.262, 1 918.168, 564.842; all P < 0.001). Further comparison showed that the wet/dry ratio, TNF-α, IL-1β and NF-κB p65expressions of lung tissue in the model group and treatment group were all significantly higher than those in the sham operation group (all P < 0.05); in the same time, the wet/dry ratio, TNF-α, IL-1β and NF-κB p65 expressions in the treatment group were all significantly lower than those in the model group (all P < 0.05). The oxygenation index of rats in the model group and treatment group was significantly lower than that in the sham operation group (both P < 0.05), while the oxygenation index in the treatment group was significantly higher than that in the model group (P < 0.05). The results of real-time quantitative PCR showed that there was significant difference in the expression of TLR4 mRNA among these three groups (F = 224.538, P < 0.001). Compared with the sham operation group, the expression of TLR4 mRNA of lung tissue in the model group and treatment group were significantly higher (both P < 0.05), while the expression of TLR4 mRNA in the treatment group was significantly lower than that in the model group (P < 0.05).

Conclusions

Sulforaphane can reduce lung tissue inflammatoryreaction and pulmonary edema and improve anoxic state in rats with acute lung injury caused by sepsis. It has a protective effect on lung tissue, and its mechanism may be related to the intervention of TLR4/NF-κB signaling pathway.

Key words:

Sepsis; Acute lung injury; Toll like receptor 4; Sulforaphane; Inflammatory reaction; Nuclear factor-kappa B

Contributor Information

Luming Tang