TOLL样受体信号转导通路在内质网应激致炎症反应中的作用

doi:10.3877/cma.j.issn.1674-1358.2017.03.004

中华实验和临床感染病杂志(电子版) ›› 2017, Vol. 11 ›› Issue (03) : 222 -227. doi: 10.3877/cma.j.issn.1674-1358.2017.03.004

基础论著

TOLL样受体信号转导通路在内质网应激致炎症反应中的作用

杨思园¹, 李辉², 李鑫², 蒋荣猛², 马成杰², 魏红山³, 李兴旺²^,^†(

)

^1. 100015　北京，北京大学地坛医院教学医院，北京大学医学部
^2. 100015　北京首都医科大学附属北京地坛医院感染性疾病诊治与研究中心
^3. 100015　北京首都医科大学附属北京地坛医院传染病研究所

收稿日期:2016-03-16 出版日期:2017-06-15
通信作者: 李兴旺
基金资助:
北京市医院管理局重点医学专业发展计划-肝炎专业(No. ZY201402)

Role of TOLL-like receptor signal transduction pathway in inflammatory reaction induced by endoplasmic reticulum stress

Siyuan Yang¹, Hui Li², Xin Li², Rongmeng Jiang², Chengjie Ma², Hongshan Wei³, Xingwang Li²^,^†()

^1. Beijing Ditan Hospital, Peking University Teaching Hospital, Beijing 100015, China
^2. Department of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China
^3. Institute of Infection Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China

Received:2016-03-16 Published:2017-06-15
Corresponding author: Xingwang Li

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引用本文：

杨思园, 李辉, 李鑫, 蒋荣猛, 马成杰, 魏红山, 李兴旺. TOLL样受体信号转导通路在内质网应激致炎症反应中的作用[J/OL]. 中华实验和临床感染病杂志(电子版), 2017, 11(03): 222-227.

Siyuan Yang, Hui Li, Xin Li, Rongmeng Jiang, Chengjie Ma, Hongshan Wei, Xingwang Li. Role of TOLL-like receptor signal transduction pathway in inflammatory reaction induced by endoplasmic reticulum stress[J/OL]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2017, 11(03): 222-227.

目的

探讨TOLL样受体信号转导通路在衣霉素（TM）致人肝癌细胞系HpeG2和人正常肝细胞系L02内质网应激及炎症反应过程中的作用。

方法

利用实时荧光定量PCR检测衣霉素作用后HepG2和L02细胞中细胞因子IRAK1、TAK1和NF-κB的基因表达水平，利用Western blot检测相应细胞因子的表达水平。

结果

两种细胞系经TM刺激后，内质网应激标志物GRP78蛋白表达增高；炎症反应标志因子NF-κB在基因水平和蛋白水平表达均呈现增高趋势；TOLL样受体信号转导通路中关键细胞因子IRAK1、TAK1在基因水平和蛋白水平表达呈现增高趋势，差异均具有统计学意义（P均< 0.05）。

结论

TM刺激HepG2和L02细胞系之后，可诱导出现内质网应激状态并引起细胞内的炎症反应；在内质网应激致炎症反应过程中伴TOLL样受体信号转导通路的激活，TOLL样受体信号转导通路对其发生、发展可能具有一定的调节作用。

关键词: 内质网应激, 炎症反应, TOLL样受体信号转导通路, 衣霉素

Objective

To investigate the role of Toll-like receptor signal transduction pathway on the process of endoplasmic reticulum stress and inflammatory reaction induced by tunicamycin (TM).

Methods

The transcription levels of IRAK1, TAK1 and NF-κB genes in HepG2 and L02 cells treated by TM were detected by real-time reverse transcription PCR, and the expression level of protein related cytokines was detected by Western blot.

Results

The levels of GRP78 (marker of endoplasmic reticulum stress) in HepG2 cells and L02 cells significantly increased after treated by TM. The gene transcription and protein expression level of NF-κB (marker of inflammatory response) were both significantly increased in accordance with the concentration and action time compared with those untreated by TM. In Toll-like receptor signal transduction pathway, the expression of key cytokines like IRAK1 and TAK1 showed an increasing trend at gene and protein levels, all with significant differences (all P < 0.05).

Conclusions

After the stimulation of TM, endoplasmic reticulum stress and inflammatory reaction were induced in HepG2 and L02 cell lines. In the endoplasmic reticulum stress induced activation inflammatory reaction associated with Toll-like receptor signaling pathway, Toll-like receptor signal transduction pathway in the occurrence and development may have a regulatory role.

Key words: Endoplasmic reticulum stress, Inflammatory reaction, Toll-like receptor signal transduction pathway, Tunicamycin

表1 引物序列

引物名称	引物序列（5´→3´）
β-actin	F：attggcaatgagcggttc
β-actin	R：cgtggatgccacaggact
IRAK1	F：tgccaggaccaagtatctga
IRAK1	R：gtgcttctcaaagccactcc
TAK1	F：agctttatggagcctgcttg
TAK1	R：agagcccccttcagcatatt
NF-κB	F：atgcgcttccgctacaagt
NF-κB	R：tggtggtatctgtgctccttc

表1 引物序列

引物名称	引物序列（5´→3´）
β-actin	F：attggcaatgagcggttc
β-actin	R：cgtggatgccacaggact
IRAK1	F：tgccaggaccaagtatctga
IRAK1	R：gtgcttctcaaagccactcc
TAK1	F：agctttatggagcctgcttg
TAK1	R：agagcccccttcagcatatt
NF-κB	F：atgcgcttccgctacaagt
NF-κB	R：tggtggtatctgtgctccttc

图1 HepG2和L02细胞经不同浓度、衣霉素处理不同时间后mRNA的表达

表2 各组细胞因子mRNA相对表达水平（ ± s）

组别	目的基因相对表达量
	IRAK1		TAK1		NF-Κb
	HepG2	L02	HepG2	L02	HepG2	L02
空白对照组	0.111 ± 0.113	0.130 ± 0.027	0.197 ± 0.007	0.213 ± 0.036	0.129 ± 0.028	0.090 ± 0.013
5 mg/ml刺激12 h组	0.527 ± 0.190	0.346 ± 0.101	0.412 ± 0.061	0.452 ± 0.100	0.401 ± 0.032	0.415 ± 0.059
5 mg/ml刺激24 h组	1.106 ± 0.215	0.903 ± 0.081	0.894 ± 0.081	0.824 ± 0.052	0.766 ± 0.048	0.756 ± 0.060
10 mg/ml刺激24 h组	1.263 ± 0.249	1.073 ± 0.189	0.983 ± 0.115	0.994 ± 0.112	1.007 ± 0.122	0.920 ± 0.029
P₁	0.003	0.010	0.068	0.545	0.631	0.051
P₂	0.015	0.002	0.016	0.020	0.033	0.018
P₃	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

表2 各组细胞因子mRNA相对表达水平（ ± s）

组别	目的基因相对表达量
	IRAK1		TAK1		NF-Κb
	HepG2	L02	HepG2	L02	HepG2	L02
空白对照组	0.111 ± 0.113	0.130 ± 0.027	0.197 ± 0.007	0.213 ± 0.036	0.129 ± 0.028	0.090 ± 0.013
5 mg/ml刺激12 h组	0.527 ± 0.190	0.346 ± 0.101	0.412 ± 0.061	0.452 ± 0.100	0.401 ± 0.032	0.415 ± 0.059
5 mg/ml刺激24 h组	1.106 ± 0.215	0.903 ± 0.081	0.894 ± 0.081	0.824 ± 0.052	0.766 ± 0.048	0.756 ± 0.060
10 mg/ml刺激24 h组	1.263 ± 0.249	1.073 ± 0.189	0.983 ± 0.115	0.994 ± 0.112	1.007 ± 0.122	0.920 ± 0.029
P₁	0.003	0.010	0.068	0.545	0.631	0.051
P₂	0.015	0.002	0.016	0.020	0.033	0.018
P₃	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

图2 HepG2和L02细胞内GRP78表达

图3 HepG2和L02细胞内IRAK1、TAK1、pp65蛋白表达

表3 HepG2细胞系GRP78、IRAK1、TAK1和pp65蛋白相对表达水平（ ± s）

组别	灰度分值比较
组别	IRAK1	TAK1	pp65	GRP78
空白对照组	1.000	1.000	1.000	1.000
5 mg/ml 12 h组	1.033 ± 0.023	1.432 ± 0.372	1.020 ± 0.012	1.038 ± 0.033
5 mg/ml 24 h组	1.925 ± 0.073	2.095 ± 0.481	1.236 ± 0.048	1.326 ± 0.271
10 mg/ml 24 h组	2.016 ± 0.049	2.882 ± 0.715	1.507 ± 0.092	1.926 ± 0.249
P₁值	0.007	0.015	0.399	0.001
P₂值	< 0.001	0.010	0.001	0.005
P₃值	< 0.001	0.019	< 0.001	< 0.001

表3 HepG2细胞系GRP78、IRAK1、TAK1和pp65蛋白相对表达水平（ ± s）

组别	灰度分值比较
组别	IRAK1	TAK1	pp65	GRP78
空白对照组	1.000	1.000	1.000	1.000
5 mg/ml 12 h组	1.033 ± 0.023	1.432 ± 0.372	1.020 ± 0.012	1.038 ± 0.033
5 mg/ml 24 h组	1.925 ± 0.073	2.095 ± 0.481	1.236 ± 0.048	1.326 ± 0.271
10 mg/ml 24 h组	2.016 ± 0.049	2.882 ± 0.715	1.507 ± 0.092	1.926 ± 0.249
P₁值	0.007	0.015	0.399	0.001
P₂值	< 0.001	0.010	0.001	0.005
P₃值	< 0.001	0.019	< 0.001	< 0.001

表4 L02细胞系GRP78、IRAK1、TAK1、pp65蛋白相对表达水平（ ± s）

组别	灰度分值
组别	IRAK1	TAK1	pp65	GRP78
空白对照组	1.000	1.000	1.000	1.000
5 mg/ml 12 h组	1.844 ± 0.115	1.952 ± 0.090	1.015 ± 0.029	1.140 ± 0.114
5 mg/ml 24 h组	2.038 ± 0.145	2.523 ± 0.052	1.952 ± 0.063	1.483 ± 0.341
10 mg/ml 24 h组	2.227 ± 0.089	2.934 ± 0.102	2.002 ± 0.049	1.873 ± 0.189
P₁值	0.002	0.001	0.059	0.041
P₂值	< 0.001	0.001	< 0.001	< 0.001
P₃值	< 0.001	< 0.001	0.001	< 0.001

表4 L02细胞系GRP78、IRAK1、TAK1、pp65蛋白相对表达水平（ ± s）

组别	灰度分值
组别	IRAK1	TAK1	pp65	GRP78
空白对照组	1.000	1.000	1.000	1.000
5 mg/ml 12 h组	1.844 ± 0.115	1.952 ± 0.090	1.015 ± 0.029	1.140 ± 0.114
5 mg/ml 24 h组	2.038 ± 0.145	2.523 ± 0.052	1.952 ± 0.063	1.483 ± 0.341
10 mg/ml 24 h组	2.227 ± 0.089	2.934 ± 0.102	2.002 ± 0.049	1.873 ± 0.189
P₁值	0.002	0.001	0.059	0.041
P₂值	< 0.001	0.001	< 0.001	< 0.001
P₃值	< 0.001	< 0.001	0.001	< 0.001

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