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J Zhejiang Univ (Med Sci)  2020, Vol. 49 Issue (6): 758-764    DOI: 10.3785/j.issn.1008-9292.2020.12.11
    
IL-17A activates mouse lung fibroblasts through promoting chemokine CXCL12 secretion
WANG Huaying(),LYU Jiapei,CHEN Liping,YU Wanjun*()
Department of Respiratory and Critical Care Medicine, People's Hospital Affiliated to Ningbo University, Yinzhou People's Hospital, Ningbo 315040, Zhejiang Province, China
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Abstract  

Objective: To investigate the role of IL-17A in promoting the activation of lung fibroblasts and the secretion of chemokine CXCL12, and to explore the possible mechanism. Methods: Lung tissues of BALB/c mice were collected after intraperitoneal injection of recombinant mouse IL-17A (rmIL-17A). Real-time RT-PCR and Western blotting were used to detect the expression levels of α-smooth muscle actin (α-SMA) and collagen I in lung tissues, and immunohistochemical staining and real-time RT-PCR were used to determine the expression of CXCL12. Normal mouse primary lung fibroblasts were isolated and cultured, and identified by immunofluorescence staining with optical microscopy. Cells and supernatant of culture medium were collected after stimulation with rmIL-17A at different concentrations. mRNA levels of α-SMA, collagen I, and CXCL12 in the cells were determined by real-time RT-PCR, and the levels of collagen I and CXCL12 in the supernatant of culture medium were determined by ELISA. Results: The mRNA and protein levels of α-SMA and collagen I in the lung tissue of mice injected with rmIL-17A were significantly increased compared with the control group (all P < 0.01). The mRNA levels of α-SMA, collagen I and CXCL12 in mice primary lung fibroblasts were increased after stimulation of rmIL-17A at different concentrations (all P < 0.01), and the concentration of collagen Ⅰ and CXCL12 in the supernatants of culture medium were also increased in a dose-dependent manner (all P < 0.01). Conclusions: IL-17A can promote the activation of lung fibroblasts and translation into myofibroblast. The secretion of collagen is increased, which promote the deposition of extracullular matrix, and leads to the occurrence and development of lung fibrosis. CXCL12, a chemokine secreted by activated fibroblasts, may be involved in this process.



Key wordsInterleukin-17A      Chemokine      CXCL12      Fibroblast      α-smooth muscle actin      TypeⅠ collagen      Mice     
Received: 15 May 2020      Published: 14 January 2021
CLC:  R363  
Corresponding Authors: YU Wanjun     E-mail: yingmeire@163.com;nbyuwanjun@163.com
Cite this article:

WANG Huaying,LYU Jiapei,CHEN Liping,YU Wanjun. IL-17A activates mouse lung fibroblasts through promoting chemokine CXCL12 secretion. J Zhejiang Univ (Med Sci), 2020, 49(6): 758-764.

URL:

http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2020.12.11     OR     http://www.zjujournals.com/med/Y2020/V49/I6/758


趋化因子CXCL12可能参与白细胞介素17A对小鼠肺成纤维细胞的活化

目的: 研究IL-17A促进小鼠肺成纤维细胞活化及趋化因子CXCL12在其中的作用。方法: 以雄性BALB/c小鼠为研究对象,小鼠腹腔注射重组小鼠IL-17A后取小鼠肺组织。采用实时逆转录PCR和蛋白质印迹法检测小鼠肺组织α平滑肌肌动蛋白(α-SMA)和Ⅰ型胶原蛋白的mRNA和蛋白表达,采用免疫组织化学法和实时逆转录PCR法测定肺组织中趋化因子CXCL12的表达。分离培养正常小鼠原代肺成纤维细胞,采用免疫荧光染色法和光学显微镜观察鉴定原代肺成纤维细胞。将分离的肺成纤维细胞与不同浓度的重组小鼠IL-17A共培养后收集细胞及上清液,采用实时逆转录PCR法测定细胞中α-SMA、Ⅰ型胶原蛋白和CXCL12的mRNA水平,采用ELISA法测定培养上清液中Ⅰ型胶原蛋白和CXCL12蛋白水平。结果: 与对照组比较,重组小鼠IL-17A处理后小鼠肺组织中α-SMA、Ⅰ型胶原蛋白的mRNA和蛋白水平均升高(均P < 0.01)。与不同浓度的重组小鼠IL-17A共培养后,小鼠肺成纤维细胞表达α-SMA、Ⅰ型胶原蛋白和CXCL12的mRNA明显增加(均P < 0.01),其培养基上清液中Ⅰ型胶原蛋白、CXCL12的浓度也明显升高(均P < 0.01),且呈剂量依赖性。结论: IL-17A能促进肺成纤维细胞的活化并转化为肌成纤维细胞,分泌Ⅰ型胶原蛋白明显增加,促进细胞外基质的沉积,从而导致肺纤维化的发生和发展,而活化的成纤维细胞分泌的趋化因子CXCL12可能参与了这个过程。


关键词: 白细胞介素17A,  趋化因子,  CXCL12,  成纤维细胞,  α平滑肌肌动蛋白,  Ⅰ型胶原蛋白,  小鼠 
引物名称 引物序列(5′→3′)
α-SMA:α平滑肌肌动蛋白;collagen Ⅰ:Ⅰ型胶原蛋白.
CXCL12 正向:GGAGGATAGATGTGCTCTGGAAC
反向:AGTGAGGATGGAGACCGTGGTG
α-SMA 正向:CCCAGACATCAGGGAGTAATGG
反向:TCTATCGGATACTTCAGCGTCA
collagenⅠ 正向:TAAGGGTCCCCAATGGTGAGA
反向:GGGTCCCTCGACTCCTACAT
β-actin 正向:GTGACGTTGACATCCGTAAAGA
反向:GCCGGACTCATCGTACTCC
Tab 1 Primer sequences for real-time RT-PCR
Fig 1 Expression of collagen I and α-smooth muscle actin in mouse lung tissue with or without rmIL-17A treatment
Fig 2 mRNA expression of CXCL12, α-smooth muscle actin and collagen I in lung tissue in mice with or without rmIL-17A treatment
Fig 3 Expression of CXCL12 in lung tissues from normal mice and mice with rmIL-17A injection (immunohistochemical staining)
Fig 4 Observation of primary lung fibroblast from BALB/c mice under the microscope
Fig 5 IL-17A promotes the expression of α-smooth muscle actin and collagen I of mouse primary lung fibroblasts
Fig 6 IL-17A promotes the expression of CXCL12 in mouse primary lung fibroblasts
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