IL-17 A对系统性硬化病小鼠模型皮肤和肺部纤维化病变的作用

Effects of IL-17A on fibrosis of skin and lung in a mouse model of systemic sclerosis

目的 研究白细胞介素（IL）-17A在博莱霉素（BLM）致系统性硬化病（SSc）小鼠模型中的表达及对皮肤、肺部炎症和纤维化病变的作用.方法 24只雌性BALB/c小鼠随机分为4组,包括正常对照组（小鼠背部皮下注射磷酸盐缓冲液）、模型组（小鼠背部皮下注射BLM）、抗体组（BLM＋抗IL-17A单克隆抗体）、同型对照组（BLM＋同型对照）.观察各组小鼠背部注射部位皮肤和肺部的病理改变、炎症和纤维化评分,免疫组化和实时荧光定量PCR（RT-PCR）检测小鼠皮肤和肺部的IL-17A、TGF-β1、Ⅰ型胶原mRNA的表达.体外培养小鼠肺成纤维细胞（FB）,分别加入IL-17A细胞因子和单克隆抗体,检测各组细胞Ⅰ型胶原mRNA、TGF-β1 mRNA的表达,酶联免疫吸附试验（ELISA）检测培养液上清IL-6、TGF-β1的水平.结果 （1）抗体组小鼠皮肤厚度、皮肤炎症、肺组织炎症和纤维化评分明显低于模型组及同型对照组（P＆lt;0.05）.（2）免疫组化提示模型组IL-17A在皮肤和肺组织的表达明显高于正常对照组,抗体组皮肤和肺组织IL-17A蛋白的表达均低于模型组和同型对照组（P＆lt;0.05）.（3）模型组IL-17A mRNA在皮肤和肺组织的表达明显高于正常对照组,抗体组小鼠皮肤和肺组织中IL-17A、TGF-β1、Ⅰ型胶原mRNA的表达均低于模型组和同型对照组（P＆lt;0.05）.（4）体外细胞培养SSc小鼠肺FB,IL-17A单克隆抗体阻断组TGF-β1、Ⅰ型胶原mRNA水平、 细胞上清液IL-6、TGF-β1水平均低于IL-17A细胞因子刺激组（P＆lt;0.05）,而均高于对照组（P＆lt;0.05）.结论 IL-17A可促进SSc小鼠模型皮肤和肺脏炎症和纤维化的发展,阻断IL-17A可能通过抑制TGF-β1、IL-6和Ⅰ型胶原的产生进而抑制SSc纤维化病变.

Objective To analyze the expression of interleukin （ IL）-17A in a mouse model of bleomycin （ BLM）-induced systemic sclerosis （ SSc） and to evaluate its effects on inflammation and fibrosis in skin and lung tissues. Methods Twenty-four female BALB/c mice were randomly divided into four groups：normal control group （ mice were subcutaneously injected with phosphate buffer ） , model group （subcutaneously injected with BLM）, antibody group （injected with BLM ＋ IL-17A monoclonal antibody）, homotypic control group （ injected with BLM ＋ isotype control） . Pathological changes in skin and lung tis-sues of those mice were observed;inflammatory and fibrotic scores were assessed. Immunohistochemistry and real-time fluorescent quantitative PCR （ RT-PCR） were used to detect the expression of IL-17A, TGF-β1 and typeⅠ collagen in skin and lung tissues of those mice at mRNA level. Mouse lung fibroblasts （ FB） de-rived from the mice of model group were cultured in vitro and then were cultured with IL-17A cytokines with or without the interference of monoclonal antibodies. Expression of typeⅠ collagen and TGF-β1 at mRNA level and levels of IL-6 and TGF-β1 in the culture supernatants were detected by RT-PCR and enzyme-linkedimmunosorbent assay （ ELISA） , respectively. Results Compared with the mice of model and homotypic control groups, those of the antibody group showed mild skin thickening, skin inflammation and lung inflam-mation as well as lower fibrosis scores （P＆lt;0. 05）. The expression of IL-17A at both protein and mRNA lev-els and the expression of TGF-β1 and collagen typeⅠat mRNA level in skin and lung tissues of mice of the antibody group were significantly lower than those of the model and homotypic control group （P＆lt;0. 05）. Re-sults of the in vitro cell culture of SSc mice-derived lung FB with IL-17A showed that the expression of TGF-β1 and typeⅠ collagen at mRNA level and the levels of IL-6 and TGF-β1 in the culture supernatants were decreased with the interference of anti-IL-17A monoclonal antibody （P＆lt;0. 05）, but were still higher than those of the control group （P＆lt;0. 05）. Conclusion IL-17A promotes the development of inflammation and fibrosis in skin and lung tissues in the mouse model of SSc. Blocking IL-17A might inhibit fibrosis in SSc by inhibiting the production of TGF-β1, IL-6 and typeⅠ collagen.