CCN1在脂多糖诱导急性肺损伤中的表达调控和在炎症反应中的作用  

Regulatory role of CCN1 in lipopolysaccharide-induced mouse acute lung injury

作　　者：董年 王蓓蓓 宋晨剑 陈俊杰[1] 陈成水[1] 石林 DONG Nian;WANG Bei-bei;SONG Chen-jian;CHEN Jun-jie;CHEN Cheng-shui;SHI Lin(Department of Respiratory and Critical Care Medicine,The First Affiliated Hospital,Wenzhou Medical University,Wenzhou 325000,China;Department of Respiratory and Critical Care Medicine,Zhongshan Hospital,Fudan University,Shanghai 200000,China)

机构地区：[1]温州医科大学附属第一医院呼吸与危重症医学科,浙江温州325000 [2]复旦大学附属中山医院呼吸与危重症医学科,上海200000

出　　处：《中国病理生理杂志》2020年第3期507-513,共7页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81800034,No.81770074);浙江省医药卫生面上项目(No.2018264229);温州市科技局课题(No.Y20180125);复旦大学附属中山医院青年科学基金(No.2018ZSQN01)。

摘　　要：目的:体内观察富半胱氨酸蛋白61(CYR61/CCN1)在正常肺组织中的表达定位和在脂多糖(LPS)诱导小鼠急性肺损伤中的表达变化,体外研究LPS调控CCN1表达的分子机制和CCN1在LPS诱导炎症介质表达中的作用。方法:分别通过免疫组化(IHC)染色及免疫荧光法观察CCN1在小鼠肺组织和气道上皮细胞16HBE中的表达定位;气道滴注LPS建立小鼠急性肺损伤模型,IHC观察CCN1在肺组织中的表达变化;体外研究中,分别予气道上皮16HBE细胞以ERK1/2、JNK、P38和PI3K信号通路特异性抑制剂预处理2 h后加入LPS刺激,通过RT-qPCR和Western blot检测CCN1的mRNA和蛋白表达变化;分别通过CCN1-siRNA和重组CCN1蛋白刺激16HBE细胞,qPCR检测炎症介质白细胞介素6(IL-6)、IL-8、转化生长因子β(TGF-β)和血管内皮生长因子(VEGF)的mRNA水平。结果:CCN1在正常肺组织中以气道上皮表达为主,在LPS诱导的急性肺损伤小鼠气道上皮细胞中CCN1表达升高;LPS可刺激16HBE细胞中CCN1的表达水平升高,其中ERK1/2、JNK、P38和PI3K信号通路特异性抑制剂可以不同程度逆转LPS诱导的CCN1表达升高。重组CCN1蛋白可以诱导16HBE细胞中IL-6、IL-8、TGF-β和VEGF的mRNA合成,干扰CCN1可以部分逆转LPS刺激后IL-6、IL-8、TGF-β和VEGF的mRNA合成。结论:气道上皮来源的CCN1在急性肺损伤中表达升高,其表达调控涉及ERK1/2、JNK、P38和PI3K信号通路;CCN1在介导LPS诱导的炎症介质表达过程中发挥了重要作用。本研究为未来急性肺损伤诊治的潜在靶点提供了一定的理论基础。AIM: To observe the cellular location and expression change of cysteine-rich protein 61(CYR61/CCN1) in lung tissues of the mice with lipopolysaccharide(LPS) intratracheal instillation, and to clarify the regulatory role of CCN1 expression in mediating inflammatory response. METHODS: The expression change of CCN1 in the lung tissues in vivo was observed by the method of immunohistochemistry, and immunofluorescence was employed to certify the cellular location of CCN1 in bronchial epithelial cells. Bronchial epithelial 16 HBE cells were cultured in vitro, and the expression of CCN1 under the condition of LPS stimulation was quantified by RT-qPCR and Western blot with or without specific inhi-bitors of ERK1/2, JNK, P38 and PI3 K signaling pathways. The mRNA expression levels of interleukin-6(IL-6), IL-8, transformrg growth factor-β(TGF-β) and vascular endothlial growth factor(VEGF) were measured by RT-qPCR under the condition of recombinant CCN1 exposure or transfection with CCN1-siRNA. RESULTS: The results of immunohistochemistry indicated that CCN1 was primarily located in bronchial epithelium. The results of immunofluorescence revealed that CCN1 was localized in the cytoplasm. The specific inhibitors of ERK1/2, JNK, P38 and PI3 K signaling pathways reversed the up-regulation of CCN1 upon LPS stimulation. Exposure to recombinant CCN1 resulted in the up-regulation of IL-6, IL-8, TGF-β and VEGF, while LPS-related up-regulation of IL-6, IL-8, TGF-β and VEGF was blocked by silencing of CCN1.CONCLUSION: Airway epithelium-derived CCN1 is up-regulated under the condition of lung injury and the regulatory mechanism involves ERK1/2, JNK, P38 and PI3 K signal transduction pathways. CCN1 acts as an inflammatory mediator in amplification of inflammatory response, laying theoretical basis for the potential molecular therapeutic target of acute lung injury.

关 键 词：富半胱氨酸蛋白61 气道上皮细胞 脂多糖 急性肺损伤 炎症反应 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学] R363.2[医药卫生—基础医学]