Wang Na, Yan Di, Liu Yi, Liu Yao, Gu Xianmin, Sun Jian, Long Fei, Jiang Shujuan
Department of Pulmonary Medicine, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, 250021, China.
Respir Res. 2016 Sep 22;17(1):117. doi: 10.1186/s12931-016-0437-1.
Asthma is a worldwide health burden with an alarming prevalence. For years, asthma-associated airway injury remains elusive. Transforming growth factor β1 (TGF-β1) is a pleiotropic cytokine that has been shown to be involved in the synthesis of the matrix molecules associated with airway remodeling. Human antigen R (HuR), the member of the Hu RNA-binding protein family, can bind to a subset of short-lived mRNAs in their 3' untranslated regions (UTR). However, the functional roles and relevant signaling pathways of HuR in airway remodeling have not been well illustrated. Thus, we aim to explore the relationship between HuR and TGF-β1 in platelet derived growth factor(PDGF)-induced airway smooth muscle (ASM) cells and asthmatic animal.
Cultured human ASM cells were stimulated by PDGF for 0, 6, 12 and 24 h. Western blotting, RT-PCR and immunofluoresence were used to detect the expression of HuR, TGF-β1, α-smooth muscle actins (α-SMA) and collagen type I (Col-I). Then knockdown of HuR, flow cytomerty was used to detect the morphological change and western blotting for functionally change of ASM cells. Furthermore, the interference of TGF-β1 and exogenous TGF-β1 were implemented to testify the influence on HuR. A murine OVA-driven allergic model based on sensitization and challenge was developed. The inflammatory response was measured by bronchoalveolar lavage fluid (BALF), airway damage was analyzed by hematoxylin and eosin staining, airway remodeling was assessed by sirius red staining and periodic acid-schiff staining, the expression level of HuR, TGF-β1 and α-SMA were measured by RT-PCR, western blotting and immunohistochemistry.
Here, we found that PDGF elevated HuR expression both at mRNA and protein level in cultured ASM cells at a time-dependent manner, which was simultaneously accompanied by the enhanced expression of TGF-β1, α-SMA and Col-I. Further study revealed that the knockdown of HuR significantly increased the apoptosis of ASM cells and dampened TGF-β1, Col-I and α-SMA expression. However, interfering TGF-β1 with siRNA or extra addition of TGF-β1, HuR could restore its production as well as Col-I. Compared with normal mice stimulating with PBS, OVA-induced mice owned high amount of inflammatory cells, such as eosinophils, lymphocytes and neutrophils except macrophages. HE staining showed accumulation of inflammatory cells surrounding bronchiole and sirius red staining distinguished collagen type I and III deposition around the bronchiole. Higher abundance of HuR, TGF-β1 and α-SMA were verified in OVA-induced mice than PBS-induced mice by RT-PCR, western blotting and immunohistochemistry.
A HuR/TGF-β1 feedback circuit was established to regulate airway remodeling in vivo and in vitro and targeting this feedback has considerable potential for the intervention of asthma.
哮喘是一项全球性的健康负担，其患病率令人担忧。多年来，哮喘相关的气道损伤一直难以明确。转化生长因子β1（TGF-β1）是一种多效性细胞因子，已被证明参与与气道重塑相关的基质分子的合成。人抗原R（HuR）是Hu RNA结合蛋白家族的成员，可在其3'非翻译区（UTR）与一部分短寿命mRNA结合。然而，HuR在气道重塑中的功能作用和相关信号通路尚未得到充分阐明。因此，我们旨在探讨血小板衍生生长因子（PDGF）诱导的气道平滑肌（ASM）细胞和哮喘动物中HuR与TGF-β1之间的关系。
用PDGF刺激培养的人ASM细胞0、6、12和24小时。采用蛋白质免疫印迹法、逆转录-聚合酶链反应（RT-PCR）和免疫荧光法检测HuR、TGF-β1、α平滑肌肌动蛋白（α-SMA）和I型胶原（Col-I）的表达。然后敲低HuR，用流式细胞术检测ASM细胞的形态变化，并用蛋白质免疫印迹法检测其功能变化。此外，实施TGF-β1干扰和外源性TGF-β1以验证对HuR的影响。建立基于致敏和激发的小鼠卵清蛋白（OVA）驱动的过敏性模型。通过支气管肺泡灌洗液（BALF）测量炎症反应，通过苏木精和伊红染色分析气道损伤，通过天狼星红染色和过碘酸-希夫染色评估气道重塑，通过RT-PCR、蛋白质免疫印迹法和免疫组织化学测量HuR、TGF-β1和α-SMA的表达水平。
在此，我们发现PDGF以时间依赖性方式在培养的ASM细胞中升高HuR在mRNA和蛋白质水平的表达，同时伴有TGF-β1、α-SMA和Col-I表达的增强。进一步研究表明，敲低HuR显著增加ASM细胞的凋亡，并抑制TGF-β1、Col-I和α-SMA的表达。然而，用小干扰RNA（siRNA）干扰TGF-β1或额外添加TGF-β1，HuR可恢复其产生以及Col-I的产生。与用磷酸盐缓冲盐水（PBS）刺激的正常小鼠相比，OVA诱导的小鼠除巨噬细胞外有大量炎症细胞，如嗜酸性粒细胞、淋巴细胞和中性粒细胞。苏木精和伊红染色显示细支气管周围有炎症细胞聚集，天狼星红染色可区分细支气管周围I型和III型胶原的沉积。通过RT-PCR、蛋白质免疫印迹法和免疫组织化学验证，OVA诱导的小鼠中HuR、TGF-β1和α-SMA的丰度高于PBS诱导的小鼠。
建立了HuR/TGF-β1反馈回路以在体内和体外调节气道重塑，针对该反馈进行干预在哮喘治疗中具有相当大的潜力。
