Yes相关蛋白1在血管紧张素Ⅱ诱导的大鼠肺纤维化中的作用及其机制

Role of Yes-associated protein 1 in angiotensinⅡ -induced pulmonary fibrosis in rats

目的 探讨Yes相关蛋白1（Yap1）在血管紧张素Ⅱ（AngⅡ）诱导大鼠肺纤维化中的作用及其机制。方法 体内实验将18只Wistar大鼠按随机数字表法分成对照组、博来霉素（BLM）组、BLM＋AngⅡ组各6只；造模后28 d后取肺组织行HE、Masson染色及Yap1免疫组化评价。体外分离培养原代肺成纤维细胞，予10－7 mmol/L AngⅡ或AngⅡ拮抗剂伊贝沙坦作用后，PCR及Western印迹法检测不同时间段Yap1、PDZ结合相关性转录共激活因子（TAZ）、Ⅰ型胶原的mRNA、蛋白相对表达量及Yap1、TAZ核质转位情况。随后设置空载质粒（vector）、vector＋AngⅡ、Yap1 RNA干扰（shRNA）、Yap1 shRNA＋AngⅡ四个组，Western印迹法检测Yap1、TAZ、Smad3、Ⅰ型胶原等蛋白相对表达量，CCK-8及EdU实验检测细胞增殖力。结果 BLM组肺纤维化明显，Yap1免疫组化表达增加，BLM＋AngⅡ组肺纤维化程度和Yap1表达均高于BLM组（均P〈0.05）。体外实验中，AngⅡ不同刺激时间组Yap1、TAZ及Ⅰ型胶原的mRNA及蛋白相对表达量均显著高于0 h（均P〈0.05）；其中，磷酸化Yap1蛋白相对表达量在2 h时达峰值。核质转位方面，24 h时AngⅡ组胞核中Yap1、TAZ相对表达量显著高于空白组（0.382±0.007比0.031±0.001，1.097±0.030比0.357±0.015），胞质中相对表达量显著低于空白组（0.323±0.058比0.418±0.044，0.858±0.059比1.201±0.015），AngⅡ＋伊贝沙坦组胞核中Yap1、TAZ相对表达量显著低于AngⅡ组（0.323±0.058比0.418±0.044，0.858±0.059比1.201±0.015），胞质中相对表达量显著高于AngⅡ组（0.598±0.060比0.323±0.058，1.495±0.052比0.858±0.059）（均P〈0.05）。Yap1 shRNA＋AngⅡ组Yap1、TAZ、Smad3及Ⅰ型胶原蛋白相对表达量均显著低于vector＋AngⅡ组（均P〈0.05）。vector＋AngⅡ组吸光度、EdU阳性细胞百分比均显著高于vector组，Yap1 shRNA＋AngⅡ组吸光度、EdU阳性细胞百分比均显著低于vector＋AngⅡ组（均P〈0.05）。结论 AngⅡ通过活化Yap1促进原代肺成纤维细胞增殖及Ⅰ型胶原合成，进而诱导大鼠肺纤维化。

Objective To explore the mechanism of Yes-associated protein 1 （Yap1） in angiotensinⅡ（AngⅡ）-induced pulmonary fibrosis.Methods In vivo, 18 male Wistar rats were randomly divided into three equal groups with 6 rats in each group, including control group, bleomycin-treated group （BLM）, and BLM＋ AngⅡ group. 28 days later, the lung tissues in all groups were harvested for the HE and Masson staining as well as the immunohistochemical （IHC） staining for Yap1. In vitro, the isolated fibroblasts were treated with 10-7 mmol/L AngⅡor the AngⅡ-targeted inhibitor irbesartan for the scheduled time for mRNA and protein expressions of Yap1, PDZ-binding motif （TAZ）, and collagen Ⅰusing PCR and Western blot, as well as the translocation test from the nucleus to the cytoplasm of Yap1 and TAZ. Subsequently, the fibroblasts were assigned into 4 groups： the empty plasmid （vector） group, the vector＋ AngⅡ group, the Yap1 shRNA group, and the Yap1 shRNA＋ AngⅡ group. Western blot was used to detect the relative expressions of Yap1, TAZ, Smad3 and collagen Ⅰ. The CCK-8 and EdU assays were performed to determine the proliferative capacity.Results In vivo, severe lung fibrosis and increased Yap1 expression of IHC staining were found in BLM group. Additionally, more severe lung fibrosis and higher Yap1 expression were detected in the BLM＋ AngⅡ group than the BLM group （both P〈0.05）. In vitro, both the mRNA and protein relative expressions of Yap1, TAZ and collagenⅠ were markedly higher in AngⅡ-treated groups than the control group （all P〈0.05）. Meanwhile, the relative expression of phosphorylated Yap1 reached its peak at 2 h after AngⅡ stimulation. In the protein translocation tests, after treated with AngⅡ for 24 h, the relative protein levels of Yap1 and TAZ in the nucleus of the AngⅡ group were significantly higher than those in the control group （0.382±0.007 vs 0.031±0.001, 1.097±0.030 vs 0.357±0.015）. However, the relative protein expressions in the cytoplasm of the AngⅡ group were obviously less than that in the control group （0.323±0.058 vs 0.418±0.044, 0.858±0.059 vs 1.201±0.015）. Compared with the AngⅡ group, the expressions of Yap1 and TAZ in the AngⅡ＋ irbesartan group were higher in cytoplasm （0.598±0.060 vs 0.323±0.058, 1.495±0.052 vs 0.858±0.059）, while lower in the nucleus （0.323±0.058 vs 0.418±0.044, 0.858±0.059 vs 1.201±0.015） （all P〈0.05）. Furthermore, the relative protein expressions of Yap1, TAZ, Smad3 and collagenⅠin Yap1 shRNA＋ AngⅡ group were distinctly lower than the vector＋ AngⅡ group （all P〈0.05）. In the cell proliferation tests, the absorbance and the percentage of EdU positive cells of vector＋ AngⅡ group exceeded that of vector group （both P〈0.05）. However, the absorbance and the percentage of EdU positive cells in the Yap1 shRNA＋ AngⅡgroup were less than the vector＋ AngⅡ group （both P〈0.05）.Conclusion AngiotensinⅡ promoted the collagen synthesis and cell proliferation in primary lung fibroblasts by increasing the Yap1 activity, leading to the progress of fibrosis.