摘要
目的：探讨视黄醇X受体（RXR）激动剂9-顺式视黄酸（9-cis-RA）干预对缺氧条件下转化生长因子β1（TGF-β1）诱导的大鼠心肌成纤维细胞（CFs）胶原合成的影响和分子机制。方法：心肌组织块干涸法培养大鼠CFs,持续通氮气法建立细胞缺氧环境,评价9-cis-RA和TGF-β1对CFs胶原合成的影响;ELISA法测CFs上清液Ⅰ、Ⅲ型胶原水平,Western blot法检测胞浆、胞核和细胞的Smad2及p-Smad2水平,细胞免疫化学法观察p-Smad2的亚细胞定位。结果：TGF-β1（0.01-10μg/L）在缺氧条件下呈浓度依赖性地诱导CFs合成Ⅰ型和Ⅲ胶原,当浓度达到5μg/L时,Ⅰ、Ⅲ型胶原的合成水平显著增高（P〈0.01）。9-cis-RA（10^-9-10^-6mol/L）则呈浓度依赖性抑制TGF-β1在缺氧条件下诱导的CFs胶原合成,当浓度为10^-7mol/L时,Ⅰ型和Ⅲ型胶原的合成水平显著降低（P〈0.01）。Smad2抑制剂（20 nmol/L）亦可显著抑制TGF-β1在缺氧条件下诱导的CFs Ⅰ型和Ⅲ型胶原的合成。免疫杂交和细胞免疫化学结果显示,与TGF-β1干预相比,TGF-β1和9-cis-RA联合干预组的CFs其胞浆内p-Smad2的水平显著增加,但胞核内p-Smad2的水平明显降低（P〈0.05）。结论：RXR激动剂9-cis-RA显著下调TGF-β1在缺氧条件下诱导的CFs Ⅰ型和Ⅲ型胶原的合成,其机制与其抑制TGF-β1诱导的p-Smad2细胞核转位有关。
AIM：To investigate the effect of activation of retinoid X receptor（ RXR） on transforming growth factor β1（ TGF-β1） induced collagen synthesis under hypoxic environment in rat cardiac fibroblasts（ CFs） and underlying molecular mechanisms.METHODS：CFs were cultured using myocardial tissue with dry method.Hypoxic environment was established for CFs by continuous nitrogen supplement.Type Ⅰ and type Ⅲ collagens in supernatants were detected by ELISA.Nuclear and cytoplasmic extractions were prepared using NE-PER nuclear and cytoplasmic extraction reagents.The protein levels of Smad2 and p-Smad2 were determined by Western blot and immunocytochemical staining.RESULTS：Under hypoxic condition,TGF-β1（ 0.01 - 10 μg/L） increased the synthesis of type Ⅰ and type Ⅲ collagens in a dose-dependent manner in the CFs.At the concentration of 5 μg/L,the synthesis of collagen Ⅰ and Ⅲ was significantly increased as compared with control group（ P〈0.01）.RXR agonist 9-cis-retinoic acid（ 9-cis-RA; 10^-9- 10^-6mol/L） decreased TGF-β1（ 5 μg/L）-induced synthesis of type Ⅰ and Ⅲ collagens in a dose-dependent manner in the CFs under hypoxic condition.The synthesis of type Ⅰ and type Ⅲ collagens was significantly inhibited by 9-cis-RA（ P〈0.01）.Smad2 inhibitor（ 20 nmol/L） showed similar inhibitory effect on the synthesis of type Ⅰ and Ⅲ collagens induced by TGF-β1 under hypoxic condition.Compared with TGF-β1 intervention group,the cytoplasmic level of p-Smad2 in the CFs was significantly increased in TGF-β1 ＋ 9-cis-RA group,but the nuclear p-Smad2 level was significantly decreased（ P〈0.05）.CONCLUSION：Retinoid X receptor agonist 9-cis-RA inhibits TGF-β1-induced synthesis of type Ⅰ and type Ⅲ collagens in the CFs by repressing p-Smad2 nuclear translocation under hypoxic condition.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2016年第12期2228-2232,共5页
Chinese Journal of Pathophysiology
基金
福建省卫生系统中青年骨干人才培养重点项目(No.2013-ZQN-ZD-18)
