摘要
目的探讨血管紧张素（Ang）1-7对博来霉素诱导大鼠肺纤维化的抑制作用。方法18只Wister雄性大鼠按随机数字表法随机分为对照组、博来霉素组和博来霉素＋Angl-7组。均以微量泵0．29μl／h皮下24h恒速持续泵注4周，具体为：博来霉素组以博来霉素气管内滴入并泵内注入注射用水，博来霉素＋Angl-7组以博来霉素气管内滴入并泵内注入Angl-7（25μg·kg-1·h-1），对照组以生理盐水气管内滴入并泵内注入注射用水。4周后HE、Masson染色评价纤维化程度，测定羟脯氨酸含量，Western印迹法分析检测I型胶原蛋白，实时定量聚合酶链反应（RT-PCR）检测转化生长因子（TGF）-β及a-I型胶原mRNA含量。培养人胚肺成纤维细胞（HFL-1），设置无刺激对照组、AnglI刺激组（10-7mol／L的Angll刺激）、Angl-7刺激组（10-7mol／L的Ang1-7刺激）、Ang11＋Angl-7刺激组（上述浓度的Angl-7和AngⅡ刺激）、Ang11＋Angl-7＋A779刺激组（10-6mol／L的A779干预1h后予上述浓度Angl-7和AngⅡ刺激）。利用QuantiGene多基因定量检测下游因子TGF-β1、RT-PCR检测a-I型胶原mRNA表达情况。结果体内实验：与对照组比较，博来霉素组纤维化程度增高，组织中羟脯氨酸的含量明显增多[（3．69±0．18）比（0．50±0．15）mg／g，P〈0．05]；博来霉素＋Angl-7组纤维化程度减轻，羟脯氨酸含量[（2．14±0．29）mg／g]明显减少（P〈0．05）；博来霉素组TGF-β1mRNA、a-I型胶原mRNA和d-I型胶原蛋白相对表达量分别为4．45±0．45、5．14±0．55和1．48±0．34，均高于对照组的1．00±0．20、1．00±0．08和0．23±0．11（均P〈0．05）；而博来霉素＋Angl-7组上述表达量（2．80±0．35、3．10±0．52和0．49±0．11）均低于博来霉素组（均P〈0．05）。体外实验：AngⅡ刺激组TGF-β1mRNA和a-I型胶原mRNA的相对表达量（1．67±0．26和4．86±1．36）均高于无刺激对照组（1．00±0．10和1．46±0．54）（均P〈0．05）；而Angl-7刺激组上述表达量（1．30±0．22和2．07±1．05）与无刺激对照组差异均无统计学意义（均P〉0．05）；AngⅡ＋Angl-7刺激组上述表达量（0．91±0．30和1．57±0．27）均低于AngllⅡ激组（均P〈0．05）；AngⅡ＋Angl-7＋A-779刺激组上述表达量（1．25±0．14和1．29±0．49）与AngⅡ＋Angl-7刺激组差异均无统计学意义（均P〉0．05）。结论Ang1-7对博来霉素所致的肺纤维化具有抑制作用，该作用可能与Ang1-7抑制AngⅡ诱导TGF-β1的表达有关。
Objective To explore the anti-fibrotic effects of angiotensin （Ang） 1-7 on bleomycin （BLM） -induced pulmonary fibrosis in rats. Methods Eighteen Wister male rats were randomly divided into 3 groups, including control group （ intratracheal instillation with physiological saline and subcutaneous micro- pump with hi-distilled water at the rate of 0. 29 μl/h）, BLM group （ intratracheal instillation with bleomycin and subcutaneous micro-pump with bi-distilled water at the same rate） and BLM ＋ Angl-7 group （ intratracheal instillation with bleomycin and subcutaneous micro-pump with Angl-7 at a dose of 25 μg ·kg-1· h-1 at the same rate）. At Day 28, lung tissues were collected. Histological changes of lungs were evaluated by hematoxylin and eosin and Masson＇s trichrome stains. Collagen content of lung tissues was assessed by hydroxyprolin concentration. Then the products of protein and RNA were collected. And Western blot and tealtime polymerase chain reaction （RT-PCR） were used to detect the protein or mRNA of TGF-β1 and a-collagen I . Human embryonic lung fibroblast （ HFL-1 ） was divided into 5 groups： （ 1 ） control group： no stimulation; （2） Ang U group： stimulation of Ang H （ 10-7 moL/L） ; （3） Angl-7 group： stimulation of Angl-7 （ 10 -7 mol/L） ; （4） Angl-7 plus Aug Ⅱ group ： stimulation by AngⅡ （ 10 -7 mol/L） with Angl-7 （10-7mol/L） pre-treatment; （5） Angl-7 ＋ Ang Ⅱ ＋ A-779 group： stimulation by Ang Ⅱ and Angl-7 （ 10-7 mol/L） with Mas receptor inhibitor A-779 （10-6mol/L） pre-treatment. Then the products of protein and RNA were collected. And QuantiGene and RT-PCR were used to detect the activation of TGF-β1, and a-collagen I mRNA. Results Compared with control group, fibrosis score and hydroxyproline concentrations increased significantly in BLM group, but declined in BLM ＋ Angl-7 group. The difference was statistically significant （P 〈 0.05 ）. TGF-β1 mRNA, a-collagen 1 mRNA and a-collagen [ protein level were up-regulated by BLM （4.45 ±0. 45 vs 1.00 ±0. 20, 5. 14 ±0. 55 vs 1.00 ±0. 08, 1.48 ±0. 34 vs 0.. 23 ± 0. 11 ） （ all P 〈 0. 05） ; while compared with BLM group, those of BLM ＋ Ang1-7 group were down-regulated （2. 80 ± 0.35, 3. 10 ±0.52, O. 49± O. 11 ） （ all P 〈 0.05 ）. In vitro ： TGF-β1 mRNA and a-collagen I mRNA level were up-regulated by Ang Ⅱ （ 1.67± 0. 26 vs 1.00 ±0. 10, 4.86±1.36 vs 1.46 ±0. 54） （all P 〈0. 05） ; while those of Ang Ⅱ ＋ Angl-7 group were down-regulated （0. 91 ± 0. 30, 1.57 ± 0. 27） compared with Ang Ⅱgroup （ all P 〈 0. 05 ） ; no significant difference existed between the Ang Ⅱ ＋ Angl-7 ＋ A-779 group （1.25 ±0. 14, 1.29 ±0.49） and Ang Ⅱ ＋Angl-7 group （P〉 0.05 ）. Conclusion Ang1-7 has anti-fibrous effect upon bleomycin-indueed pulmonary fibrosis in rats andsuch an effect of Angl-7 may be associated with Ang Ⅱ -induced expression of TGF-β1.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2013年第20期1585-1589,共5页
National Medical Journal of China
基金
国家自然科学基金（30900659）
广东省科技计划项目（20098060300021）
