血管紧张素1-7对博来霉素诱导大鼠肺纤维化的抑制作用
혈관긴장소1-7대박래매소유도대서폐섬유화적억제작용
Anti-fibrotic effects of angiotensin1-7 on bleomycin-induced pulmonary fibrosis in rats
  • 万方数据
    中华医学杂志 중화의학잡지
    National Medical Journal of China
    2013年 20期 1585-1589 ,共5页

    孟莹%余常辉%蔡绍曦%李旭

    맹형%여상휘%채소희%리욱

    血管紧张素类%转化生长因子β%肺纤维化%博来霉素 血管緊張素類%轉化生長因子β%肺纖維化%博來黴素
    혈관긴장소류%전화생장인자β%폐섬유화%박래매소
    Angiotensins%Transforming growth factor beta%Pulmonary fibrosis%Bleomycin
    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+Ang1-7组.均以微量泵0.29 μl/h皮下24 h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+ Ang1-7组以博来霉素气管内滴入并泵内注入Ang1-7(25 μg·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α-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、AngⅡ刺激组(10-7 mol/L的AngⅡ刺激)、Ang1-7刺激组(10-7mol/L的Ang 1-7刺激)、AngⅡ+Ang1-7刺激组(上述浓度的Ang1-7和AngⅡ刺激)、AngⅡ+Ang1-7+ A779刺激组(10-6mol/L的A779干预1h后予上述浓度Ang1-7和AngⅡ刺激).利用QuantiGene多基因定量检测下游因子TGF-β1、RT-PCR检测α-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+ Ang1-7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-β1 mRNA、α-Ⅰ型胶原mRNA和α-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+ Ang1-7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-β1 mRNA和α-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Ang1-7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+ Ang1-7 刺激组上述表达量(0.91±0.30和1.57±0.27)均低于AngⅡ刺激组(均P＜0.05)；AngⅡ+ Ang1-7+A-779刺激组上述表达量(1.25 ±0.14和1.29±0.49)与AngⅡ+Ang1-7刺激组差异均无统计学意义(均P＞0.05).结论 Ang 1-7对博来霉素所致的肺纤维化具有抑制作用,该作用可能与Ang 1-7抑制AngⅡ诱导TGF-β1的表达有关.
    목적 탐토혈관긴장소(Ang) 1-7대박래매소유도대서폐섬유화적억제작용.방법 18지Wister웅성대서안수궤수자표법수궤분위대조조、박래매소조화박래매소+Ang1-7조.균이미량빙0.29 μl/h피하24 h항속지속빙주4주,구체위:박래매소조이박래매소기관내적입병빙내주입주사용수,박래매소+ Ang1-7조이박래매소기관내적입병빙내주입Ang1-7(25 μg·kg-1·h-1),대조조이생리염수기관내적입병빙내주입주사용수.4주후HE、Masson염색평개섬유화정도,측정간포안산함량,Western인적법분석검측Ⅰ형효원단백,실시정량취합매련반응(RT-PCR)검측전화생장인자(TGF)-β급α-Ⅰ형효원mRNA함량.배양인배폐성섬유세포(HFL-1),설치무자격대조조、AngⅡ자격조(10-7 mol/L적AngⅡ자격)、Ang1-7자격조(10-7mol/L적Ang 1-7자격)、AngⅡ+Ang1-7자격조(상술농도적Ang1-7화AngⅡ자격)、AngⅡ+Ang1-7+ A779자격조(10-6mol/L적A779간예1h후여상술농도Ang1-7화AngⅡ자격).이용QuantiGene다기인정량검측하유인자TGF-β1、RT-PCR검측α-Ⅰ형효원mRNA표체정황.결과 체내실험:여대조조비교,박래매소조섬유화정도증고,조직중간포안산적함량명현증다[(3.69±0.18)비(0.50±0.15) mg/g,P＜0.05]；박래매소+ Ang1-7조섬유화정도감경,간포안산함량[(2.14±0.29) mg/g]명현감소(P＜0.05)；박래매소조TGF-β1 mRNA、α-Ⅰ형효원mRNA화α-Ⅰ형효원단백상대표체량분별위4.45 ±0.45、5.14±0.55화1.48±0.34,균고우대조조적1.00±0.20、1.00±0.08화0.23 ±0.11(균P＜0.05)；이박래매소+ Ang1-7조상술표체량(2.80±0.35、3.10±0.52화0.49±0.11)균저우박래매소조(균P＜0.05).체외실험:AngⅡ자격조TGF-β1 mRNA화α-Ⅰ형효원mRNA적상대표체량(1.67±0.26화4.86±1.36)균고우무자격대조조(1.00±0.10화1.46±0.54)(균P＜0.05)；이Ang1-7자격조상술표체량(1.30±0.22화2.07±1.05)여무자격대조조차이균무통계학의의(균P＞0.05)；AngⅡ+ Ang1-7 자격조상술표체량(0.91±0.30화1.57±0.27)균저우AngⅡ자격조(균P＜0.05)；AngⅡ+ Ang1-7+A-779자격조상술표체량(1.25 ±0.14화1.29±0.49)여AngⅡ+Ang1-7자격조차이균무통계학의의(균P＞0.05).결론 Ang 1-7대박래매소소치적폐섬유화구유억제작용,해작용가능여Ang 1-7억제AngⅡ유도TGF-β1적표체유관.
    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 micropump with bi-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 + Ang1-7 group (intratracheal instillation with bleomycin and subcutaneous micro-pump with Ang1-7 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 realtime polymerase chain reaction (RT-PCR) were used to detect the protein or mRNA of TGF-β1 and α-collagen Ⅰ.Human embryonic lung fibroblast (HFL-1) was divided into 5 groups:(1) control group:no stimulation; (2) AngⅡ group:stimulation of AngⅡ (10-7mol/L) ; (3) Ang1-7 group:stimulation of Ang1-7 (10-7mol/L) ; (4) Ang1-7 plus AngⅡ group:stimulation by AngⅡ (10-7mol/L) with Ang1-7 (10-7mol/L) pre-treatment; (5) Ang1-7 +AngⅡ + A-779 group:stimulation by AngⅡ and Ang1-7 (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 α-collagen Ⅰ mRNA.Results Compared with control group,fibrosis score and hydroxyproline concentrations increased significantly in BLM group,but declined in BLM + Ang1-7 group.The difference was statistically significant (P ＜ 0.05).TGF-β1 mRNA,α-collagen Ⅰ mRNA and α-collagen Ⅰ 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 ± 0.52,0.49 ± 0.11) (all P ＜ 0.05).In vitro:TGF-β1 mRNA and α-collagen Ⅰ mRNA level were up-regulated by AngⅡ (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 + Ang1-7 group were down-regulated (0.91 ± 0.30,1.57 ± 0.27) compared with Ang Ⅱ group (all P ＜ 0.05) ; no significant difference existed between the Ang Ⅱ + Ang1-7 + A-779 group (1.25 ± 0.14,1.29 ± 0.49) and Ang Ⅱ + Ang1-7 group (P ＞ 0.05).Conclusion Ang1-7 has anti-fibrous effect upon bleomycin-induced pulmonary fibrosis in rats andsuch an effect of Ang1-7 may be associated with Ang Ⅱ-induced expression of TGF-β1.
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