银杏叶提取物抗大鼠肺纤维化机制的研究
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摘要
肺纤维化是多种原因所致的严重肺损伤的共同结局，也是患者死亡的主要原因，其机制尚未完全阐明，且缺乏有效的治疗手段。目前认为，氧化应激导致肺上皮损伤、炎症细胞浸润及随后细胞因子网络的平衡的打破，最终导致基质重塑和胶原大量堆积。在众多的细胞因子中，转化生长因子β1(Transforming growth factor-β1,TGF-β1)在肺纤维化形成过程中由多种炎症细胞和肺组织细胞分泌，它具有趋化成纤维细胞、促进其增殖并刺激其分泌胶原增多的作用。银杏叶提取物(Ginkgo biloba Extract,GbE)是一种传统的中药，具有抗炎、降血脂、抗过敏等多重药理作用。已有文献报道，GbE具有抗大鼠实验性肝纤维化和肺纤维化的作用，且可能与减低TGF-β1的表达有关。TGF-β1在博莱霉素(Bleomycin,BLM)诱导的肺纤维化中的表达是广泛的，在纤维化形成的不同阶段，GbE对肺部不同细胞类型和不同部位影响TGF-β1表达的详细机制还不清楚。
泡沫细胞是巨噬细胞吞噬了大量的脂质过氧化物而形成的。泡沫细胞出现在大鼠整个肺纤维化形成发展的过程中，且与Ⅰ、Ⅲ型胶原相伴行。泡沫细胞在动脉粥样硬化及其他纤维化疾病，可以促进炎症反应，但是它与纤维化形成的关系还不是很确定。此外，体外实验证明泡沫细胞与巨噬细胞相比由于细胞膜表达尿激酶型血浆素原激活剂，纤溶酶原可以刺激泡沫细胞比巨噬细胞表达更多的TGF-β1。银杏
中文摘要
叶提取物的主要成分黄酮是一种有效的抗氧化剂，而另一种
主要成分菇内醋可以减少血小板的积聚，减轻其活性，同时
还是血小板激活因子受体的有效拮抗剂。过氧化和血小板聚
集都有效的刺激了泡沫细胞的形成。所以，我们欲通过GbE
干预泡沫细胞的形成，来观察GbE是否可以通过减少泡沫细
胞的数量来减少TGF一困蛋白的表达。也为阐明氧化应激导
致TGF一印增多的细胞机制提供实验证据。
1 GbE减少TGF一pl蛋白表达及其抗大鼠肺纤维化作用的
观察
目的通过观察GbE对大鼠TGF一困蛋白的表达来阐述
GbE减轻博莱霉素诱导肺纤维化的机制。
方法将30只雄性SD大鼠分成五组(n二6):①Ctd组:
不作特殊处理;②BLM14天组:一次性气管内注射博莱霉
素AS(BLMAS)造模，于14天取材;③BLM30天组:一
次性气管内注射BLMAS造模，30天取材;④GbE14天组:
造模后，每日灌胃给予GbE，直到14天时处死;⑤GbE 30
天组:造模后，每日灌胃给予GbE，直到30天时处死。
通过检测大鼠肺组织轻脯氨酸观察肺胶原含量的变化;
利用Masson染色观察纤维化程度的变化;利用HE染色观
察肺组织损伤的变化。利用免疫组织化学的方法观察注射
BLMA530天时，肺组织各部分TGF一卿蛋白表达的变化;利
用免疫细胞化学的方法观察注射BLMA514天及30天时，各
组BALF细胞TGF一印蛋白表达的变化。
结果给予GbE30天后，肺组织经脯氨酸(巧.46士0.52
mg.g.’lung weight)含量较BLM组(1 5.87士0.62 mg.g”lung
weight)显著减少(p<0.01)，肺组织胶原含量减少。Masson
中文摘要
染色观察:Ctd组，肺泡结构正常，胶原窄小，衬于肺泡细
胞下方;BLM 30天组，肺泡隔增厚明显，胶原呈粗条索状、
甚至出现纤维斑块;而GbE30天组肺泡隔增厚较轻，胶原
条带较BLM30天组的窄小、且不连续。纤维化分级系数:
BLM 30天组(4.5士0.43)t匕Ctrl组(0.33士0.21)显著增加
(P<0.01)，而GbE 30天组(3.38士0.26)比BLM 30天组
显著减低(P<0.01)。HE染色进行形态学观察:Ctri组，肺
组织结构清晰，无炎症表现;BLM30天组，肺泡壁明显增
厚，肺泡结构消失，有一定量的炎性细胞浸润，大量的成纤
维细胞增殖并出现大量的胶原积聚;GbE30天组，炎细胞浸
润、肺泡隔增厚程度、肺实变等肺损伤程度较BLM 30天组
有所减轻。肺泡炎分级结果如下:BLM30天组(4.00士0.26)
与Ctrl组(0.33士0.21)相比显著增加(P<0.01);GbE 30
天组(3.25士0.16)与BLM30天组相比显著减少(P<0.05)。
以上结果证明GbE对博莱霉素引起的肺损伤和纤维化发生
有保护作用。
免疫组化结果显示:Ctri组，TGF一pl支气管粘膜上皮细
胞、血管内皮细胞和肺内出现的少量的炎细胞为阳性表达。
BLM组30天，TGF一邵除了在以上部位表达外，在肺增厚的
间隔及肺实变区的出现大量的强阳性表达，且成纤维样细胞
强表达。与Ctrl组(6.33士0.68%)相t匕，BLM 30天组TGF一pl
阳性细胞数(44.88士1 .84%)显著增高(P<0.01)，而GbE 30
天组TGF一pl阳性细胞数(3 2.3士1 .24%)与BLM 30天组相
比显著减少(尸<0.01)。BALF中的细胞表达TGF一pl的情况
如下:BLM 14、30天组的TGF一pl阳性率(69.22士1 .53、46.42
士4.39%)均明显高于Ctrl组(1 0.93士0.82%)(P<0.01)，
中文摘要
且BLM 30天组比BLM 14天组显著减少(P<0.01)。GbE 14
天组(55.55士1.91%)与BLM一4天组相比，ToF一pl阳性率
显著降低(p<0.01);而GbE 30天组TGF一pl阳性率(41.18
士3.31%)与BLM 30天组相比，无显著性差异(尸>0.05)。
两部分免疫组织化学的结果综合分析:在肺组织的炎症
反应期(应用BLM14天)，GbE可以减少BALF细胞TGF一pl
的蛋白表达;在肺组织的纤维化期(应用BLM 30天)，GbE
并未显著减少BALF细胞TGF一哪的表达，
The mechanisms of pulmonary fibrosis, a consequence of complex and severe lung injury, are still uncertain. The irreversible end-stage organ failure is a major cause of morbidity and mortality, and treating the progressive fatal diseases is a major challenge in modern medicine. The destruction of alveolar epithelium barrier, inflammatory cell infiltration and disequilibrium on the cytokine-network, and subsequent collagen accumulation may be the common pathway in different types of pulmonary fibrosis. Among numerous cytokines contributing to promote pulmonary fibrosis, transforming growth factorpi (TGF-P1) that is expressed by many inflammatory cells and intrinsic cells, is a fibroblast chemoattractant and is able to exert an effect on fibroblast proliferation. Moreover, it is also the most potent stimulator of fibroblast collagen production. Ginkgo biloba, a traditional Chinese medicine, has many pharmacological activities such as anti-inflammation, anti-allergy and lowering blood viscosity. It has been rep
orted that GbE has protective effect against many fibrotic diseases, which are thought to be related to the reduction of TGF protein expression. Since TGF- was extensively
expressed in lung tissue during fibrosis development, it is still unknown about the detailed mechanisms of the effect of GbE on the reduction of TGF-pl in different cell type and in various stage of pulmonary fibrosis.
Noticeably, foam cells, which swallow mass of lipid peroxides, are present coincidently with collagen during the development of pulmonary fibrosis. Foam cell , present in atherosclerosis and other chronic fibrotic disease, has many different characteristics from macrophage during the pathologic process , which pushes forward the chronic inflammation process. However, the relationship between the foam cell and fibrosis is still uncertain. Additionally, in vitro foam cells expressing more membrane uPA (urokinase-type plasminogen activator), can release more TGF-(31 than macrophages in presence of plasminogen. One griedient of GbE, flavonoid, is functioned as an effective scanvager of reactive oxygen species and lipid peroxides. Another componet of GbE, ginkgolide is able to suppress platelet aggregation and activation. Moreover ginkgolide is also an specific antagonist to PAF(platelet activating factor) receptor. Lipid peroxidation , as well as platelet aggregation and activation are both the stimulating factors to the foam cell formation. Therefore, we try to apply GbE to bleomycin-induced rats to intervene the formation of foam cells in lung. We investigate wether GbE can down-regulate the expression of TGF-J31 in BALF by decreasing the foam cells number, thereby providing
experimental evidence for clarifying the cell mechanisms of up-regulation of TGF-01 induced by oxidative stress. 1 The investigation on effect of GbE on reduction of TGF-pl expression and against pulmonary fibrosis in rats.
Aim To investigate the effect of GbE on the bleomycin-induced pulmonary fibrosis and TGF-01 expression in rats.
Methods 32 rats were randomly divided into five groups: control group, BLM 14th group , BLM 30th group , BLM 14th group and GbE 30th group. Firstly, the content of lung hydroxproline was measured and the fibrosis and alveolitis grade was observed to investigate the effect of GbE on the bleomycin-induced lung injury and fibrosis. Using immunocytochemical and immunohistochemical method, we observed the changes of TGF- ]3 1 immunoreactive cell percentage and in BALF and in tissue of rats treating with GbE after intratracheal instillation of BLMA5 on different time point.
Results The content of lung hydroxproline (collagen content) in GbE group (15.46?.52 mg.g"1 lung weight) was significantly less than that in BLM group (18.87?.62 mg.g"1 lung weight, P<0.01) on 30th days. In Masson staining, lungs from the control group showed normal construction and few collagenosis. Lungs from BLM group on 30th day showed remarkable tile-like collagen accumulation. While lungs from GbE group showed fewer fibrotic area. Changes of pulmo
泡沫细胞是巨噬细胞吞噬了大量的脂质过氧化物而形成的。泡沫细胞出现在大鼠整个肺纤维化形成发展的过程中，且与Ⅰ、Ⅲ型胶原相伴行。泡沫细胞在动脉粥样硬化及其他纤维化疾病，可以促进炎症反应，但是它与纤维化形成的关系还不是很确定。此外，体外实验证明泡沫细胞与巨噬细胞相比由于细胞膜表达尿激酶型血浆素原激活剂，纤溶酶原可以刺激泡沫细胞比巨噬细胞表达更多的TGF-β1。银杏
中文摘要
叶提取物的主要成分黄酮是一种有效的抗氧化剂，而另一种
主要成分菇内醋可以减少血小板的积聚，减轻其活性，同时
还是血小板激活因子受体的有效拮抗剂。过氧化和血小板聚
集都有效的刺激了泡沫细胞的形成。所以，我们欲通过GbE
干预泡沫细胞的形成，来观察GbE是否可以通过减少泡沫细
胞的数量来减少TGF一困蛋白的表达。也为阐明氧化应激导
致TGF一印增多的细胞机制提供实验证据。
1 GbE减少TGF一pl蛋白表达及其抗大鼠肺纤维化作用的
观察
目的通过观察GbE对大鼠TGF一困蛋白的表达来阐述
GbE减轻博莱霉素诱导肺纤维化的机制。
方法将30只雄性SD大鼠分成五组(n二6):①Ctd组:
不作特殊处理;②BLM14天组:一次性气管内注射博莱霉
素AS(BLMAS)造模，于14天取材;③BLM30天组:一
次性气管内注射BLMAS造模，30天取材;④GbE14天组:
造模后，每日灌胃给予GbE，直到14天时处死;⑤GbE 30
天组:造模后，每日灌胃给予GbE，直到30天时处死。
通过检测大鼠肺组织轻脯氨酸观察肺胶原含量的变化;
利用Masson染色观察纤维化程度的变化;利用HE染色观
察肺组织损伤的变化。利用免疫组织化学的方法观察注射
BLMA530天时，肺组织各部分TGF一卿蛋白表达的变化;利
用免疫细胞化学的方法观察注射BLMA514天及30天时，各
组BALF细胞TGF一印蛋白表达的变化。
结果给予GbE30天后，肺组织经脯氨酸(巧.46士0.52
mg.g.’lung weight)含量较BLM组(1 5.87士0.62 mg.g”lung
weight)显著减少(p<0.01)，肺组织胶原含量减少。Masson
中文摘要
染色观察:Ctd组，肺泡结构正常，胶原窄小，衬于肺泡细
胞下方;BLM 30天组，肺泡隔增厚明显，胶原呈粗条索状、
甚至出现纤维斑块;而GbE30天组肺泡隔增厚较轻，胶原
条带较BLM30天组的窄小、且不连续。纤维化分级系数:
BLM 30天组(4.5士0.43)t匕Ctrl组(0.33士0.21)显著增加
(P<0.01)，而GbE 30天组(3.38士0.26)比BLM 30天组
显著减低(P<0.01)。HE染色进行形态学观察:Ctri组，肺
组织结构清晰，无炎症表现;BLM30天组，肺泡壁明显增
厚，肺泡结构消失，有一定量的炎性细胞浸润，大量的成纤
维细胞增殖并出现大量的胶原积聚;GbE30天组，炎细胞浸
润、肺泡隔增厚程度、肺实变等肺损伤程度较BLM 30天组
有所减轻。肺泡炎分级结果如下:BLM30天组(4.00士0.26)
与Ctrl组(0.33士0.21)相比显著增加(P<0.01);GbE 30
天组(3.25士0.16)与BLM30天组相比显著减少(P<0.05)。
以上结果证明GbE对博莱霉素引起的肺损伤和纤维化发生
有保护作用。
免疫组化结果显示:Ctri组，TGF一pl支气管粘膜上皮细
胞、血管内皮细胞和肺内出现的少量的炎细胞为阳性表达。
BLM组30天，TGF一邵除了在以上部位表达外，在肺增厚的
间隔及肺实变区的出现大量的强阳性表达，且成纤维样细胞
强表达。与Ctrl组(6.33士0.68%)相t匕，BLM 30天组TGF一pl
阳性细胞数(44.88士1 .84%)显著增高(P<0.01)，而GbE 30
天组TGF一pl阳性细胞数(3 2.3士1 .24%)与BLM 30天组相
比显著减少(尸<0.01)。BALF中的细胞表达TGF一pl的情况
如下:BLM 14、30天组的TGF一pl阳性率(69.22士1 .53、46.42
士4.39%)均明显高于Ctrl组(1 0.93士0.82%)(P<0.01)，
中文摘要
且BLM 30天组比BLM 14天组显著减少(P<0.01)。GbE 14
天组(55.55士1.91%)与BLM一4天组相比，ToF一pl阳性率
显著降低(p<0.01);而GbE 30天组TGF一pl阳性率(41.18
士3.31%)与BLM 30天组相比，无显著性差异(尸>0.05)。
两部分免疫组织化学的结果综合分析:在肺组织的炎症
反应期(应用BLM14天)，GbE可以减少BALF细胞TGF一pl
的蛋白表达;在肺组织的纤维化期(应用BLM 30天)，GbE
并未显著减少BALF细胞TGF一哪的表达，
The mechanisms of pulmonary fibrosis, a consequence of complex and severe lung injury, are still uncertain. The irreversible end-stage organ failure is a major cause of morbidity and mortality, and treating the progressive fatal diseases is a major challenge in modern medicine. The destruction of alveolar epithelium barrier, inflammatory cell infiltration and disequilibrium on the cytokine-network, and subsequent collagen accumulation may be the common pathway in different types of pulmonary fibrosis. Among numerous cytokines contributing to promote pulmonary fibrosis, transforming growth factorpi (TGF-P1) that is expressed by many inflammatory cells and intrinsic cells, is a fibroblast chemoattractant and is able to exert an effect on fibroblast proliferation. Moreover, it is also the most potent stimulator of fibroblast collagen production. Ginkgo biloba, a traditional Chinese medicine, has many pharmacological activities such as anti-inflammation, anti-allergy and lowering blood viscosity. It has been rep
orted that GbE has protective effect against many fibrotic diseases, which are thought to be related to the reduction of TGF protein expression. Since TGF- was extensively
expressed in lung tissue during fibrosis development, it is still unknown about the detailed mechanisms of the effect of GbE on the reduction of TGF-pl in different cell type and in various stage of pulmonary fibrosis.
Noticeably, foam cells, which swallow mass of lipid peroxides, are present coincidently with collagen during the development of pulmonary fibrosis. Foam cell , present in atherosclerosis and other chronic fibrotic disease, has many different characteristics from macrophage during the pathologic process , which pushes forward the chronic inflammation process. However, the relationship between the foam cell and fibrosis is still uncertain. Additionally, in vitro foam cells expressing more membrane uPA (urokinase-type plasminogen activator), can release more TGF-(31 than macrophages in presence of plasminogen. One griedient of GbE, flavonoid, is functioned as an effective scanvager of reactive oxygen species and lipid peroxides. Another componet of GbE, ginkgolide is able to suppress platelet aggregation and activation. Moreover ginkgolide is also an specific antagonist to PAF(platelet activating factor) receptor. Lipid peroxidation , as well as platelet aggregation and activation are both the stimulating factors to the foam cell formation. Therefore, we try to apply GbE to bleomycin-induced rats to intervene the formation of foam cells in lung. We investigate wether GbE can down-regulate the expression of TGF-J31 in BALF by decreasing the foam cells number, thereby providing
experimental evidence for clarifying the cell mechanisms of up-regulation of TGF-01 induced by oxidative stress. 1 The investigation on effect of GbE on reduction of TGF-pl expression and against pulmonary fibrosis in rats.
Aim To investigate the effect of GbE on the bleomycin-induced pulmonary fibrosis and TGF-01 expression in rats.
Methods 32 rats were randomly divided into five groups: control group, BLM 14th group , BLM 30th group , BLM 14th group and GbE 30th group. Firstly, the content of lung hydroxproline was measured and the fibrosis and alveolitis grade was observed to investigate the effect of GbE on the bleomycin-induced lung injury and fibrosis. Using immunocytochemical and immunohistochemical method, we observed the changes of TGF- ]3 1 immunoreactive cell percentage and in BALF and in tissue of rats treating with GbE after intratracheal instillation of BLMA5 on different time point.
Results The content of lung hydroxproline (collagen content) in GbE group (15.46?.52 mg.g"1 lung weight) was significantly less than that in BLM group (18.87?.62 mg.g"1 lung weight, P<0.01) on 30th days. In Masson staining, lungs from the control group showed normal construction and few collagenosis. Lungs from BLM group on 30th day showed remarkable tile-like collagen accumulation. While lungs from GbE group showed fewer fibrotic area. Changes of pulmo
引文
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