肺纤方干预肺纤维化大鼠微血管新生的实验研究
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摘要
目的：观察肺纤方治疗博莱霉素诱发大鼠肺纤维化的疗效,探讨肺纤维化的发病机制和肺纤方治疗肺纤维化的作用机理。
方法：Wistar雄性大鼠80只,体重200±20g,随机数字表法分为空白组、模型组、激素组、中药组,每组20只。大鼠置于屏障级动物室适应性喂养1周。模型组、激素组、中药组气管插管后一次性推注博莱霉素1mg/kg剂量,空白组气管内一次性推注等体积的生理盐水。造模后第2天开始灌胃给药。中药组肺纤方给药剂量为1.25g／(kg·d),激素组强的松给药剂量为3mg/(kg·d).模型组和空白组给予等体积0.5%羧甲基纤维素钠溶液。14d、28d分批处死动物。用3.5%水合氯醛1 m1／100g体重,腹腔注射麻醉大鼠后。以动脉血气针取腹主动脉血2ml,15min内送检。
打开胸腔,行肺组织灌注。待大鼠头部、上肢、肺叶变硬时,停止灌注。取出肺脏,置于中性福尔马林中固定24h,常规脱水、石蜡包埋,连续组织切片(厚5μm),进行苏木精-伊红(HE)染色和三色(Masson)染色。
HE染色切片分为无染区、深染区、浅染区。OLYMPUS显微数码下采集图像。以Image-Pro Plus 6.0进行图像分析,测出各部分所占面积,取其平均值(mm2)。计算出各组肺泡炎和肺纤维化分级。
免疫组化SABC法检测大鼠肺组织切片血管内皮细胞CD34表达状况,用CD34抗体标记血管内皮细胞,镜下计数单位面积中的微血管数目,即微血管密度(MVD)。
免疫组化SABC法检测大鼠bFGF、VEGF、FLT-1、FLK-1、ANG-2的表达情况,结合图像分析技术,比较各组大鼠微血管新生促进因子表达情况。
结果：HE染色结果：空白组肺组织形态无明显病变。14d模型组肺泡隔明显增宽,肺泡隔和肺泡壁见较多炎细胞侵润。纤维结缔组织增生,出现实变区。14d激素组肺泡隔增宽,部分肺泡实变和大量炎性细胞浸润。14d中药组可见不同程度的炎症和纤维化灶,部分小支气管周围可见实变区。28d模型组、激素组、中药组炎症表现均较14d同组减轻。28d模型组肺泡隔明显增宽,胶原纤维显著增多,纤维组织呈斑片状分布,部分肺泡结构消失,萎缩闭塞,个别肺泡囊性扩张。肺泡隔和肺泡壁伴炎细胞侵润,较14d模型组减轻。28d激素组肺泡隔轻度纤维化。细小支气管周围可见纤维灶,部分肺泡和细支气管内可见炎性细胞。28d中药组肺泡隔纤维增生灶较少,肺泡内可见散在炎性细胞。
Masson染色结果：空白组肺组织形态无明显病变。14d模型组肺泡隔增宽,各级支气管和血管周围可见蓝色胶原纤维增生。14d中药组肺泡隔,各级支气管周围见少量纤细的胶原纤维。28d模型组肺泡壁、肺泡隔、终末细支气管、呼吸性细支气管周围和小支气管管壁可见大量胶原纤维增生。28d中药组胶原纤维增生较轻。激素组胶原纤维增生程度介于模型组和中药组之间。
HE染色形态定量分析结果：14d激素组深染区较模型组减少,差异有显著性意义(P<0.05)；14d中药组深染区较激素组明显减少,差异有非常显著性意义(P<0.01)。14d激素组浅染区与模型组比较,差异无显著性意义(P>0.05)；14d中药组浅染区较模型组明显减少,差异有非常显著性意义(P<0.01)。28d激素组深染区与模型组比较,差异无显著性意义(P>0.05)；28d中药组深染区较模型组明显减少,差异有非常显著性意义(P<0.01)。28d激素组浅染区与模型组比较,差异无显著性意义(P>0.05)；28d中药组浅染区较模型组明显减少,差异有非常显著性意义P<0.01)。
MVD结果：14d模型组、激素组MVD均较空白组增高,差异均有显著性意义(P<0.01,P<0.05)。28d模型组、激素组、中药组MVD均较同组14d降低,差异均有显著性意义(P<0.01,P<0.05,P<0.05)。激素组MVD与模型组比较,差异均无显著性意义(P>0.05)。14d中药组MVD较模型组降低,差异有显著性意义(P<0.05),28d中药组MVD较模型组明显降低,差异有非常显著性意义(P<0.01)。
微血管新生促进因子表达结果：模型组bFGF、ANG-2、FLK-1的表达均较空白组明显增强,差异均有非常显著性意义(P<0.01)；模型组VEGF表达均较空白组增强,差异均有显著性意义(P<0.05)；14d模型组FLT-1表达较空白组增强,差异有显著性意义(P<0.05),28d模型组FLT-1表达较空白组明显增强,差异有非常显著性意义(P<0.01)。中药组bFGF、ANG-2、FLK-1的表达均较模型组明显减弱,差异均有非常显著性意义(P<0.01)；中药组VEGF表达较模型组减弱,差异均有显著性意义(P<0.05)；14d中药组FLT-1表达较模型组减弱,差异有显著性意义(P<0.05),28d中药组FLT-1表达较模型组明显减弱,差异有非常显著性意义(P<0.01)。激素组bFGF、VEGF、FLT-1、FLK-1、ANG-2的表达与模型组比较,差异均无显著性意义(P>0.05)。
血气分析结果：模型组、激素组、中药组在14d和28d均可出现低氧血症。激素组Pa02与模型组比较,差异均无显著性意义(P>0.05)。中药组Pa02较模型组均明显升高,差异均有非常显著性意义(P<0.01)。
结论：1以博莱霉素1mg/kg剂量气管内推注成功复制了肺纤维化大鼠模型。
2博莱霉素诱发大鼠肺纤维化病变呈早期肺泡炎,晚期肺纤维化的演变过程。
3肺纤方具有改善肺纤维化大鼠肺泡炎症和肺纤维化的作用,疗效优于强的松。
4肺纤方可改善肺纤维化大鼠的低氧血症,这可能是通过肺纤方改善大鼠肺朝百脉的功能来实现的。
5博莱霉素诱发大鼠肺纤维化发病过程中存在病理性微血管新生,微血管新生以14d尤为活跃,随肺纤维化的加重病理性微血管新生逐渐减弱,肺组织微血管密度进行性减少。
6博莱霉素诱发大鼠肺纤维化发病过程中微血管新生促进因子bFGF、VEGF、FLT-1、FLK-1、ANG-2的表达上调,肺组织微血管新生活跃。微血管新生可能是肺纤维化发病过程中一个重要环节,抑制微血管新生可能会从根本上延缓肺纤维化进展。
7肺纤方治疗肺纤维化的机理可能是通过抑制微血管新生促进因子bFGF、VEGF、FLT-1、FLK-1、ANG-2的表达,进而抑制肺组织微血管新生,从而改善肺纤维化病情,延缓肺纤维化进展。
8肺纤方可能是通过改善肺朝百脉的功能,改善肺纤维化低氧血症,从而使微血管新生促进因子表达下调的。
Objectives:To observe effect of treating rats pulmonary fibrosis induced by BLM on Fei Xian formula, and explore the pathogenesis of pulmonary fibrosis and mechanism of pulmonary fibrosis treated by formula of Fei Xian.
Method:According to random number table,80 Wistar male rats, with the weight of 200±20g, were divided into four groups:blank group, model group, hormone group and TCM group, n= 20. Rats were placed in barrier-level animal house to be adaptively fed for 1 week. After tracheal intubation of model group, hormone group, and TCM group, a dosage of lmg/kg of bleomycin was bolus-injected respectively. For the blank group, it was bolus-injected equivalent volume of saline into trachea. Intragastric administration would begin in the second day after modeling. The dose of administration of Fei Xian formula in TCM group was 1.25g/(k·d); the dose of administration of prednisone in hormone group was 3mg/(kg·d); and for model group and blank group, they were given the equivalent volume of 0.5% sodium carboxymethyl cellulose. In the 14th and 28th days, these rats were sacrificed in batches. After anaesthetizing rats with an intraperitoneal injection of 3.5% chloral hydrate 1ml/100g weight, the volume of 2ml of abdominal aorta blood was withdrawn by arterial blood gas needle. It should be sent for inspection within 15 minutes.
Open the chest to perfuse the lung tissues. Stop infusing until rat's head, upper limbs, and pulmonary lobes became stiff. Before hematoxylin-eosin (HE) staining and three-color (Masson) staining, a serial preparations should be carried out, like remove of the lungs to neutral formalin for fixation for 24 hours; conventional dehydration, paraffin embedding, and serial tissue sections (thickness of 5μm).
HE staining section was divided into non-staining area, dark staining area, and light staining area. The images were pictured by digital microscope of OLYMPUS. Use Image-Pro plus 6.0 to analyze, to measure the area of each part, and choose their average (mm2). Calculate classification of each group's alveolitis and pulmonary fibrosis.
Expression of CD34 in vascular endothelial cells of lung tissue sections of rats was detected by immunohistochemistry SABC. The vascular endothelial cells labeled with CD34 antibody were counted the number of microvessel in unit area under microscope, which was called microvessel density (MVD).
Expressions of bFGF, VEGF, FLT-1, FLK-1, and ANG-2 were detected by immunohistochemistry SABC. Combined with image analysis, expression of promoting factor of microvascular angiogenesis of each group's rats could be compared.
Result:Results of HE staining:There was no obvious pathological change in pulmonary morphology in bland group. In 14d model group, the alveolar septum was obviously widened, also a large number of infiltrated inflammatory cells found in alveolar septum and wall, as well as hyperplasia of fibrous connective tissue, even consolidation. In 14d hormone group, the alveolar septum was widened, and part of alveoli changed into consolidation and large quantities of inflammatory cells infiltrated. Inflammation and fibrosis foci at different degrees could be found in 14d TCM group and also consolidation around the part of small bronchi. Compared with 14d model group, hormone group, and TCM group, inflammatory signs in the same groups of 28d greatly abated. In 28d model group, alveolar septa obviously widened, collagen fibers significantly increased, fibrous tissues distributed in patch, the structure of part alveoli disappeared, shrank, and occluded, and several alveoli expanded in cyst. Compared with 14d model group, infiltration of inflammatory cells in alveolar septum and wall is in greater reduction. Mild fibrosis of alveolar septum was found in 28d hormone group. Fibrosis foci could be seen around the small bronchioles and inflammatory cells appeared in part of alveoli and bronchioles. In 28d TCM group, fibroplasias foci of alveolar septum were less, and the scattered inflammatory cells could be found in alveoli.
Results of Masson staining:There was no obvious pathological change in pulmonary morphology in bland group. In 14d model group, alveolar septum widened, and the hyperplasia of blue collagen fibers could be seen around each bronchus and blood vessel. In 14d TCM group, a scanty of fine collagen fibers could be found in alveolar septa and around each bronchus. In 28d model group, a large number of hyperplasia of collagen fibers appeared in alveolar walls and septa, terminal bronchioles, walls of small bronchus, and around respiratory bronchioles. The hyperplasia of collagen fibers is slight in 28d TCM group. In hormone group, the degree of collagen fiber hyperplasia was between model group and TCM group.
The result of morphological and quantitative analysis of HE staining:At the fourteenth day, the deep-staining region of hormone group is smaller than that of model group; and there is a significant difference (P<0.05). At the same stage, the deep-staining region of TCM group is much smaller than that of hormone group; and there is a more significant difference (P<0.01). At the fourteenth day, there is no significance to the difference between the light-staining region of hormone group and that of model group (P>0.05). At the same time, the light-staining region of TCM group is much smaller than that of model group; and there is a obvious significant difference (P<0.01). At the 28th day, to the comparison between the deep-staining region of hormone group and that of model group, there is no significance (P>0.05). At this stage, the deep-staining region is much smaller than that of model group, and there is an obvious significant difference (P<0.01). At the 28th day, to the comparison between the light-staining region of hormone group and that of model group, there is no significance (P>0.05). At the same stage, compared with the light-staining region of model group, the region of TCM group is evidently reduced; the difference is obviously significant (P<0.01).
MVD result:At the 14th day, MVDs of model group and hormone group are both higher than that of blank group; and there is significance in the differences (P<0.01, P<0.05). The MVDs of model group, hormone group and TCM group at the 28th day are all lower than theirselve at the 14th day; and the differences are all significant (P<0.01, P<0.05, P<0.05). To the comparison between the MVD of hormone group and that of model group, there is no obvious significance in the difference (P>0.05). Compared with the MVD of model group, the MVD of TCM group is reduced at the 14th day, the difference is significant (P<0.05); and it is distinctly reduced at the 28th day, the difference is obviously significant (P<0.01).
The results for the expressions of angiogenesis factors:Compared with blank group, the expressions of bFGF, ANG-2 and FLK-1 in model group are all increased obviously; and the differences are all obviously significant (P<0.01). The VEGF expression in model group is increased compared with that in blank group, there is significant meaning in the difference (P<0.05). For FLT-1 expression, that of model group is increased than that of blank group at 14th day, the difference is significant (P<0.05); and distinctly increased at the 28th day, the difference is obviously significant (P<0.01). the expressions of bFGF、ANG-2、FLK-1 in TCM group are all reduced than these expressions in model group, and the differences are all quite significant (P<0.01). The VEGF expression in TCM group is reduced than that in model group, and the differences are all significant (P<0.05). For FLT-1 expression, that of TCM group is increased than that of model group at 14th day, the difference is significant (P<0.05); and distinctly increased at the 28th day, the difference is obviously significant (P<0.01). for the expressions of bFGF、VEGF、FLT-1、FLK-1、ANG-2, there is no significance in the difference between hormone group and model group (P>0.05).
The results of qi-blood analysis:Hypoxemia appeared in model group, hormone group and TCM group at both the 14th day and the 28th day. For the PaO2, there is no significance in the difference between the hormone group and model group (P>0.05). However, for this norm, that of TCM is obviously increased than that of model group, and the difference is distinctly significant (P<0.01).
Conclusion:
1. Successfully modeled the rats with pulmonary fibrosis by injecting bleomycin into trachea in the dose of lmg/kg.
2. Bleomycin triggers pulmonary fibrosis of rats; at the early episode, it manifested as pulmonary alveolitis; and pulmonary fibrosis at the end-stage.
3. Fei Xian formula can relieve pulmonary alveolitis and pulmonary fibrosis of rats; and it is more effective than prednisone.
4. Fei Xian formula can relieve hypoxaemia of rats with pulmonary fibrosis. This may work by improving the function of lung faceing all the vessels.
5. The pathological change of microvessel vascularization in the process of pulmonary fibrosis was triggered by bleomycin in rats. The active stage of this change is around the 14th day. Then as pulmonary fibrosis exacerbated, the pathological microvessel vascularization reduced gradually, and the density of microvessel in lung tissue lowered progressively.
6. Bleomycin caused the ascending in the expressions of bFGF, VEGF, FLT-1, FLK-1 and ANG-2, which are microvessel angiogenesis-promoting factors in the process of rats pulmonary fibrosis. Then microvessel vascularization was active. Probably, microvessel vascularization is the important point in the process of pulmonary fibriosis. In that case, that restraint the microvessel vascularization may keep down the progress of pulmonary fibrosis radically.
7. The mechanism of treating pulmonary fibrosis through Fei Xian fomula may are as follows:restrainting the expressions of microvessel angiogenesis-promoting factors such as bFGF, VEGF, FLT-1, FLK-1 and ANG-2, to keep down microvessel vascularization in the lung tissue, further to relieve the pulmonary fibrosis and slow its progress.
8. Fei Xian fomula may work in the way of improving the function of lung facing all the vessels and relieving hypoxaemia in the disease of pulmonary fibrosis to downward the exprssions of microvessel angiogenesis-promoting factors.
方法：Wistar雄性大鼠80只,体重200±20g,随机数字表法分为空白组、模型组、激素组、中药组,每组20只。大鼠置于屏障级动物室适应性喂养1周。模型组、激素组、中药组气管插管后一次性推注博莱霉素1mg/kg剂量,空白组气管内一次性推注等体积的生理盐水。造模后第2天开始灌胃给药。中药组肺纤方给药剂量为1.25g／(kg·d),激素组强的松给药剂量为3mg/(kg·d).模型组和空白组给予等体积0.5%羧甲基纤维素钠溶液。14d、28d分批处死动物。用3.5%水合氯醛1 m1／100g体重,腹腔注射麻醉大鼠后。以动脉血气针取腹主动脉血2ml,15min内送检。
打开胸腔,行肺组织灌注。待大鼠头部、上肢、肺叶变硬时,停止灌注。取出肺脏,置于中性福尔马林中固定24h,常规脱水、石蜡包埋,连续组织切片(厚5μm),进行苏木精-伊红(HE)染色和三色(Masson)染色。
HE染色切片分为无染区、深染区、浅染区。OLYMPUS显微数码下采集图像。以Image-Pro Plus 6.0进行图像分析,测出各部分所占面积,取其平均值(mm2)。计算出各组肺泡炎和肺纤维化分级。
免疫组化SABC法检测大鼠肺组织切片血管内皮细胞CD34表达状况,用CD34抗体标记血管内皮细胞,镜下计数单位面积中的微血管数目,即微血管密度(MVD)。
免疫组化SABC法检测大鼠bFGF、VEGF、FLT-1、FLK-1、ANG-2的表达情况,结合图像分析技术,比较各组大鼠微血管新生促进因子表达情况。
结果：HE染色结果：空白组肺组织形态无明显病变。14d模型组肺泡隔明显增宽,肺泡隔和肺泡壁见较多炎细胞侵润。纤维结缔组织增生,出现实变区。14d激素组肺泡隔增宽,部分肺泡实变和大量炎性细胞浸润。14d中药组可见不同程度的炎症和纤维化灶,部分小支气管周围可见实变区。28d模型组、激素组、中药组炎症表现均较14d同组减轻。28d模型组肺泡隔明显增宽,胶原纤维显著增多,纤维组织呈斑片状分布,部分肺泡结构消失,萎缩闭塞,个别肺泡囊性扩张。肺泡隔和肺泡壁伴炎细胞侵润,较14d模型组减轻。28d激素组肺泡隔轻度纤维化。细小支气管周围可见纤维灶,部分肺泡和细支气管内可见炎性细胞。28d中药组肺泡隔纤维增生灶较少,肺泡内可见散在炎性细胞。
Masson染色结果：空白组肺组织形态无明显病变。14d模型组肺泡隔增宽,各级支气管和血管周围可见蓝色胶原纤维增生。14d中药组肺泡隔,各级支气管周围见少量纤细的胶原纤维。28d模型组肺泡壁、肺泡隔、终末细支气管、呼吸性细支气管周围和小支气管管壁可见大量胶原纤维增生。28d中药组胶原纤维增生较轻。激素组胶原纤维增生程度介于模型组和中药组之间。
HE染色形态定量分析结果：14d激素组深染区较模型组减少,差异有显著性意义(P<0.05)；14d中药组深染区较激素组明显减少,差异有非常显著性意义(P<0.01)。14d激素组浅染区与模型组比较,差异无显著性意义(P>0.05)；14d中药组浅染区较模型组明显减少,差异有非常显著性意义(P<0.01)。28d激素组深染区与模型组比较,差异无显著性意义(P>0.05)；28d中药组深染区较模型组明显减少,差异有非常显著性意义(P<0.01)。28d激素组浅染区与模型组比较,差异无显著性意义(P>0.05)；28d中药组浅染区较模型组明显减少,差异有非常显著性意义P<0.01)。
MVD结果：14d模型组、激素组MVD均较空白组增高,差异均有显著性意义(P<0.01,P<0.05)。28d模型组、激素组、中药组MVD均较同组14d降低,差异均有显著性意义(P<0.01,P<0.05,P<0.05)。激素组MVD与模型组比较,差异均无显著性意义(P>0.05)。14d中药组MVD较模型组降低,差异有显著性意义(P<0.05),28d中药组MVD较模型组明显降低,差异有非常显著性意义(P<0.01)。
微血管新生促进因子表达结果：模型组bFGF、ANG-2、FLK-1的表达均较空白组明显增强,差异均有非常显著性意义(P<0.01)；模型组VEGF表达均较空白组增强,差异均有显著性意义(P<0.05)；14d模型组FLT-1表达较空白组增强,差异有显著性意义(P<0.05),28d模型组FLT-1表达较空白组明显增强,差异有非常显著性意义(P<0.01)。中药组bFGF、ANG-2、FLK-1的表达均较模型组明显减弱,差异均有非常显著性意义(P<0.01)；中药组VEGF表达较模型组减弱,差异均有显著性意义(P<0.05)；14d中药组FLT-1表达较模型组减弱,差异有显著性意义(P<0.05),28d中药组FLT-1表达较模型组明显减弱,差异有非常显著性意义(P<0.01)。激素组bFGF、VEGF、FLT-1、FLK-1、ANG-2的表达与模型组比较,差异均无显著性意义(P>0.05)。
血气分析结果：模型组、激素组、中药组在14d和28d均可出现低氧血症。激素组Pa02与模型组比较,差异均无显著性意义(P>0.05)。中药组Pa02较模型组均明显升高,差异均有非常显著性意义(P<0.01)。
结论：1以博莱霉素1mg/kg剂量气管内推注成功复制了肺纤维化大鼠模型。
2博莱霉素诱发大鼠肺纤维化病变呈早期肺泡炎,晚期肺纤维化的演变过程。
3肺纤方具有改善肺纤维化大鼠肺泡炎症和肺纤维化的作用,疗效优于强的松。
4肺纤方可改善肺纤维化大鼠的低氧血症,这可能是通过肺纤方改善大鼠肺朝百脉的功能来实现的。
5博莱霉素诱发大鼠肺纤维化发病过程中存在病理性微血管新生,微血管新生以14d尤为活跃,随肺纤维化的加重病理性微血管新生逐渐减弱,肺组织微血管密度进行性减少。
6博莱霉素诱发大鼠肺纤维化发病过程中微血管新生促进因子bFGF、VEGF、FLT-1、FLK-1、ANG-2的表达上调,肺组织微血管新生活跃。微血管新生可能是肺纤维化发病过程中一个重要环节,抑制微血管新生可能会从根本上延缓肺纤维化进展。
7肺纤方治疗肺纤维化的机理可能是通过抑制微血管新生促进因子bFGF、VEGF、FLT-1、FLK-1、ANG-2的表达,进而抑制肺组织微血管新生,从而改善肺纤维化病情,延缓肺纤维化进展。
8肺纤方可能是通过改善肺朝百脉的功能,改善肺纤维化低氧血症,从而使微血管新生促进因子表达下调的。
Objectives:To observe effect of treating rats pulmonary fibrosis induced by BLM on Fei Xian formula, and explore the pathogenesis of pulmonary fibrosis and mechanism of pulmonary fibrosis treated by formula of Fei Xian.
Method:According to random number table,80 Wistar male rats, with the weight of 200±20g, were divided into four groups:blank group, model group, hormone group and TCM group, n= 20. Rats were placed in barrier-level animal house to be adaptively fed for 1 week. After tracheal intubation of model group, hormone group, and TCM group, a dosage of lmg/kg of bleomycin was bolus-injected respectively. For the blank group, it was bolus-injected equivalent volume of saline into trachea. Intragastric administration would begin in the second day after modeling. The dose of administration of Fei Xian formula in TCM group was 1.25g/(k·d); the dose of administration of prednisone in hormone group was 3mg/(kg·d); and for model group and blank group, they were given the equivalent volume of 0.5% sodium carboxymethyl cellulose. In the 14th and 28th days, these rats were sacrificed in batches. After anaesthetizing rats with an intraperitoneal injection of 3.5% chloral hydrate 1ml/100g weight, the volume of 2ml of abdominal aorta blood was withdrawn by arterial blood gas needle. It should be sent for inspection within 15 minutes.
Open the chest to perfuse the lung tissues. Stop infusing until rat's head, upper limbs, and pulmonary lobes became stiff. Before hematoxylin-eosin (HE) staining and three-color (Masson) staining, a serial preparations should be carried out, like remove of the lungs to neutral formalin for fixation for 24 hours; conventional dehydration, paraffin embedding, and serial tissue sections (thickness of 5μm).
HE staining section was divided into non-staining area, dark staining area, and light staining area. The images were pictured by digital microscope of OLYMPUS. Use Image-Pro plus 6.0 to analyze, to measure the area of each part, and choose their average (mm2). Calculate classification of each group's alveolitis and pulmonary fibrosis.
Expression of CD34 in vascular endothelial cells of lung tissue sections of rats was detected by immunohistochemistry SABC. The vascular endothelial cells labeled with CD34 antibody were counted the number of microvessel in unit area under microscope, which was called microvessel density (MVD).
Expressions of bFGF, VEGF, FLT-1, FLK-1, and ANG-2 were detected by immunohistochemistry SABC. Combined with image analysis, expression of promoting factor of microvascular angiogenesis of each group's rats could be compared.
Result:Results of HE staining:There was no obvious pathological change in pulmonary morphology in bland group. In 14d model group, the alveolar septum was obviously widened, also a large number of infiltrated inflammatory cells found in alveolar septum and wall, as well as hyperplasia of fibrous connective tissue, even consolidation. In 14d hormone group, the alveolar septum was widened, and part of alveoli changed into consolidation and large quantities of inflammatory cells infiltrated. Inflammation and fibrosis foci at different degrees could be found in 14d TCM group and also consolidation around the part of small bronchi. Compared with 14d model group, hormone group, and TCM group, inflammatory signs in the same groups of 28d greatly abated. In 28d model group, alveolar septa obviously widened, collagen fibers significantly increased, fibrous tissues distributed in patch, the structure of part alveoli disappeared, shrank, and occluded, and several alveoli expanded in cyst. Compared with 14d model group, infiltration of inflammatory cells in alveolar septum and wall is in greater reduction. Mild fibrosis of alveolar septum was found in 28d hormone group. Fibrosis foci could be seen around the small bronchioles and inflammatory cells appeared in part of alveoli and bronchioles. In 28d TCM group, fibroplasias foci of alveolar septum were less, and the scattered inflammatory cells could be found in alveoli.
Results of Masson staining:There was no obvious pathological change in pulmonary morphology in bland group. In 14d model group, alveolar septum widened, and the hyperplasia of blue collagen fibers could be seen around each bronchus and blood vessel. In 14d TCM group, a scanty of fine collagen fibers could be found in alveolar septa and around each bronchus. In 28d model group, a large number of hyperplasia of collagen fibers appeared in alveolar walls and septa, terminal bronchioles, walls of small bronchus, and around respiratory bronchioles. The hyperplasia of collagen fibers is slight in 28d TCM group. In hormone group, the degree of collagen fiber hyperplasia was between model group and TCM group.
The result of morphological and quantitative analysis of HE staining:At the fourteenth day, the deep-staining region of hormone group is smaller than that of model group; and there is a significant difference (P<0.05). At the same stage, the deep-staining region of TCM group is much smaller than that of hormone group; and there is a more significant difference (P<0.01). At the fourteenth day, there is no significance to the difference between the light-staining region of hormone group and that of model group (P>0.05). At the same time, the light-staining region of TCM group is much smaller than that of model group; and there is a obvious significant difference (P<0.01). At the 28th day, to the comparison between the deep-staining region of hormone group and that of model group, there is no significance (P>0.05). At this stage, the deep-staining region is much smaller than that of model group, and there is an obvious significant difference (P<0.01). At the 28th day, to the comparison between the light-staining region of hormone group and that of model group, there is no significance (P>0.05). At the same stage, compared with the light-staining region of model group, the region of TCM group is evidently reduced; the difference is obviously significant (P<0.01).
MVD result:At the 14th day, MVDs of model group and hormone group are both higher than that of blank group; and there is significance in the differences (P<0.01, P<0.05). The MVDs of model group, hormone group and TCM group at the 28th day are all lower than theirselve at the 14th day; and the differences are all significant (P<0.01, P<0.05, P<0.05). To the comparison between the MVD of hormone group and that of model group, there is no obvious significance in the difference (P>0.05). Compared with the MVD of model group, the MVD of TCM group is reduced at the 14th day, the difference is significant (P<0.05); and it is distinctly reduced at the 28th day, the difference is obviously significant (P<0.01).
The results for the expressions of angiogenesis factors:Compared with blank group, the expressions of bFGF, ANG-2 and FLK-1 in model group are all increased obviously; and the differences are all obviously significant (P<0.01). The VEGF expression in model group is increased compared with that in blank group, there is significant meaning in the difference (P<0.05). For FLT-1 expression, that of model group is increased than that of blank group at 14th day, the difference is significant (P<0.05); and distinctly increased at the 28th day, the difference is obviously significant (P<0.01). the expressions of bFGF、ANG-2、FLK-1 in TCM group are all reduced than these expressions in model group, and the differences are all quite significant (P<0.01). The VEGF expression in TCM group is reduced than that in model group, and the differences are all significant (P<0.05). For FLT-1 expression, that of TCM group is increased than that of model group at 14th day, the difference is significant (P<0.05); and distinctly increased at the 28th day, the difference is obviously significant (P<0.01). for the expressions of bFGF、VEGF、FLT-1、FLK-1、ANG-2, there is no significance in the difference between hormone group and model group (P>0.05).
The results of qi-blood analysis:Hypoxemia appeared in model group, hormone group and TCM group at both the 14th day and the 28th day. For the PaO2, there is no significance in the difference between the hormone group and model group (P>0.05). However, for this norm, that of TCM is obviously increased than that of model group, and the difference is distinctly significant (P<0.01).
Conclusion:
1. Successfully modeled the rats with pulmonary fibrosis by injecting bleomycin into trachea in the dose of lmg/kg.
2. Bleomycin triggers pulmonary fibrosis of rats; at the early episode, it manifested as pulmonary alveolitis; and pulmonary fibrosis at the end-stage.
3. Fei Xian formula can relieve pulmonary alveolitis and pulmonary fibrosis of rats; and it is more effective than prednisone.
4. Fei Xian formula can relieve hypoxaemia of rats with pulmonary fibrosis. This may work by improving the function of lung faceing all the vessels.
5. The pathological change of microvessel vascularization in the process of pulmonary fibrosis was triggered by bleomycin in rats. The active stage of this change is around the 14th day. Then as pulmonary fibrosis exacerbated, the pathological microvessel vascularization reduced gradually, and the density of microvessel in lung tissue lowered progressively.
6. Bleomycin caused the ascending in the expressions of bFGF, VEGF, FLT-1, FLK-1 and ANG-2, which are microvessel angiogenesis-promoting factors in the process of rats pulmonary fibrosis. Then microvessel vascularization was active. Probably, microvessel vascularization is the important point in the process of pulmonary fibriosis. In that case, that restraint the microvessel vascularization may keep down the progress of pulmonary fibrosis radically.
7. The mechanism of treating pulmonary fibrosis through Fei Xian fomula may are as follows:restrainting the expressions of microvessel angiogenesis-promoting factors such as bFGF, VEGF, FLT-1, FLK-1 and ANG-2, to keep down microvessel vascularization in the lung tissue, further to relieve the pulmonary fibrosis and slow its progress.
8. Fei Xian fomula may work in the way of improving the function of lung facing all the vessels and relieving hypoxaemia in the disease of pulmonary fibrosis to downward the exprssions of microvessel angiogenesis-promoting factors.
引文
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