【作者】 孙晓芳；

【导师】 牛建昭； 段斐；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2011， 博士

【摘要】 肺纤维化是一种病因复杂的疾病,以肺上皮细胞的过度凋亡、成纤维细胞过度增殖及胶原蛋白沉积为主要特征。目前临床上也缺乏行之有效的治疗方法,该病的预后不良。因此,研究肺纤维化发病机制,寻找有效的治疗药物具有重大意义。PNS是从三七中提取的有效活性成份,是三七发挥药理作用的主要成分之一。现代药理研究表明PNS具有广泛的药理作用,如止血、活血化瘀、消肿、补血、调节细胞凋亡等作用。为探讨PNS对肺纤维化小鼠的防治作用,我们以BLM诱导小鼠肺纤维化为模型,从整体与分子水平阐明PNS的作用和作用机制,以期为临床防治肺纤维化提供实验基础。1 PNS防治肺纤维化作用的观察1.1方法C57BL/6性小鼠180只,分为模型组；PNS高、中和低剂量三组；阳性药物对照组(醋酸强的松片)；假手术组；共6组,每组30只。除假手术组外其余各组小鼠气管内一次性滴注BLM,假手术组小鼠气管内一次性滴注等体积的生理盐水。造模后第二天开始给药。PNS高、中和低剂量组分别为120、60、30mg/kg/d,醋酸泼尼松组0.56mg/kg/d,假手术组,模型组分别灌服等体积的生理盐水。各组动物于7、14、28d摘眼球取血,分离血清,观察血清中Ⅲ型胶原(Ⅲ-C),Ⅳ型胶原(Ⅳ-C)及层黏连蛋白(LN)和透明质酸(HA)的含量,同时取肺组织进行HE, Mallory染色,检测肺组织羟脯氨酸(HYP)的变化。1.2结果PNS可降低肺纤维化小鼠血清中Ⅲ-C、Ⅳ-C型胶原、LN及HA的含量,降低肺纤维化小鼠肺组织HYP的含量,减轻肺组织胶原的增生。因此提示：PNS具有防治BLM诱导的小鼠肺纤维化的作用。2 PNS防治肺纤维化作用机制的实验研究2.1 PNS对肺纤维化小鼠肺组织细胞凋亡的影响通过PNS对肺纤维化小鼠肺组织细胞凋亡影响的研究,探讨PNS防治肺纤维化的作用机制。肺组织来源于实验一。利用TUNEL染色法测定小鼠肺组织中细胞的凋亡情况。结果表明：肺纤维化小鼠肺组织内凋亡细胞明显增加,凋亡的细胞主要为支气管上皮细胞、肺泡上皮细胞和巨噬细胞,还有少量肺间质细胞。PNS中剂量组能显著降低肺纤维化小鼠肺组织上皮细胞的凋亡。因此提示,PNS能通过抑制肺上皮细胞的凋亡而发挥其抗纤维化的作用。2.2 PNS对肺纤维化小鼠肺组织CatB和CatD的影响观察PNS对肺纤维化小鼠肺组织CatB和CatD的影响,探讨PNS防治肺纤维化的作用机制。肺组织来源于实验研究一。实验采用免疫组织化学和Westem blotting的方法,研究PNS对肺组织中CatB和CatD的影响。结果表明：PNS中剂量组防治后,各时间点小鼠肺组织中CatB和CatD含量和表达均有一定程度的下降,说明PNS能够从蛋白水平调节CatB和CatD的活性。提示抑制CatB和CatD的表达和活性可能是PNS防治肺纤维化的机制之一2.3 PNS对肺纤维化小鼠肺组织Bax和Bcl-2的影响观察PNS对肺纤维化小鼠肺组织Bax和Bcl-2的影响,探讨PNS抗肺纤维化的作用机制。肺组织来源于实验研究一。实验方法采用免疫组织化学法和RT-PCR的方法,研究PNS对肺组织中Bax和Bcl-2的蛋白和基因影响。结果表明：①Bax在肺纤维化小鼠肺组织中的含量明显升高,并且主要表达于肺损伤区的细支气管上皮细胞、肺泡上皮细胞和巨噬细胞,而在肺间质细胞少有表达,因此提示肺内实质细胞是Bax的主要来源之一,Bax在肺纤维化实质细胞的过度凋亡中发挥重要作用；②Bcl-2在肺纤维化小鼠肺组织中的含量亦明显升高,虽然细支气管上皮细胞和肺泡上皮细胞有表达,但在肺损伤区的肺间质细胞表达却明显增多,并且有的已进入细胞核,因此提示增多的Bcl-2主要是由肺间质细胞分泌的,Bcl-2在抑制成纤维细胞凋亡中发挥重要作用；③肺纤维化小鼠肺组织BaxmRNA和Bcl-2mRNA的表达亦明显升高,表明Bax和Bcl-2对肺纤维化小鼠肺组织细胞凋亡的调控至少是在蛋白转录或翻译水平进行的；④结果提示：PNS发挥其抗肺纤维化作用的机制之一在于从蛋白转录和翻译水平调节肺组织中Bax和Bcl-2的活性。2.4 PNS对肺纤维化小鼠肺组织Fas和FasL的影响观察PNS对肺纤维化小鼠肺组织Fas和FasL的影响,探讨PNS抗肺纤维化的作用机制。肺组织来源于实验研究一。用免疫组织化学法,研究对肺组织Fas和FasL的表达。结果表明：PNS能降低肺纤维化小鼠肺组织中Fas和FasL的含量。提示：抑制Fas和FasL的表达和活性可能是PNS防治肺纤维化的机制之一结论1 PNS可以调节BLM诱导的肺纤维化小鼠细胞外基质的含量,降低肺组织炎症和纤维化的程度,发挥防治肺纤维化的作用。2 PNS防治肺纤维化的作用机理2.1抑制肺上皮细胞的凋亡。2.2抑制肺组织蛋白CatB和CatD的活性.2.3调节肺组织蛋白Bax和Bcl-2的活性。2.4抑制肺组织蛋白Fas和FasL的活性。总之,PNS具有防治肺纤维化的作用,其作用机理可能在于通过抑制肺组织CatB.CatD和Fas/FasL的活性,调节Bax/Bcl-2的活性,抑制肺上皮细胞的凋亡,促进成纤维细胞的凋亡,来实现PNS防治肺纤维化的作用。更多还原

【Abstract】 The pathogenesis of pulmonary fibrosis is complicated, it is characterized with fibroblast hyperplasia, Epithelial cells apoptosis and deposition of collagen protein. The clinically effective therapy is deficient, the prognosis of this disease is not so better. Therefore, it is important to study the pathogenetic mechanisms and creating new treating scheme.PNS is one of the effective element extracted from notoginseng. It is one of the major components for notoginseng playing pharmacology.It owes various biological functions, including stop bleeding, remove blood stasis, detumescence, anemia, adjust the cell apoptosis functions and so on. To discuss the preventing and treating effect of PNS on idiopathic pulmonary fibrosis,mice model induced by bleomycin (BLM) was adopted. This thesis describes the effect and mechanisms of PNS on PF from aspects of integral and molecular level which can provides experimental foundation for more exact and scientific therapy. Methods and Results1 Role of observation on the prevention of PNS on pulmonary fibrosis 1.1 Methods 180 male C57BL/6 were randomly divided into 6 groups(30 mice in each group).Mice in the model control group;high,moderate and low PNS groups were injected. with a single dose of bleomycin by trachea,and mice in sham-model control group with same volume normal saline.1 day after the injection, oral administration of drug daily until they were killed 1 day before.PNS solution of different dosages(120mg,60mg,30mg/kg/d) was respectively given to mice in the high,moderate and low PNS group by gavages, while equal volume of normal saline was given to those in the sham-model control group and model control group, and an equal volume of prednisone(0.56mg·kg-1·d-1) was saline was given to those in positive medicine control group. Each animal in 7,14,28d pick eyeball take blood serum, observation, separationⅢserum collagen type (Ⅲ-C), IV collagen type (Ⅳ-C) and layer ankylosis protein (LN) and hyaluronic acid (HA), and take the content of lung tissue for HE Mallory stain, detection, lung tissue hydroxyproline (HYP) changes.1.2 Results PNS can modulate the level ofⅢ-collagen,Ⅳ-collagen,laminin and hyaluronic acid in serum compared with those levels in model control group and it can also lessen the hyperplasia of pulmonary fibrosis. It is indicated that PNS can prevent and treat pulmonary fibrosis2 Experimental study on mechanisms of PNS on pulmonary fibrosis2.1 The effect of PNS on cell apoptosis in pulmonary tissues of miceMechanisms of PNS on pulmonary fibrosis were discussed by the study on the level of cell apoptosis.By the method of TUNEL, observing the apoptosis of the lung. From the results, Pulmonary fibrosis in lung tissue apoptotic cells in mice increased significantly, mainly for the cell apoptosis of bronchial epithelium cells, alveolar epithelial cells, macrophages,and a few interstitial lung cells. In PNS dose group of mice could significantly reduce pulmonary fibrosis apoptosis of epithelial cells of lung tissue. It is suggested that PNS can play the role of the antioxidant fibrosis through inhibiting lung epithelial cell apoptosis.2.2 The effect of PNS on CatB and CatD in pulmonary tissues of miceMechanims of PNS on pulmonary fibrosis were discussed by observing the effect on CatB and CatD.The activition of CatB and CatD in the protein of pulmonary tissues was studied by means of immuno-histochemistry and Western blotting. It is showed that PNS can reduce the content of CatB and CatD in nucleoprotein. which means that one of mechanisms of PNS on pulmonary fibrosis is by inhibiting the activity of CatB and CatD.2.3 The effect of PNS on Bax and Bcl-2 in pulmonary tissues of miceThe effect of PNS on the lung tissue was investigated by detecting the activity of bax and bcl-2 at the protein and gene transcription level by using immuno-histochemistry and Real-time-PCR methods. The possible mechanism of PNS as anti-fibrosis is fully studied. The lung tissue is originated from experimentⅠ.①The expression of Bax is significantly increas-ed in the lung tissue of pulmonary fibrosis mice, it mainly expressed in the damaged lung bronchial epithelium cells, alveolar epithelial cells, and macrophages. However, the expres-sion of Bax in the interstitial lung cells are less significant, which means that hepatocytes is one of the main sources of Bax, bax plays an important role in the apoptosis of pulmonary fibrosis.②The expression of bcl-2 in the lung tissue of pulmonary fibrosis mice is signi-ficantly enhanced. Although there is expression in the fine bronchial epithelium cells and alveolar epithelial cells, the expression of bcl-2 in the interstitial lung cells is significantly increased, and some already enter the cell nucleus. It shows that bcl-2 are secreted by interstitial lung cells, bcl-2 plays an important role in inhibiting fibroblasts apoptosis.③The mRNA of Bax and bcl-2 significantly increased in the lung tissue of pulmonary fibrosis mice. The result shows that Bax and bcl-2 regulate the apoptosis of lung tissue cells of pulmonary fibrosis mice at least in protein transcription or translation level.④The results shows that the mechanism of PNS as the anti- pulmonary fibrosis is due to regulate the activity of Bax and bcl-2 of the lung tissue at the protein transcription and translation level2.4 The effect of PNS on Fas and FasL in pulmonary tissues of mice Mechanims of PNS on pulmonary fibrosis were discussed by observing the effect on Fas/FasL.The activition of CatB and CatD in the protein of pulmonary tissues was studied by means of immuno-histochemistry. It is showed that PNS can reduce the content of Fas/FasL in nucleoprotein. which means that one of mechanisms of PNS on pulmonary fibrosis is by inhibitting the activity of Fas/FasL.1 PNS can reduce the degree of pulmonary inflammation and fibrosis, and thus prevent and treat pulmonary fibrosis by adjusting the content of extracellular matrix in fibrotic mice by bleomycin-induced.2 Mechanisms of PNS on IPF lie in such aspects2.1 PNS can inhibit the activity of CatB and CatD in protein of pulmonary tissues.2.2 PNS can regulate the activity of Bax and Bcl-2 in protein of pulmonary tissues.2.3 PNS can inhibit the activity of Fas/FasL in protein of pulmonary tissues.In conclusion, we can conclude that PNS has a certain effect on the prevention and treatment of pulmonary fibrosis. The possible mechanism is due to inhibit the activity of CatB、CatD and Fas/FasL, regulate the activity of Bax/Bcl-2, inhibit the apoptosis of lung epithelial cells, and induce the apoptosis of fiber cells.更多还原