The Effet Of PAI-1SiRNA In Bleomycin-induced Pulmonary Fibrosis In Rats And Its Mechanism

Idiopathic pulmonary fibrosis(IPF) is a kind of chronic and fatal disorders of fibrinogen. It is characterized by the thickening of the alveolar wall and the disappearance of the alveolar interval. Eventually it leads to the diseases characterized by loss of lung function. The main phenomena are abnormal fibroblasts proliferation and excessive collagen deposition in the extracellular matrix(ECM).The pathological characteristics mainly includes interstitial cell hyperplasia, excessive deposition of ECM and the reconstruction of lung tissue structure.A large number of cytokines can be interacted mutually, also can with a variety of inflammatory mediators and the lung tissue cells interacted with each other in the process of pulmonary fibrosis formation, then a complex series of reactions occured, lung tissue inflammation or immune injury was aggravated and fibroblast proliferation division was stimulated, finally the deposition of the ECM was promoted. The two main enzymes of degradation of extracellular matrix are fibrinolytic enzyme and a matrix metalloproteinases. The abnormal expression of PAs/PAIs and MMPs/TIMPs plays an important role in the degradation of the ECM and lung fibrosis.Profibrinolysin is one of the key components of the human fibrinolytic system, which activated by plasminogen activator and translated into fibrinolytic enzyme,The fibrinolytic enzyme not only Playing the fibrinolytic function, but also to participate in a series of procession related to protein hydrolysis in the body physiological and pathological process, such as inflammation, tissue reconstruction and tumor,etc[1]. PAI- 1 is a kind of main inhibitors of the urokinase type fiber protease original(u- PA), which also plays the main regulator in the vascular injury, thrombosis and fibrinolysis [2]. The mechanism of tissue fibrosis promoted by PAI- 1 is unknown. Animal models confirmed that in overexpression PAI-1 mice, bleomycin-induced pulmonary fibrosis was significantly increased.The balance of the MMPs and TIMPs system plays a key role in the ECM synthesis and degradation. MMPs can degrade of ECM and TIMPs is an important inhibitor of MMPs.TIMPs can promote collagen deposition and reduce the extracellular matrix degradation. Found in patients with IPF in recent years, the expression of TIMPS has obvious advantages to the MMPS, so as to promote the occurrence and development of pulmonary fibrosis.The discovery of RNA interference(RNAi) heralded a revolution in the domain of biology. RNAi is a sequence-specific posttranscription gen silencing mechanism. It has been extensively applied in the gene function and therapy through synthesizing of specific small RNAS realize target genes inhibit.Objective:In present study, The activity of PAI-1 in alveolar lavage fluid was determined and the expression of α-SMA, collagen type III, matrix metalloproteinases inhibitor-1(TIMP-1) and transforming growth factor-beta(TGF- beta) in lung tissues in bleomycin induced pulmonary fibrosis in rats were observed by RT-PCR. Furtherly, whether PAI-1si RNA participated in the adjustment process of pulmonary fibrosis and its mechanism would be discussed.Methods:A total of 72 healthy male Sprague-Dawley(SD) rats(provided by Experiment Animal of Hebei Medical University) weighed 140±20g were divided into four groups with random number table: sham group, BLM group(B),BLM+PAI-1si RNA group(B+P)and BLM+Non-specfic si RNA(NCsi RNA) group(B+N). 18 rats were included in each group. Anhydrous ether was used for local anesthesia, the rats were fixed in supine position, the neck skins were disinfect, the middle of the neck skins were cut, the tissues were separated, tracheas were expose. Lung fibrosis models were induced by intratracheal injection BLM(5mg/kg) 0.2~0.3ml in B, B+N and B+P groups, while equal volume 0.9% Na Cl were injected into trachea in Sham group. Intratracheal injection was carried out twice a week after making models: PAI-1si RNA dissolved in DEPC was intratracheal injection in B+P group; NCsi RNA was injected in B+N group; 0.2 m L 0.9% Na Cl was injected in Sham and BLM groups. 6 rats were sacrificed on 7d, 14 d and 28 d in each group. The left lung for determine the activity of PAI-1 in bronchoalveolar lavage fluid(BALF). Right middle lobe lung for Real time PCR. The lung tissues for Real time PCR were stored in liquid nitrogen at-80℃. The expression of α-SMA, collagen type III, matrix metalloproteinases inhibitor-1(TIMP-1) and transforming growth factor-beta(TGF-beta) in lung tissues in bleomycin induced pulmonary fibrosis in rats were observed by RT-PCR.Statistical analysis of values was performed with SPSS13.0 software.Results:(1)The results of PAI-1 in alveolar lavage fluid: The activity of PAI-1 in group B increasing on 7. 14 and 28 days and two times a week endotracheal inject PAI-1si RNA can reduce its activity. It has statistically significant differences respectively when to compare the BLM group on same point(P< 0.05).(2)PCR results:The expression of m RNA of α-smooth muscle actin(α-SMA), collagen type III, matrix metalloproteinases inhibitor-1(TIMP- 1) in group B was significantly higher than that in the Sham group(P<0.01)on 7th day,14 th day and 28 th day, The expression of m RNA of α-SMA, collagen type III, TIMP-1 in group B+P was significantly lower than that in the group B on the three time points and has statistically significant differences(P<0.01), The expression of m RNA of TGF-β in group B was significantly higher than that in the Sham group(P<0.01)on the 7th day and 28 th day, and the expression of m RNA of TGF-β in group B+P was lower than that in the group B(P<0.01)on the 7th day and 28 th day, but there were no significant differences on 14 th day(P > 0.05).There were no significant differences between group B and B+N(P>0.05) on each time point.Conclusions:1 Intratracheal injection of PAI-1si RNA could decrease collagen synthesis,facilitate ECM degradation and inhibit lung fibrosis on bleomycin-induced lung fibrosis in rats.2 Perhaps PAI-1si RNA played a role in the treatment of pulmonary fibrosis by downregulation the expression of TIMP-1 and TGF-β.