| Objective: Idiopathic pulmonary fibrosis (IPF) is a disease characterizedby diffused alveolitis, alveolar structural disorder, and eventually leading toexacerbation of lung function. It is a dynamic pathological process to damagedlung tissue. The pathological features includes interstitial cell hyperplasia,extracellular matrix（ECM）hyperplasia deposition and lung tissue remodeling.Among, ECM metabolic imbalance plays an important role in thedevelopment of pulmonary fibrosis. Fibroblasts, versatile connective tissuecells and key effectors in wound-repair responses, are recruited to sites oftissue injury where ECM components and soluble factors, play an importantrole in development of injury lung tissue. Myofibroblasts secrete, organize,remodel, and contract the ECM, facilitating wound closure andreepithelialization. With the resolution of normal wound repair,fibroblast/myofibroblast decrease substantially due to apoptosis. Thephysiologic stimuli that trigger fibroblast/myofibroblast apoptosis. Failure ofmyofibroblast apoptosis is associated with pathologic wound repaircharacterized by tissue fibrosis. The plasmin and matrix metalloproteinases(MMP) exerts a major role in regulating ECM degradation. Plasmin, a serineprotease with potent fibrinolytic activity, is generated through the proteolyticcleavage of its zymogen, plasminogen. Plasmin is capable of cleavingextracellular matrix components such as fibronectin and activating growthfactors and other matrix proteases. Inhibition of plasminogen activation byplasminogen activator inhibitor-1(PAI-1) has been causally implicated in thepathogenesis of pulmonary fibrosis and other fibrotic disease. Themechanisms by which PAI-1promotes tissue fibrosis, while plasminogenactivation protects against fibrogenesis, have not been fully defined. Animalmodels confirmed that in overexpression PAI-1mice, bleomycin-induced pulmonary fibrosis was significantly increased.The discovery of RNA interference (RNAi) heralded a revolution in thedomain of biology. RNAi is a sequence-specific posttranscription gensilencing mechanism. It has been extensively applied in the gene function andtherapy.In present study, we used PAI-1siRNA to silence PAI-1gene ofbleomycin-induced pulmonary fibrosis in rats. We observed the degree of lungfibrosis and the expression of casepase-3. Further, we investigated thepathogenesis of pulmonary fibrosis in apoptosis.Methods:A total of72healthy male Sprague-Dawley (SD) rats (providedby Experiment Animal of Hebei Medical University) weighed200±20g weredivided into four groups with random number table: sham group, BLM group(B), BLM+PAI-1siRNA group (B+P), and BLM+Non-specfic siRNA(NCsiRNA) group (B+N).18rats were included in each group. Anhydrousether was used for local anesthesia, the rats were fixed in supine position, theneck skins were disinfect, the middle of the neck skins were cut, the tissueswere separated, tracheas were expose. Lung fibrosis models were induced byintratracheal injection BLM (5mg/kg)0.2～0.3ml in B, B+N and B+P groups,while equal volume0.9%NaCl were injected into trachea in Sham group.Intratracheal injection was carried out twice a week after making models:PAI-1siRNA dissolved in DEPC was intratracheal injection in B+P group;NCsiRNA was injected in B+N group;0.2mL0.9%NaCl was injected inSham and BLM groups.6rats were sacrificed on7d,14d and28d in eachgroup. Right lower lung was fixed by4%paraformaldehyde, paraffin forhistological examination (HE staining, Masson staining) andimmunohistological; right lower lung for Real time PCR andhydroxyproline(HYP) determination. The lung tissues for Real time PCR werestored in liquid nitrogen and the lung tissues for HYP determination werestored at-80℃. Alveolitis and pulmonary fibrosis were divided by the methodof Szapiel. The therapeutic effect of PAI-1siRNA was determined via HEstaining, Masson staining and determination of concentration of HYP of lung tissu. The expression of caspase-3in lung was measured byimmunohistological The integral optical density of the sum analysis canevaluate the degree of expression of caspase-3. The relative concentration ofCaspase-3mRNA in lung tissue was determined by Real time PCR. Statisticalanalysis of values was performed with SPSS13.0software.Results:1The results of pulmonary pathology: the deree of alveolitis of group B wasmore serious than that of the Sham group on each point (P<0.01）. The level offibrosis of group B was more serious than that of the Sham group（P<0.01）onthe14th day and28th day. These indicated that the model of pulmonaryfibrosis was successfully. The level of alveolitis in group B+P wassignificantly ameliorated than that of group B on each point (P＜0.01）. Thedegree of fibrosis in group B+P was ameliorated significantly than that of thegroupB（P<0.01）on the14th day and28th day, but there were no significantdifferences between group B and B+N(P>0.05). These indicated thatPAI-1siRNA could ameliorate pulmonary fibrosis in the BLM-induced ratmodel.2The change of the content of HYP in lung tissue: the content of HYP in thegroup B showed an increase tendency on day7,14, and28. The content ofHYP in the group B a was increased significantly than that of the Sham group（P<0.01）. The content of HYP was increased on a low lever in group B+P.That of There was significant decreasion in group B+P than in group B oneach time point（P<0.01）, but there were no significant differences betweengroup B and B+N (P>0.05).3Immunohistochemical results: the expression of Caspase-3in group B wasnot significantly increased than the expression in group Sham on7th day, butthere was significantly increasing on14th day and28th day（P<0.01）. Therewere no significant differences between group B+P and B on7th day, but therewas significant decreasion in group B+P than in group B on14th day and28thday（P<0.01）. There were no significant differences between group B andB+N(P＞0.05). 4Real time PCR results: The expression of mRNA in group B+P wassignificantly higher than that in the group B（P<0.01）on7th day, there was thesame tendency on14th day and28th day (P＜0.01). There were no significantdifferences between group B and B+N (P＞0.05) on each time point.Conclusions:1Intratracheal injection of PAI-1siRNA could decrease collagen synthesisand inhibit lung fibrosis on bleomycin-induced lung fibrosis in rats.2Perhaps PAI-1siRNA played a role in the treatment of pulmonary fibrosisby upregulation caspase-3. |
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