Plasminogen Activator Inhibitor-1Promoted Proliferation And Inhibited Apoptosis Of Embryonic Lung Fibroblasts And Its Mechanisms In Rats

Background and Objective：Idiopathic pulmonary fibrosis （IPF） is themost common type of idiopathic interstitial pneumonia. IPF is about47%-71%in idiopathic interstitial pneumonia. The prognosis of IPF is poor. It isreported that the median survival time is only3to4years. It is characterizedpathologically by the damage of type-â…  alveolar epithelial cells, vascularendothelial cells and basement membrane, accompanied with infiltration ofinflammatory cells such as neutrophils, macrophages and lymphocytes, etc,and the hyperplasia of type-â…¡ alveolar epithelial cells and collagendeposition.The exact mechanisms underlying the development of IPF remain unknown. Itwas previously reported that the inflammation and fibrosis of lung wereassociated with a variety of cytokines such as transforming growth factor-β（TGF-β）, tumor necrosis, metalloproteinase MMP, etc.The main role of plasminogen activator inhibitor-1（PAI-1） is theinhibition of urokinase-type plasminogen activator （uPA） and tissue-typeplasminogen activators （t-PA）, which inactivated plasminogen by theformation of1:1complex with the u-PA or t-PA. In recent research, PAI-1could regulate adhesion and migration of a variety of cells such as leukocyteand fibroblasts, enter the damaged organs, and interact with many cytokinessuch as TGF-β, TNF-α, PDGF in the development of pulmonary fibrosis.Meanwhile, the expression of PAI-1was up-regulated, its activity wasincreased and the activity of u-PA was decrease. Then, the fibrinolytic systemwas damaged, the fibrin could not be completely eliminated, and thepulmonary fibrosis was accelerated. Although the latent mechanism of thedevelopment of IPF is unknown, it was reported that over-expression of PAI-1played an important role in the pathogenesy of IPF. It was confirmed that the extent of pulmonary fibrosis and PAI-1gene density was positively correlatedwith Bleomycin （BLM）-induced pulmonary fibrosis in transgenic rat model.Results of recent studies have suggested that down-regulating PAI-1expression with siRNA could be an effective way to decrease liver fibrosis.PAI-1can promote proliferation of hepar stellate cell and vascularsmooth muscle cell, inhibit their apoptosis. In order to investigate the effect ofPAI-1, the present study was undertaken to observe changes of proliferation,conversion and collagen synthesis to extrinsic source PAI-1in lung fibroblast.In addition, the anti-Fas antibody induced apoptotic effect of PAI-1and itsmechanisms were also observed in fibroblasts sensitized by TNF-α.Methods:1The effect of PAI-1on proliferation of fibroblasts and its signal pathwayThe embryonic lung fibroblasts were recovered from the liquid nitrogenand cultured. Three groups were designed: Control, PAI-1and TGF-β. PAI-1or TGF-β was added into the culture solution and the final concentration ofthem was20ng/ml （PAI-1） or2ng/ml （TGF-β）, respectively. Thisconcentration was proved to be optimal in our preliminary research. Theprotein expression of PAI-1, α-SMA, ERK, p-ERK, AKT, p-AKT in24h,48hand72h were determined with Western blot. The mRNA expression of PAI-1,α-SMA, collagen type-â…  and type-â…¢ in24h were determined by RT-PCRand Real time PCR. The intracellular concentration of Ca²⁺on24h and48hwas determined by laser scanning confocal microscope.2The effect of PAI-1on apoptosis of fibroblasts and its signal pathway.The embryonic lung fibroblasts were recovered from the liquid nitrogenand cultured. Four groups were designed: Control, PAI-1, TNF-α+anti-Fas,TNF-α+anti-Fas+PAI-1. The corresponding substance was respectivelyadded into the culture solution in a concentration as follwing: PAI-1:20ng/ml;TNF-α:20ng/ml; anti-Fas:1μg/ml. The expressions of PAI-1, Caspase-3,NF-κB, ERK, p-ERK, AKT, p-AKT were determined with Western blot.3Statistical analysisData were expressed as mean±SD（X±SD） and statistically treated with statistical software of Statistical Program for Social Sciences13.0. One-wayANOVA was applied to compare between groups. If there is significantdifference, least significant difference was used to test difference betweengroups. P<0.05was considered statistically significant.Results:1Fibroblasts were globular after recovered. Fibroblasts adhered and expandedafter4-5hours. The shape of the fully expanded cell liked the spindle. Thecells were lucency, compaction, smooth of cell confine, and gradually radiateout in growth and connected to each other. The cells which were successlyrecovered were the second generation. The third to fifth generations were usedfor experiment.2The expression of PAI-1in fibroblast was upregulated at24h and theupregulation lasted to72h after the stimulation of extrinsic source PAI-1.Meanwhile, the expression of α-SMA, collagen type-â…  and â…¢, the concentration of intra-cellar Ca²⁺, p-AKT and p-ERK expression were up-regulated（P<0.05）. The above effects of PAI-1were similar to those of TGF-β.3The extrinsic source PAI-1down-regulated Caspase-3expression in controlfobroblasts and inhibited the upregulation of Caspase-3in the fibroblastssensitized with TNF-α and anti-Fas antibody. Meanwhile, the proteinexpression of NF-κB, the phosphorylation protein of ERK and AKT wereup-regulated as well after the the extrinsic source PAI-1（P<0.05）.Conclusions:1The extrinsic source PAI-1could promote proliferation, conversion andcollagen synthesis of fibroblast, it may be relate to increasing the intracellularconcentration of Ca²⁺and activating the AKT and ERK signal pathways.2The extrinsic source PAI-1could inhibit spontaneous apoptosis and TNF-αinduced apoptosis, it may be relate to activating NF-κB, ERK and AKT signalpathway.