Amelioration Of Bleomycin-induced Lung Fibrosis In Rat By SiRNA Against Plasminogen Activator Inhibitor-1 And Its Mechanisms

Background and Objective: Idiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of excess extracellular matrix (ECM). The pathological change is alveolitis in the early stage and fibrosis in the late stage. In this processes, fibroblast converses myofibroblast which expresses alpha-smooth muscle actin (α-SMA). Decreasing ECM deposition and enhancing its degradation might be particularly valuable in the therapy of lung fibrosis. The plasmin and matrix metalloproteinases (MMP) exerts a major role in regulating ECM degradation. Plasminogen Activator Inhibitor-1 (PAI-1) is the major inhibitor of urokinase type plasminogen activator and tissue-type plasminogen activator, inhibits plasmin and MMP, and results in ECM deposion and lung fibrosis. Previous study showed upregulation of PAI-1 might promote organ fibrosis. Tai-Lan et al observed that overexpression PAI-1 produced elevated collagen accumulation in normal and Keloid fibroblasts. Sheila et al observed that reduced fibrotic tissue formation in PAI-1 deficient mice. Therefore we hypothesized downregulation of PAI-1 expression would exert a potential treatment for lung 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.In present study, the proliferation effect of upregulation or downregulation PAI-1expression in fibroblast was observed in vitro first, and then lung fibrosis was therapy by PAI-1 siRNA.Methods:1 Construct the models of fibroblasts which downregulated and upregulated PAI-1 expression1.1 Fibroblasts were isolated and cultured from embryo lung in pregnancy rats. Vimentin andα-SMA immunohistochemistry identified.1.2 Screening of effective PAI-1 siRNAThree pairs of siRNAs directing against PAI-1 mRNA were synthesized according to reference. They were 219 siRNA, 559 siRNA and 1061 siRNA. These siRNAs were respectively transfected into fibroblast by liposomes. Nonspecific siRNA was used as negative control. The inhibition efficiency of siRNA on PAI-1gene expression on 24 h and protein expression on 48 h and 72 h were determined by Real time RT-PCR and Western blot.1.3 Eukaryon plasmid PCDNA-PAI-1 which include PAI-1 gene was transfected into fibroblast. The carrier PCDNA3.1 was used as negative control. The expression of PAI-1 mRNA on 24h was determined by Real time RT-PCR, the protein expression on 48 h and 72 h was determined by Western blot.2 The effect of PAI-1 on proliferation, transformation, collagen synthesis and the concentration of Ca²⁺ in fibroblast and signal transduction pathway.The cells'proliferation were estimated with MTT assay after transfecting in Normal, NC, 559 siRNA, PCDNA3.1, PCDNA-PAI-1 groups on 24h, 48h and 72h; the concentration of Ca²⁺on 24h and 48h was determined by Laser Scanning Confocal Microscope; the cells were divided into 2 different groups: Normal, NC, 559 siRNA groups and Normal, PCDNA3.1, PCDNA-PAI-1 groups, the mRNA expression ofα-SMA, collagen type-1and type-3 on 24h were determined respectively by Real time RT-PCR; the protein expression of ERK, p-ERK, AKT, p-AKT on 48h and 72h were determined by Western blot.3 The effect of 559 siRNA on bleomycin(BLM)induced lung fibrosis in rats and its signal mechanisms.72 Wister male rats weighed 130-140g in four groups:Sham, BLM(B), BLM+559 siRNA(B+P) and BLM+NC siRNA (B+N) groups. Lung fibrosis models were induced by intratracheal injection BLM (5mg/kg) in B, B+N and B+P groups, while 0.2 mL 0.9%NaCl were injected into trachea in Sham group. Intratracheal injection was carried out twice a week after making models: 559 siRNA dissolved in DEPC was intratracheal injection in B+P group; NC siRNA was injected in B+N group; 0.2 mL 0.9% NaCl was injected in Sham and BLM groups. 6 rats were sacrificed on 7d, 14d and 28d in each group. The inhibition efficiency of siRNA on PAI-1gene and protein expression were evaluated by Real time RT-PCR and Western blot; the histological changes were detected by HE staining; the changes ofα-SMA were determined by Real time RT-PCR, Western blot and immunohistochemistry; the effect of 559 siRNA in collagen synthesis was determined by Masson'trichrome staining and hydroxyproline content assay; the expression of collagen type-1and type-3 were evaluated by Real time RT-PCR and immunohistochemistry in order to identify the type of collagen; the expression of ERK, p-ERK, AKT, p-AKT were evaluated by Western blot.4 Statistical analysisStatistical analysis of values was performed with SAS8.0 software. Measurement data were indicated by (X|-)±S. One-Way ANOVA was applied, P<0.05 was considered statistically significant.Results:1 Successfully construct the models of fibroblasts which downregulated and upregulated PAI-1 expression1.1 Fibroblasts were identified by positive expression of Vimentin and negative expressionα-SMA by immunohistochemistry, 2-4 generations were tested.1.2 Screening of PAI-1 siRNA by the methods of Molecular Biology559 siRNA significantly downregulated PAI-1 mRNA expression 70±7% in 24h, while 219 siRNA downregulated 25±13% by Real time RT-PCR. 559 siRNA downregulated PAI-1 protein expression 73.5±10% and 42±3% on 48h and 72h respectively, while 219 siRNA downregulated 47±20% and 29.3±1% by Western blot, whereas 1061 siRNA seemed to have no effect on PAI mRNA and protein expression. These data indicated 559 siRNA exerted the most effective inhibition effect. 1.3 The fibroblasts which upregulated PAI-1expression more than 72h were set up successfullyThe expression of PAI-1 mRNA was upregulated 1818±6.10 %(P<0.01)in 24h by Real time RT-PCR after transfection PCDNA-PAI-1 into fibroblast. The protein expression was upregulated 280.6±49% and 306.6±32.4% on 48h and 72h.2 The effect of upregulation or downregulation PAI-1 expression on proliferation, transformation, collagen synthesis and the concentration of Ca²⁺ in fibroblast and signal pathway2.1 As showed by MTT assay, 559 siRNA could inhibit the proliferation of fibroblasts compared with NC group, and the inhibition effect peaked on 48h and descended on 72h. Overexpression PAI-1 could promote the proliferation of fibroblasts significantly, which lasted 72h (P<0.01).2.2 The concentration of Ca²⁺ in fibroblast was decreased after transfected 559 siRNA, while increased after transfected PCDNA-PAI-1(P<0.05).2.3 The mRNA expression ofα-SMA and collagen type-1 decreased and increased after downregulation or upregulation the expression of PAI-1(P<0.05) whereas collagen type-3 seemed not to be affected(P>0.05).2.4 The phosphorylation protein expression of ERK1/2, AKT were downregulated or upregulated accordingly after downregulation or upregulation the expression of PAI-1(P<0.01) by Western blot dates. Perhaps PAI-1 improved proliferation, conversion of fibroblast by AKT, ERK signal mechanism.3 The effect of 559 siRNA on BLM induced lung fibrosis3.1 After intratracheal injection 559 siRNA, the expression of PAI-1 mRNA were downregulated 29±9%, 58±9% and 67±7% on 7d, 14d and 28d. The protein expression were downregulated 46±11%, 51.9±5.3% and 65.5±4%, while immunohistochemistry indicated the expression of PAI-1 in fibroblast decreased significantly(P<0.01). 559 siRNA could downregulate the gene and protein expression of PAI-1 efficiently in the fibrotic lung. 3.2 In B and B+N groups, there were many neutrophils, fibroblasts, macrophages were observed in mesenchymal on 7d by HE staining, alveolitis were alleviated but the alveolar wall became thicker with the time went by. In B+P groups, alveolitis and the alveolar structure damage was significantly alleviated on 7d, 14d, 28d compared with B+N groups.3.3 The effect of 559 siRNA in mRNA and protein expression ofα-SMA significantly decreased on 7d, 14d and 28d in B+P group compared with B+N groups, while immunohistochemistry showed the expression in endochylema and cytomembrane significantly decreased.3.4 The effect of 559 siRNA in the expression of collagen3.4.1 The Masson'trichrome staining showed there were only focal collagen deposition on 14d in B and B+N groups, while a sheet of collagen deposition were observed on 28d. In B+P groups, the collagen deposition was significantly decreased on 14d and 28d compared with B+N groups(P<0.05).3.4.2 The hydroxyproline content had no increasing on 7d, it constantly increased from 14d to 28d. In B+P groups, the hydroxyproline content significantly decreased compared with B+N groups(P<0.01).3.4.3 The effect of 559 siRNA in the expression of collagen type-1559 siRNA inhibited collagen type-1 mRNA on 7d and 14d. There was no significantly difference on 28d compared with B+N groups. Microphotographs showed immunoparticles of collagen type-1 in fibroblast and mesenchyma in B+P group decreased significantly in 7d, 14d and 28d.3.4.4 The effect of 559 siRNA in the expression of collagen type-3559 siRNA inhibited collagen type-3 mRNA on 14d and 28d. There was no significantly difference on 7d compared with B+N groups. Microphotographs showed that immunoparticles of collagen type-3 in fibroblast and mesenchyma continually increased on 14d and 28d in B and B+N groups, the expression significantly decreased in B+P group on 14d and 28d.3.5 The phosphorylation protein expression of ERK1/2, AKT were downregulated on 7d, 14d and 28d, as demonstrated by Western blot, P<0.05. Conclusions:1 In vitro, 559 siRNA and PCDNA-PAI-1 had excellent transfection effect in embryo lung fibroblast in rats, PAI-1 were expressed downregulation or upregulation more than 72 h; downregulation the expression of PAI-1 could inhibit proliferation, conversion and collagen synthesis of fibroblast, decrease concentration of Ca²⁺, and inhibit phosphorylation protein expression of ERK1/2 and AKT; upregulation the expression of PAI-1 could improve proliferation, conversion and collagen synthesis of fibroblast, increase concentration of Ca²⁺, and active AKT, ERK signal pathway.2 In vivo, the expression of PAI-1 was remarkably upregulated during lung fibrosis, and PAI-1siRNA could downregulated its expression; 559 siRNA could decrease collagen synthesis, facilitate ECM degradation and inhibit lung fibrosis; perhaps 559 siRNA inhibited lung fibrosis by downregulation phosphorylation protein expression of ERK1/2 and AKT in fibroblast.