SiRNA Against Plasminogen Activator Inhibitor-1 Ameliorates Hepatic Fibrosis In Rats

[Background and Objective]Hepatic fibrosis is characterized by the accumulation of excess extracellular matrix (ECM), regardless of the underlying etiology. Decreasing ECM deposition and enhancing its degradation may prove particularly valuable for the treatment of hepatic fibrosis. The plasminogen activator (PA)/plasmin system, including urokinase-type plasminogen activator (uPA), plasmin, plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinases (MMPs) and their inhibitors, exerts a major role in regulating ECM degradation. It locates upstream of the fibrolysis system and is capable of directly degrading matrix components, and indirectly inhibits the deposition of ECM by activating latent MMPs. PAI-1 is the major physiological inhibitor of both uPA and tPA, molecules that cleave plasminogen to plasmin. This inhibition decreases both fibrinolysis and matrix proteolysis, which will result in ECM deposition and hepatic fibrogenesis.Normally, there is a delicate balance between uPA and PAI-1. During liver fibrosis, the activity of uPA is gradually decreased, whereas the expression of PAI-1 is increased. Increased expression of PAI-1 weakened the activity of uPA, thus inhibiting plasmin generation and leading to excess ECM degradation. Our previous study showed that upregulation of uPA during liver fibrogenesis significantly attenuated ECM deposition in rats. Additionally, there was substantial evidence indicating that PAI-1 deficiency had a protective effect against organ fibrosis, whereas PAI-1 overexpression exacerbated fibrosis. Therefore, we postulated that downregulation of PAI-1 expression will exert as a potential treatment for hepatic fibrogenesis.Small interfering RNA (siRNA) is a sequence-specific, posttranscriptional gene-silencing mechanism through double-stranded RNA molecules homologous to the sequence of the target gene, which has opened new avenues in gene therapy. Compared with other antisense strategies, RNAi has been proved to be much more simple-handled and potent in terms of gene knockdown.In the present study, we used PAI-1 as the targeted gene, and evaluated the antifibrotic effect of PAI-1 siRNA on liver fibrosis in vitro and in vivo, which would present as a new target and solution for the treatment of hepatic fibrosis. [Methods]1. Screening of PAI-1 siRNAFour pairs of 21 nucleotide siRNAs directing against PAI-1 mRNA 219,559,1061 and 2665 targets were designed using RNA design software and synthesized. These siRNAs were separately transfected into activated hepatic stellate cell line (HSC-T6). Untransfected HSC-T6 and HSC-T6 transfected with nonspecific siRNA were set as blank and negative controls respectively. The inhibition efficiency of siRNA on PAI-1 expression was determined by real time reverse transcription polymerase chain reaction (RT-PCR) and Western blot.2. The effect of PAI-1 siRNA on HSC-T6The selected PAI-1 siRNA was transfected into HSC-T6 by either cell-suspension transfection method or stable cell line. Expression of fibrosis markers were evaluated by real-time RT-PCR. Collagen types I and III contents in the supernatants were measured by enzyme linked immunosorbent assay (ELISA). Cell proliferation and the cell cycle were determined by the methyl thiazolyl tetrazolium (MTT) method and flow cytometry.3. Construction of recombinant adenovirus vector AdshPAIpSUPER-shPAI, which was constructed previously was used to provide the HI promoter and shRNA sequence and cloned into the AdEasyTM system to get the recombinant adenoviral plasmid-pAdshPAI by homologous recombination. Finally, the recombinant adenovirus AdshPAI was successfully generated after being packaged in 293 cells. The control adenovirus AdNC which expresses the negative control (NC) shRNA and AdGFP which expresses green fluorescent protein (GFP), were prepared as well. The expression of PAI-1 in HSC-T6 cell line was detected by RT-PCR on the second day after infection with AdshPAI or AdNC.4. The effect of AdshPAI on experimental hepatic fibrosisTwo distinct models of experimental hepatic fibrosis were established in rats, either by bile duct ligation (BDL) or dimethylnitrosamine (DMN) administration. In BDL-induced fibrosis,40 rats were divided into four groups randomly (10 rats per group). Group 1 served as the control with sham surgery, and the rats in next three groups were hepatic fibrosis models induced by BDL for 3 w. Common bile ducts of models were dissected bluntly and ligated.48 h before BDL, rats in group 3 and 4 were infused with 4×109 plaque forming unit (PFU) AdNC or 4×109 PFU AdshPAI via the tail vein, respectively. Rats were sacrificed 3 w after BDL. In DMN-induced fibrosis, Group 1 served as a normal control, while the remaining rats were injected intraperitoneally with 1%DMN (10μg/kg body weight) for three consecutive days per week for up to 4w. At the end of the second week, rats in groups 2,3 and 4 were infused with a single dose of 4×109 PFU AdNC or 4×109 PFU AdshPAI via the tail vein, respectively. Rats were sacrificed 4 w after DMN. The effect of AdshPAI on fibrosis was evaluated by histological examination. Sirius red staining and Masson's trichrome staining were used to determine collagen deposition. Immunohistochemical examination was carried out to detect the expression of PAI-1,α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), MMP-9, TIMP-1, Brdu and PCNA in liver tissues. The image analyzer was used to do the semiquantitative analysis. The mRNA level of PAI-1,α-SMA,TGF-β1,collagen typeⅠ,smad3,smad7,uPA,uPAR,tPA,MMP-2,MMP-9,MMP-13 and TIMP-1 were evaluated by real-time RT-PCR. The liver samples were subjected to acid hydrolysis to determine the amount of hydroxyproline. Immunofluorescence staining of apoptotic cells was performed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL).5. Statistical AnalysisResults are presented as means of three independent experiments (±SD). Statitical analysis of values was performed with SPSS software (11.0 version), with a P<0.05 considered significant and P<0.01 as very significant.[Results]1. Screening of PAI-1 siRNA and construction of the stable cell lineBoth of the real time RT-PCR and Western blot results revealed that 219 siRNA exert the highest repression activity, which significantly downregulated PAI-1 mRNA expression 24 h and 48 h after transfection (P<0.05), and reduced the protein expression up to 85% (P<0.05).559 siRNA and 2665 siRNA also suppressed PAI-1 mRNA expression by 72% and 66%, and protein expression by 57%and 63%(P<0.05), respectively, whereas 1061 siRNA seemed to have no effect on PAI-1 expression.2. The effect of PAI-1 siRNA on the biological characteristic of HSC-T6Expression of collagen typesⅠandⅢ.α-SMA,TGF-β1 and TIMP-1 mRNA decreased at the 219 siRNA and 559 siRNA-transfection froup (P<0.05). And the collagen contents in the supernatant of HSC-T6 also decreased in PAI-1 gene silenced HSC-T6 (P<0.05), compared with those of the negative control. Moreover, compared with the NC group, PAI-1 siRNA significantly inhibited HSC proliferation as determined by the MTT assay (P<0.05). And Cell cycle study also indicated that cells were arrested at the G0/G1 phase (57.61±0.19%vs.45.49±0.93%, P<0.05) and cells at the S phase were significantly reduced (30.29±2.17%vs.40.68±1.65%, P<0.05) after downregulating PAI-1 in HSCs.3. Establishment of AdshPAIThe plasmid pSUPER-shPAI, which was constructed previously was used to provide the H1 promoter and shRNA sequence and cloned into the plasmid pShuttle, which is devoid of a promoter. The recombinant pAdshPAI was established by homologous recombination and confirmed by restriction endonuclease digestion and sequencing. AdshPAI was amplified in 293 cells and then purified by cesium chloride gradient ultracentrifugation. Titers of viral stocks were determined by plaque assays using 293 cells, and 1×1010 pfu/ml titer of AdshPAI was obtained after amplification. After 2 days infection by the viruses, the expression of PAI-1 in HSC-T6 was confirmed by RT-PCR. The expression of PAI-1 was inhibited by over 90%, suggesting that the adenovirus AdshPAI was successfully constructed and adequate to downregulate PAI-1 expression.4. The effect of AdshPAI on experimental hepatic fibrosis1. The effect of AdshPAI on the expression of PAI-1:Compared with the normal rats, the PAI-1 mRNA level in the fibrotic liver was increased by 6.69±0.15-fold and 2.62±0.08-fold in BDL and DMN models, respectively (P<0.01), which was significantly reduced after AdshPAI treatment (P<0.01). Moreover, compared with the control and AdNC group, the PAI-1 protein level was significantly reduced after AdshPAI treatment in terms of both quantity and intensity as determined by immunohischemistry.2. The effect of AdshPAI on collagen deposition:Quantitative analysis indicated that the hydroxyproline content in AdshPAI treated group was strongly lower than that in the AdNC group (BDL:205.97±4.68μg/g vs.271.33±10.71μg/g; DMN:201.15±1.51μg/g vs.303.20±15.11μg/g, P<0.05). Additionally, the AdshPAI group had a significant reduction in fibrosis area, as demonstrated by Masson's trichrome staining and Sirus Red staining (P<0.05).3. The effect of AdshPAI on collagen synthesis:The expression of collagen I, a-SMA, TGF-β1 and smad3 mRNA were all increased in fibrotic livers (P<0.01), and were all significantly downregulated after AdshPAI treatment as demonstrated by real time RT-PCR (P<0.05). Furthermore, immunohistochemistry results revealed that a-SMA and TGF-β1-positive cells were significantly reduced by AdshPAI administration. 4. The effect of AdshPAI on the fibrolysis system:Compared with the AdNC group, the expression of MMP9 and MMP13 were upregulated and TIMP-1 was downregulated (P<0.01). However, PAI-1 siRNA did not affect the mRNA levels of uPA, tPA, uPAR and MMP2. Meanwhile, the immunostaining for MMP9 and TIMP-1 showed the same pattern of results.5. The effect of AdshPAI on cell proliferation and apoptosis:Compared with the AdNC group, Brdu immunostaining and PCNA indices of hepatocytes were almost 5-fold higher in the AdshPAI group (P<0.01). And there were numerous hepatocellular mitotic figures and binucleated hepatocytes after AdshPAI treatment. Moreover, TUNEL staining results revealed that the liver specimens from the control rats showed many apoptotic hepatocytes around the periportal region and fibrotic area, and AdshPAI treatment had a strong protective effect on the hepatocytes against apoptosis (P<0.05).[Conclusion]1. Chemically synthesized 219 siRNA targeting PAI-1 mRNA had excellent silencing effect on PAI-1 expression in HSC-T6.2. The inhibition of PAI-1 expression could significantly suppress the activation, proliferation of HSC-T6 and its synthesis and secretion of ECM.3. The expression of PAI-1 was remarkably upregulated during hepatic fibrogenesis, and AdshPAI could downregulate PAI-1 expression.4. AdshPAI administration facilitated matrix degradation and inhibited ECM deposition and had a protection effect on hepatic fibrosis.5. AdshPAI treatment stimulated hepatocellular proliferation and inhibited cellular apoptosis.