Inhibition Of Extracellular Signal-Regulated Kinase 1 By Adenovirus Mediated SiRNA Attenuates Hepatic Fibrosis In Rats

[Background and Objective]Hepatic fibrosis is the response of the liver to different chronic insults, and is characterized by the excess production and deposition of extracellular matrix (ECM) components, leading to tissue scarring and the destruction of normal hepatic parenchyma.A very large number of studies have identified the hepatic stellate cells (HSCs) as the predominant source of myofibroblasts. Nevertheless, recent studies suggest that myofibroblasts can be generated from a variety of sources including resident mesenchymal cells, epithelial and endothelial cells. In particular, substantial advances have been made to highlight that myofibroblasts can be supplemented from cholangiocytes and hepatocytes by epithelial-mesenchymal transition (EMT) during hepatic fibrosis, indicating that the parenchymal epithelial cells of the liver have a significant role in the perpetuation of hepatic fibrosis.Extracellular regulation kinase 1 (ERK1) is an important kinase in the mitogen-activated protein kinase (MAPK) signal transduction pathway. In unstimulated fibroblasts, the majority of ERK proteins are associated with the microtubule cytoskeleton. Mitogenic stimulation causes a significant proportion of ERK1/2 to accumulate in the nucleus, in contrast to their relative exclusion from the nucleus in resting cells. It has been proven that of ERK was involved in the transforming growth factor-β1 (TGF-β1) or aldosterone-induced EMT of the proximal tubular epithelial cells and renal fibrosis. ERK1/2 pathway also participates in the development of liver fibrosis. For example, ERK1/2 mediates platelet-derived growth factor-D (PDGF-D) and leptin-related activation of moyfibroblasts. Activatd ERK1/2 can induce JunD-mediated elevation of tissue inhibitor of metalloproteinase-1 expression. Our previous cDNA array work reveals that ERK1 expression was remarkably upregulated during the development of hepatic fibrosis. Transfection of ERK1 small interfering RNA (siRNA) into an active HSC cell line HSC-T6 led to a significant inhibition of the proliferation of HSC, accompanied by the induction of HSC apoptosis and reduction of collagen synthesis and deposition. Taken together, all of these studies suggest that ERK1 is involved in the development of hepatic fibrosis, and selective inhibition of ERK1 might be a novel strategy for the treatment of hepatic fibrosis. In the present study, we aimed to investigate the effect of ERK1 siRNA on hepatic fibrosis and clarify its mechanism. Our results demonstrated that inhibition of ERK1 by adenovirus-delivered siRNA significantly attenuated hepatic fibrosis in rats induced by both bile duct ligation (BDL) and dimethylnitrosamine (DMN) through inhibition of HSC proliferation and activation, meanwhile, reversing the EMT of biliary epithelial cells (BECs) in BDL-induced biliary fibrosis or EMT of hepatocyte in DMN-induced chemical fibrosis. These results suggest that gene-silencing therapy with ERK1 siRNA might lead to new therapeutic options for hepatic fibrosis.[Methods]1. Construction of siRNA expressing adenovirus vector and cell infectionThree siRNAs targeting rat ERK1 mRNA and a scrambled siRNA used as a negative control (NC) were designed with software on www.ambion.com and synthesized by GenePharma. The adenovirus vectors, containing ERK1 siRNA (AdshERK1) and the NC (AdshNC), were constructed using pShuttle as previously described. HSC-T6 cells were infected with recombinant virus for 72h or 96h.2. Determination of HSC proliferationTo test the effect of ERK1 siRNA on HSC proliferation, HSC-T6 were plated in triplicate wells on a 96-well plate at 1.5×103/well and cultured for 24 h. The cells were then infected with adenovirus at a MOI of 1200. The number of metabolically active mitochondria and viable cells was determined colorimetrically at 490 nm using the MTT assay as described.3. Treatment of hepatic fibrosis in ratsTwo distinct models of hepatic fibrosis were induced either by BDL or injection of DMN. Group 1 served as the control with sham surgery. Two days before or 3 days after the BDL operation, rats in groups 2,3, and 4 (8 rats in each group) were infused with a single dose of PBS,2×109 pfu AdshNC, or AdshERKl via the tail vein, respectively. For DMN model, group 1 served as a normal control that received intraperitoneal saline injection. The remaining rats were injected intraperitoneally with 1%DMN (100μl/100g) for 3 consecutive days per week up to 4 weeks and infused with PBS,2×109 pfu AdshNC, or the same amount of AdshERKl after 6 DMN injections via the tail vein, respectively. The animals were sacrificed 3 weeks after BDL or 4 weeks after DMN administration.4. Histological examination and immunohistological staining All paraffin-embedded liver tissues were stained with H&E, Masson's trichrome, and Sirius red staining. For the semiquantitative analysis, connective tissues stained blue with Masson's trichrome staining were measured on an image analyzer by a technician blinded to the samples. Three fields were selected randomly from each of two sections, and six rats from each group were examined. The percentage was calculated as previously described.Immunohistochemistry and double immunostaining were performed on paraffin-embedded liver sections according to manufacturer's recommendation.5. Measurement of hepatic hydroxyproline contentTotal hepatic hydroxyproline levels were determined in the hydrolysates of liver samples as previously described.6. Statistical analysisThe arithmetic mean and standard deviation (s.d.) were calculated for the data, and statistically evaluated using 2-tailed unpaired t-test. P< 0.05 was considered statistically significant.[Results]1. AdshERKl reduces the expression of ERK1 in HSC-T6According to the real-time RT-PCR and Western Blot results, the siRNA 189 showed the best inhibition of ERK1 expression in HSC-T6, and was selected for subsequent experiments. AdshERKl decreased ERK1 mRNA in HSC-T6 by 74% and 77% at 72 h and 96 h after the infection respectively, compared with AdshNC. In addition, AdshERKl reduced both ERK1 protein and phosphorylated ERK1 by approximately 65%, but ERK2 expression was not affected. Suppression of ERK protein expression by AdshERK1 was also detected by immunofluorescence.2. AdshERKl inhibits cell proliferation and fibrosis-associated gene expressionMTT analysis revealed that AdshERKl significantly suppressed HSC-T6 proliferation from the third day after infection. On the fifth day, the suppression rate was up to 50%(P< 0.001). Furthermore, compared with AdshNC, ERK1 silencing suppressed collagen typesⅠandⅢ, tissue inhibitor of metalloproteinase-1, and TGF-β1 mRNA levels 96 h after the infection by 44%(P< 0.01),57%(P< 0.01),35%(P< 0.05), and 47%(P< 0.05), respectively.3. ERK1 siRNA attenuates hepatic fibrosis induced by BDL or DMN in ratsWe then tested the effect of AdshERKl in two distinct rat models of hepatic fibrosis, BDL or DMN administration. Only faint ERK1 staining and normal collagen distribution was detected in sham-operated animals. Overexpression of ERK1 and aniline blue-stained fibrils were associated with proliferating bile ducts that formed a continuous meshwork of connective tissue infiltrating the hepatic parenchyma with loss of the lobular architecture 3 weeks after BDL, suggesting that hepatic fibrosis was successfully established. Interestingly, a single dose of AdshERKl suppressed ERK1 expression and prevented the development of hepatic fibrosis, as confirmed by HE, Masson's trichrome, and Sirius red staining. AdshERKl reduced the ECM area (Masson's staining) by 62% after injection (P < 0.01). Moreover, hydroxyproline content reduced in the AdshERKl-treated group (199.84±4.20μg/g), compared with AdshNC group (272.59±7.8μg/g, P< 0.05) and model group (288.28±11.46μg/g, P< 0.05), while the hydroxyproline content in sham group was 176.91±13.04μg/g liver tissue. In addition, AdshERKl significantly suppressed collagen types I and III in liver.To further evaluate the effect of ERK1 siRNA on fibrosis induced by BDL, we injected the virus 3d after BDL in which hepatic fibrosis has already developed as demonstrated by Beaussier' study (6). The results showed that AdshERKl could ameliorate liver fibrosis as well. The hydroxyproline content in AdshERKl group (230.12±24.35μg/g) decreased by 41% as compared with AdshNC group (392.05±19.69μg/g, P< 0.01) and by 40% as compared with model group (383.52±12.04μg/g, P<0.05). The ECM area (Masson's staining) reduced by 64%(P<0.05) as compared with AdshNC group.As expected, DMN injection induced prominent hepatic fibrosis in rats as shown by Masson's trichrome and Sirius red staining and increased ERK1 expression. AdshERK1 treatment blocked this increase in ERK1 and reduced ECM (Masson's staining) by 74%(P <0.01). The hydroxyproline content in AdshERK1-treated group (198.94±26.33μg/g liver tissue) also decreased compared with that in AdshNC group (355.11±54.82μg/g, P<0.05) and model group (359.41±59.10μg/g, P<0.05).4. ERK1 is associated with the activation of myofibroblastsTo address the possible role of ERK1 in the activation of myofibroblasts, double-immunostaining was performed to detect the expression profile of ERK1 and a-smooth muscle actin (α-SMA) in both fibrosis models. There was no overlap of ERK1 and a-SMA in normal liver. ERK1 staining dramatically increased in the cytoplasm of proliferative BECs after bile duct ligation. Interestingly, ERK1 expression was also detected in the nuclei of the cells around peribiliary region and colocalized withα-SMA. Similarly, increased staining of ERK1 was found around portal tracts and in fibrotic septa 4 weeks after DMN injection, especially in the nuclear of interstitial cells, which was accompanied by increased expression ofα-SMA, implying that ERK1 is implicated in the activation of myofibroblasts. AdshERKl, however, prevented the coexpression of ERK1 andα-SMA in the two kinds of liver fibrosis and decreased mRNA levels of ERK1 andα-SMA in both models.5. BECs and hepatocytes contribute to the accumulation of myofibroblasts in fibrogenesis in vivoTo further investigate the activation of myofibroblasts in fibrogenesis, immnostaining was performed in normal and fibrotic liver using serial section. As shown in Fig.4A, the signal forα-SMA, desmin and glial fibrillary acidic protein (GFAP) was restricted to the wall of blood vessels in sham-operated control rats. Nevertheless, the expression ofα-SMA, desmin and GFAP was increased around the bile ducts in BDL rats and in fibrotic septa in DMN-induced hepatic fibrosis, which was remarkably attenuated by AdshERKl. Interestingly, we noticed that the desmin or GFAP labeled HSCs only accounted for approximately 50% of theα-SMA positive myofibroblasts, implying that not all of the myofibroblasts were derived from HSCs.To detect the other source of the myofibroblasts in fibrotic liver, double immunostaining for the biliary epithelial marker cytokeratin-19 (CK19) andα-SMA was carried out in the BDL model. As shown in Fig 5A, CK19 staining was clearly detected in BECs. Additionally, a large amount of CK19 positive cells were also detected around proliferative bile ducts and in fibrotic septa, although with weaker signal than those within the basement membrane. Interestingly, the cells with weaker CK19 signals were colocalized withα-SMA, while CK19-positive BECs within the basement membrane wereα-SMA negative. AdshERKl, however, inhibited the coexpression of CK19 andα-SMA in biliary epithelium.To seek the evidence that hepatocyts can convert into matrix-producing myofibrolasts, double immunofluorescence staining was used to show the colocalization of albumin andα-SMA in the DMN model. Besides definite albumin staining in hepatocytes, a large amount of fusi-shaped cells with weaker albumin expression were also detected in fibrotic septa that were alsoα-SMA positive, while hepatocytes in the liver parenchyma wereα-SMA negative. Similarly, AdshERKl attenuated the coexpression of albumin andα-SMA in hepatocytes. These observations suggested that both BECs and hepatocytes contribute to the accumulation of matrix-producing myofibrolasts in fibrogenesis and AdshERKl protects the epithelial cells from this transformation.6. ERKl siRNA attenuates experimental hepatic fibrosis through inhibiting EMTWe then analyzed the levels of EMT markers by immunohistochemistry. E-cadherin decreased on the membranes of the parenchymal and nonparenchymal cells in both fibrosis models, whereas vimentin and snail were up-regulated. These changes were reversed by AdshERKl treatment. The localization of TGF-βin fusi-shaped myofibroblasts was also blocked by AdshERKl. In consistent with the immunohistochemical results, the enhanced expression of TGF-β1 mRNA level was substantially decreased by AdshERKl in both models. In addition, the expression of PDGF-BB, bone morphogenetic protein 4 (BMP4) and p-Smadl increased along with the upregulation of ERK1 in DMN-induced hepatic fibrosis, which was reduced after AdshERKl treatment.. Proliferating cell nuclear antigen (PCNA) expression decreased in the fibrotic liver, and AdshERKl treatment increased the levels of PCNA-positive hepatocytes in both models.[Conclusion]All of our results revealed that:1. Adenovirus delivered ERK1 siRNA effectively inhibites ERK1 expression in vitro, and the fibrotic characteristic of activated HSCs.2. Gene delivery of ERK1 siRNA in vivo was an effective strategy to attenuate experimental hepatic fibrosis induced by BDL or DMN.3. Myofibroblasts could be generated from hepatocytes and billiary epithelial cells besides HSCs.4. ERK1 is related with the activation of moyfibroblasts, and ERK1 siRNA could inhibite the activation of moyfibroblasts.5. ERK1 siRNA ameliorates hepatic fibrosis by inactivating HSCs, as well as interrupting EMT of hepatocytes and biliary epithelial cells.