| BACKGROUNDHepatic fibrosis, a common disease, is considered as an outcome of extracellular matrix (ECM) deposition because of chronic liver injury during a long-term wound healing response. It eventually leads to cirrhosis and later HCC. Hepatic fibrosis is a worldwide disease which seriously threatens people's health. Recent reports show that fibrotic liver can be returned to near-normal architecture under effective treatments but not cirrhosis. Thus the phage of fibrosis is a critical stage for treatment.The only effective treatment is liver transplantation for liver fibrosis and cirrhosis.But the donor liver is limited and patients who underwent liver transplantation have to receive life-long immune therapy. Therefore, it is urgent to ascertain the liver fibrosis development mechanism in order to provide effective treatmentLiver fibrosis is the predominant pathologic feature of end-stage liver disease which causes increasing amounts of extracellular matrix (ECM) deposition in the liver. All kinds of liver damage factor cause liver cell injury. During liver injury, the activated HSC adopts a myofibroblastic phenotype and is capable of vigorous proliferation, matrix protein synthesis. The activated HSC expresses a-SMA and produces a large number of ECM and causes the imbalance of matrix synthesis and degradation which leading to changes in liver structure. Extracellular matrix components include collagen,glycoproteins, proteoglycans and a variety of cytokines,such as TGF?1, TNF-alpha, interferon-y and leukocyte interleukin-la. TGF?1 is the major signaling factor to stimulate HSC produce extracellular matrix. With liver fibrosis, the balance is broken among various signaling pathways and TGF?1 expression increases significantly in the liver tissue. Then TGF?1 stimulates a variety of extracellular matrix production and inhibit their degradation.TGF?1-Smad3 is one of the most important signalings to cause liver fibrosis.The course of TGF?1 activates HSC as followed: Briefly, two types of transmembrane protein kinases, types ? and ? receptors, mediate signaling by TGF?1.Ligands interact with a homodimer of type ? receptors (TbR?), which recruit and activate type ? receptors (TbR?, activin receptor-like kinases, ALKs). The TbR?kinase then phosphorylates TbRI, which is one of the critical events in TGF?1 signaling and serves as the initiation point for the downstream events. Activated ALKs phosphorylate a subset of the downstream signaling molecules, the receptor-activated Smads (Smad3 and Smad2),which enables their binding to the common Smad (Smad4). And Smad3 or Smad2/Smad4 complex is shuttled into the nucleus where it can interact with various transcription factors and regulate transcription of many target genes. T?R? can be inhibited by the small molecule compounds SB431542 through specific binding with ALK5 kinase in ATP binding site. Therefore, phosphorylation of Smad3 and aggregation of Smad complex in the nuclear is inhibited and that slows the liver fibrosis progression.Reports have pointed out Slit2-Robol signaling pathway is multiple functional such as inhibiting neutrophil chemotaxis, the migration of vascular smooth muscle cells and langerhans cells in skin lesion, involving in the tumor angiogenesis,tumorigenesis and metastasis of glioma, lung cancer, breast cancer, colorectal cancer,hepatocellular carcinoma. Slit2-Robol signaling pathway is very important in hepatocellular carcinoma and inflammation, but liver cancer mostly bases on the hepatic cirrhosis and hepatic fibrosis is caused by chronic inflammation. So we speculate that Slit2-Robol signaling pathway would play an important role in the process of chronic inflammation-liver fibrosis-hepatic cirrhosis-hepatocellular carcinoma.OBJECTIVETo find the role of Slit2-Robol in liver fibrosis, we detected the expression of Slit2-Robol and TGF?1 signaling molecules in different types of fibrosis mice. LX-2 cell lines and clinical liver tissues. It will be helpful to understand the molecular basis of liver fibrosis, and to establish Slit2-Robol as a new way for therapeutic strategies.METHODS1. Establishment of liver fibrosis model induced by CCl4C57 and Slit2-Tg mice received an intraperitoneal injection of 2ml/kg of CCl4 in olive oil (1:4 ratio),2 times a week. After several weeks' injections (2, 4, 6, and 8 weeks, respectively), mice were sacrificed and liver tissues were collected. We selected the time point that mice liver presented obvious fibrosis and the degree of fibrosis was different between C57 and Slit2-Tg mouse as the modeling time.2. Expression of Slit2 and Robol in mice liver tissueExpression of Slit2 and Robol was detected by immunohistochemistry,immunofluorescense, Real time RT-PCR and Western blot in C57 and Slit2-Tg mice.3. Establishment of R5 blocking Slit2-Robol in liver fibrosis model induced by CCl4R5, a mouse monoclonal IgG antibody against the first immunoglobulin domain of Robo1 that neutralizes the interaction of Robo1 with Slit2 and mIgG is its iso-type control antibody. C57 mice received an intraperitoneal injection of 2ml/kg of CC14 in olive oil (1:4). Simultaneouly, R5 and mIgG were injected intraperitoneally.4. The comparison of liver fibrosis degree in C57 and Slit2-Tg mice after R5 blocking Slit2-Robol and before blocking and expression of a -SMAThe structure, content of collagen fibers and reticular fibers in the liver were detected by H&E, Sirius red and Reticulin staining respectively. Expression of a-SMA and Robol were detected by immunofluorescence.5. Expression of TGF?1 in C57/con, C57/CC14, Slit2-Tg/con, Slit2-Tg/CC14,C57/CC14/mIgG, C57/CC14/R5 groupExpression of Robo1,TGF?1, p-Smad3, Smad2/3,a -tubulin were detected by Western blot.6. The comparison of liver fibrosis degree and expression of p-Smad3, a-SMA,Robol in C57 and Slit2-Tg mice after SB431542 blocking TGF?1-Smad3 signaling and before blockingC57 and Slit2-Tg mice received an intraperitoneal injection of 2ml/kg of CCl4 in olive oil (1:4 ratio), 2 times a week. Simultaneously, they received an intraperitoneal injection of 5mg/kg of SB431542, 1 times a week. After 6 weeks' injections, mice were sacrificed and liver tissues were collected. The structure, content of collagen fibers and reticular fibers in the liver were detected by H&E, Sirius red and Reticulin staining respectively. Expression of ?-SMA was detected by immunofluorescence.7. The expression of ?-SMA,Robol,TGF?1,p-Smad3 in LX-2 cell lines after Slit2-Robol and TGF?1-Smad3 were lockedExpression of ?-SMA and Robo1 were detected by immunofluorescense and expression of TGF?1,p-Smad3 were detected by Western blot after Roobl siRNA and R5 blocking Slit2-Robol and before blocking in LX-2 cell lines. Expression of Robo1 was detected after TGF?1-Smad3 was blocked.8. The expression of a-SMA and Robo1 in human normal and hepatic fibrosis sectionsThe structure, content of collagen fibers and reticular fibers in the liver were detected by H&E, Sirius red and Reticulin staining respectively. Expression of a-SMA and Robo1 were detected by immunohistochemistry and immunofluorescense.9. The standard of liver fibrosisMouse liver fibrosis degree was evaluated according to Ishak Stage. 0, No fibrosis; 1, Fibrous expansion of some portal areas, with or without short fibrous septa; 2, Fibrous expansion of most portal areas, with or without short fibrous septa; 3,Fibrous expansion of most portal areas with occasional portal to portal bridging; 4,Fibrous expansion of portal areas with marked bridging (portal to portal as well as portal to central); 5, Marked bridging (portal - portal and/or portal - central) with occasional nodules (incomplete cirrhosis); 6, Cirrhosis, probable or definiteHuman liver fibrosis degree was evaluated according to Metavir Stage. 0, No fibrosis; 1, Fibrous portal expansion; 2, Few bridges or septa; 3, Numerous bridges or septa; 4, Cirrhosis.10. Statistical treatmentSPSS version 13.0 for Windows was used for all analyses. The statistical significance between experimental groups was determined by t test. Correlation was analyzed by Spearmans. All values are expressed as mean ± standard deviation (S.D.).P values of less than 0.05 were considered as statistically significant.RESULTSThe main results and findings are as follows:1. Establishment of liver fibrosis model induced by CCl4Mice were sacrificed and liver tissues were collected after being injected 20%CCl4 in C57 and Slit2-Tg mice. The degree of liver fibrosis increased with the prolonged induction time and the Ishake score was higher in Slit2-Tg mouse than C57 mouse (F=83.932, P=0.000). The collagen fibers content of liver increased with the prolonged induction time and it was higher in Slit2-Tg mouse than C57 mouse. It was statistically significant when mice were induced 6 week (F=1 81.763, P=0.000).Moreover, the collagen fibers content of liver in C57 and Slit2-Tg mice were much higher than control and solvent control group (F=61.027,P=0.000). Hepatic fibrosis can not occur when mice were injected olive oil because the collagen fibers content of liver was similar in solvent control with in control. So, we decided to inject 20% CCl4,2 times a week for 6 weeks as the liver fibrosis model.2. Activation of Slit2-Robol in fibrotic liver tissueExpression of Slit2 and Robol was detected by immunohistochemistry and the results as follows: The IOD of Slit2 increased from 0.01% in control C57 group to 0.29% in hepatic fibrosis C57 group. The IOD of Slit2 increased from 0.02% in control Slit2-Tg group to 0.64% in hepatic fibrosis Slit2-Tg group. The IOD of Slit2 was markedly higher in Slit2-Tg group than C57 group (F=33.690, P=0.000). The IOD of Robo1 increased from 0.02% in control C57 group to 0.37% in hepatic fibrosis C57 group. The IOD of Robo1 increased from 0.04% in control Slit2-Tg group to 0.76% in hepatic fibrosis Slit2-Tg group. The IOD of Robol was markedly higher in Slit2-Tg group than in C57 group (F=14.788, P=0.001). The expression of Slit2 and Robo1 were higher in liver of hepatic fibrosis Slit2-Tg group than hepatic fibrosis C57 group detected by Real time RT-PCR (P=0.027, P=0.044). The expression of Slit2 and Robo1 were consistent when they were detected by Real time RT-PCR and Western blot in C57 and Slit2-Tg mice. These results suggest that Slit2-Robo1 signaling was activated in fibrotic liver. The results of Real time RT-PCR showed Slit2 and Robol increasing by 2.75±1.34 and 11.29±1.05 fold in CCl4-induced C57 mice; by 7.60±15.10 and 14.36±3.91 fold in CCl4-induced Slit2-Tg mice (t=-1.874, P=0.027; t=-1.544, P=0.044) . The expression trend of Slit2 and Robol detecting by western is consistent with immunohistochemistry and immunofluorescense.3.Slit2-Robol signaling promoted hepatic fibrosisIn normal C57 and Slit2-Tg control group, H&E staining showed a normal liver construction and Ishake score was 0. CCl4 - induced model of C57 and Slit2-Tg mice showed different pathological changes from control group, besides hepatocyte necrosis, aberrant construction was the major feature and Ishake score were 3.10±0.99 and 4.40±0.84 respectively (t=-3.153, P=0.006) . Ishake score were 2.20±0.92 and 3.70±0.82 in C57/CCl4/R5 and C57/CCl4/mIgG group respectively(P=0.003). Sirius red staining showed a normal distribution of collagen in a small amount in the portal tracts and around the terminal hepatic veins and the collagen fibers content were 0.05%±0.02% and 0.06±0.02% in C57/con and Slit2-Tg/con.CCl4-induced model showed complete fibrotic septum, forming a pattern of micronodular cirrhosis to the parenchyma and the collagen fibers content were 1.95±1.01% and 4.06±1.58% in C57/CCl4 and Slit2-Tg/CCl4. In C57 mouse treated with R5, Sirius red staining showed a smaller collagen deposition in the portal tracts and lobules. Only collagen fibers around the terminal hepatic veins were observed and collagen fibers content were 0.65±0.34% and 1.87±0.99% in C57/CCl4/R5 and C57/CCl4/mIgG group respectively (P=0.019). Reticulin staining showed a normal distribution of reticular fibers around the hepatocyte and the reticular fibers content were 1.13%±0.14% and 1.11 ±0.18% in C57/con and Slit2-Tg/con.CCl4-induced model showed complete fibrotic septum and the reticular fibers content were 18.58±1.75% and 42.07±1.22% in C57/CC14 and Slit2-Tg/CCl4. In C57 mouse treated with R5, Reticulin staining showed a small reticular deposition and reticular fibers content were 7.52±0.99% and 20.77±1.19% in C57/CCl4/R5 and C57/CCl4/mIgG group respectively (P=0.000). Immunofluorescence staining showed a distribution of?-SMA around the vessels and IOD were 0.10±0.03% and 0.01±0.00% in C57/con and Slit2-Tg/con. CCl4-induced model The IOD were 0.54±0.20% and 1.24±0.32% in C57/CCl4 and Slit2-Tg/CCl4 (P=0.018). In C57 mouse treated with R5, IOD were 0.12±0.02% and 0.52±0.19% in C57/CCl4/R5 and C57/CCl4/mIgG group respectively(P=0.018).The degree of hepatic fibrosis and the expression of a-SMA and Robol decreased in C57 mouse when Slit2-Robolwas blocked by R5. We concluded that Slit2-Robol promoted hepatic fibrosis of C57 and Slit2-Tg mice through active HSC.4. The differential expression of TGF?1 signaling molecule in fibrotic liver of C57 and Slit2-Tg mice namely pre-and post-Slit2-robo1 being blockedWestern blot showed the expression of Robo1, TGF-?1 and p-Smad3 increased significantly in fibrotic liver, especially in Slit2-Tg fibrotic liver. The expression of TGF-?1 and p-Smad3 decreased after Slit2-Robo1 being blocked by R5.5. The degree of hepatic fibrosis and expression of p-Smad3, Robo1were lower in liver of C57 and Slit2-Tg mice after TGF?1-Smad3 being blocked by SB431542,especially in Slit2-Tg miceThe relative liver weight decreased after TGF?1-Smad3 being blocked by SB431542. In fibrotic control group of C57 and Slit2-Tg, H&E staining showed a destruction of construction. Compared with the control group, the SB431542 treatment group had a significantly alleviated liver injury after treatment for 6 weeks.Ishake score were 3.30±0.82 and 2.20±1.14 in control group and SB431542 treatment group of C57 mouse respectively and Ishake score was decreased by 33.3%(t=2.480,P=0.023) . Ishake score were 4.50±0.71 and 2.20±0.79 in control group and SB431542 treatment group of Slit2-Tg mouse respectively and Ishake score was decreased by 51.1% (t=6.866, P=0.000). Sirius red staining showed that the collagen fibers content was 2.16±0.43% and 1.57±0.29% in control group and SB431542 treatment group of C57 mouse respectively, decreasing by 27.3% (t=3.259,P=0.006) . Collagen fibers content was 4.10±0.94% and 2.33±0.53% in control group and SB431542 treatment group of Slit2-Tg mouse respectively and reducing by 43.2% (t=4.624, P=0.000). Reticulin staining showed that the reticular fibers content was 22.48±2.21 % and 17.52±1.98% in control group and SB431542 treatment group of C57 mouse respectively, decreasing by 22.1% (t=3.726, P=0.006) .Reticular fibers content was 40.76±2.74% and 23.26±1.54% in control group and SB431542 treatment group of Slit2-Tg mouse respectively and reducing by 42.9% (t=12.430,P=0.000). Compared with the control, the IOD of ?-SMA of C57 and Slit2-Tg treated groups decreased from 0.70±0.10% and 0.43±0.05% (t=5.975, P=0.000) and from 1.42±0.27% and 0.71±0.10% (50%,t=5.953,P=0.000), respectively. The data indicate that the degree of hepatic fibrosis and the number of active HSC reduced after SB431542 blocking TGF?1-Smad3 especially in .Slit2-Tg group. Western blot showed that expression of ?-SMA, p-Smad3, Robo1 were decreased after TGF?1-Smad3 was blocking in C57 and Slit2-Tg mouse hepatic liver.6. The expression of ?-SMA, Robol, TGF?1, p-Smad3 in LX-2 cell lines after R5 blocking Slit2-Robol and before blockingImmunofluorescence staining showed that the expression of a-SMA and Robol increased markedly at LX-2 cell lines after stimulating with recombinant human transforming growth factor?1 compared to the untreated group. Cells with a-SMA cytoplasm positive must coexist Robol positive. The expression of a-SMA reduced in LX-2 cells treated with R5 or Robo1 siRNA and recombinant human transforming growth factor?1 simultaneously. The expression of ?-SMA and Robo1 was similar between TGF?1 treated group and TGF?1+mlgG treated group. The results showed the control antibody mIgG had no effect on LX-2 cell. The expression of a-SMA?TGF?1 and p-Smad3 were detected by Western blot and the results directed that?-SMA?TGF?1 and p-Smad3 increased in TGF?1 treated group and decreased after R5 treatment in LX-2 cell lines. Expression of Robol decreased after TGF ?1-Smad3 was blocked .Thus, Slit2-Robol promoted the LX-2 to activate through active TGF?1-Smad3 signaling and Robol may be regulated by TGF?1-Smad3.7. The expression of ?-SMA and Robol in human normal and hepatic fibrosis sectionsH&E staining showed the tissue construction was normal in control liver and fibrotic liver showed different pathological such as hepatocyte necrosis, aberrant construction and fibrotic septum. Sirius red and staining showed a normal distribution of collagen fibers in the portal tracts and around the terminal hepatic veins but fibrotic liver showed complete fibrotic septum, forming a pattern of cirrhosis to the parenchyma. Reticulin staining showed a normal distribution of reticular fibers around the hepatocyte but fibrotic liver showed the reticular fibers increased and reticular fibers deposited in septum irregularly and formed fibers-bridge or oval septum. Immunohistochemistry staining of ?-SMA showed a distribution around vessels in normal liver but distribution in septum and around portal vein in fibrotic liver. The expression of Robol were higher just as ?-SMA in fibrotic liver compared with the normal liver and colocalized expressions were observed. The positive correlation existed between ?-SMA, Robo1 and stage, between ?-SMA and Robo1.Those results suggest that Slit2-Robol promote liver fibrosis through activating the HSC in human fibrotic liver tissues.CONCLUSIONS1. Slit2-Robol signaling was activated when C57 and Slit2-Tg mice suffered liver fibrosis;2. The fibrosis level was higher in Slit2-Tg mice than C57 mice,and the fibrosis level decreased after endogenous Slit2-Robo1 being blocked with R5. So we presumed that Slit2-Robo1 signaling promoted mouse hepatic fibrosis;3. The expression changes of ?-SMA and Robo1 were consistent with the fibrosis level, but also they were co-locating expressions. So we presumed that Slit2-Robo1 signaling promoted mice hepatic fibrosis through activating HSC;4. Slit2-Robol accelerated hepatic fibrosis development through up-regulating the expression TGF?1 and p-Smad3;5. Slit2-Robol promoted human liver fibrosis by activated HSC.INNOVATIVE POINTS1. Slit2-Robol signaling promoted hepatic fibrosis through activating HSC;2. A new view was given that Slit2-Robol accelerated hepatic fibrosis development through up-regulating the expression TGF?1-Smad3 signaling;3.A method was found that R5 and SB431542 may reduce liver fibrosis. |
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