Newcastle Disease Virus Represses The Activation Of Human Hepatic Stellate Cells And Reverses The Development Of Hepatic Fibrosis In Mice

Hepatocellular carcinoma (HCC) is the most common histological type of liver cancer. About seventy to ninety percent of HCC patients are accompanied with liver cirrhosis in China. In Western countries which have a increasing occurrence tendency of HCC in recent years, the liver carcinogenesis is almost derived from the liver cirrhosis, influencing the therapeutic choice, therapeutic effect, complications, and prognosis for the treatment of HCC. The mechanisms of HCC development in cirrhosis are based on two aspects: 1) under the exist of some detriment factors, such as hepatitis virus, flavacin, metabolism disease, and alcohol, hepatocytes are damaged persistently, resulting in liver cell degeneration and necrosis followed by excess synthesis of extracellular matrix (ECM) induced by inflammatory reaction. ECM cannot be degraded and deposited in hepatic tissue, which contributs to the liver fibrogenesis finally cirrhosis characterized by nodular regeneration which is surrounded by new formed fibrous tissue. Hepatic stellate cells (HSC) transform from quiescent to activated state and transdifferentiates into myofibroblasts which secrete excessive ECM, playing an important role in the development of hepatic fibrosis and cirrhosis. 2) Reactive oxygen species are generated under the inflammatory circumstance which then induces DNA damage in hepatocytes. The damaged hepatocytes are proliferated in response to the cytokines secreted via paracrine. The alternation between damage and regeneration induces polygenic mutations initiating carcinogenesis. Liver fibrogenesis caused by HSC activation which is an important pathologic stage during the development of HCC. Immunohistochemistry shows that a number of activated HSCs exits in hepatic tissue from HCC complicating cirrhosis. While mitogenic factors secreted by HCC cells promote the activation and proliferation of HSC. The interaction between activated HSC and malignant cells aggravate the HCC development. Targeted therapy focusing on the activated HSC is noval strategy for repression of liver fibrosis. There are two methods to repress the activation of HSC: blocking transforming growth factorβ(TGF-β) signal pathway to antagonize the function of TGF-βon activation of HSC; decreasing the synthesis of ECM and degrading the excessively deposited collagen in liver tissue.Newcastle disease virus (NDV) contains nonsegmented single-stranded negative-sense RNA and is a member of the family Paramyxoviridae. Because most RNA virus have the characteristics of selective replication in tumor cells, NDV is interesting to be a new viral carrier in tumor gene therapy in recent years. In the other hand, NDV is applied clinically in tumor therapy due to the oncolytic properties. The mechanism of the NDV replication in tumor cells is based on the defective interferon signal pathway in these tansformed cells. In 1976, McGregor reported NDV replicated in activated T cells and induced the cell death. Recently, Fábián confirmed that NDV can replicate in abnormal transformed cells effectively. Activated HSC is also a kind of"abnormal"and is capable of proliferation, we presumed that NDV can replicate in these cells followed by repression of the activation of cells and reversion of hepatic fibrosis. In this dissertation my intention is focusing on the repression of HSC activation by an oncolytic strain NDV-Italien, investigation of the NDV replication in activated HSC and its effects on decrease of the levels of mRNA and protein in those genes which are related to hepatic fibrosis. We evaluated the repression of liver fibrosis by NDV in vivo through mouse hepatic fibrosis model.PartⅠNDV replication in the activated HSC induced with human hepatocellular carcinoma conditioned mediumAim: To explore the function of conditioned medium (CM) from human HCC cell line FHCC-98 on the activation of human hepatic stellate cell line LX-2 and to investigate the replication efficiency of NDV in LX-2 cells. Methods: LX-2 cells were stimulated with different concentrated CM from FHCC-98 cells and the proliferation of LX-2 cells after stimulation was detected by MTT assay in which TGF-β1 was used as positive control to evaluate the role of CM. Semi-quantitative and Real-time quantitative RT-PCR was used to measure the changes of mRNA levels ofα-SMA, collagenⅠ, TIMP-1, and TGF-β1 which are related with HSC activation in LX-2 cells after stimulation by 40% CM or TGF-β1. After infected by NDFLtag-EGFP, a recombinant NDV carring enhanced green fluorescent protein (EGFP), virus replication in LX-2 cells allows the expression of EGFP which can be observed under fluroscent microscopy and quantitated by flow cytometry (FACS). Results: Either FHCC-98 CM or TGF-β1 can stimulate the activation of LX-2 cells. Low concentrations of CM are beneficial to the cell proliferation than the high ones. The maximum proliferation rate is 25.8% with 40% CM stimulation. TGF-β1 induced the proliferation of LX-2 cells in a dose-dependent way. After stimulated by 40% CM or TGF-β1, the mRNA expression levels ofα-SMA, collagenⅠ, TIMP-1, and TGF-β1 were up-regulated comparing with the unstimulated control group. The efficiency of NDV replication was increased in the consecutive passages of LX-2 cells, FACS analysis showed that the efficiency of NDV replication in 40% CM and TGF-β1 stimulated cells were enhanced to 1.76 and 1.52 folds compared with the controls. Conclusion: CM from FHCC-98 cells induced the proliferation and activation of LX-2 cells, the replication efficiency of NDV was increased in this activated LX-2 cells.PartⅡRepression of HSC activation by NDVAim: To explore the influence of NDV replication in LX-2 cells on the cell viability and behavior of cellular biology. Methods: CM-stimulated LX-2 cells were infected by different titres of oncolytic NDV-Italien, the proliferation rate of LX-2 cells was detected by MTT assay. Immunofluorescent technique was used to detect the expression ofα-SMA in LX-2 cells. Real-time quantitative RT-PCR was used to evaluate the expression ofα-SMA, collagenⅠ, TIMP-1, and TGF-β1 in the activated LX-2 cells after NDV infection. Secretion of MMPs in LX-2 cells was determined by gelatin zymography. Results: NDV infection in LX-2 cells repressed the cell proliferation in a dose-dependent manner. Compared with control, the growth inhibition rate of activated LX-2 cells was increased. The mRNA expression of four genes,α-SMA, collagenⅠ, TIMP-1, and TGF-β1 in activated LX-2 cells were down-regulated and the secretion of MMP-2 and MMP-9 was decreased after NDV infection. Conclusion: The replication of NDV in activated LX-2 cells repressed the cell proliferation and attenuated the activation of LX-2 cells.PartⅢReversion of hepatic fibrosis by NDV in mice induced by CCl4Aim: To explore the influence of NDV treatment on the reversion of hepatic fibrosis in mice induced by CCl4 in vivo. Methods: Kunming mice (weight about 20 g) were injected intraperitoneally (i.p.) 100μl carbon tetrachloride (CCl4)/peanut oil solution (20%, V/V) twice a week, the mice were given 100μl physiological saline (PS) replacing CCl4 as PS group for 8 weeks. Each mouse was treated with 1,000 hemagglutination unit (HU) NDV-Italien in 200μl volume via tail vein for one time or 3 times with 24 hour-interval after the final infusion of CCl4. The mice were sacrificed at 24 h-post injection of NDV and liver tissues were excised for grossly examination with naked eye and taking photograph. Tissue samples were fixed in 10% formalin, then embedded in paraffin, the sections were stained by HE and masson's trichrome methods. Fresh tissue samples were lysed in RIPA lysis buffers and protein contents was determined by BCA assay. Fifty microgram total protein for each sample was subjected to 10% SDS-PAGE electrophoresis and detected the expression ofα-SMA by western blot analysis. Cryostat sections of liver tissues were fixed with cold acetone and followed by observision of NDV virions andα-SMA expression under fluorescent microscopy with double staining technique. Results: After 8 weeks of CCl4 induction, the mouse liver showed fibrosis symptom severely. Hepatic tissues become hard, coarse and there were large amounts of white plaques on liver surface. HE staining showed that tissue structure in fibrosis liver was loose and perisinusoidal space is dilated. Masson's trichrome staining revealed that collagen in fibrosis tissue was deposited abnormally. After three times injection of NDV, white plaques on liver surface were dispelled dramatically and deposition of collagen in liver tissue was suppressed. Immunofluorescence microscope showed thatα-SMA and NDV were detected in the perisinusoidal spaces and co-located in activated HSCs, while no specific adsorption was visualized in liver tissues of normal control mice. Conclusion: In CCl4-induced mouse hepatic fibrosis, NDV repressed the activation of HSC and reversed fibrosis of liver through its selective replication in the activated HSC.