| Summary: | 博士 === 國立陽明大學 === 臨床醫學研究所 === 101 === Background: Acute liver injury is manifested by different degree of hepatocyte necrosis and may have chance to recover via the process of hepatocyte regeneration. Thus an effective therapy should be able to reduce injury and promote liver repair and regeneration. Resveratrol (RSV) is a natural product with anti-inflammation, anti-oxidation and anti-ageing activities, which may have potential therapeutic effect on liver injury. However, whether RSV may promote liver regeneration is still unclear. To promote liver regeneration, stem cell therapy is most promising. The induced pluripotent stem (iPS) cells can be generated from somatic cells and differentiated into hepatocytes. But at present, the effects and the exact mechanism of iPS cells on acute liver injury haven’t been revealed. In addition, the tumorgenic effect of iPS is still under concern. Therefore, it would be helpful for developing a new and safer treatment stratagem of liver injury in the future if we could understand and adopt their beneficial mechanisms of action. Aims: The aims of this study were to investigate the therapeutic effects and underlying mechanism of iPS and RSV in liver injury, seeking for different and related mechanism. Materials and Methods: Liver injury was induced by carbon tetrachloride (CCl4) injection or bile duct ligation (BDL) inC57/Bl6 mice. Injured mice were randomly allocated to receive vehicle, RSV or iPS rescued. The blood and liver were harvest at specific time points for studies of liver biochemistry, liver histology, hepatocyte apoptosis and proliferation, and hepatic cytokines expressions. Therapeutic effects on kupffer cells (KC), hepatic stellate cells (HSC) and survival of mice were evaluated. The experiments were performed in parts to investigate (A1) the effect and mechanism of iPS on CCl4 injured mice. (A2) the effect and mechanism of RSV on CCl4 and BDL (B1) injured mice. Results: (A1) Effect of iPS on CCl4-injured mice: iPS performed better than the iPS-derived hepatocyte in reducing ALT, AST and liver necrosis area. IPS significantly reduced TNF-a but increased CXCL10 expression and promoted hepatocyte proliferation. In vitro culture study demonstrated the iPS secreted CXCL10 and increased the release of CXCL10 in CCl4-injured AML12 dose dependently. In addition, iPS treatment increased CXCL10 protein and mRNA expression in the injured liver in vivo. The hepatocytes isolated from iPS-treated injured liver expressed more CXCl10 mRNA. The administration of recombinant CXCL10 (rCXCl10) effectively reduced liver injury as iPS alone while the CXCL10-neutralizing antibody attenuated the protective effects of iPS and decreased hepatocyte proliferation. Both iPS and rCXCL10 significantly reduced the mortality rate in CCl4-injured mice. (A2) Effect of RSV on CCl4-injured mice: RSV significantly decreased ALT, AST, liver necrotic areas and liver 4-HNE. Although RSV reduced hepatocyte apoptosis (decreased the expressions of caspase 8 and 3 without affecting Bax、Bcl-xL), RSV also reduced hepatocyte proliferation around portal area. RSV reduced hepatic KC recruitment, TNF- and IL-6 but not IL-10 mRNA expressions. RSV reduced HGF, c-Met and TGF-α mRNA expressions but had no effect on collagen 1a1 and αSMA, indicated RSV did not affect HSC activation. Cytokine analysis revealed RSV decreased CXCL10 (IP-10) concentration in serum and its mRNA expressions in liver. In vitro culture study revealed that CXCL10 could promote the proliferation of mouse hepatocyte cell line (AML12). In addition, RSV inhibited the release of CXCL10 from CCl4-injred AML12. This study indicated that RSV was beneficial for CCl4-induced liver injury but could not promote hepatocyte proliferation possible due to inhibitory effect on CXCL10. (B1) the effect and mechanism of RSV on BDL injured mice: RSV significantly reduced serum ALT, AST and hepatic necrotic areas. RSV increased the survival rate of injured mice. RSV decreased the number of KC recruited in the injured liver and reduced IL-6, TNF-α mRNA expressions. In addition, RSV promoted hepatocyte proliferation. RSV inhibited the expressions of collagen Ia1 and TIMP-1 to reduced hepatic fibrosis. RSV lowered the level of 4-HNE, an index of oxidative stress, in the injured liver and increased HGF expression. RSV did not affect HSC activation. This study indicated that RSV was beneficial for cholestatic liver injury and could promote hepatocyte regeneration. Conclusions: Both iPS or RSV, through their anti-inflammatory or antioxidant effects, were beneficial for reducing the degrees of liver injury. However, they performed differently in the promotion of liver regeneration. This study found that CXCL10 acted as a hepatoprotective agent and could promote AML12 proliferation. The expressions of CXCL10 in RSV experiments were very different from those in iPS experiments. RSV did not promote hepatocyte proliferation in CCl4-injured model possibly due to its inhibitory effect on CXCL10 synthesis and release. In contrast, iPS was able to promote hepatocyte proliferation. iPS itself not only release CXCL10 and also enhance the synthesis and release of CXCL10 by hepatocyte. In conclusions, CXCL10 could be an important hepatoprotective factors to facilitate hepatocyte regeneration in CCl4-injured liver. It deserves further study and may have the potential to offer alternative therapeutic stratagem in the clinical management of liver injury in the future.
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