Chronic alcohol induced liver disease (ALD) comprises of a spectrum of disease stages progressing from fatty liver, steatohepatitis, fibrosis, to cirrhosis that may eventually lead to death. Although, the early stages of ALD are reversible, 40% of the patients develop advanced stage liver disease characterized by significant hepatic fibrosis and cirrhosis, for which, currently, liver transplantation is the only curative approach. However, the number patients waiting for liver transplantation far exceeds the meager number of available donors resulting in premature mortality of such patients. Hence, there is an urgent need for therapies for not only prevention and early intervention to stop the disease progression, but also to effectively regenerate the remaining healthy liver so that the patient can be reasonably functional before they can fully recover with a liver transplantation. Thus, any biologically natural modulator that can effectively prevent the progression of ALD until the donor liver is available for transplantation would be desirable even if it cannot completely cure the disease.
Thymosin β4 (Tβ4) is an immune modulating natural peptide secreted by thymus gland that is known to prevent inflammation and fibrosis, and promote wound healing and regeneration in the eye, skin and heart. Previous work from our laboratory has also shown that Tβ4 protects against carbon tetrachloride induced acute liver injury in rat. However, not much is known of the role of Tβ4 in alcoholic liver injury. Therefore, in this dissertation research, the role of Tβ4 was investigated in acute on chronic ethanol and lipopolysaccharide (LPS) induced hepatic oxidative stress, inflammation, and fibrosis in an in vivo mouse model, as well as its regenerative potential was studied in chronic ethanol fed mice after partial hepatectomy. Furthermore, the underlying molecular mechanism by which Tβ4 exerts its action, particularly on fibrosis was examined using human hepatic stellate cells (HSC), the main fibrogenic cells of the liver.
Based on the well accepted two-hit model for ALD, in the hepatocytes, ethanol acts as the first hit and is oxidized to acetaldehyde, the highly toxic first metabolite of ethanol oxidation by alcohol dehydrogenase (ADH) and ethanol-inducible cytochrome P450 2E1 (CYP2E1) leading to the generation of reactive oxygen species (ROS), resulting in oxidative stress. On the other hand, ethanol-induced leaky gut results in the release of endotoxin (LPS) that acts as the second hit and activates nuclear factor Kappa B (NF?B) in the Kupffer cells and the subsequent production of the pro-inflammatory cytokines that propagates liver inflammation. ROS and the pro-inflammatory cytokines act as fibrogenic stimuli for the activation of HSC and their trans-differentiation from quiescent lipid storing phenotype to activated myofibroblasts that express fibrogenic genes and proliferate and migrate to the site of injury and form a fibrous scar, resulting in fibrosis. This is essentially due to the fact that the quiescent HSC exhibit up-regulated adipogenic gene, peroxisome proliferator-activated receptor gamma (PPARγ), and down-regulated fibrogenic gene, methyl CpG binding protein (MeCP2), whereas the reverse is true upon their activation to myofibroblasts.
The experimental results showed that Tβ4 reduced the ethanol and LPS induced levels of ROS by increasing the levels of the antioxidants, glutathione and superoxide dismutase. It also inhibited the nuclear translocation of NFκB by blocking the phosphorylation of the inhibitory protein IκB and thereby prevented the up regulation of pro-inflammatory genes, TNF-α, IL-1β, and IL-6. Tβ4 also prevented the activation of HSC by up-regulating miRNA 132, thus suppressing MeCP2, that coordinately reversed the down-regulated adipogenic gene, PPARγ, and the up-regulated fibrogenic genes (α-smooth muscle actin, PDGF-β receptor, collagen 1, and fibronectin), and fibrosis. Moreover, Tβ4 also promoted liver regeneration after partial hepatectomy in chronic ethanol fed mice by increasing hepatocyte growth factor and its receptor, c-Met; α-fetoprotein; proliferation markers, proliferating cell nuclear antigen and Ki-67 as well as the liver progenitor cell marker, cytokeratin 19.
Furthermore, it was discovered that in human HSC cultures, Tβ4 prevented PDGF-BB induced fibrogenesis and also abolished PDGF-BB induced HSC proliferation and migration by blocking the phosphorylation of Akt by preventing the binding of Akt to actin. Moreover, experiments with two bioactive peptides of Tβ4, the amino terminal peptide (1-15 aa) and the actin binding peptide (17-23 aa) revealed that Tβ4 exerts most of its anti-fibrotic actions via its actin binding domain.
In conclusion, these data suggest that Tβ4 has antioxidant, anti-inflammatory, anti-fibrotic and hepatic regenerative potential against alcoholic liver injury.
|Advisor:||Lakshman, Raj, Hu, Valerie|
|Commitee:||Caldovic, Ljubica, Kumar, Ajit|
|School:||The George Washington University|
|Department:||Biochemistry and Systems Biology|
|School Location:||United States -- District of Columbia|
|Source:||DAI-B 78/08(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology, Biochemistry|
|Keywords:||Alcoholic liver disease, Hepatic stellate cells, Lipopolysaccharide, Liver fibrosis, Liver regeneration, Thymosin beta 4|
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