טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentPevzner Anna
SubjectThe Effect of Halofuginone on Rat Hepatic Stellate Cell
Activation
DepartmentDepartment of Medicine
Supervisor Professor Gad Spira
Full Thesis textFull thesis text - English Version


Abstract

The currently existing treatments for advanced liver fibrosis and cirrhosis are unsatisfactory or inefficient. Antifibrotic treatment that can prevent, halt or even reverse advanced fibrosis is thus urgently needed.

The semisynthetic plant alkaloid halofuginone (HF) was reported to downregulate collagen type I synthesis in both in vivo and in vitro models. In fibrotic rats, HF was found to partly reverse experimentally induced liver fibrosis. In all of the above, the antifibrotic effect of HF was associated with reduced number of cells positively stained for α smooth muscle actin (αSMA). The present study aimed mainly at unraveling the mechanism by which HF affects hepatic stellate cells (HSC) activation and activity. Given the heterogeneous population of HSC in the liver and its possible different responsiveness, the work was carried out using primary and three HSC lines, each presenting different responsiveness to stimuli and differentiation state.

HF affected cell proliferation at concentrations higher than 100nM. In all three lines, as well as in primary HSC line, HF's treatment was associated with downregulation of collagen type I and MMP-2 mRNA levels and increased levels of MMP-13 transcripts. The expression of αSMA, MMP-3, MMP-9, MMP-14, TIMP-1 and TIMP-2 mRNA levels varied among the cells. HF's pro-apoptotic activity was tested, using various apoptotic markers: active caspase 3 - a member of the caspase cascade, activated during apoptosis and common to all apoptotic pathways; a member of the histone family - H2AX, which is phosphorylated upon apoptotic induction, and TUNEL staining - an indication for DNA fragmentation, occurring during the last stages of cell death progression. Treatment with HF elevated all three parameters in all cell lines tested. The drug also upregulated the apoptosis related protein (ARTS) levels in treated cells, implying the intrinsic pathway involvement in HF induced apoptotic effect.

The data presented suggest that HF’s therapeutic effect on liver fibrosis is the result of multiple pathways, including inhibition of collagen type I synthesis, acceleration of ECM degradation, and induction of apoptosis of HSC.