Apoptotic Gene Expression in Sheep Hepatocytes during Fasciola hepatica Infection (Fascioliasis)

Abstract

Aims: The main objective of this work was to investigate the apoptotic genes of sheep liver hepatocytes to elucidate the apoptosis pathway mechanisms during Fasciola hepatica infection using molecular and serological techniques. Study Design: This is a laboratory based study whereby F. hepatica infected liver specimens were used. Methodology: Total RNA was extracted from fresh-frozen liver tissue. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to investigate the genes encoding the mRNA for following proteins: 18S; Bax; Bcl-2; Caspase-3. Additionally, the concentration of total protein in each sample was estimated spectrophotometrically. Polyclonal antibodies were produced by immunizing rabbits and diluted with 5% skimmed milk in PBS containing 0.1% Tween 20 (1:1000). The detection of the reacted antigen/antibody products was performed using immunoblotting technique were used to assess the quantification of protein kinetic to apoptotic genes. Data obtained from this investigation were analyzed using Portable IBM SPSS 2006 Statistics software. Results: We investigated the apoptotic gene expression in sheep liver hypatocytes during                F. hepatica infection by mRNA expression of three genes involved in apoptosis; Bax, Bcl-2 and Caspase-3 and histopathological parameter after infection with F. hepatica in the liver cells. Quantitative real-time PCR, histological examination and immunoblotting were used to quantify apoptotic genes, histopathology and protein kinetic, respectively. F. hepatica infection induces apoptosis in the liver cells via Bax, Bcl-2 and Caspase-3 genes. Conclusions: F. hepatica infection induces apoptosis in the liver cells via Bax, Bcl-2 and Caspase-3 genes and it precedes necrosis. Thus, this study suggests that the induced apoptotic gene expression was due to the outcome of F. hepatica.