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, 23 (9), 708-17

Hepatitis C Virus Dynamics and Cellular Gene Expression in uPA-SCID Chimeric Mice With Humanized Livers During Intravenous Silibinin Monotherapy

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Hepatitis C Virus Dynamics and Cellular Gene Expression in uPA-SCID Chimeric Mice With Humanized Livers During Intravenous Silibinin Monotherapy

S DebRoy et al. J Viral Hepat.

Abstract

Legalon SIL (SIL) is a chemically hydrophilized version of silibinin, an extract of milk thistle (Silybum marianum) seeds that has exhibited hepatoprotective and antiviral effectiveness against hepatitis C virus (HCV) in patients leading to viral clearance in combination with ribavirin. To elucidate the incompletely understood mode of action of SIL against HCV, mathematical modelling of HCV kinetics and human hepatocyte gene expression studies were performed in uPA-SCID-chimeric mice with humanized livers. Chronically HCV-infected mice (n = 15) were treated for 14 days with daily intravenous SIL at 469, 265 or 61.5 mg/kg. Serum HCV and human albumin (hAlb) were measured frequently, and liver HCV RNA was analysed at days 3 and 14. Microarray analysis of human hepatocyte gene expression was performed at days 0, 3 and 14 of treatment. While hAlb remained constant, a biphasic viral decline in serum was observed consisting of a rapid 1st phase followed by a second slower phase (or plateau with the two lower SIL dosings). SIL effectiveness in blocking viral production was similar among dosing groups (median ε = 77%). However, the rate of HCV-infected hepatocyte decline, δ, was dose-dependent. Intracellular HCV RNA levels correlated (r = 0.66, P = 0.01) with serum HCV RNA. Pathway analysis revealed increased anti-inflammatory and antiproliferative gene expression in human hepatocytes in SIL-treated mice. The results suggest that SIL could lead to a continuous second-phase viral decline, that is potentially viral clearance, in the absence of adaptive immune response along with increased anti-inflammatory and antiproliferative gene expression in human hepatocytes.

Keywords: anti-inflammatory; chimeric mice with humanized livers; gene expression; uPA-SCID; viral kinetic modelling.

Conflict of interest statement

Conflict of Interest Disclosures: CT is an employee of PhoenixBio Co. Ltd. None of the authors has any financial interest or conflict of interest related to this research.

Figures

Figure 1
Figure 1. Serum HCV RNA and human albumin (hAlb) kinetics with model curves
Chronically HCV infected uPA-SCID chimeric mice with humanized livers were treated intravenously with the indicated doses of SIL. Blood was drawn from mice daily. Serum HCV RNA level (solid black squares) and human albumin level (open grey triangles) are graphed as log10 IU/mL and ng/mL, respectively. HCV model curves (black lines) and hAlb linear regression (grey dashed lines) are shown. See Tables S1 and 1 for viral kinetic and estimated parameter values, respectively.
Figure 2
Figure 2. Ingenuity Pathway Analysis (IPA) canonical pathway analysis
Linear Modeling of Microarray (limma) results were analyzed using IPA. P-values were calculated using the right-tailed Fisher exact test and reflect the likelihood that the association between SIL-related genes and genes in a given canonical pathway is due to random chance. Bar length represents the –log10 P-value; therefore, the longer the bar, the less likely the association is due to chance; i.e. the pattern of differentially expressed genes in SIL-treated versus control mice suggests involvement of the antigen presentation and granulocyte adhesion pathways. Bars crossing the red threshold line have P-values less than 0.05.
Figure 3
Figure 3. Predicted TNFα and NFκB –mediated regulatory effects downstream of SIL
IPA software uses pathway information to identify potential upstream regulators that could explain the observed pattern of gene expression. The blue square in (A) and circle in (B) indicate that inhibition of the predicted upstream regulator TNFα (A) and NFκB (B) could directly or indirectly result in inhibition (blue arrows and green shapes) or activation (orange arrows and red shapes) of the target genes shown.
Figure 4
Figure 4. Predicted regulatory effects of gene expression profiles in response to SIL
Differential gene expression patterns in SIL-treated versus control mice were used to infer perturbed upstream regulators and then predict the downstream effects on hepatocytes.

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