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. 2005 May 1;387(Pt 3):601-8.
doi: 10.1042/BJ20041598.

Pregnenolone-16alpha-carbonitrile inhibits rodent liver fibrogenesis via PXR (pregnane X receptor)-dependent and PXR-independent mechanisms

Carylyn J Marek  1 , Steven J Tucker, Dimitrios K Konstantinou, Lucy J Elrick, Dee Haefner, Charalambos Sigalas, Graeme I Murray, Bryan Goodwin, Matthew C Wright
Affiliations

Pregnenolone-16alpha-carbonitrile inhibits rodent liver fibrogenesis via PXR (pregnane X receptor)-dependent and PXR-independent mechanisms

Carylyn J Marek et al. Biochem J. .

Abstract

The effect of liver growth stimulation [using the rodent PXR (pregnane X receptor) activator PCN (pregnenolone-16alpha-carbonitrile)] in rats chronically treated with carbon tetrachloride to cause repeated hepatocyte necrosis and liver fibrogenesis was examined. PCN did not inhibit the hepatotoxicity of carbon tetrachloride. However, transdifferentiation of hepatic stellate cells and the extent of fibrosis caused by carbon tetrachloride treatment was significantly inhibited by PCN in vivo. In vitro, PCN directly inhibited hepatic stellate cell transdifferentiation to a profibrogenic phenotype, although the cells did not express the PXR (in contrast with hepatocytes), suggesting that PCN acts independently of the PXR. Mice with a functionally disrupted PXR gene (PXR-/-) did not respond to the antifibrogenic effects of PCN, in contrast with wild-type (PXR+/+) mice, demonstrating an antifibrogenic role for the PXR in vivo. However, PCN inhibited the transdifferentiation of PXR-/--derived mouse hepatic stellate cells in vitro, confirming that there is also a PXR-independent antifibrogenic effect of PCN through a direct interaction with hepatic stellate cells. These data suggest that the PXR is antifibrogenic in rodents in vivo and that a PXR-independent target for PXR activators exists in hepatic stellate cells that also functions to inhibit fibrosis.

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Figures

Figure 1
Figure 1. Lack of impact of PCN administration on carbon tetrachloride (‘CCl4’)-mediated hepatotoxicity
Rats were administered carbon tetrachloride and/or PCN (25 mg/kg body weight; one injection/week) as outlined in the Experimental section for 6 weeks. (A) AST levels in serum from the indicated treatment groups; results are the means±S.D. for three to five animals/treatment group. (B) Severity scores for liver damage as determined from examination of haematoxylin-and-eosin-stained liver sections (independently scored for severity on a scale of 0–5 (normal–extensive damage) by an examiner blinded to the treatment groups. Each point represents the score for a section from a single animal; the number of individual animals is equal to the number of points per treatment group. The mean score is indicated by the line. (C) Representative liver sections from the indicated treatment groups stained with haematoxylin and eosin. (D) Western-blot immunoquantification of microsomal CYP2E levels from the indicated treatment groups; results are means±S.D. for three to five animals/treatment group. (E) Effect of PCN and CYP inhibitors (final concentrations in assay as indicated) on microsomal p-nitrophenol hydroxylase (CYP2E) activities (control levels 450±42 pmol of 4-nitrocatechol produced/min per mg of microsomal protein; means±S.D. of three separate determinations). Note the vehicle solvent DMSO is a substrate (not shown) for CYP2E and therefore markedly inhibits p-nitrophenol hydroxylase activities. Accordingly, all potential inhibitors of this assay were dissolved directly into the assay buffer. PCN was used from a saturated stock (actual concentration determined through UV–visible absorbance spectroscopy at 248 nm). * Indicates significantly different activity versus control (P>95%) using Student's t test.
Figure 2
Figure 2. PCN inhibits the carbon tetrachloride (‘CCl4’)-dependent liver fibrosis
Rats were administered carbon tetrachloride and/or PCN (25 mg/kg body weight; one injection/week) as outlined in the Experimental section for 6 weeks. (A) Representative liver sections from the indicated treatment groups immunostained for α-smooth-muscle actin. (B) Representative liver sections from the indicated treatment groups and stained with Sirius Red. (C) Severity scores for α-smooth-muscle actin and Sirius Red. Liver sections were independently scored for severity on a scale of 0–5 (normal–extensive staining/fibrosis) by an examiner blinded to the treatment groups. Each point represents the score for a section from a single animal, the number of individual animals being equal to the number of points per treatment group. The mean score is indicated by the line. * Indicates significantly lower damage versus carbon tetrachloride-treated group, P>95% using Student's t test.
Figure 3
Figure 3. Effect of PCN on the transdifferentiation of Rat HSCs in vitro
(A) Time course for the expression of α-smooth muscle actin (‘α-SMA’) and desmin in rat HSC primary culture. Freshly isolated (IC) rat HSCs and cells cultured for the indicated time were harvested and cell extracts (10 μg/lane) probed for the indicated protein by Western blotting. Std, standard. (B) Effect of PXR activator addition to rat HSC primary culture on the transdifferentiation of HSCs in vitro. Abbreviations: UT, vehicle control only (<0.5%, v/v, DMSO); RU, 10 μM RU486; PCN, 20 μM PCN; PB, 1 mM phenobarbital; DEX, 1 μM dexamethasone; MET, 500 μM metyrapone). The levels of α-smooth muscle actin were determined by Western blotting (5 μg of protein/lane) after 9 days of continuous treatment. (C) Effect of PXR activator treatment on the levels of total spectrophotometrically detectable cytochrome P450 and CYP3A1/3A23 levels in primary cultures of rat hepatocytes. The concentration of PXR activators was as given in (B), except that treatment time was 48 h. (D) Reverse-transcription PCR analysis of RNA from rat hepatocytes and HSCs. Total RNA was subjected to reverse-transcription PCR analysis for PXR expression as outlined in the Experimental section. PCR control, no 1st-strand cDNA control; RT-control, no RNA control; HC, freshly isolated hepatocyte RNA (template levels as indicated); pSG5-rPXR, a plasmid encoding the rat PXR cDNA as template (input: template plasmid in PCR reaction; output: amplified fragment after PCR reaction); HSC, 2-day quiescent HSC or profibrogenic 14-day transdifferentiated HSC RNA (template levels as indicated). The lower band corresponds to a fragment of amplified PXR mRNA (sequenced confirmed; results not shown); the upper band amplified from rat hepatocytes was cloned and sequenced and was found to be unrelated to the PXR.
Figure 4
Figure 4. Antifibrogenic effects of PCN in the liver are lost in PXR−/− mice
Mice were administered carbon tetrachloride (‘CCl4’) for 8 weeks and/or PCN (100 mg/kg body weight; one injection/week for the last 4 weeks of treatment) as outlined in the Experimental section for 8 weeks. (A) ALT levels in serum from the indicated treatment groups; results are means±S.D. for three to five animals/treatment group. (B) Liver sections stained for either α-smooth-muscle actin or desmin were independently scored for severity on a scale of 0–5 (normal–extensive staining/fibrosis) by an examiner blinded to the treatment groups. Each point represents the score for a section from a single animal, and the number of individual animals is equal to the number of points per treatment group. The mean score is indicated by the horizontal line. * Indicates significantly lower transdifferentiation versus carbon tetrachloride-treated group; P>95% using Student's t test. (C) Representative liver sections from the indicated treatment groups and stained with Sirius Red. Severity scores are given in the right-hand panel. * Indicates significantly lower fibrosis versus carbon tetrachloride-treated group; P>95% using Student's t test.
Figure 5
Figure 5. PXR genotype and mouse HSC transdifferentiation: effect of PCN
(A) RT-PCR analysis for PXR mRNA expression. Primers used are given in Table 1. (B) Western blot for α-smooth-muscle actin or total actin levels in extracts from transdifferentiating mouse HSCs. (C) HSCs were isolated from four PXR+/+ or PXR−/− mice, pooled, counted and seeded at approx. 50 cells/field of view. The number of attached cells per ×20 field were then counted in eight randomly selected fields to quantify the proliferation of the HSCs. (D) Expression of α-smooth-muscle actin, total actin and GFAP in extracts from passage-2 HSCs derived from PXR+/+ or PXR−/− mice.
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