Sustained Isoprostane E2 Elevation, Inflammation and Fibrosis after Acute Ischaemia-Reperfusion Injury Are Reduced by Pregnane X Receptor Activation

<div><p>Liver grafts donated after cardiac death are increasingly used to expand the donor pool but are prone to ischaemic-type biliary lesions. The anti-inflammatory effects of the activated pregnane X receptor have previously been shown to be beneficial in a number of inflammatory liver conditions. However, its role in reducing peri-portal inflammation and fibrosis following ischaemia-reperfusion injury has not been investigated. Hepatic injury and its response to pregnane X receptor activation was examined after partial hepatic ischaemia-reperfusion injury induced by surgically clamping the left and middle lobar blood vessels in rats. Molecular and pathological changes in the liver were examined over the following 28 days. Ischaemia-reperfusion injury resulted in transient cholestasis associated with microvillar changes in biliary epithelial cell membranes and hepatocellular injury which resolved within days after reperfusion. However, in contrast to chemically-induced acute liver injuries, this was followed by sustained elevation in isoprostane E2, peri-portal inflammation and fibrosis that remained unresolved in the ischaemic reperfused lobe for at least 28 days after clamping. Administration of pregnenolone-16α-carbonitrile—a rodent-specific pregnane X receptor activator—resulted in significant reductions in cholestasis, hepatic injury, ischaemic lobe isoprostane E2 levels, peri-portal inflammation and fibrosis. Hepatic ischaemia-reperfusion injury therefore results in inflammatory and fibrotic changes that persist well beyond the initial ischaemic insult. Drug-mediated activation of the pregnane X receptor reduced these adverse changes in rats, suggesting that the pregnane X receptor is a viable drug target to reduce ischaemic-type biliary lesions in recipients of liver transplants donated after cardiac death.</p></div

IRI results in progressive fibrosis.

<p><b>(A)</b> α-SMA immunohistochemistry—typical views from the indicated treatment group post and time point (upper panels) and quantification of α-SMA immunohistochemistry staining, scale bar represents 100μm. (<b>B)</b> quantification of α-SMA staining. <b>(C)</b> Sirius red staining–typical views from the indicated treatment group post and time point (upper panels) and quantification of Sirius red staining, scale bar represents 100μm. (<b>D</b>) quantification of Sirius red staining. <b>(E-F)</b> qRT-PCR analysis in ischaemic and sham ischaemic lobes. Data are the mean and standard deviation from 3 separate animals at each time point and treatment, *Significantly different compared to sham IRI group, p<0.05.</p

PCN treatment results in hepatic Cyp3a1 induction, reduced oxidative stress and reduced cholestasis in IRI.

<p><b>(A)</b> Quantification of Cyp3a1 mRNA levels as determined by qRT-PCR RNA was isolated from whole liver as outlined in the methods section. (<b>B)</b> Western blot of Cyp3a1 expression in whole liver homogenates on day 1 and 10 in IRI+PCN and IRI+vehicle groups. (<b>C)</b> Liver MDA levels on day 1 post clamp release. (<b>D)</b> Comparison of bile flow in the IRI+PCN and IRI+vehicle groups. (<b>E)</b> Serum bile acid levels. Data are the mean and standard deviation from 5 separate animals at each time point and treatment, *Significantly different compared to sham IRI group, p<0.05.</p

Hepatic stellate cells are responsive to stimulation by TLR ligands.

<p>(A) mRNA level of IL-6 was quantified by qRT-PCR in four separate preparations of activated rat HSCs (culture day 10) treated with TLR2 ligand (LTA, 100 ng/ml), TLR3 ligand (Poly(I∶C), 1 µg/ml), TLR4 ligand (LPS, 100 ng/ml) or TLR5 ligand (Flagellin, 1 µg/ml) for up to 24 hours (n = 4). Expression level was normalised to β actin. (B) Secreted IL-6 protein was measured by ELISA in conditioned media collected from activated HSCs treated with TLR ligands as in (A) following 2 h, 8 h or 24 h of stimulation (n = 4). (C) mRNA levels of IL-6, TIMP1, αSMA and collagen I were quantified by qRT-PCR in four separate preparations of activated rat HSCs (culture day 10) treated with TLR3 ligand (Poly(I∶C), 1 µg/ml) for up to 24 hours. Expression level was normalised to β actin. (#p<0.1, *p<0.05, **p<0.01***p<0.001).</p

IRI results in a transient cholestasis and altered biliary physiology.

<p><b>(A)</b> Partial hepatic ischaemia model with bile duct isolation (Left). Schematic representation of groups in both studies (Right). (<b>B)</b> Comparison of bile flow rates (normalised to total body weight). (<b>C)</b> Comparison of serum bile acid concentration between the IRI and sham IRI groups. (<b>D)</b> TEM images comparing BEC microvilli morphology during early (day 1) and late (day 28) reperfusion timepoints. Data are the mean and standard deviation from 3 separate animals at each time point and treatment, *Significantly different compared to sham IRI group, p<0.05.</p

PXR activation reduces IRI-induced ductal reactions and the numbers of fibrogenic cells in the liver.

<p><b>(A, C, E)</b> CK-19, α-SMA and vimentin immunohistochemistry respectively between IRI+PCN and IRI+vehicle groups–typical views from the indicated treatment group at the indicated time point after clamp release (scale bar represents 100μm) with (<b>D, E F)</b> corresponding stain quantification, data are the mean and standard deviation from 5 separate animals at each time point and treatment, *Significantly different compared to IRI + vehicle group, p<0.05.</p

Interferon gamma and IL-6 expression are TLR3 mediated and loss of ability to induce IFNγ in aHSCs is associated with transcriptional repression by Polycomb complex.

<p>(A) WT and <i>tlr3</i>^−/− activated mHSCs were treated with Poly(I∶C) (1 µg/ml) for 8 and 24 hours; subsequently IFNγ mRNA expression was measured by qPCR. Data are normalised to GAPDH and expressed as fold induction relative to control. (B) IL-6 mRNA expression was measured in WT and <i>tlr3</i>^−/− activated mHSCs in response to increasing concentrations of Poly(I∶C). (C) Wild type qHSCs were treated with transcriptional inhibitors Act D (1 µM) and DRB (80 µM) for 24 hours and IL-6 mRNA expression analysed by qRT-PCR. Data are normalised to GAPDH and expressed as fold induction relative to untreated control. (D and E) One hundred micrograms of crosslinked chromatin from quiescent and activated rat HSC was incubated with 10 µg of anti-trimethyl and anti-dimethyl H3K27 antibody and ChIP assay performed. Data are expressed as fold enrichment relative to IgG control. (*p<0.05, **p<0.01, ***p<0.001).</p

Activation of HSC decreases TLR3 mediated interferon and cytokine response.

<p>(A,B,C) HSCs were isolated from control, acute CCl₄ treated rats (single injection), or chronic CCl₄ treated rats (4 weeks twice weekly injections); cells were seeded onto plates and treated with Poly(I∶C) (1 µg/ml) for up to 24 hours. Interferon α, β and γ were measured and normalised to β actin. Data are expressed as RLTD. (D) Interferon-inducible cytokines CXCL10, TNF-α, MxA, CXCL1/KC, IL-1β and indoleamine 2,3-dioxygenase were measured and normalised to β actin (n = 4). Data are presented as fold change, Poly(I∶C) stimulated to unstimulated. Secreted IFNγ was measured by ELISA in the media of cultured HSCs isolated from rats treated with either chlodronate-liposomes (E) or empty liposome control (F). (G) Expression of MHC class II on macrophages cultured in qHSC conditioned media. Flow cytometric analysis of control macrophages (untreated), or macrophages incubated with control media, untreated qHSC conditioned media, Poly(I∶C) treated qHSC conditioned media or 100 ng/ml recombinant IFNγ as positive control. (*p<0.05, **p<0.01, ***p<0.001).</p

Table of primers.

<p>Table of primers.</p

IRI results in persistent inflammatory changes in peri-portal regions and a ductal reaction.

<p><b>(A)</b> H&E stained liver sections of a zone 3 (upper) and zone 1 (lower) region in an IRI lobe (day 1 and day 10 respectively):- BEC, biliary epithelial cell; pv, portal venule; bd, bile ductile; AIC, acute inflammatory cell; H, hepatocyte; F, fibroblast or myofibroblast; hM, haemosiderin-laden macrophage; MG, mononuclear granulocyte. Scale bar represents 100μm. <b>(B-C)</b> Comparison of inflammatory cell counts in peri-portal and centrilobular areas between the IRI and sham IRI groups. (<b>D</b>), cytokeratin 19 (CK-19) staining of liver sections–typical views from the indicated treatment group and time point. Scale bar represents 100μm. E, quantification of CK-19 staining. Data are the mean and standard deviation from 3 separate animals at each time point and treatment, *Significantly different compared to sham IRI group, p<0.05.</p