8 research outputs found
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
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
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
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
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
PXR activation reduces IRI-induced fibrosiss in the liver.
<p><b>(A)</b> Liver sections stained with Sirius red stain, typical views from the indicated treatment group at the indicated time point after clamp release (scale bar represents 100μm), with (<b>B)</b>, corresponding stain quantification. (<b>C-D)</b> qRT-PCR analysis for TGF-β1 and Col1A1 mRNA transcripts. 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
IRI results in increases in biliary and serum cytokine expression.
<p>(<b>A-B)</b> Comparison of biliary MCP-1 and RANTES concentrations between IRI and sham IRI groups. (<b>C-D)</b> Comparison of serum MCP-1 and RANTES levels between IRI and sham IRI groups. (<b>E)</b> Comparison of the isoprostane F2 IV (left) and E2 (right) levels in the ischaemic lobe of IRI and sham IRI livers. 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