Dysregulation of antioxidant responses in patients diagnosed with concomitant Primary Sclerosing Cholangitis/Inflammatory Bowel Disease

doi:10.1016/j.yexmp.2017.11.012

Experimental and Molecular Pathology

Volume 104, Issue 1, February 2018, Pages 1-8

https://doi.org/10.1016/j.yexmp.2017.11.012 Get rights and content

Highlights

•
Elevated cell specific peri-portal carbonylation occurs in PSC/IBD.
•
PSC/IBD patients exhibit dysregulation of anti-oxidant responses.
•
SOD2 expression is upregulated in PSC/IBD.
•
In PSC/IBD catalase expression is not evident in cholangiocytes.
•
Patients with PSC/IBD possess elevated Akt activation.

Abstract

Objective

Primary Sclerosing Cholangitis (PSC) is a chronic cholestatic liver disease that is characterized by severe peri-biliary tract inflammation and fibrosis, elevated oxidative stress and hepatocellular injury. A hallmark of PSC patients is the concurrent diagnosis of Inflammatory Bowel Disease occurring in approximately 70%–80% of PSC patients (PSC/IBD). The objective of this study was to determine the impact of end stage PSC/IBD on cellular antioxidant responses and the formation of protein carbonylation.

Methods

Using hepatic tissue and whole cell extracts isolated from age-matched healthy humans and patients diagnosed with end stage PSC/IBD, overall inflammation, oxidative stress, and protein carbonylation were assessed by Western blotting, and immunohistochemistry.

Results

Increased immunohistochemical staining for CD3 + (lymphocyte), CD68 (Kupffer cell) and myeloperoxidase (neutrophil) colocalized with the extensive Picrosirius red stained fibrosis confirming the inflammatory aspect of PSC. Importantly, the increased inflammation also colocalized with elevated periportal post-translational modification by the reactive aldehydes 4-HNE, MDA and acrolein. 4-HNE, MDA and acrolein IHC all displayed a significant component in hepatocytes adjacent to fibrotic regions. Furthermore, acrolein was also elevated within the nuclei of periportal inflammatory cells whereas MDA staining was increased in hepatocytes across the lobule. Prussian Blue staining, when compared to the positive controls (ALD, NASH), did not display any evidence of iron accumulation in PSC/IBD livers. Western analysis of PSC/IBD anti-oxidant responses revealed elevated expression of SOD2, GSTπ as well as upregulation of Akt Ser473 phosphorylation. In contrast, expression of GSTμ, GSTA4, catalase, Gpx1 and Hsp70 were suppressed. These data were further supported by a significant decrease in measured GST activity. Dysregulation of anti-oxidant responses in the periportal region of the liver was supported by elevated SOD2 and GSTπ IHC signals in periportal hepatocytes and cholangiocytes. Expression of the Nrf2-regulated proteins HO-1, NAD(P)H quinone reductase (NQO1) and Gpx1 was primarily localized to macrophages. In contrast, catalase staining decreased within periportal hepatocytes and was not evident within cholangiocytes.

Conclusions

Results herein provide additional evidence that cholestasis induces significant increases in periportal oxidative stress and suggest that there are significant differences in the cellular and subcellular generation of reactive aldehydes formed during cholestatic liver injury. Furthermore, these data suggest that anti-oxidant responses are dysregulated during end-stage PSC/IBD supporting pathological data. This work was funded by NIH 5R37AA009300-22 D.R.P.

Section snippets

Background

The orphan disease, Primary Sclerosing Cholangitis (PSC) is a progressive cholestatic liver disease of unknown etiology characterized by biliary inflammation, fibrosis, and stricturing of the intra and/or extra-hepatic bile ducts (Eaton et al., 2013). The incidence rate is ~ 1:100,000 person-years with no medical therapies currently available. Long-term disease progression leads to biliary obstruction, repeated bouts of cholangitis, and secondary biliary cirrhosis with a median time of survival

Sample procurement

To determine the status of protein carbonylation and acetylation during cholestasis, paraffin embedded and frozen hepatic tissue from normal and end stage PSC/IBD patients (N = 9 PSC/IBD, 8 normal) procured during transplantation (ages 25–62, Male/Female) were obtained from the University of Minnesota Liver Tissue cell Distribution Center NIH Contract #HHSN276201200017C. Whole cell extracts (WCE) of each sample was prepared by dounce homogenization (10 ×) of tissue resuspended in 50 mM tricine pH

Results

For this study, fresh frozen human hepatic tissue and formalin fixed tissue from normal and end stage PSC/IBD patients was obtained prior to transplant from the University of Minnesota Liver Tissue cell Distribution Center (NIH Contract #HHSN276201200017C). The mean age of patients was 45.67 years old. For each patient relevant hepatic parameters was provided (Model for End-stage Liver Disease (MELD) (Kamath et al., 2007), aspartate aminotransferase (AST), International Normalized Ratio of

Discussion and conclusions

Previous studies have shown that oxidative stress markers such as 4-HNE and MDA are elevated in murine models of cholestasis such as the bile duct ligation model as well as in Mdr2^−/− mice (Fickert et al., 2006, Parola et al., 1996, Peres et al., 2000). In addition, elevated lipid peroxidation and decreased anti-oxidant capacity in the form of suppressed concentrations of serum GSH have been demonstrated in patients with cholestatic liver disease (Aboutwerat et al., 2003). The data presented in

Financial support and acknowledgements

This research was supported by the following grants from the National Institutes of Health; R37AA009300-22 D.R.P. The authors wish to thank E. Erin Smith, HTL(ASCP)CMQIHC of the University of Colorado Denver Cancer Center Research Histology Core for assistance in preparing histology slides. The UCDCCRHC is supported in part by NIH/NCRR Colorado CTSI Grant Number UL1 RR025780 and the University of Colorado Cancer Center Grant (P30 CA046934).

Conflict of interest

The authors have no conflict of interest to report.

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Power SYBR™ Green PCR Master Mix, and a 7900HT Fast Real-Time PCR System with 384-Well Block Module (Applied Biosystems™/ThermoFisher) running 40 cycles at 95 °C for 15 s, 60 °C for 60 s. Biotin hydrazide purification: Biotin hydrazide derivatization and purification of aldehyde modified proteins was performed as previously described using 500 μg of LE protein from each group (n = 6 WT, 6 Mdr2KO, 8 Normal, 8 PSC [16,17]) [21]. Determination of GST and SOD2 activity: Enzymatic activity for GST and SOD2 was determined using LE prepared from fresh frozen hepatic tissue as previously described [22,23].
Cholangiopathies such as primary sclerosing cholangitis (PSC) are chronic liver diseases characterized by increased cholestasis, biliary inflammation and oxidative stress. The objective of this study was to elucidate the impact of cholestatic injury on oxidative stress-related factors. Using hepatic tissue and whole cell liver extracts (LE) isolated from 11-week old C57BL/6J (WT) and Mdr2^KO mice, inflammation and oxidative stress was assessed. Concurrently, specific targets of carbonylation were assessed in LE prepared from murine groups as well as from normal and human patients with end-stage PSC. Identified carbonylated proteins were further evaluated using bioinformatics analyses. Picrosirius red staining revealed extensive fibrosis in Mdr2^KO liver, and fibrosis colocalized with increased periportal inflammatory cells and both acrolein and 4-HNE staining. Western blot analysis revealed elevated periportal expression of antioxidant proteins Cbr3, GSTμ, Prdx5, TrxR1 and HO-1 but not GCLC, GSTπ or catalase in the Mdr2^KO group when compared to WT. From immunohistochemical analysis, increased periportal reactive aldehyde production colocalized with elevated staining of Cbr3, GSTμ and TrxR1 but surprisingly not with Nrf2. Mass spectrometric analysis revealed an increase in carbonylated proteins in the Mdr2^KO and PSC groups compared to respective controls. Gene ontology and KEGG pathway analysis of carbonylated proteins revealed a propensity for increased carbonylation of proteins broadly involved in metabolic processes as well more specifically in Rab-mediated signal transduction, lysosomes and the large ribosomal subunit in human PSC. Western blot analysis of Rab-GTPase expression revealed no significant differences in Mdr2^KO mice when compared to WT livers. In contrast, PSC tissue exhibited decreased levels of Rabs 4, 5 and increased abundance of Rabs 6 and 9a protein. Results herein reveal that cholestasis induces stage-dependent increases in periportal oxidative stress responses and protein carbonylation, potentially contributing to pathogenesis in Mdr2^KO. Furthermore, during early stage cholestasis, there is cell-specific upregulation of some but not all, antioxidant proteins.
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A complete biochemical description of each patient is reported in (Shearn et al., 2017). Liver extracts (LE) of each sample was prepared by dounce homogenization (10×) of tissue resuspended in 50 mM tricine pH 8.0, 1 mM NaCl plus phosphatase and protease inhibitors (SIGMA ALDRICH, St. Louis, MO) followed by sonication (3 × 15 seconds@ 4 °C) and processing as previously described (Shearn et al., 2017). Formalin fixed slides prepared from normal and end stage PSC/IBD patients (N = 9 PSC, 8 Normal) were used.
Primary Sclerosing Cholangitis (PSC) is a severe cholestatic liver disease characterized by progressive peri-biliary tract inflammation, elevated oxidative stress and hepatocellular injury. A hallmark of PSC patients is the concurrent diagnosis of Inflammatory Bowel Disease occurring in approximately 70%–80% of PSC patients (PSC/IBD). We previously reported dysregulation of key anti-oxidant pathways in PSC/IBD. The objective of this study was to expand previous data by examining the abundance of thioredoxins (Trx) in PSC/IBD.
Using hepatic tissue and whole cell extracts isolated from age-matched healthy humans and patients diagnosed with end stage PSC/IBD, the protein abundance of thioredoxin, thioredoxin reductase (TrxR1), and their downstream substrates peroxiredoxins was assessed.
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Dysregulation of antioxidant responses in patients diagnosed with concomitant Primary Sclerosing Cholangitis/Inflammatory Bowel Disease

Highlights

Abstract

Objective

Methods

Results

Conclusions

Section snippets

Background

Sample procurement

Results

Discussion and conclusions

Financial support and acknowledgements

Conflict of interest

Biochim. Biophys. Acta

Clin. Res. Hepatol. Gastroenterol.

J. Hepatol.

Cell Mol. Gastroenterol. Hepatol.

FEBS Lett.

Gastroenterology

Toxicol. Appl. Pharmacol.

Gastroenterology

Lancet

Cell

Am. J. Gastroenterol.

J. Clin. Densitom.

Liver Transpl.

J. Biol. Chem.

J. Hepatol.

Free Radic. Biol. Med.

J. Hepatol.

Am. J. Pathol.

J. Autoimmun.

Free Radic. Biol. Med.

J. Nutr. Biochem.

J. Biol. Chem.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Gastroenterology

J. Hepatol.

Alcohol, oxidative stress and free radical damage

Proc. Nutr. Soc.

Mitochondrial dysfunction in cholestatic liver diseases

Front. Biosci. (Elite Ed).

Serum metabolic signatures of primary biliary cirrhosis and primary sclerosing cholangitis

Liver Int.

Detoxication of base propenals and other alpha, beta-unsaturated aldehyde products of radical reactions and lipid peroxidation by human glutathione transferases

Proc. Natl. Acad. Sci. U. S. A.

Alkylation of the tumor suppressor PTEN activates Akt and beta-catenin signaling: a mechanism linking inflammation and oxidative stress with cancer

PLoS One