Proteomic approach to identify molecular signatures during experimental hepatic fibrosis and resveratrol supplementation,☆☆

https://doi.org/10.1016/j.ijbiomac.2018.08.062Get rights and content

Highlights

  • Proteomic study on fibrotic (HF) and resveratrol-supplemented rats is carried out.

  • 2D-MALDI-MS analysis revealed 10 major protein spots including 2 uncharacterized.

  • Carbohydrate, lipid and Ca2+ signaling pathway are inter-linked during HF.

  • Oxidative pathway appears principal to mediate liver damage.

  • Proteomic evidences suggest strong hepatoprotective potential of resveratrol.

Abstract

We document the changes in sera and liver proteome during experimental hepatic fibrosis (HF) and its protection by resveratrol (Rsvtrl) in rats. HF was induced by N′-Nitrosodimethylamine (NDMA) administration thrice a week for 21-days. Compared to the control group, significant (P < 0.05) elevations in ALT, AST, ALP, γGT and bilirubin occurred during fibrosis. H&E, IHC for α-SMA and reticulin stainings of liver demonstrated that collagen amassing and HF were concurrent. Rsvtrl-supplementation refurbished hepatic architecture and lessened collagen deposition. 2DE proteome analyses showed ~398 (pH, 3–10) and ~129 signatures (pH, 4–7) within 5–201 kDa range in liver and sera, respectively. Nearly, 45 spots in liver and 18 in sera were differentially expressed in fibrotic and Rsvtrl-supplemented animals. MALDI−TOF–MS/MS analysis revealed at least ten signatures as the potential biomarkers for HF, because of the significant changes (>2 fold) in their expression. Out of them, two signatures have not so far been characterized and we have accomplished it in this study. Our proteomics data provides new evidence on an overall involvement of carbohydrate, lipid, Ca2+ signaling and oxidative pathways during NDMA-induced HF. More importantly, the oxidative pathway appears to be the principal mediator in the NDMA-induced HF as well as the hepato-protection by Rsvtrl.

Introduction

Rodent model for liver fibrosis, usually generated by bile duct ligation (BDL) or administration of environmental toxicants like N′-Nitrosodimethylamine (NDMA), N′-Nitrosodiethylamine (NDEA), carbon tetrachloride (CCl4) or thioacetamide (TAA) are among the most commonly studied fibrosis models [[1], [2], [3], [4], [5]]. Besides, numerous other causes such as viral/protozoal insult, alcohol abuse, autoimmune diseases etc. [6] may also contribute to the generation of fibrosis. During the present study, NDMA was used to induce liver fibrosis in rats within a short span of 21 days in the animals exposed to its repeated lower doses, with a histological appearance that perfectly imitates liver fibrosis in human beings. The metabolism of NDMA proceeds through the intermediate radical [CH3(CH2radical dot)NBNdouble bondO] generation by the action of cytochrome CYP2E1-dependent oxidases, either via α-hydroxylation or denitrosation of the nitrosamine [7,8]. During α-hydroxylation pathway, the hydroxymethylnitrosamine (HOCH2CH3HBNdouble bondO) decomposes to formaldehyde and monomethylnitrosamine (CH3NHNdouble bondO) which undergo rearrangement and form methyldiazonium ion (CH3N+triple bondN). Methyldiazonium alkylates biological macromolecules like DNA, RNA and proteins. On the other hand, metabolic translation of the intermediate radical via denitrosation leads to the formation of methylamine (CH3NH2) and formaldehyde. α-Hydroxylation pathway is believed to form the active metabolites responsible for the genotoxic and carcinogenic potential of NDMA [7]. Moreover, NDMA biotransformation stimulates generation of free radicals and reactive oxygen species (ROS) by hepatocyte, Kupffer cells and neutrophils which further enhance the oxidative damage to contribute hepatic fibrosis [9,10]. The net consequence of these changes is transdifferentiation of quiescent hepatic stellate cells (HSCs) into myofibroblast-like activated HSCs. The highly proliferative activated HSCs are characterized by the loss of vitamin-A droplets and the release of proinflammatory, profibrogenic and promitogenic cytokines such as transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), endothelin-1 (ET-1) and interferon-γ (INF-γ) [11]. The characteristic features of liver fibrosis are irregular proliferation and deposition of types-I, -III and -IV collagen-rich tough fibrous connective tissue, which create an imbalance between synthesis and degradation of extracellular matrix (ECM) within liver [1,[12], [13], [14]]. The details of underlying biochemical and molecular mechanisms and integrated pathway of NDMA-induced hepatic fibrosis are yet to be elucidated.

Owing to the significant role of ROS in liver diseases, increased levels of intracellular antioxidants should be one of the options in any strategy to avert liver damage. In the present study, we have examined the hepatoprotective potential of resveratrol which reportedly possesses anti-inflammatory, anti-cancerous/pro-apoptotic, anti-microbial and anti-oxidative properties [[15], [16], [17], [18], [19], [20]]. Literature also suggests that the hydrogen electron donation from hydroxyl group of Rsvtrl counters several types of oxidative damage [21]. The in vivo use of Rsvtrl as a restorative medicine has always been a subject of research against different ailments including hepatic disorders [22]. A number of reports are available indicating remedial role of Rsvtrl in non-alcoholic fatty liver diseases (NAFLD), heart diseases, neurological disorders, diabetes and obesity [23,24]. While a definite progress has been made in researching friendly alternatives to treat liver fibrosis, the molecular mechanism of action of many of them, in general, and resveratrol in particular, is yet to be explained in aggressive fibrotic process.

Broadly, the purpose of present study was to measure and identify changes in the serum and liver proteomic profiles during fulminant stages of liver fibrosis caused by NDMA and subsequent recovery due to resveratrol treatment. In addition to biochemical markers which reflect changes in metabolism, we also compared differentially expressed proteins in the liver and sera proteome in NDMA-induced fibrotic rats with those which receive Rsvtrl supplement. For identifying differentially expressed proteomic markers, two-dimensional electrophoresis (2-DE) coupled with mass spectrometric analysis (MS), has gained wide recognition [25]. It has quality attributes of precision and rapid analysis when integrated with data obtained by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) [26]. Considering the scarcity of literature, it is realized that further studies shall be carried out to provide a better understanding of underlying mechanism of hepatic fibrosis by way of identifying differentially expressed protein candidates for predicting hepatic fibrosis [27]. Identifying changes in a larger perspective of protein expression of serum and liver will strengthen our understanding of NDMA-induced hepatic fibrosis and ameliorative potential of resveratrol in animal models with possible relevance to human exposure.

Section snippets

Chemicals and reagents

N′-Nitrosodimethylamine (NDMA), resveratrol, acrylamide, bis-acrylamide, ammonium persulphate (APS) and TEMED were purchased from Sigma-Aldrich. Hematoxylin and eosin stains were obtained from SRL, India. α-Smooth muscle actin (α-SMA) antibodies and goat anti-mouse IgG-HRP conjugated antibodies were procured from Trends Bio-product Pvt. India and CALTAG laboratories, Bangkok respectively. All the other chemicals, reagents and salts used were of analytical grade.

Animals

Adult male albino rats (Rattus

Blood biochemistry and histopathology

Changes in liver function enzymes, which were obtained for livers of rats out of various groups treated with NDMA and Rsvtrl, are given in Fig. 1. Fibrotic group exhibited significantly higher levels of serum AST, ALT, ALP and Hyp than the control group (P < 0.05). As per estimates, per cent disruption in the levels of AST, ALT, ALP and Hyp were of the order of ~29.6, ~33.33, ~55.44, and ~24.1, respectively within three weeks. In comparison, Rsvtrl-supplement significantly alleviated these

Conclusions

NDMA-induced liver fibrosis results in the enhanced oxidative stress leading to the generation of oxyradicals which can bind to proteins and cellular constituents. The majority of the proteins identified here showing differential expression were enzymes involved in carbohydrate (UTP glucose-1-phosphate uridylyl transferase isomerase-1, C-type lectin domain family 2)/lipid (inositol monophosphatase-1) metabolism and redox balance (GST-Mu 2, CA-III, RGN via Ca2+ signaling pathway, C-type lectin

Acknowledgements

The authors sincerely thank the Chairmen, Department of Zoology and Department of Pathology (JN Medical College) for necessary facilities. Authors are grateful to Dr. Absar-ul Hasnain, AMU Aligarh; Dr. Irfan Qureishy, Jamia Millia Islamia (New Delhi, India) and Dr. Padmavathi Ramanujam, Alagappa University (Karaikudi, India) for useful exchange of views and suggestions. Facilities extended by SANDOR Proteomics, Hyderabad, India are gratefully acknowledged. Thanks are also due to Hadiya Husain

Conflicts of interest

The authors declare that there are no conflicts of interest in this article.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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    Conflicts of interest: The authors declare that there are no conflicts of interest regarding the publication of this article.

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    Author's contributions: Both the authors contributed equally in the preparation of this MS.

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