Modulatory effects of NDP-MSH in the regenerating liver after partial hepatectomy in rats

Peptides 50 (2013) 145–152

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Modulatory effects of NDP-MSH in the regenerating liver after partial hepatectomy in rats Caterina Lonati a , Andrea Carlin a,c , Patrizia Leonardi a,c , Franco Valenza b,c , Silvano Bosari a,c , Anna Catania a,c,∗ , Stefano Gatti a,c a

Centro di Ricerche Chirurgiche Precliniche, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Italy Dipartimento di Anestesia, Rianimazione ed Emergenza Urgenza, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Italy c Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Università degli Studi di Milano, 20122 Milano, Italy b

a r t i c l e

i n f o

Article history: Received 27 September 2013 Received in revised form 17 October 2013 Accepted 17 October 2013 Available online 25 October 2013 Keywords: Melanocortin peptides 70% partial hepatectomy (PH) Liver regeneration Interleukin 6 (IL-6) Suppressor of cytokine signaling (SOCS)

a b s t r a c t Melanocortins are endogenous peptides that exert protective actions on the host physiology. The broad modulatory effects of these molecules suggest that they might influence the mediator network induced during liver regeneration. The research aim was to determine if melanocortin treatment alters liver molecular changes induced by partial hepatectomy (PH). Rats under isoflurane anesthesia were subjected to standard 70% PH or sham surgery. Animals received a single i.v. injection of Nle4,DPhe7-␣-melanocyte stimulating hormone (NDP-MSH) or saline 30 min before surgery. Sacrifice was performed at time intervals between 4 and 72 h. A preliminary screening based on TaqMan low-density array (TLDA) identified 71 transcripts altered by PH. Real-time PCR analysis revealed that NDP-MSH modulated the expression of a substantial proportion of these transcripts including several chemokines and their receptors. The critical signaling pathway interleukin-6 (IL-6)/signal transducer and activator of transcription (STAT)/suppressor of cytokine signaling (SOCS) was significantly enhanced by NDP-MSH. Further, peptide treatment considerably reduced the decline of I␬B␣ protein caused by PH. Although the final organ regeneration was not substantially affected, NDP-MSH modulated expression of cell cycle mediators and exerted subtle influences on hepatocyte replication. Most of the changes brought about by NDP-MSH, a peptide approved for clinical use, should be salutary during liver regeneration. © 2013 Elsevier Inc. All rights reserved.

1. Introduction Following hepatectomy, quiescent hepatocytes re-enter the cell cycle and divide until the liver mass is restored [13,28]. A complex network of mediators including cytokines, growth factors, and

Abbreviations: ␣-MSH, ␣-melanocyte stimulating hormone; Atf3, activating transcription factor 3; Ccr2, chemokine (C–C motif) receptor 2; Ctf1, cardiotrophin 1; Cxcl1, chemokine (C–X–C motif) ligand 1; Egr1, early growth response 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Hsp70-1, heat shock protein 70-1; IFN-␥, interferon gamma; I␬B␣, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha; LBWR, liver-to-body weight ratio; Lif, leukemia inhibitory factor; MC1–5 , melanocortin receptors 1–5; MCP-1, monocyte chemoattractant protein-1; MIP-1␣, macrophage inflammatory protein 1 alpha; NDP-MSH, Nle4,DPhe7-␣-melanocyte stimulating hormone; NF-␬B, nuclear factor kappa B; Nos2, nitric oxide synthase 2; Osm, oncostatin M; PH, partial hepatectomy; PCNA, proliferating cell nuclear antigen; Rrm2, ribonucleotide reductase M2; RT-qPCR, reverse transcription quantitative polymerase chain reaction; Socs1–3, suppressor of cytokine signaling 1–3; STAT, signal transducer and activator of transcription; TLDA, TaqMan low-density array. ∗ Corresponding author at: IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Via F. Sforza 35, 20122 Milano, Italy. Tel.: +39 02 55033318; fax: +39 02 55033318. E-mail address: [email protected] (A. Catania). 0196-9781/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2013.10.014

cyclins participate in this process. Control of hepatocyte replication during liver regeneration requires that signals promoting regeneration are limited both in strength and duration [13]. Conversely, in the presence of injury or liver disease, a more sustained stimulation may be required because of impaired regenerative capacity [1]. The general idea underlying the present research was that melanocortins, endogenous peptides that exert modulatory actions on many cell pathways [4,5], could influence the mediator network induced by partial hepatectomy (PH). Melanocortin ligands and their receptors form a highly conserved evolutionary system that participates in control of disparate functions [3,10,16]. Effects on host physiology span from modulation of fever and inflammation to control of food intake, autonomic functions, melanogenesis, and exocrine secretions [2,4,5,16,31,35]. The signal transduction occurs through five melanocortin (MC1 through MC5 ) receptors functionally coupled to adenylyl cyclase [20]. ␣-Melanocyte stimulating hormone (␣-MSH) is a 13 amino acid melanocortin peptide that has been broadly studied for its modulatory effects [3,6,25]. A major contribution of ␣-MSH to the host physiology resides in its capacity to prevent tissue injury induced by harmful stimuli, including endotoxin [8,15], reperfusion injury [30], blood loss [18,26], and oxidative stress [11].

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The aim in the present research was to determine effects of ␣-MSH administration on the regenerating liver. The model used consisted of 70% PH in rats, an established preclinical method to study liver regeneration and its control in normal and pathological conditions [19]. Treatment was performed using the synthetic ␣MSH analogue Nle4,DPhe7-␣-MSH (NDP-MSH), a non-specific MC agonist that exerts similar effects relative to the natural ␣-MSH and is generally preferred for its greater chemical stability [35]. Similar to ␣-MSH, NDP-MSH recognizes all the known MC receptors with the exception of the corticotropin receptor MC2 [20]. The research focus was on pathways known to exert key functions during liver regeneration [13]. 2. Materials and methods 2.1. Animals and partial hepatectomy All experiments were performed in accordance with the Principles of Laboratory Animal Care (NIH Publication No. 86-23, revised 1985) and approved by the Local Committee for Experimental Animal Research. Adult Sprague-Dawley male rats (Charles River, Calco, Italy) weighing 250–300 g were housed individually in a ventilated cage system (Tecniplast, Buguggiate, VA, Italy) at 22 ± 1 ◦ C, 55 ± 5% humidity, on a 12 h dark/light cycle. Rats under isoflurane anesthesia were subjected to 70% PH according to the classical model of Higgins and Anderson (medial and left lateral lobes resection) [19]. The zero time point for PH experiments was set at ligation of the liver lobes before excision. In the sham procedure, livers were briefly exposed outside the peritoneal cavity and gently palpated to mimic the surgical stress of the PH procedure. 2.2. Treatments and liver sampling Our previous observations indicate that 750 ␮g/kg NDP-MSH induce a preconditioning-like phenotype in the heart [7]. It appears that several expression changes in resting tissues are promoted early after peptide administration. As these changes could form the basis for preventive defense against subsequent injuries, we elected to administer this same peptide dose 30 min before PH. Thirty-five rats received i.v. injections of 750 ␮g/kg of NDP-MSH dissolved in 400 ␮l saline or an equal volume of saline (N = 35). Rats were sacrificed by exsanguination under deep anesthesia at 4, 8, 24, 48 or 72 h (N = 7 per group). Seven baseline control animals received no treatment and were sacrificed under deep anesthesia. The remnant liver was immediately collected and weighed. Tissue samples (right lateral lobe) were removed and snap frozen in liquid nitrogen and stored at −80 ◦ C for molecular biology analysis. For immunohistochemistry examination, tissue biopsies were fixed for 24 h in 4% phosphate-buffered formaldehyde solution (Sigma–Aldrich, St. Louis, MO), embedded in paraffin wax using conventional techniques, sectioned at 4 ␮m thickness and stained with hematoxylin–eosin (H–E). Blood was withdrawn from the abdominal aorta and plasma was separated by centrifugation (3000 rpm, 20 min). 2.3. Total RNA isolation Frozen liver specimens weighing 100–150 mg were rapidly homogenized in lysis buffer (Applied Biosystems, Life Technologies, Foster City, CA) with an Ultra-Turrax tissue homogenizer (IKA Labortechnik, Staufen, Germany) and tissue lysates were digested with proteinase K (Applied Biosystems). Total RNA was isolated on an ABI Prism 6100 Nucleic Acid PrepStation using Total RNA Chemistry method (Applied Biosystems). Genomic DNA contamination was removed by on-column DNase I treatment (Applied

Biosystems). RNA samples were quantified by optical density measurement using a Nanodrop ND-100 spectrophotometer (Nanodrop Technologies, Wilmington, DE). Each sample showed a 260/280 ratio between 1.8 and 2. RNA integrity was assessed by electrophoresis on denaturing agarose–formaldehyde gels. 2.4. Gene expression analysis using TaqMan low-density array (TLDA) We designed a custom TaqMan low-density array (TLDA, P/N 4342259, Applied Biosystems) to investigate transcriptional changes occurring in the liver after PH. Arrays included transcripts involved in liver function and metabolism, immune response, stress response, cell cycle, and regeneration-specific signal transduction (the complete gene list is provided in Supplemental Table 1). Seven samples from the same treatment group were pooled and RNA pools were reverse transcribed using High Capacity cDNA Archive kit (Applied Biosystems), following manufacturer’s protocol. An amount of cDNA corresponding to 300 ng of RNA pool were mixed with an appropriate volume of TaqMan Universal Master Mix 2× (Applied Biosystems) and loaded into each port of the array. Amplification reactions were performed on an ABI PRISM 7900HT Sequence Detection System with TaqMan Array Upgrade (Applied Biosystems) using the following thermal cycling conditions: 50 ◦ C for 2 min, 94.5 ◦ C for 10 min and then 45 cycles of 97 ◦ C for 30 s and 59.7 ◦ C for 1 min. Raw data were analyzed with SDS 2.3 software (Applied Biosystems) and then imported in a RQ study (RQ Manager 1.2, Applied Biosystems) to convert fluorescence intensities into threshold cycles (Ct ). Relative quantification was obtained with the comparative Ct method (Ct ), using the average Ct across all samples as calibrator for every gene. mRNA relative quantities (RQ) were divided by a normalization factor calculated for each sample with the geNorm VBA applet version 3.4 for Excel. RQ were log2-transformed and differences among groups were investigated by unsupervised method using DNA-chip analyzer program (www.dChip.org). Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.peptides. 2013.10.014. 2.5. cDNA synthesis and real-time PCR analysis Two micrograms of total RNA were reverse transcribed to single stranded cDNA by random priming using the High Capacity cDNA Archive kit (Applied Biosystems), according to standard protocol. A negative control of RT reaction (no MMLV reverse transcriptase) was run for each sample. Based on TLDA expression data, we arbitrarily selected genes with a fold change ≥2 or ≤0.5 relative to basal expression at one or more intervals after PH. Seventy-one genes that met this criterion were used for the subsequent real-time PCR analysis to investigate NDP-MSH effects on expression of individual genes during liver regeneration. Real-time PCR was performed using 40 ng of retrotranscribed RNA, 1× FastStart Universal Probe Master (Roche Diagnostics GmbH, Mannheim, Germany), and predesigned 900 nM primers and 250 nM probe mix (TaqMan Gene Expression Assays, Applied Biosystems) in a final volume of 10 ␮l. Assay IDs for target and reference genes are reported in the online supporting material (Supplemental Table 1). The same procedure was followed using 2 ␮l of RT negative control of each sample to exclude any genomic DNA contamination for a correct evaluation of melanocortin receptor expression. Amplification reactions were carried out on an ABI PRISM 7900HT sequence detection system (Applied Biosystems) with the following thermal profile settings: an initial step of 10 min at 95 ◦ C, then 50 cycles of 95 ◦ C for 10 s and 60 ◦ C for 30 s. Three independent PCR amplification experiments were performed for each transcript. Fluorescence intensities were

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converted in threshold cycles using ABI Prism SDS 2.3 software (Applied Biosystems); baseline and threshold parameters were set by automatic analysis. Relative quantification of target gene expression was calculated with the comparative Ct method (Ct ), using the average Ct across basal samples as calibrator for each gene. Analysis of reference genes stability, performed with the geNorm software, indicated Actb, Hprt, Gapdh, and Rplp2 as the most stable genes. 2.6. Circulating IL-6 Plasma IL-6 concentration was determined using a commercially available ELISA method (Endogen Inc., Cambridge, MA) following manufacturer’s instructions. 2.7. Western blotting Frozen liver samples were rapidly immerged in tissue protein extraction reagent (T-PER) buffer (Pierce, Rockford, IL), supplemented with protease and phosphatase inhibitor cocktail (Sigma–Aldrich), and homogenized on ice using an Ultra-Turrax tissue homogenizer (IKA Labortechnik). Protein concentration was determined using the BCA Protein Assay (Thermo, Whaltam, MA). Fractionated proteins were transferred onto Trans-Blot nitrocellulose membranes (Bio-Rad, Hercules, CA) and blocked for 1 h at room temperature with 5% NFDM (Bio-Rad) for detection of MC1 , MC5 , phospho-STAT1, and STAT1; ECL advance blocking agent (GE Healthcare, Buckinghamshire, UK) was used for detection of I␬B␣; SuperBlock Blocking Buffer 10× (Thermo) in TBST (Tris Buffered Saline, 0.1% Tween 20; Bio-Rad) for STAT3 and phospho-STAT3. Membranes were probed overnight at 4 ◦ C with the following rabbit polyclonal antibodies: anti-MC1R (1:500; Alomone Labs), anti-MC5R (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-STAT3 (1:1000; Cell Signaling Technology Inc.), anti-STAT3 (1:1000; Cell Signaling Technology Inc.), anti-phospho-STAT1 (1:1000; Cell Signaling Technology Inc.), antiSTAT1 (1:2000; Cell Signaling Technology Inc.), and anti-IkB␣ (1:10,000; Cell Signaling Technology Inc.). After extensive washing with TBST, blots were incubated with a HRP-conjugated secondary antibody for 1 h at RT. Finally, membranes were developed using enhanced chemiluminescence (ECL) Advanced Western Blotting System (GE Healthcare) reagents, following the manufacturer’s instructions. Loading control was performed using a goat monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:2000; Santa Cruz Biotechnology), a secondary HRP-conjugated sheep anti-goat IgG (1:5000; Santa Cruz Biotechnology), and ECL reagents. Luminescent signals of protein bands were detected with a Kodak Gel Logic 2200 Digital Imaging System (Eastman Kodak, Rochester, NY). Densitometric analysis was performed using the Kodak Molecular Imaging Software version 4.0.5. 2.8. Statistical analysis Data are presented as mean ± SEM. Statistical analysis was performed using two way analysis of variance (ANOVA) followed by Bonferroni multicomparison post hoc analysis using SigmaStat software3.5 (Systat Software Inc., San Jose, CA). A probability value <0.05 was considered statistically significant. 3. Results 3.1. PH-induced transcriptional changes A preliminary investigation evaluated transcriptional changes that occurred in the liver after PH. Gene expression analysis was

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performed using a custom-designed TLDA (Fig. 1). Transcript selection in the TLDA was based on an accurate review of literature in order to include the most representative genes (see [32,42] for review). Hierarchical cluster analysis identified 6 gene clusters according to expression pattern similarities. Cluster 1 included genes that were down-regulated at 4 h and slowly returned to basal levels at 72 h. Genes in cluster 2 were steadily reduced after PH. Cluster 3 contained genes that increased at 4 h after PH and returned to basal level thereafter. Genes in cluster 4 were upregulated at both 4 and 8 h. Cluster 5 genes progressively increased reaching a peak at 8 h. Finally, transcripts of cluster 6 increased at 24 h post-PH. Cluster 1 mostly included genes encoding for cell junction proteins; cluster 2 liver metabolism and function; and cluster 3 immediate-early genes. Several genes grouped in cluster 4 consisted of IL-6-related molecules, whereas inflammatory genes accounted for a substantial proportion of transcripts in cluster 5. Finally, genes involved in cell cycle progression sorted in cluster 6.

3.2. Modulatory effects of NDP-MSH on liver phenotype during regeneration An investigation on melanocortin receptor expression in the liver documented presence of both mRNA and protein for MC1 and MC5 receptors, whereas MC2 , MC3 , and MC4 were not detected (Supplemental Fig. 1). Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.peptides. 2013.10.014. Based on TLDA expression data, we arbitrarily selected genes with a fold change ≥2 or ≤0.5 relative to basal expression at one or more intervals during regeneration. Seventy-one genes that met this criterion were evaluated using real-time PCR analysis to investigate NDP-MSH effects on their expression. Expression of a substantial number of molecules altered by PH was modulated by NDP-MSH administration at different intervals (Table 1). At 4 h after PH, transcripts of heat shock protein (Hsp) 70-1, nitric oxide synthase (Nos) 2, activating transcription factor 3 (Atf3), chemokine (C–X–C motif) ligand (Cxcl) 1, and early growth response (Egr) 1 were induced by peptide administration, whereas nuclear receptor subfamily 4, group A, member 1 (Nr4a1, Nur77) expression was attenuated by NDP-MSH relative to the saline group. At 8 h post PH, peptide treatment was associated with reduced expression of interferon (IFN)-␥, fibrinogen ␣ (Fga), and of several chemokines and their receptors. NDP-MSH administration exerted significant influences on the IL-6/STAT/SOCS pathway, known to be crucial during liver regeneration. The rise in plasma IL-6 concentration, observed in saline group at 4 h after PH, was amplified by NDP-MSH treatment at 8 h (Fig. 2A). In addition, peptide administration further increased hepatic Il6 mRNA expression at 4 and 8 h post-PH (Fig. 2B). Except for Oncostatin M that was induced by NDP-MSH at 4 h after PH (Table 1), the expression of other members of the gp130 cytokine family (cardiotrophin 1 and leukemia inhibitory factor) was not altered by the peptide (data not shown). STAT1 phosphorylation, markedly induced by PH, was further enhanced by NDP-MSH at 4 and 8 h (Fig. 2C). PH-induced STAT3 phosphorylation was greater in NDP-MSH-treated rats at 8 h relative to saline-treated animals. Finally, PH induced significant mRNA expression of the inhibitory proteins Socs1 and Socs3 (Fig. 2B). Relative to saline treated animals, NDP-MSH treatment elicited a further, significant induction of both Socs1 and Socs3 expression at 4 h post-PH (Fig. 2B). There was no significant expression change in sham- operated rats relative to saline-treated PH animals at any time interval.

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Fig. 1. Hierarchical cluster analysis of gene expression data in the regenerating liver. Heatmap showing global profiles at 4, 8, 24, 48, and 72 h after PH. Columns denote expression data for each sample; rows show individual genes. Up-regulation is indicated in red, down-regulation in green; the degree of color saturation reflects magnitude of expression changes. Algorithm grouped genes into 6 major clusters, according to similar expression patterns. Individual panels show the average expression pattern for each gene cluster, calculated using log ratios mean of expression abundance of every gene. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Table 1 Modulatory effects of NDP-MSH on PH-induced gene expression changesa in the liver. Time after PH

Gene symbol

Treatment PH + saline

4h

Hsp70-1 Nos2 Egr1 Atf3 Cxcl1 Osm p21 Nur77

8h

p21 Ccnd1 Ccr2 MIP-1␣ Ccr5 MCP-1 IFN-␥ Fga

24 h

CcnA2 Ccnb1 Ccne1

48 h

Cyp21a1

NDP-MSH vs saline PH + NDP-MSH

FC

p valueb

2.12 10.80 2.01 15.70 18.54 5.53 10.74 22.71

± ± ± ± ± ± ± ±

0.28 2.60 0.36 4.16 1.65 0.57 0.12 5.31

5.12 20.60 3.26 26.60 23.48 7.92 12.27 12.67

± ± ± ± ± ± ± ±

1.13 3.90 0.36 7.06 1.99 0.93 0.11 2.59

2.41 1.91 1.62 1.46 1.27 1.43 1.14 0.55

0.001 0.030 0.030 0.036 0.016 0.008 0.042 0.004

2.29 2.15 2.07 4.86 2.00 2.26 1.50 6.94

± ± ± ± ± ± ± ±

0.17 0.35 0.68 1.33 0.21 0.89 0.22 0.77

4.32 1.06 0.73 2.50 0.89 0.66 0.52 4.50

± ± ± ± ± ± ± ±

0.41 0.13 0.09 0.62 0.08 0.08 0.08 0.44

1.89 0.49 0.35 0.51 0.44 0.29 0.34 0.64

0.049 0.018 0.018 0.049 0.001 0.005 0.001 0.035

17.03 ± 1.90 10.04 ± 1.75 20.90 ± 2.57

23.93 ± 3.06 14.97 ± 1.94 16.51 ± 2.61

1.44 1.49 0.78

0.030 0.002 0.026

6.98 ± 0.49

4.66 ± 0.25

0.66

0.033

Values are expressed as mean ± SE, N = 7 animals per group. a Denoted as relative expression; FC, fold change. b Two-way ANOVA followed by Bonferroni multicomparison test.

Fig. 2. NDP-MSH given as a single i.v. injection 30 min before hepatic resection enhanced PH-induced IL-6 production and signaling during liver regeneration. (A) PH caused a significant increase in circulating IL-6; such increase was significantly greater in NDP-MSH-treated animals at 8 h post-PH. (B) Following PH, there was prominent STAT1 and STAT3 phosphorylation. Peptide treatment further induced p-STAT1 at 4 and 8 h, and p-STAT3 at 8 h post-PH. Western blot representative gels are shown. (C) Hepatic IL-6 mRNA expression markedly rose at 4 and 8 h post-PH; this increase was significantly more pronounced in NDP-MSH treated animals. Further, peptide administration elicited a significant increase in Socs1 and Socs3 transcripts relative to the saline group. Data are expressed as mean (N = 7 per group) ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001, PH + NDP-MSH vs PH + saline at the same interval.

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Fig. 3. Effect of NDP-MSH administration on I␬B␣ degradation after PH. Western blot analysis indicates that NDP-MSH treatment prevented the I␬B␣ degradation that occurred after PH in the saline group. GAPDH was used as loading control for densitometry. A representative gel is shown above densitometric analysis. Bars denote mean (N = 7 per group) ± SEM; **p < 0.01, PH + NDP-MSH vs PH + saline at the same interval.

Only the immediate-early gene Nur77, significantly increased at 4 h (2.03 ± 0.41, p < 0.05 vs basal). 3.3. Inhibitory effect of NDP-MSH on PH-induced IB˛ degradation I␬B␣ protein expression was examined in liver tissue at 4, 8, and 24 h after PH. Western Blot analysis indicated that NDP-MSH treatment prevented the significant decline of I␬B␣ that occurred at 4 h after PH (Fig. 3). 3.4. Effect of NDP-MSH on liver regeneration after PH NDP-MSH treatment did not substantially influence liver regeneration after PH. However, liver-to-body weight ratio measurements indicated that the mass recovery tended to be lower in peptide- treated animals at later intervals (Fig. 4A). Consistent with a moderately slower regeneration rate, the proliferative index Ki-67 was reduced in NDP-treated animals (Fig. 4B). Ki-67 immunostaining was significantly reduced in the NDP-MSH group at 48 h after PH. Analysis of cyclin expression indicated that G1/S cyclins D1 and E1 were induced by PH at 8 and 24 h, respectively, whereas Mcyclins A2 and B1 production peaked at 24 h. In the NDP-MSH group there was delayed induction of Cyclin D1 at 4 h, reduced increase of Cyclin E1 at 24 h, and enhanced expression of Cyclin A2 and B1 at 24 h (Fig. 4C). Finally, p21expression, induced by PH at 4 and 8 h, was significantly amplified by peptide treatment, whereas p27

mRNA accumulation was similar in saline and NDP-MSH groups at all time points after PH (not shown). 4. Discussion The data show that NDP-MSH exerts modulatory influences on liver molecular phenotype during regeneration after experimental PH. Treatment with the peptide enhanced hepatoprotective pathways and reduced potentially harmful mediators in the absence of significant changes of liver regeneration. A preliminary gene expression analysis on transcripts involved in liver metabolism and function, stress response, IL-6-signaling, inflammation, and cell cycle progression, identified broad transcriptional changes in the regenerating liver after 70% PH. Expression of a substantial number of molecules altered by PH was modulated by NDP-MSH treatment. A significant effect of NDP-MSH during liver regeneration likely resides in its capacity to enhance IL-6 both in the liver and in the circulation. Indeed, although IL-6 likely exerts only a marginal influence on hepatocyte replication, it is clear that this cytokine exerts protective effects on hepatocytes [17,24,39,41]. IL-6 activates multiple signaling pathways, promotes cell survival, and modulates expression of hepatocyte-specific metabolic factors. Several investigations showed that IL-6 is protective in a variety of liver injuries, including ischemia associated with transplantation, CCl4-induced damage, and FAS-activated apoptosis [31,38,39]. Trautwein and coworkers showed that mice knockout for IL-6 or with defective IL6 signaling (gp130 deletion) were more vulnerable to LPS-induced stress [43]. Further, IL-6 improves metabolic functions in the liver

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Fig. 4. Liver mass, hepatocellular replication, and expression of genes involved in cell cycle progression after 70% PH in saline- and NDP-MSH-treated rats. (A) Peptide treatment did not significantly influence liver mass recovery. Regeneration index, assessed as liver-to-body weight ratio calculated at 4, 8, 24, 48, and 72 h after hepatic resection, was similar in the two groups although it tended to be lower in NDP-MSH treated animals. Points denote mean (N = 7 per group) ± SEM. (B) NDP-MSH administration was likewise associated with a reduction in Ki-67 positivity with a significant difference at 48 h. Results are expressed as the percentage of Ki-67 positive cells per 1000 hepatocytes in randomly selected high-power fields (100× magnification, 10 fields). Points denote mean (N = 7 per group) ± SEM. *p < 0.05, PH + NDP-MSH vs PH + saline at the same time interval. (C) NDP-MSH modulated expression of genes encoding cell cycle mediators. At 8 h post-PH, peptide treatment delayed the PH-induced increase in Cyclin D1, whereas at 24 h NDP-MSH reduced Cyclin E1 production. M-cyclins A2 and B1, that were induced at 24 h after PH, were further enhanced by NDP-MSH at the same time point. Relative mRNA expression was determined using RT-qPCR. Data were normalized using a normalization factor calculated with 6 reference genes. Histograms denote mean (N = 7 per group) ± SEM. *p < 0.05; PH + NDP-MSH vs PH + saline at the same interval.

remnant after PH [23]. Thus, the additional increase in IL-6 production induced by NDP-MSH during liver regeneration is likely protective and could render hepatocytes more resistant to stress signals. NDP-MSH treatment was associated with enhanced activation of the STAT1 and STAT3 signaling pathways and increased induction of the inhibitory molecules SOCS1 and SOCS3. This observation is significant as previous studies reported that the STAT3/SOCS3 pathway exerts protective actions against liver injury [24,38,43]. Further, STAT3 signaling is required for survival in the acute stage following 70% PH and reduces the inflammatory reaction associated with hepatocyte necrosis [29]. Among modulatory effects exerted by NDP-MSH, reduced expression of monocyte chemotactic protein 1 (MCP-1/CCL2) and its receptor CCR2 appear particularly significant, as activation of the MCP-1/CCR2 axis mediates infiltration by monocytes and macrophages from the periphery and bone marrow [34,44].

Production of MCP-1 by hepatocytes and Kupffer cells has been observed in different experimental models of liver injury and in human acute liver failure [44]. NDP-MSH-associated reduction in interferon ␥ (IFN-␥) could likewise be protective during regeneration because of the well-known role of this cytokine in promoting hepatic inflammation [37]. Preclinical studies in KO mice or in animals treated with IFN-␥ neutralizing antibodies showed that loss of IFN-␥ signaling is protective in experimental liver injury [9,22]. Reduced phosphorylation of the inhibitory molecule I␬B␣ and, consequently, reduced NF-␬B activation is a recognized mechanism in the anti-inflammatory action of melanocortins [10,21]. In the present research, NDP-MSH treatment caused substantial protection of I␬B␣ from degradation induced by PH. In addition to the anti-inflammatory effects, this action could partly explain the subtle changes exerted on liver regeneration markers in the absence of eventual organ mass recovery. Indeed, hepatocyte proliferation is promoted early after PH by inflammatory mediators, including

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TNF-␣, released by Kupffer cells [14]. Therefore, the delay of cell cycle progression observed in the NDP-MSH group could be caused by an initial reduction of cytokine expression associated with I␬B␣ protection and, consequently, diminished NF-␬B-dependent transcription. On the other hand, NDP-MSH had no effect on the eventual organ mass recovery. This is consistent with recent observations that NF-␬B signaling is not required for successful liver regeneration after PH [33]. It is also important to consider that prolonged NF-␬B stimulation causes hepatocyte death and tissue damage due to excessive production of cytokines and chemokines [12]; NF-␬B inhibitors such as A20 exerted antiapoptotic functions after extended hepatectomy [27]. Therefore, the general idea is that NDP-MSH treatment, despite reduced hepatocyte replication, did not modify substantially the eventual liver mass recovery because of concomitant apoptosis reduction. In this regard, several investigations indicate that ␣-MSH and related melanocortins exert antiapoptotic influences in different organs and cell types [3,36,40]. In conclusion, NDP-MSH, a peptide approved for clinical use, exerted wide regulatory effects on several cell pathways during liver regeneration. Modulation of different cell cycle mediators together with enhancement of IL-6/STAT3/SOCS3 signaling suggest that NDP-MSH, in addition to its known anti-inflammatory effects, promotes a controlled regenerative response. Most of the observed changes could be salutary in conditions of impaired regeneration, particularly in the presence of inflammatory disorders. Acknowledgments Authors thank Nicola Fusco for Ki-67 immunohistochemical analysis. This research was supported by funds of Ricerca Corrente Ospedale Maggiore Policlinico Milano and Fondazione Fiera Milano. References [1] Bernuau D, Rogier E, Moreau A, Bernuau J, Feldmann G. Inhibitory effect of the acute inflammatory reaction on liver regeneration after partial hepatectomy in the rat. Gastroenterology 1986;90:268–73. [2] Brzoska T, Bohm M, Lugering A, Loser K, Luger TA. Terminal signal anti-inflammatory effects of alpha-melanocyte-stimulating hormone related peptides beyond the pharmacophore. Adv Exp Med Biol 2010;681:107–16. [3] Brzoska T, Luger TA, Maaser C, Abels C, Bohm M. Alpha-melanocyte-stimulating hormone and related tripeptides: biochemistry, antiinflammatory and protective effects in vitro and in vivo, and future perspectives for the treatment of immune-mediated inflammatory diseases. Endocr Rev 2008;29:581–602. [4] Catania A. The melanocortin system in leukocyte biology. J Leukoc Biol 2007;81:383–92. [5] Catania A, Gatti S, Colombo G, Lipton JM. Targeting melanocortin receptors as a novel strategy to control inflammation. Pharmacol Rev 2004;56:1–29. [6] Catania A, Lipton JM. Alpha-melanocyte stimulating hormone in the modulation of host reactions. Endocr Rev 1993;14:564–76. [7] Catania A, Lonati C, Sordi A, Leonardi P, Carlin A, Gatti S. The peptide NDP-MSH induces phenotype changes in the heart that resemble ischemic preconditioning. Peptides 2010;31:116–22. [8] Chiao H, Foster S, Thomas R, Lipton J, Star RA. Alpha-melanocyte-stimulating hormone reduces endotoxin-induced liver inflammation. J Clin Invest 1996;97:2038–44. [9] Dong Z, Zhang C, Wei H, Sun R, Tian Z. Impaired NK cell cytotoxicity by high level of interferon-gamma in concanavalin A-induced hepatitis. Can J Physiol Pharmacol 2005;83:1045–53. [10] Eves PC, Haycock JW. Melanocortin signalling mechanisms. Adv Exp Med Biol 2010;681:19–28. [11] Eves PC, MacNeil S, Haycock JW. Alpha-melanocyte stimulating hormone, inflammation and human melanoma. Peptides 2006;27:444–52. [12] Fan C, Yang J, Engelhardt JF. Temporal pattern of NFkappaB activation influences apoptotic cell fate in a stimuli-dependent fashion. J Cell Sci 2002;115:4843–53. [13] Fausto N. Liver regeneration. J Hepatol 2000;32:19–31. [14] Fausto N, Campbell JS, Riehle KJ. Liver regeneration. Hepatology 2006;43:S45–53. [15] Gatti S, Carlin A, Sordi A, Leonardi P, Colombo G, Fassati LR, et al. Inhibitory effects of the peptide (CKPV)2 on endotoxin-induced host reactions. J Surg Res 2006;131:209–14.

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