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Heme oxygenase-1 inhibitor tin-protoporphyrin improves liver regeneration after partial hepatectomy
Life Sciences 204 (2018) 9–14
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Heme oxygenase-1 inhibitor tin-protoporphyrin improves liver regeneration after partial hepatectomy Monica Pibiri, Vera Piera Leoni, Luigi Atzori
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Department of Biomedical Sciences, Oncology and Molecular Pathology Unit, University of Cagliari, Via Porcell 4, 09124 Cagliari, Italy
A R T I C LE I N FO
A B S T R A C T
Keywords: Heme oxygenase Liver regeneration Tin protoporphyrin IX
Aims: This study investigates the effects of the heme oxygenase-1 (HO-1) inhibitor tin protoporphyrin IX (SnPP), on rat liver regeneration following 2/3 partial hepatectomy (PH) in order to clarify the controversial role of HO1 in the regulation of cellular growth. Main methods: Male Wistar rats received a subcutaneous injection of either SnPP (10 μmoles/kg body weight) or saline 12 h before PH and 0, 12 and 24 h after surgery. Rats were killed from 0.5 to 36 h after PH. Bromodeoxyuridine (BrdU) incorporation was used to analyze cell proliferation. Immunohistochemistry, Western blot analysis and quantitative Real Time-PCR were used to assess molecular and cellular changes after PH. Key findings: Data obtained have shown that administration of SnPP caused an increased entry of hepatocytes into S phase after PH, as demonstrated by labeling (L.I.) and mitotic (M.I.) indexes. Furthermore, enhanced cell cycle entry in PH-animals pre-treated with SnPP was associated with an earlier activation of IL-6 and transcription factors involved in liver regeneration, such as phospho-JNK and phospho-STAT3. Significance: Summarizing, data here reported demonstrate that inhibition of HO-1 enhances rat liver regeneration after PH which is associated to a very rapid increase in the levels of inflammatory mediators such as IL-6, phopsho-JNK and phospho-STAT3, suggesting that HO-1 could act as a negative modulator of liver regeneration. Knowledge about the mechanisms of liver regeneration can be applied to clinical problems caused by delayed liver growth, and HO-1 repression may be a mechanism by which cells can faster proliferate in response to tissue damage.
1. Introduction The ability of the liver to regenerate after 2/3 partial hepatectomy (PH) typifies the capacity of an organ to change instantly from an essentially quiescent state to a rapidly growing one [1,2] providing a robustexperimentalin vivo model to study cell cycle entry and cell proliferation. Although considerable advance has been made in the comprehension of the molecular mechanisms associated to the control of liver cell growth, their exact nature has not been fully elucidated yet. At present, the molecular signals triggering liver regeneration are thought to include cytokines, growth factors, reactive oxygen species, complement factors C3 and C5 and, possibly, bacterial endotoxin [3]. The priming phase of liver regeneration, which corresponds to the G0/ G1 transition, is controlled by a cytokine network which acts through activation of pre-existing latent transcription factors, such as STAT3,NF-kB and AP-1, which, in turn, mediate the transcription of immediate early genes, such as c-jun, c-fos, c-myc, responsible for cell cycle progression [1,4,5]. A major role in signal transduction pathway ⁎
Corresponding author. E-mail address: [email protected] (L. Atzori).
https://doi.org/10.1016/j.lfs.2018.05.011 Received 12 February 2018; Received in revised form 24 April 2018; Accepted 4 May 2018 Available online 05 May 2018 0024-3205/ © 2018 Published by Elsevier Inc.
induction has been attributed to a condition of oxidative stress [6–8], which may contribute to activation of oxidant-sensitive transcription factors (e.g. NF-kB and STAT3) and cytokines. It appears that the initial increase of cytokines associated with oxidative stress is beneficial for liver regeneration, as demonstrated by the finding that the response to PH is impaired in interleukin 6 (IL-6)-deficient mice [9,10]. Heme oxygenase (HO; EC 1.14.99.3), a member of the heat-shock protein family, plays a protective role in inflammation and oxidative stress [11,12]. Like other antioxidant enzymes, HO-1 contains the antioxidant response elements (ARE) which is a binding site for the transcription factor Nrf2 [13,14] whose activity is regulated by redox status [15]. HO-1 gene product, is a heme-catabolising enzyme that converts heme yielding carbon monoxide (CO), iron (Fe2+) and biliverdin. HO-1, the inducible form of HO, is upregulated during oxidative stress and has been proposed to have a role in the regulation of inflammatory processes [16]. The enzymatic activity of HO-1 results in decreased oxidative stress and attenuation of the inflammatory response due to removal of heme, a potent prooxidant and
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antibodies directed against, E2F-1 (C20), p107 (C-18), cyclin A (C-19) and Lamin A/C(H-110) (Santa Cruz Biotechnology), SAPK/JNK, phospho SAPK/JNK, STAT3 and phospho STAT3 (Tyr705) (Cell Signalling Technology, Beverly, MA, USA) and goat monoclonal antibody directed against albumin (Bethyl Laboratories, Montgomery, TX).
proinflammatory agent, and to the generation of biologically anti-inflammatory active products, such as CO, bilirubin and ferritin. In addition, HO-1 exerts anti-inflammatory effects by inhibition of tumor necrosis factor-α and interleukin-1βand by upregulation of interleukin10 [17]. Recent data suggest a key role for HO-1 in the regulation of cellular homeostasis and growth [18], although its effects are cell-type specific and can be opposite. Accordingly, while in vascular smooth muscle cells and in smooth muscle cells induction of HO-1inhibits proliferation [19,20], its over-expression in endothelial cells protects against apoptosis TNF-α-induced and promotes proliferation [21]. Moreover, HO-1 deficiency in humans is associated with susceptibility to oxidative stress and increased pro-inflammatory state in epithelium [22], fibroblasts [23], and T lymphocytes [24]. The synthetic heme analogue tin protoporphyrin IX (SnPP), is an extreme potent HO-1 inhibitor which exerts its action by a dual mechanism that involves competitive inhibition of the enzyme for the natural substrate heme and simultaneous enhancement of new enzyme synthesis in liver cells that, as a consequence, results completely inhibited [25,26]. Secondary increases of HO-1 mRNA and protein levels by SnPP can be due to enzyme “feedback inhibition” or to induction of a mild pro-oxidant state with counterbalancing HO-1 production [27]. However, this could potentially be offset by SnPP-induced HO-1 inhibition which lasts until SnPP elimination [27]. Inhibition of HO-1 activity by SnPP has been successfully used to avoid heme degradation to bilirubin in vivo and thus, to suppress various forms of jaundice [28,29]. Based on the potential role of HO modulation on cell proliferation, and on the strict association between HO-1 and inflammatory cytokines, key mediators of liver regeneration, the present study was designed to investigate whether inhibition of HO-1 by SnPP could affect liver regeneration after PH in rats.
2.3. Immunohistochemistry BrdU-positive hepatocytes were stained with the peroxidase method as described previously [32]. Labelling index (L.I.) was expressed as number of BrdU positive hepatocyte nuclei/100 nuclei. Mitotic index (M.I.) was determined as number of mitoses/1000 hepatocytes. Results are expressed as means ± SE of four to five rats per group. At least 5000 hepatocyte nuclei per liver were scored. 2.4. Quantitative real time-PCR Total RNA was extracted from frozen tissue with Trizol Reagent (Invitrogen, San Giuliano, Milan, Italy) and RNeasy MiniElute Cleanup Kit (Qiagen, Valencia, CA). Equal amounts of at last 3 samples for each group were used to create pools that were reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Gene expression analysis was performed with Realtime PCR analysis of 10 ng of cDNA mixed with 2× TaqMan Gene expression Master Mix and 20× specific TaqMan gene expression assays (Rn00561420_m1,Il6; Rn00477784_m1, HO-1; Rn00566528_m1, Nqo1) (Applied Biosystems) with an ABI PRISM 7300 Thermocycler (Applied Biosystems). Each sample was run in triplicate and gene expression analysis of Actin-beta or GAPDH was used as endogenous control. Relative quantification analysis for each gene was calculated by 2-ΔΔCt method.
2. Materials and methods 2.5. Statistical analysis 2.1. Animals and treatment Results are expressed as the mean ± Standard Deviation (SD). Differences between groups were performed using either unpaired twotail Student's t test or ANOVA for multiple groups comparison.
Male Wistar rats (180–220 g), purchased from Charles River (Milano, Italy), were maintained on a standard laboratory chow diet (Ditta Mucedola, Milano, Italy). The animals were provided food and water ad libitum, whit a 12 h light/dark daily cycle and were acclimated for 1 week before starting the experiment. Guidelines for the Care and Use of Laboratory Animals were used during the investigation. Two thirds hepatectomy was performed according to Higgins and Anderson [30]. SnPP (Frontier Scientific, Carnfort, UK) was dissolved in 0.2 N NaOH and then back titrate with HCl solution. Animals received subcutaneous injections of either SnPP (10 μmoles/kg of body weight) or vehicle 12 h before PH and 0, 12, and 24 h after PH. Rats were killed from 0.5 to 36 h after surgery. Control groups also include animal treated with SnPP or untreated and sacrificed at the same time points without been subjected to PH. To score S phase hepatocytes, rats received BrdU (SIGMA Chemical, St. Louis, MO) either in drinking water (1 mg/ml) all throughout the experimental period or in a single dose (50 mg/kg body weight i.p.) 2 h before sacrifice. Immediately after sacrifice, sections of the liver were fixed in 10% buffered formalin and processed for staining with hematoxylin-eosin or immunohistochemistry. The remaining liver was snap-frozen in liquid nitrogen and kept at −80 °C until use.
3. Results To investigate whether inhibition of HO-1 by SnPP could affect liver regeneration response, male Wistar rats were subjected to 70% PH and injected s.c. with either 10 μmoles/kg body weight (bw) of SnPP or vehicle 12 h before surgery, at the time of PH and 12 h after. Animals were sacrificed 24 h after PH. Animals treated with 10 SnPP or untreated and sacrificed 12 h later, without been subjected to PH, served as control groups. To monitor the hepatocytes S-phase-entry, BrdU dissolved in drinking water (1 mg/ml) was given throughout the experimental period. As shown in Fig. 1A and B, the HO-1 inhibitor SnPP significantly increased the number of BrdU-positive hepatocytes during liver regeneration, without signs of liver toxicity. Indeed, 24 h after PH labelling index was 36%in SnPP pre-treated animals vs 23% of PH group (Fig. 1B). In agreement with previous studies [25], we found that SnPP caused enhanced gene transcription of HO-1(Fig. 1C). Indeed, Real time PCR analysis revealed that SnPP treatment was associated to enhanced HO-1 mRNA levels, in both control and hepatectomized rats (Fig. 1C). This effect was specific, as the expression of Nqo1, anotherNrf2 target gene, was unaffected by the treatment with SnPP (Fig. 1D). One sample (SnPP +PH 30 min) resulted out of range in both HO-1 and Nqo1 RT-PCR analyses. To further characterize the effect of SnPP on liver regeneration, the kinetics of hepatocyte proliferation in the presence or absence of the inhibitor was examined by BrdU pulse labeling. Animals injected with SnPP or untreated prior to PH were given a single dose of BrdU (50 mg/kg bw, i.p.) 2 h before sacrifice at 18, 24, 30 and 36 h after surgery. As shown in Fig. 2C, while SnPP pre-treatment did not
2.2. Western blot analysis Preparation of total cell extracts and western blot analysis were performed as described previously [31]. Nuclear and cytosolic cell extracts were prepared according to the method of Timchenko et al. [32]. For immunoblotting experiments the following antibodies were used: mouse monoclonal antibodies directed against, PCNA (PC-10) (Santa Cruz Biotechnology, CA, USA), Cyp1/WAF-1/p21 (Upstate Biotech, New York) and actin (clone AC-40)(SIGMA); rabbit polyclonal 10
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Fig. 1. Wistar rats subjected to 70% PH were treated with SnPP or vehicle (control) 12 h prior to surgery, at the time of PH and 12 h after PH. Animal were sacrificed 24 h after PH. Controls animal, injected s.c. either with vehicle or with SnPP, were sacrificed 12 h later without been subjected to PH. (A) Representative microphotograph (×10 magnification, section counterstained with hematoxylin) which illustrates the presence of BrdU-positive hepatocytes in the liver of rats sacrificed 24 h after PH, with or without pre-treatment with SnPP (10 μmoles/kg body weight, s.c.). To label the hepatocytes, BrdU (1 mg/ml) was given all throughout the experimental period dissolved in drinking water, starting 2 h after PH. (B) Labeling Index. At least 5000 hepatocyte nuclei per liver were scored. The labeling index was expressed as number of BrdU-positive hepatocyte nuclei/100 nuclei. Results are expressed as means ± S.E. of 4–5 rats per group. * = statistically different from PH alone; p < 0.05. (C and D) Real time PCR analysis of HO-1 (C) and Noq1 (D) mRNA levels in the livers from Wistar rats treated with a single dose of SnPP (10 μmoles/kg body weight) or vehicle 12 h prior to PH and at the time of PH and sacrificed 30 min or 2 h post surgery. Each lane represents a pool of three samples per group.
and growth [17,19,36], although its role in modulating cell growth is still unclear. Indeed, while a positive correlation between HO-1 induction and proliferation was found in epidermal keratinocytes, in vascular endothelium [37,38] and in various tumor cells [39,40], the induction of HO-1 was also reported to inhibit cell growth in rat kidney epithelial cells [21] and vascular smooth muscle cells [41,42] and in human pulmonary or renal tubular cells [43,44]. Taken together, all these evidences suggest that the effect of HO-1 on cell growth is essentially cell type-dependent. In the present study, to investigate the role of HO-1 on hepatocyte proliferation we analyzed the effect of SnPP, an inhibitor of HO-1 activity, in rat liver regeneration after 2/3 PH. The results showed that administration of SnPP prior to PH led to increased entry of hepatocytes into S phase compared to PH alone, as demonstrated by the increased number of BudR-positive hepatocytes and the higher expression of cellcycle proteins. To search for possible mechanisms responsible for the increased regenerative response observed following SnPP administration, we evaluated the expression/activation of transcription factors, such asSTAT3 and JNK, known to be involved in triggering the very early events of liver regeneration [2,3,9]. Our present findings showing that inhibition of HO-1 by SnPP leads to a more rapid activation of JNK and STAT3, suggest that HO-1 might inhibit liver regeneration by negatively activating cytokine-induced transduction pathways needed for the priming phase of hepatocytes. According to our hypothesis, it has been demonstrated that mice
significantly modify the number of BrdU-positive hepatocytes 18 h after PH respect to PH alone, a striking increase of the L.I. was evident at 24 h in SnPP+PH group (L.I. was 46% vs 24% of PH alone) (Fig. 2A and C). At later time points, the L.I. decreased in both groups. The increased entry of hepatocytes into S phase observed in SnPP + PH rats compared to PH alone 24 h after surgery, was accompanied by a significant enhancement of mitoses (Fig. 2Band 2D). Next, the expression of cell cycle proteins was examined by western blot. As shown in Fig. 3, SnPP induced increased levels of the G1-S regulators PCNA (18 h after surgery), p107 and E2F (starting from 24 h after PH),compared to SnPP-untreated group. No significant changes in the expression of the cyclinCDK inhibitor p21 were observed between the two experimental groups. In keeping with previous reports [16,33], pre-treatment with SnPP resulted in a significant increase of IL-6 mRNA levels at 30 min and 2 h after PH (Fig. 4A). Cytokine release during inflammation is known to activateSTAT3 and JNK-mediated pathways [34,35]. Accordingly, an earlier enhancement of JNK and STAT3 phosphorylation was observed in animals pre-treated with the HO-1 inhibitor (Fig. 4B). 4. Discussion Alteration of redox balance homeostasis can lead to the activation of various survival pathways. An increased synthesis of HO-1 occurs as a general response to inflammation or stress in biological systems. Recent data suggest a key role of HO-1 in the regulation of cellular homeostasis 11
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Fig. 2. Wistar rats subjected to 70% PH were treated with SnPP or vehicle 12 h prior to surgery, at the time of PH and 12 and 24 h after PH. Animals were sacrificed at the indicated time points after PH. SnPP or vehicle groups were sacrificed at the same time points without been subjected to PH. (A) Representative microphotograph (×10, section counterstained with hematoxylin) which illustrates the presence of BrdU-positive hepatocytes in the liver of rats sacrificed 24 h after PH, with or without pre-treatment with SnPP (10 μmoles/kg body weight, s.c.). (B) Representative microphotograph (×20 magnification, containing a small section with ×40 magnification) which illustrates H&E (hematoxylin and eosin) staining to detect the presence of mitosis in the liver of rats sacrificed 30 h after PH, with or without pre-treatment with SnPP (10 μmoles/kg body weight, s.c.). Arrows indicate some mitotic cells. (C) Labeling Index. To label the hepatocytes, BrdU (50 mg/Kg b.w., i.p.) was given 2 h before sacrifice. At least 5000 hepatocyte nuclei per liver were scored. The L.I. was expressed as number of BrdU-positive hepatocyte nuclei/100 nuclei. Results are expressed as means ± S.E. of 4–5 rats per group. *** = statistically different from PH alone; p < 0.0009. (D) Mitotic Index in livers of rats sacrificed 24 h after PH. The mitotic index was expressed as number of hepatocytes entering mitosis/1000 hepatocyte nuclei. At least 5000 hepatocyte nuclei per liver were scored. Results are expressed as means ± S.E. of 4–5 rats per group. * = statistically different from PH alone; p < 0.05.
Fig. 3. Expression of cell cycle related proteins in the liver of Wistar subjected to 70% PH and treated with SnPP or vehicle 12 h prior to surgery, at the time of PH and 12 and 24 h after PH. PH animals were killed from 18 to30 hours after PH, with or without SnPP, while SnPP or vehicle (CO) groups were sacrificed at the same time points without been subjected to PH. (A) Western blot analysis of nuclear protein extracts prepared from the livers; the analysis was performed as described in Materials and Methods. Appropriate loading was confirmed by laminin A/C detection. Each lane represents a pool of 3 samples; CO: vehicle; Sn: SnPP·(B) Densitometric analysis of cell cycle-related protein expression normalized to A/C laminin. 12
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Fig. 4. Analysis of inflammation markers in the livers from Wistar rats treated with a single dose of SnPP (10 μmoles/kg body weight) or vehicle 12 h prior to PH and at the time of PH and sacrificed 30 min or 2 h post surgery. (A) Real time PCR analysis of IL-6 mRNA levels. Each lane represents a pool of three samples. (B) Western blot analysis of phospho-JNK and phospho-STAT3. Total protein extracts (100–150 μg/lane) were prepared from the livers and Western analysis was performed as described in the Materials and Methods. Appropriate loading was confirmed by Actin detection. Each lane represents a single sample; CO: vehicle; Sn: SnPP.
withNrf2−/− genotype, lacking of Nrf2-dependent HO-1 activation, show increased severity of inflammatory response to different factors [45] which was correlated to increased cell proliferation and cancer development [46]. Indeed, Yokoo et al. [46], reported that Nrf2 deficiency increased susceptibility to oxidative stress-induced small intestinal carcinogenesis in mice, through overexpression of COX2 followed by stimulation of cell cycle progression. Our data are apparently in contrast with those of Glanemann et al. [47] showing an improvement of liver regeneration following HO-1 induction by CoPP pre-treatment. In the latter study, the increased regenerative capacity of the liver was attributed to HO-1 mediated decrease of oxidative stress and inflammatory response. However, this conclusion has not been fully confirmed by other reports [48] which showed that no enhanced liver regeneration after HO-1 induction by CoPP treatment occurs unless NO production was blocked by the NOS inhibitor L-NAME. Thus, from this study clearly emerges that HO-1 induction by CoPP alone was not able to enhance hepatocyte proliferation after PH. More recently, Marinic et al. [49] reported that pre-exposure to the olive oil polyphenols extract (PFE) stimulated liver regeneration through a transient increase in oxidant load within the first hours after hepatectomy, and the associated induction of stress response genessuch as HO-1 and GCSc under the control of Nrf2.Based on these results, the authors postulated that antioxidant Nrf2-induced genes, such as HO-1, play the key role in increasing liver regeneration. However, it is likely that in PFE-pretreated hepatectomyzed rats the initial increased oxidative stress per se, rather than HO-1 activation, could enhance liver regeneration post-surgery. If so, it follows that induction of antioxidant genes could only represent a compensatory response and not the causative agent of the enhanced hepatocyte proliferation, supporting our present findings showing that increased oxidative stress due to HO-1 inhibition, results in enhanced liver regeneration. Although much remains to be learned about the molecular mechanisms involved in cell proliferation after PH, elucidation of the signalling pathways modulated by HO-1 may provide new insights for improved understanding and possibly treatment of pathologies where hepatocyte proliferation is important (e.g. acute hepatitis, liver tumor, living related donor grafts). Knowledge about the mechanisms of liver regeneration can be applied to clinical problems caused by delayed liver growth, and HO-1 repression may be a mechanism by which cells can faster proliferate in response to tissue damage.
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Heme oxygenase-1 inhibitor tin-protoporphyrin improves liver regeneration after partial hepatectomy Monica Pibiri, Vera Piera Leoni, Luigi Atzori
T
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Department of Biomedical Sciences, Oncology and Molecular Pathology Unit, University of Cagliari, Via Porcell 4, 09124 Cagliari, Italy
A R T I C LE I N FO
A B S T R A C T
Keywords: Heme oxygenase Liver regeneration Tin protoporphyrin IX
Aims: This study investigates the effects of the heme oxygenase-1 (HO-1) inhibitor tin protoporphyrin IX (SnPP), on rat liver regeneration following 2/3 partial hepatectomy (PH) in order to clarify the controversial role of HO1 in the regulation of cellular growth. Main methods: Male Wistar rats received a subcutaneous injection of either SnPP (10 μmoles/kg body weight) or saline 12 h before PH and 0, 12 and 24 h after surgery. Rats were killed from 0.5 to 36 h after PH. Bromodeoxyuridine (BrdU) incorporation was used to analyze cell proliferation. Immunohistochemistry, Western blot analysis and quantitative Real Time-PCR were used to assess molecular and cellular changes after PH. Key findings: Data obtained have shown that administration of SnPP caused an increased entry of hepatocytes into S phase after PH, as demonstrated by labeling (L.I.) and mitotic (M.I.) indexes. Furthermore, enhanced cell cycle entry in PH-animals pre-treated with SnPP was associated with an earlier activation of IL-6 and transcription factors involved in liver regeneration, such as phospho-JNK and phospho-STAT3. Significance: Summarizing, data here reported demonstrate that inhibition of HO-1 enhances rat liver regeneration after PH which is associated to a very rapid increase in the levels of inflammatory mediators such as IL-6, phopsho-JNK and phospho-STAT3, suggesting that HO-1 could act as a negative modulator of liver regeneration. Knowledge about the mechanisms of liver regeneration can be applied to clinical problems caused by delayed liver growth, and HO-1 repression may be a mechanism by which cells can faster proliferate in response to tissue damage.
1. Introduction The ability of the liver to regenerate after 2/3 partial hepatectomy (PH) typifies the capacity of an organ to change instantly from an essentially quiescent state to a rapidly growing one [1,2] providing a robustexperimentalin vivo model to study cell cycle entry and cell proliferation. Although considerable advance has been made in the comprehension of the molecular mechanisms associated to the control of liver cell growth, their exact nature has not been fully elucidated yet. At present, the molecular signals triggering liver regeneration are thought to include cytokines, growth factors, reactive oxygen species, complement factors C3 and C5 and, possibly, bacterial endotoxin [3]. The priming phase of liver regeneration, which corresponds to the G0/ G1 transition, is controlled by a cytokine network which acts through activation of pre-existing latent transcription factors, such as STAT3,NF-kB and AP-1, which, in turn, mediate the transcription of immediate early genes, such as c-jun, c-fos, c-myc, responsible for cell cycle progression [1,4,5]. A major role in signal transduction pathway ⁎
Corresponding author. E-mail address: [email protected] (L. Atzori).
https://doi.org/10.1016/j.lfs.2018.05.011 Received 12 February 2018; Received in revised form 24 April 2018; Accepted 4 May 2018 Available online 05 May 2018 0024-3205/ © 2018 Published by Elsevier Inc.
induction has been attributed to a condition of oxidative stress [6–8], which may contribute to activation of oxidant-sensitive transcription factors (e.g. NF-kB and STAT3) and cytokines. It appears that the initial increase of cytokines associated with oxidative stress is beneficial for liver regeneration, as demonstrated by the finding that the response to PH is impaired in interleukin 6 (IL-6)-deficient mice [9,10]. Heme oxygenase (HO; EC 1.14.99.3), a member of the heat-shock protein family, plays a protective role in inflammation and oxidative stress [11,12]. Like other antioxidant enzymes, HO-1 contains the antioxidant response elements (ARE) which is a binding site for the transcription factor Nrf2 [13,14] whose activity is regulated by redox status [15]. HO-1 gene product, is a heme-catabolising enzyme that converts heme yielding carbon monoxide (CO), iron (Fe2+) and biliverdin. HO-1, the inducible form of HO, is upregulated during oxidative stress and has been proposed to have a role in the regulation of inflammatory processes [16]. The enzymatic activity of HO-1 results in decreased oxidative stress and attenuation of the inflammatory response due to removal of heme, a potent prooxidant and
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antibodies directed against, E2F-1 (C20), p107 (C-18), cyclin A (C-19) and Lamin A/C(H-110) (Santa Cruz Biotechnology), SAPK/JNK, phospho SAPK/JNK, STAT3 and phospho STAT3 (Tyr705) (Cell Signalling Technology, Beverly, MA, USA) and goat monoclonal antibody directed against albumin (Bethyl Laboratories, Montgomery, TX).
proinflammatory agent, and to the generation of biologically anti-inflammatory active products, such as CO, bilirubin and ferritin. In addition, HO-1 exerts anti-inflammatory effects by inhibition of tumor necrosis factor-α and interleukin-1βand by upregulation of interleukin10 [17]. Recent data suggest a key role for HO-1 in the regulation of cellular homeostasis and growth [18], although its effects are cell-type specific and can be opposite. Accordingly, while in vascular smooth muscle cells and in smooth muscle cells induction of HO-1inhibits proliferation [19,20], its over-expression in endothelial cells protects against apoptosis TNF-α-induced and promotes proliferation [21]. Moreover, HO-1 deficiency in humans is associated with susceptibility to oxidative stress and increased pro-inflammatory state in epithelium [22], fibroblasts [23], and T lymphocytes [24]. The synthetic heme analogue tin protoporphyrin IX (SnPP), is an extreme potent HO-1 inhibitor which exerts its action by a dual mechanism that involves competitive inhibition of the enzyme for the natural substrate heme and simultaneous enhancement of new enzyme synthesis in liver cells that, as a consequence, results completely inhibited [25,26]. Secondary increases of HO-1 mRNA and protein levels by SnPP can be due to enzyme “feedback inhibition” or to induction of a mild pro-oxidant state with counterbalancing HO-1 production [27]. However, this could potentially be offset by SnPP-induced HO-1 inhibition which lasts until SnPP elimination [27]. Inhibition of HO-1 activity by SnPP has been successfully used to avoid heme degradation to bilirubin in vivo and thus, to suppress various forms of jaundice [28,29]. Based on the potential role of HO modulation on cell proliferation, and on the strict association between HO-1 and inflammatory cytokines, key mediators of liver regeneration, the present study was designed to investigate whether inhibition of HO-1 by SnPP could affect liver regeneration after PH in rats.
2.3. Immunohistochemistry BrdU-positive hepatocytes were stained with the peroxidase method as described previously [32]. Labelling index (L.I.) was expressed as number of BrdU positive hepatocyte nuclei/100 nuclei. Mitotic index (M.I.) was determined as number of mitoses/1000 hepatocytes. Results are expressed as means ± SE of four to five rats per group. At least 5000 hepatocyte nuclei per liver were scored. 2.4. Quantitative real time-PCR Total RNA was extracted from frozen tissue with Trizol Reagent (Invitrogen, San Giuliano, Milan, Italy) and RNeasy MiniElute Cleanup Kit (Qiagen, Valencia, CA). Equal amounts of at last 3 samples for each group were used to create pools that were reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Gene expression analysis was performed with Realtime PCR analysis of 10 ng of cDNA mixed with 2× TaqMan Gene expression Master Mix and 20× specific TaqMan gene expression assays (Rn00561420_m1,Il6; Rn00477784_m1, HO-1; Rn00566528_m1, Nqo1) (Applied Biosystems) with an ABI PRISM 7300 Thermocycler (Applied Biosystems). Each sample was run in triplicate and gene expression analysis of Actin-beta or GAPDH was used as endogenous control. Relative quantification analysis for each gene was calculated by 2-ΔΔCt method.
2. Materials and methods 2.5. Statistical analysis 2.1. Animals and treatment Results are expressed as the mean ± Standard Deviation (SD). Differences between groups were performed using either unpaired twotail Student's t test or ANOVA for multiple groups comparison.
Male Wistar rats (180–220 g), purchased from Charles River (Milano, Italy), were maintained on a standard laboratory chow diet (Ditta Mucedola, Milano, Italy). The animals were provided food and water ad libitum, whit a 12 h light/dark daily cycle and were acclimated for 1 week before starting the experiment. Guidelines for the Care and Use of Laboratory Animals were used during the investigation. Two thirds hepatectomy was performed according to Higgins and Anderson [30]. SnPP (Frontier Scientific, Carnfort, UK) was dissolved in 0.2 N NaOH and then back titrate with HCl solution. Animals received subcutaneous injections of either SnPP (10 μmoles/kg of body weight) or vehicle 12 h before PH and 0, 12, and 24 h after PH. Rats were killed from 0.5 to 36 h after surgery. Control groups also include animal treated with SnPP or untreated and sacrificed at the same time points without been subjected to PH. To score S phase hepatocytes, rats received BrdU (SIGMA Chemical, St. Louis, MO) either in drinking water (1 mg/ml) all throughout the experimental period or in a single dose (50 mg/kg body weight i.p.) 2 h before sacrifice. Immediately after sacrifice, sections of the liver were fixed in 10% buffered formalin and processed for staining with hematoxylin-eosin or immunohistochemistry. The remaining liver was snap-frozen in liquid nitrogen and kept at −80 °C until use.
3. Results To investigate whether inhibition of HO-1 by SnPP could affect liver regeneration response, male Wistar rats were subjected to 70% PH and injected s.c. with either 10 μmoles/kg body weight (bw) of SnPP or vehicle 12 h before surgery, at the time of PH and 12 h after. Animals were sacrificed 24 h after PH. Animals treated with 10 SnPP or untreated and sacrificed 12 h later, without been subjected to PH, served as control groups. To monitor the hepatocytes S-phase-entry, BrdU dissolved in drinking water (1 mg/ml) was given throughout the experimental period. As shown in Fig. 1A and B, the HO-1 inhibitor SnPP significantly increased the number of BrdU-positive hepatocytes during liver regeneration, without signs of liver toxicity. Indeed, 24 h after PH labelling index was 36%in SnPP pre-treated animals vs 23% of PH group (Fig. 1B). In agreement with previous studies [25], we found that SnPP caused enhanced gene transcription of HO-1(Fig. 1C). Indeed, Real time PCR analysis revealed that SnPP treatment was associated to enhanced HO-1 mRNA levels, in both control and hepatectomized rats (Fig. 1C). This effect was specific, as the expression of Nqo1, anotherNrf2 target gene, was unaffected by the treatment with SnPP (Fig. 1D). One sample (SnPP +PH 30 min) resulted out of range in both HO-1 and Nqo1 RT-PCR analyses. To further characterize the effect of SnPP on liver regeneration, the kinetics of hepatocyte proliferation in the presence or absence of the inhibitor was examined by BrdU pulse labeling. Animals injected with SnPP or untreated prior to PH were given a single dose of BrdU (50 mg/kg bw, i.p.) 2 h before sacrifice at 18, 24, 30 and 36 h after surgery. As shown in Fig. 2C, while SnPP pre-treatment did not
2.2. Western blot analysis Preparation of total cell extracts and western blot analysis were performed as described previously [31]. Nuclear and cytosolic cell extracts were prepared according to the method of Timchenko et al. [32]. For immunoblotting experiments the following antibodies were used: mouse monoclonal antibodies directed against, PCNA (PC-10) (Santa Cruz Biotechnology, CA, USA), Cyp1/WAF-1/p21 (Upstate Biotech, New York) and actin (clone AC-40)(SIGMA); rabbit polyclonal 10
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Fig. 1. Wistar rats subjected to 70% PH were treated with SnPP or vehicle (control) 12 h prior to surgery, at the time of PH and 12 h after PH. Animal were sacrificed 24 h after PH. Controls animal, injected s.c. either with vehicle or with SnPP, were sacrificed 12 h later without been subjected to PH. (A) Representative microphotograph (×10 magnification, section counterstained with hematoxylin) which illustrates the presence of BrdU-positive hepatocytes in the liver of rats sacrificed 24 h after PH, with or without pre-treatment with SnPP (10 μmoles/kg body weight, s.c.). To label the hepatocytes, BrdU (1 mg/ml) was given all throughout the experimental period dissolved in drinking water, starting 2 h after PH. (B) Labeling Index. At least 5000 hepatocyte nuclei per liver were scored. The labeling index was expressed as number of BrdU-positive hepatocyte nuclei/100 nuclei. Results are expressed as means ± S.E. of 4–5 rats per group. * = statistically different from PH alone; p < 0.05. (C and D) Real time PCR analysis of HO-1 (C) and Noq1 (D) mRNA levels in the livers from Wistar rats treated with a single dose of SnPP (10 μmoles/kg body weight) or vehicle 12 h prior to PH and at the time of PH and sacrificed 30 min or 2 h post surgery. Each lane represents a pool of three samples per group.
and growth [17,19,36], although its role in modulating cell growth is still unclear. Indeed, while a positive correlation between HO-1 induction and proliferation was found in epidermal keratinocytes, in vascular endothelium [37,38] and in various tumor cells [39,40], the induction of HO-1 was also reported to inhibit cell growth in rat kidney epithelial cells [21] and vascular smooth muscle cells [41,42] and in human pulmonary or renal tubular cells [43,44]. Taken together, all these evidences suggest that the effect of HO-1 on cell growth is essentially cell type-dependent. In the present study, to investigate the role of HO-1 on hepatocyte proliferation we analyzed the effect of SnPP, an inhibitor of HO-1 activity, in rat liver regeneration after 2/3 PH. The results showed that administration of SnPP prior to PH led to increased entry of hepatocytes into S phase compared to PH alone, as demonstrated by the increased number of BudR-positive hepatocytes and the higher expression of cellcycle proteins. To search for possible mechanisms responsible for the increased regenerative response observed following SnPP administration, we evaluated the expression/activation of transcription factors, such asSTAT3 and JNK, known to be involved in triggering the very early events of liver regeneration [2,3,9]. Our present findings showing that inhibition of HO-1 by SnPP leads to a more rapid activation of JNK and STAT3, suggest that HO-1 might inhibit liver regeneration by negatively activating cytokine-induced transduction pathways needed for the priming phase of hepatocytes. According to our hypothesis, it has been demonstrated that mice
significantly modify the number of BrdU-positive hepatocytes 18 h after PH respect to PH alone, a striking increase of the L.I. was evident at 24 h in SnPP+PH group (L.I. was 46% vs 24% of PH alone) (Fig. 2A and C). At later time points, the L.I. decreased in both groups. The increased entry of hepatocytes into S phase observed in SnPP + PH rats compared to PH alone 24 h after surgery, was accompanied by a significant enhancement of mitoses (Fig. 2Band 2D). Next, the expression of cell cycle proteins was examined by western blot. As shown in Fig. 3, SnPP induced increased levels of the G1-S regulators PCNA (18 h after surgery), p107 and E2F (starting from 24 h after PH),compared to SnPP-untreated group. No significant changes in the expression of the cyclinCDK inhibitor p21 were observed between the two experimental groups. In keeping with previous reports [16,33], pre-treatment with SnPP resulted in a significant increase of IL-6 mRNA levels at 30 min and 2 h after PH (Fig. 4A). Cytokine release during inflammation is known to activateSTAT3 and JNK-mediated pathways [34,35]. Accordingly, an earlier enhancement of JNK and STAT3 phosphorylation was observed in animals pre-treated with the HO-1 inhibitor (Fig. 4B). 4. Discussion Alteration of redox balance homeostasis can lead to the activation of various survival pathways. An increased synthesis of HO-1 occurs as a general response to inflammation or stress in biological systems. Recent data suggest a key role of HO-1 in the regulation of cellular homeostasis 11
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Fig. 2. Wistar rats subjected to 70% PH were treated with SnPP or vehicle 12 h prior to surgery, at the time of PH and 12 and 24 h after PH. Animals were sacrificed at the indicated time points after PH. SnPP or vehicle groups were sacrificed at the same time points without been subjected to PH. (A) Representative microphotograph (×10, section counterstained with hematoxylin) which illustrates the presence of BrdU-positive hepatocytes in the liver of rats sacrificed 24 h after PH, with or without pre-treatment with SnPP (10 μmoles/kg body weight, s.c.). (B) Representative microphotograph (×20 magnification, containing a small section with ×40 magnification) which illustrates H&E (hematoxylin and eosin) staining to detect the presence of mitosis in the liver of rats sacrificed 30 h after PH, with or without pre-treatment with SnPP (10 μmoles/kg body weight, s.c.). Arrows indicate some mitotic cells. (C) Labeling Index. To label the hepatocytes, BrdU (50 mg/Kg b.w., i.p.) was given 2 h before sacrifice. At least 5000 hepatocyte nuclei per liver were scored. The L.I. was expressed as number of BrdU-positive hepatocyte nuclei/100 nuclei. Results are expressed as means ± S.E. of 4–5 rats per group. *** = statistically different from PH alone; p < 0.0009. (D) Mitotic Index in livers of rats sacrificed 24 h after PH. The mitotic index was expressed as number of hepatocytes entering mitosis/1000 hepatocyte nuclei. At least 5000 hepatocyte nuclei per liver were scored. Results are expressed as means ± S.E. of 4–5 rats per group. * = statistically different from PH alone; p < 0.05.
Fig. 3. Expression of cell cycle related proteins in the liver of Wistar subjected to 70% PH and treated with SnPP or vehicle 12 h prior to surgery, at the time of PH and 12 and 24 h after PH. PH animals were killed from 18 to30 hours after PH, with or without SnPP, while SnPP or vehicle (CO) groups were sacrificed at the same time points without been subjected to PH. (A) Western blot analysis of nuclear protein extracts prepared from the livers; the analysis was performed as described in Materials and Methods. Appropriate loading was confirmed by laminin A/C detection. Each lane represents a pool of 3 samples; CO: vehicle; Sn: SnPP·(B) Densitometric analysis of cell cycle-related protein expression normalized to A/C laminin. 12
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Fig. 4. Analysis of inflammation markers in the livers from Wistar rats treated with a single dose of SnPP (10 μmoles/kg body weight) or vehicle 12 h prior to PH and at the time of PH and sacrificed 30 min or 2 h post surgery. (A) Real time PCR analysis of IL-6 mRNA levels. Each lane represents a pool of three samples. (B) Western blot analysis of phospho-JNK and phospho-STAT3. Total protein extracts (100–150 μg/lane) were prepared from the livers and Western analysis was performed as described in the Materials and Methods. Appropriate loading was confirmed by Actin detection. Each lane represents a single sample; CO: vehicle; Sn: SnPP.
withNrf2−/− genotype, lacking of Nrf2-dependent HO-1 activation, show increased severity of inflammatory response to different factors [45] which was correlated to increased cell proliferation and cancer development [46]. Indeed, Yokoo et al. [46], reported that Nrf2 deficiency increased susceptibility to oxidative stress-induced small intestinal carcinogenesis in mice, through overexpression of COX2 followed by stimulation of cell cycle progression. Our data are apparently in contrast with those of Glanemann et al. [47] showing an improvement of liver regeneration following HO-1 induction by CoPP pre-treatment. In the latter study, the increased regenerative capacity of the liver was attributed to HO-1 mediated decrease of oxidative stress and inflammatory response. However, this conclusion has not been fully confirmed by other reports [48] which showed that no enhanced liver regeneration after HO-1 induction by CoPP treatment occurs unless NO production was blocked by the NOS inhibitor L-NAME. Thus, from this study clearly emerges that HO-1 induction by CoPP alone was not able to enhance hepatocyte proliferation after PH. More recently, Marinic et al. [49] reported that pre-exposure to the olive oil polyphenols extract (PFE) stimulated liver regeneration through a transient increase in oxidant load within the first hours after hepatectomy, and the associated induction of stress response genessuch as HO-1 and GCSc under the control of Nrf2.Based on these results, the authors postulated that antioxidant Nrf2-induced genes, such as HO-1, play the key role in increasing liver regeneration. However, it is likely that in PFE-pretreated hepatectomyzed rats the initial increased oxidative stress per se, rather than HO-1 activation, could enhance liver regeneration post-surgery. If so, it follows that induction of antioxidant genes could only represent a compensatory response and not the causative agent of the enhanced hepatocyte proliferation, supporting our present findings showing that increased oxidative stress due to HO-1 inhibition, results in enhanced liver regeneration. Although much remains to be learned about the molecular mechanisms involved in cell proliferation after PH, elucidation of the signalling pathways modulated by HO-1 may provide new insights for improved understanding and possibly treatment of pathologies where hepatocyte proliferation is important (e.g. acute hepatitis, liver tumor, living related donor grafts). Knowledge about the mechanisms of liver regeneration can be applied to clinical problems caused by delayed liver growth, and HO-1 repression may be a mechanism by which cells can faster proliferate in response to tissue damage.
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