RESEARCH ARTICLE
`
`IL-11 Attenuates Liver Ischemia/Reperfusion
`Injury (IRI) through STAT3 Signaling
`Pathway in Mice
`
`Miao Zhu1☯, Bo Lu2☯, Qinhong Cao1, Zhenfeng Wu1, Zhe Xu1, Weisu Li1, Xuequan Yao1,
`Fukun Liu1*
`
`1 Department of Surgical Oncology, Affiliated Hospital of Nanjing University of TCM, 155 Hanzhong Road,
`Nanjing, Jiangsu Province, P. R China, 2 Department of General Surgery, Yixing People’s Hospital, 75
`Tongzhenguan Road, Yixing, Jiangsu Province, P. R China
`
`☯ These authors contributed equally to this work.
`* fukunliu@126.com
`
`Abstract
`
`Background
`
`The protective role of IL-11, an IL-6 family cytokine, has been implicated in ischemia/reper-
`fusion injury (IRI) in the heart and kidney, but its role has not been elucidated in liver IRI.
`This study was designed to evaluate the effects of IL-11 and its mechanism of action on
`liver IRI in a mouse model.
`
`Methods
`
`A partial (70%) warm liver IRI was induced by interrupting the artery/portal vein blood supply
`to the left/middle liver lobes. IL-11 mRNA expression of ischemic liver after reperfusion was
`analyzed. Signal transducer and activator of transcription 3 (STAT3) was analyzed following
`IL-11 treatment in vivo and in vitro. Next, IL-11 was injected intraperitoneally (ip) 1 hour be-
`fore ischemia. Liver injury was assessed based on serum alanine aminotransferase levels
`and histopathology. Apoptosis and inflammation were also determined in the ischemic liver.
`To analyze the role of STAT3 in IL-11 treatment, STAT3 siRNA or non-specific (NS) siRNA
`was used in vitro and in vivo.
`
`Results
`
`OPEN ACCESS
`
`Citation: Zhu M, Lu B, Cao Q, Wu Z, Xu Z, Li W, et
`al. (2015) IL-11 Attenuates Liver Ischemia/
`Reperfusion Injury (IRI) through STAT3 Signaling
`Pathway in Mice. PLoS ONE 10(5): e0126296.
`doi:10.1371/journal.pone.0126296
`
`Academic Editor: Anindita Das, Virginia
`Commonwealth University, UNITED STATES
`
`Received: November 23, 2014
`
`Accepted: March 30, 2015
`
`Published: May 6, 2015
`
`Copyright: © 2015 Zhu et al. This is an open access
`article distributed under the terms of the Creative
`Commons Attribution License, which permits
`unrestricted use, distribution, and reproduction in any
`medium, provided the original author and source are
`credited.
`
`Data Availability Statement: All relevant data are
`within the paper.
`
`Funding: The authors have no support or funding to
`report.
`
`Competing Interests: The authors have declared
`that no competing interests exist.
`
`IL-11 mRNA expression was significantly increased after reperfusion in the ischemic liver.
`STAT3, as a target of IL-11, was activated in hepatocytes after IL-11 treatment in vivo and
`in vitro. Next, effects of IL-11/STAT3 signaling pathway were assessed in liver IRI, which
`showed IL-11 treatment significantly attenuated liver IRI, as evidenced by reduced hepato-
`cellular function and hepatocellular necrosis/apoptosis. In addition, IL-11 treatment signifi-
`cantly inhibited the gene expressions of pro-inflammatory cytokines (TNF-α and IL-10) and
`chemokines (IP-10 and MCP-1). To determine the role of STAT3 in the hepatoprotective
`Singapore Exhibit 2007
`Lassen v. Singapore et al.
`PGR2019-00053
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`IL-11 in Liver Ischemia/Reperfusion Injury
`
`effects of IL-11, STAT3 siRNA or NS siRNA was used prior to IL-11 treatment. The results
`showed STAT3 knockdown abrogated the protective effects of IL-11 in vitro and in vivo.
`
`Conclusions
`
`This work provides first-time evidence for the protective effect of IL-11 treatment on hepato-
`cyte in liver IRI, through the activation of the STAT3 pathway.
`
`Introduction
`
`Ischemia-reperfusion injury (IRI) is a key contributing factor in liver dysfunction and failure
`after hepatic trauma, resection, liver transplantation, and circulatory shock [1–4]. An effective
`method for preventing or minimizing liver IRI is urgently needed in liver surgery. The factors/
`pathways have been involved in the hepatic IRI process include anaerobic metabolism, mito-
`chondria damage, oxidative stress, endoplasmic reticulum stress, intracellular calcium over-
`load, Kupffer cell (KC) activation, neutrophil infiltrations, and production of cytokines and
`chemokines [1–3]. The adverse factors mentioned above finally lead to cell death/apoptosis,
`which indicates that cell death/apoptosis is a significant and perhaps principal contributor to
`liver IRI [5–7]. Thus, understanding the sequence of events central to the cell death/apoptosis
`mechanism may potentially lead to treatments for liver IRI.
`IL-11 is a hematopoietic IL-6 family cytokine with multifunctional effects. Indeed, IL-11 has
`thrombopoietic activity, and recombinant human IL-11 has been used for thrombocytopenia
`in clinical settings [8]. Different from other IL-6 family cytokines, IL-11 holds anti-inflamma-
`tory function against chronic inflammatory diseases, lipopolysaccharide-induced sepsis, etc
`[9–11]. Kimura’s group reported that IL-11 played a cardioprotective role, and conferred resis-
`tance to heart IRI in a mouse model by enabling significant anti-necrotic/apoptotic effects [12].
`In addition, IL-11 pretreatment also reduces IR-induced cell death/apoptosis by up-regulating
`Bcl-2 [13]. More importantly, IL-11 shares some similar effects to other IL-6 family members.
`It has been reported that IL-11 binding with gp130 receptor induces activation of STAT3,
`which is involved in many physiological and pathological processes [14]. Inhibitors of STAT3
`phosphorylation or dominant-negative STAT3 mutants facilitate the expression of pro-apopto-
`sis factors, suggesting that STAT3 plays a critical role in regulating cell proliferation and anti-
`apoptosis [15]. Furthermore, STAT3 knockout mice exhibit complete embryonic lethality [16].
`Conditional ablation of STAT3 in myocardial cells leads to higher susceptibility to drug-in-
`duced heart failure [17]. To the best of our knowledge, there has been no report on IL-11 pre-
`conditioning before liver IRI. In the present study, we tested the hypothesis that exogenous IL-
`11 attenuates liver IRI by STAT3-mediated anti-necrotic/apoptotic effects.
`
`Materials and Methods
`
`Animals
`
`Male C57BL/6 mice were purchased from the Laboratory Animal Resources Center of Nanjing
`Medical University (NMU). The animals were fed a laboratory diet with water and food and
`kept under constant environmental conditions with 12h light–dark cycles. Procedures were
`carried out in accordance with the Guidelines for Laboratory Animal Care. The animal proto-
`col had been approved by the Institutional Animal Care & Use Committee (IACUC) of Nan-
`jing Medical University (Protocol Number NMU08-092).
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`IL-11 in Liver Ischemia/Reperfusion Injury
`
`Surgical procedure and IL-11 treatment
`
`The present study used a well-established mouse model of partial (70%) warm hepatic IRI [18].
`Anesthesia was induced by 10% chloral hydrate (0.3g/kg, intraperitoneally). Mice were injected
`with heparin (100U/kg), and an atraumatic clip was used to interrupt the artery/portal vein
`blood supply to the left/middle liver lobes. After 90 minutes of ischemia, the clip was removed,
`and the mice were sacrificed (cervical dislocation) at required times after reperfusion. Some
`mice received a single injection of recombinant human IL-IL-11 (500μg/kg, ip) (PeproTech,
`Rocky Hill, NJ) or medium (PBS) 1 hour prior to ischemia. PBS injection was used as a control.
`Sham-operated controls underwent the same procedure but without vascular occlusion. To ac-
`cess effects of STAT3 on IL-11 treatment, STAT3 siRNA or NS siRNA (2mg/kg) was given in-
`travenously 4 hours prior to ischemia [19]. Reports have previously documented the efficacy of
`this siRNA approach in the liver, with>40% of intravenously infused siRNA accumulating in
`the ischemic mouse livers [20].
`
`Serum biochemical examination
`
`Blood samples collected 6h after reperfusion was centrifuged to obtain serum. The serum level
`of alanine aminotransferase (sALT) or supernatant level of lactate dehydrogenase (LDH) was
`measured to assess the extent of hepatocyte damage using an automated chemical analyzer
`(Olympus Automated Chemistry Analyzer AU5400, Tokyo, Japan).
`
`Histopathologic study
`
`Liver specimens were fixed with 10% neutral formaldehyde and then embedded in paraffin.
`The specimens were sectioned at 4μm and stained with hematoxylin and eosin. The sections
`were used in histopathologic analysis by light microscopy. Sections were scored from 0 to 4 for
`sinusoidal congestion, vacuolization of hepatocyte cytoplasm, and parenchymal, as described
`by Suzuki et al [21].
`
`Caspase-3 activity assay
`
`Caspase-3 activity was assayed in liver tissues 6h after reperfusion. Frozen samples of ischemic
`tissues were homogenized with a Polytron homogenizer and centrifuged at 16,000g for 20 min-
`utes. Activity was measured with an assay kit (Calbiochem) according to the
`manufacturer’s instructions.
`
`Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)
`staining
`
`Paraffin sections (4μm in thickness) were deparaffinized in toluene and then dehydrated in a
`graded series of ethanol solutions. Sections were stained by TUNEL using a commercially avail-
`able kit (in situ cell death detection kit, Roche-Boehringer Mannheim, Germany).
`
`Western blot analysis
`
`Proteins were extracted from liver tissues subjected to ischemia or from cell lysates, and their
`concentrations were determined by the Bradford assay (Bio-Rad, CA). About 30 μg protein
`was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to
`nitrocellulose membranes (Sunshine Biotechnology, China). These membranes were blocked
`in skim milk powder (5% wt/vol) with phosphate buffered saline containing 0.1% Tween 20
`(PBS-T) at 4°C overnight. Membranes were then incubated with primary antibodies for cleaved
`Caspase-3, P-STAT3, Bcl-2, Bax, β-actin (Cell Signaling Technology, Danvers, MA), and
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`IL-11 in Liver Ischemia/Reperfusion Injury
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`STAT3 (Santa Cruz Biotechnology, Santa Cruz, CA). Following three washes with PBS-T, the
`membranes were incubated for 1h at room temperature with peroxidase-conjugated secondary
`antibody (Cell Signaling Technology, Danvers, MA). The final results were obtained by expo-
`sure to autoradiographic film (Kodak XAR film), and then visualized via a chemiluminescent
`detection system (ECL Substrate Western blot detection system, Pierce, IL).
`
`Quantitative real-time PCR
`
`Quantitative real-time PCR was performed using the DNA Engine with a Chromo 4 Detector
`(MJ Research, Waltham, MA). In a final reaction volume of 25μl, the following were added:
`1×SuperMix (Platinum SYBR Green qPCR Kit; Invitrogen), cDNA, and 2.5μM of each primer.
`The amplification conditions were as follows: 50°C (2 min), 95°C (5 min), followed by 50 cycles
`at 95°C (15 sec) and 60°C (30 sec). The expression of the target genes (IL11, TNF-α, IL-6, IP-
`10 and MCP-1) (Invitrogen, Shanghai, China) was calculated based on the ratio of the gene of
`interest to the housekeeping gene HPRT. Primer sets (sense sequence and antisense sequence,
`respectively) for the following genes were: HPRT forward, 5’- TCA ACG GGG GAC ATA
`AAA GT-3’, reverse, 5’- TGC ATT GTT TTA CCA GTG TCA A’; IL-11 forward: 5’- CTG
`CCC ACC TTG GCC ATG AG-3’; IL-11 reverse: 5’- CCA GGC GAG ACA TCA AGA AAG
`A-3’; TNF-α forward, 5’- GCC TCT TCT CAT TCC TGC TTG T-3’, reverse, 5’- TTG AGA
`TCC ATG CCG TTG-3’; IL-6 forward, 5’- GCT ACC AAA CTG GAT ATA ATC AGG A-3’,
`reverse, 5’- CCA GGT AGC TAT GGT ACT CCA GAA-3’; IP-10 forward. 5’-GCT GCC GTC
`ATT TTC TGC-3’, reverse, 5’-TCT CAC TGG CCC GTC ATC-3’; MCP-1 forward, GGT GAT
`AAC CGC CCT AGC-3’, reverse, 5’-TGT GTC GGC TGG ATA GGC-3’.
`
`Cell culture and treatment
`
`Mouse hepatocytes were isolated using a two-step in situ collagenase perfusion procedure [18].
`Livers from the C57BL/6 mice were perfused in situ through the portal vein with ethylene
`glycol tetraacetic acid (EGTA) buffer (0.5mM EGTA, 137mM NaCl, 4.7mM KCl, 1.2mM
`KH2PO4, 0.65mM MgSO4, and 10.07mM HEPES at pH 7.4) at a flow rate of 5ml/min for
`10 min, followed by collagenase buffer (67mM NaCl, 6.7mM KCl, 4.76mM CaCl2, 0.035% col-
`lagenase type II, and 10.07mM HEPES at pH 7.6) at a flow rate of 5ml/min for 15 min. After
`centrifugation, the hepatocytes were collected and seeded in DMEM containing 10% FBS,
`100units/ml penicillin, and 100μg/ml streptomycin. Cells were preincubated with IL-11
`(1μg/ml for 1h), then H2O2 (200μm for 24h) to induce cell death.
`
`Knockdown of STAT3 expression using STAT3 siRNA transfection
`
`Hepatocytes were grown and transiently transfected with STAT3 siRNA or NS siRNA using
`Transfection Reagent LipofectamineTM RNAiMAX (Invitrogen, CA, USA) according to the
`manufacturer’s instructions. In brief, cells were seeded at 1 x 106 per well in 1.5ml of OPTI-me-
`dium (Invitrogen, CA, USA) in a 6-well plate. After 20h, the cells were transfected with
`20nmol/ml STAT3 siRNA or NS siRNA. About 6h after transfection, the medium was changed
`to a regular medium, and the cells were treated as described above after 24h.
`
`Statistical analysis
`
`The data are presented as the mean ± SEM from at least three independent experiments. One-
`way analysis of variance test [—] was used in comparisons of three groups. Student’s t-test [∏]
`was used for comparison of two groups. All P values were two-sided, and P<0.05 was consid-
`ered to be statistically significant.
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`IL-11 in Liver Ischemia/Reperfusion Injury
`
`Fig 1. IL-11 expression was increased after IR. Mice were subjected to 90min of partial liver ischemia,
`followed by 0h, 1h, 3h and 6h reperfusion. Kinetics of IL-11 gene expression was analyzed in ischemic liver
`by RT-PCR. Expression of IL-11 was normalized with that of HPRT. Data are expressed as mean±SD (n = 6/
`group). *P<0.05, **P<0.001 vs sham group.
`
`doi:10.1371/journal.pone.0126296.g001
`
`Results
`
`IL-11 is elevated in IR-stressed liver
`
`To determine the effects of IL-11 on liver IRI, we first analyzed the gene expression of IL-11 in
`ischemic livers after various reperfusion time points. As shown in Fig 1, the expression of IL-11
`was increased at 0h post reperfusion and reached its peak 3h post reperfusion. The data indi-
`cate that IL-11 was present in the liver 3h after reperfusion.
`
`IL-11 activates STAT3 in liver and hepatocytes
`
`IL-11, an IL-6 family cytokine, was supposed to activate STAT3. Here we determined whether
`administration of IL-11 stimulates STAT3 in liver by western blotting with antibody (Fig 2A).
`Phosphorylation of STAT3 was rapidly induced and recovered nearly to baseline after 2 hours.
`To further ascertain that phosphorylation of STAT3 occurred in hepatocytes, we analyzed the
`effects of IL-11 on STAT3 activity of hepatocytes in vitro. Fig 2B shows phosphorylation of
`STAT3 was also significantly elevated after IL-11 treatment, indicating that IL-11 administra-
`tion may activate STAT3 within parenchymal cells in the liver.
`
`IL-11 attenuates liver IRI
`
`Next, we analyzed effects of IL-11 administration in liver IRI. Mouse livers were subjected to
`90 min of warm ischemia 6h after reperfusion. sALT levels in each group were analyzed (Fig
`3A). sALT levels were markedly increased in the IR group compared with that of the sham
`group (33.33±5.49 and 10610.00±1393.00, respectively; P<0.01). By contrast, when mice were
`pretreated with IL-11, sALT levels (3832.00±834.90, P<0.01) were significantly decreased com-
`pared with those in the IR control. Liver serum enzyme data were in line with liver pathological
`analysis (Fig 3B and 3C). The histological parameters in the sham (0.25±0.25), IR (3.20±0.37),
`and IL-11 administration (1.8±0.37) groups were observed according to Suzuki et al [26].
`These data indicate that IL-11 treatment significantly attenuates IR-induced liver injury.
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`IL-11 in Liver Ischemia/Reperfusion Injury
`
`Fig 2. IL-11 activated STAT3 in hepatocytes in vivo and in vitro. (A) IL-11 was administered in mice for the indicated time. The lysates from liver were
`immunoblotted with anti-p-STAT3 and anti-STAT3 antibody. Representative data are shown (lower). Quantative analyses of p-STAT3 are shown (upper).
`Data are exprresed as mean±SD (n = 6/group). **P<0.001. (B) Hepatocytes were treated by IL-11 for the indicated time. The lysates from hepatocytes were
`immunoblotted with anti-p-STAT3 and anti-STAT3 antibody. Representative data are shown (lower). Quantative analyses of p-STAT3 are shown (upper).
`Data are expressed as mean±SD (n = 6/group), **P<0.001.
`
`doi:10.1371/journal.pone.0126296.g002
`
`IL-11 exhibits anti-apoptotic functions and reduces apoptosis
`
`Apoptosis/necrosis is a central mechanism of cell death in liver IRI. In this study, hepatocellu-
`lar apoptosis was analyzed in ischemic livers by TUNEL staining 6 hours after reperfusion. Our
`results showed TUNEL-positive cells were significantly lower in liver sections of the IL-11
`treatment group compared with those in IR control group (Fig 4A and 4B). TUNEL-positive
`cells in the total hepatocytes of the three groups were (0.60±0.25)%, (10.20±1.28)%, and
`(4.00±0.63)%, respectively, indicating that hepatocellular apoptosis was significantly reduced
`by IL-11 administration. In addition, anti-apoptotic protein Bcl-2 and pro-apoptotic protein
`Bax were analyzed in ischemic liver, which showed that IL-11 treatment effectively upregulated
`Bcl-2 and inhibited Bax expression (Fig 4C). Caspase-cascade system, especially the compo-
`nent Caspase-3, plays a critical role during liver IRI. Caspase-3 affects apoptosis by component
`cleaved Caspase-3. Next, we assessed the expression of cleaved Caspase-3 by immune analysis,
`suggesting that IL-11 administration effectively inhibited expression of cleaved Caspase-3 dur-
`ing liver IRI (Fig 4C). Apoptotic active caspase-3 directly caused hepatocellular apoptosis and
`reflected the progress of apoptosis in the ischemic liver. Along with the TUNEL assay and ex-
`pression of cleaved Caspases-3, Fig 4D shows that the activity of caspase-3 was significantly re-
`pressed after IL-11 preconditioning in ischemic liver tissue compared with the IR group (1.38
`±0.08 and 4.44±0.289, respectively; P<0.001).
`
`IL-11 regulates inflammatory program and MPO activity in IR-stressed
`liver
`
`A complex inflammatory program involving cytokines and chemokines is engaged in liver IRI.
`Inflammatory cytokines (TNF-α and IL-6) and chemokines (IP-10 and MCP-1) displayed
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`IL-11 in Liver Ischemia/Reperfusion Injury
`
`Fig 3. IL-11 treatment attenuated liver injury induced by IR. Mice were administered with recombinant human IL-IL-11 (500μg/kg, ip) or medium (PBS) 1
`hour prior to ischemia, followed by 6h reperfusion. (A) sALT. (B) Histopathalogic analysis of livers harvested 6 hours after reperfusion. Sham group: Normal
`hepatic architecture; IR group: severe hepatic lobule distortion, sinusoidal congestion, apparent edema, vacuolization and massive necrosis; IL-11+IR group:
`mild vacuolization, punctate necrosis and edeman. (C) The severity of liver IRI by Suzuki’s histological grading. Data are expressed as mean±SD (n = 6/
`group), **P<0.001.
`
`doi:10.1371/journal.pone.0126296.g003
`
`proinflammatory and proapoptotic roles in ischemic liver post-reperfusion. To further assess
`the hepatoprotective effects of IL-11 treatment, mRNA expressions of TNF-α, IL-6, IP-10 and
`MCP-1 were determined in ischemic liver after 6h of reperfusion by qRT-PCR. Fig 5A shows a
`significantly lower level of TNF-α (1.13.±0.13 and 0.31±0.04, respectively; P<0.001), IL-6
`(6.73±1.12 and 2.43±0.33, respectively; P<0.001), IP-10 (9.69±1.63 and 2.93±0.39, respectively;
`P<0.001) and MCP-1 (4.08±0.70 and 1.22±0.27, respectively; P<0.001) in the IL-11 adminis-
`tration group compared with that in the IR group. These data indicated that IL-11 effectively
`inhibited the expression of inflammatory cytokines in ischemic liver after reperfusion. In addi-
`tion, serum TNF-α was further analyzed by ELISA, which showed IL-11 treatment significantly
`decreased TNF-α secretion after liver IRI (Fig 5B). The protein level was consistent with gene
`expression. The MPO activity, reflecting neutrophil activity and infiltration, was reduced after
`reperfusion in IL-11 treated liver compared with controls (5.40±0.81U/g and 2.40±0.25U/g, re-
`spectively; P<0.001) (Fig 5C).
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`IL-11 in Liver Ischemia/Reperfusion Injury
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`Fig 4. IL-11 treatment decreased hepatocellular apoptosis induced by IR. (A)Liver apoptosis was examined by TUNEL staining: Sham group, IR group
`and IL-11+IR group. (B) Apoptotic cells were quantified in six high-power fields (400x), and expressed as percentages of apoptotic cells among total cells. (C)
`Western blot-assisted detection of Bcl-2, Bax, Caspase-3 and β-actin. (D) Caspase-3 activity. Data are expressed as mean±SD (n = 6/group), **P<0.001.
`
`doi:10.1371/journal.pone.0126296.g004
`
`Activation of STAT3 is essential for IL-11-mediated protective role
`
`To evaluate the effect of STAT3 activation in an IL-11-mediated protective role, primary hepa-
`tocytes were transiently transfected with STAT3 siRNA or NS siRNA. Next, STAT3 gene ex-
`pression was assessed after IL-11 treatment by qRT-PCR, which showed STAT3 gene
`expression was significantly repressed compared with the NS siRNA control (Fig 6A). These
`data indicated that STAT3 expression was successfully knocked down in hepatocytes. Then,
`these transfected cells were treated with H2O2 to induce cell death. The released LDH level
`was checked in the supernatant after H2O2 treatment for 24h. Fig 6B shows that IL-11 treat-
`ment remarkably inhibited LDH release of hepatocytes after H2O2 treatment (14.00±4.32 and
`14.80±1.99, respectively; P<0.001). However, STAT3 knockdown almost restored the de-
`creased LDH release after IL-11 treatment (14.80±1.99 and 45.80±7.14, respectively; P<0.001).
`These data indicated STAT3 activation is necessary for IL-11-mediated protective role.
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`IL-11 in Liver Ischemia/Reperfusion Injury
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`Fig 5. IL-11 treatment inhibited pro-inflammatory responeses induced by IR. (A) Cytokine gene (TNF-a, IL-6, IP-10 and MCP-1) expressions were
`analyzed in ischemic livers by RT-PCR analysis. Expressions of cytokine gene were normalized with that of HPRT. (B) TNF-α secretion was examined in
`serum by ELISA. (C) MPO activity in ischemic liver. Data are expressed as mean±SD (n = 6/group), *P<0.05, **P<0.001.
`
`doi:10.1371/journal.pone.0126296.g005
`
`Knockdown of STAT3 restores liver IRI in IL-11-treated mice
`
`To further assess the effect of STAT3 activation in IL-11-mediated hepoto-protective role dur-
`ing liver IRI, mice were injected intravenously with STAT3 siRNA or NS siRNA prior to ische-
`mia. As shown in Fig 7A, IL-11 treatment significantly increased expression of P-STAT3,
`which was reversed in STAT3 siRNA group. Fig 7B shows STAT3 siRNA treatment effectively
`neutralized the reduced sALT levels after IL-11treatment (P<0.001) (3795±879U/L versus
`11460±1941U/L, respectively). These data correlated with Suzuki’s histological grading of liver
`IRI (Fig 7C and 7D). Indeed, IL-11 resulted in minimal liver sinusoidal congestion, vacuoliza-
`tion without edema, or necrosis [Fig 7C (c); score = 1.80±0.37]. In contrast, livers in untreated
`or STAT3 siRNA-treated mice displayed moderate to severe edema and extensive hepatocellu-
`lar necrosis [Fig 7C (b and d), Fig 7D (panels b and d); score = 3.60±0.25 and 3.40±0.40, re-
`spectively]. In addition, cleaved Caspase-3 was further analyzed in ischemic liver, which
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`IL-11 in Liver Ischemia/Reperfusion Injury
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`Fig 6. STAT3 knockdown abolished IL-11 protective effects in vitro. (A) STAT3 gene expression was analyzed in transfected hepatocytes by RT-PCR
`analysis. Expressions of gene were normalized with that of HPRT. (B) The released LDH level of hepatocytes after H2O2 treatment. Data are expressed as
`mean±SD (n = 4-6/group), **P<0.001, ***P<0.0001.
`
`doi:10.1371/journal.pone.0126296.g006
`
`showed IL-11 treatment partially inhibited IR-triggered cleaved Caspase-3 upregulataion (Fig
`7A). These data indicated knockdown of STAT3 recreates liver IRI in IL-11-treated mice.
`
`Discussion
`
`IL-11 has displayed protective roles during ischemia-reperfusion injury (IRI) in the heart, kid-
`ney and intestine, but a similar role has not been elucidated in liver IRI [12, 13, 22, 23]. In this
`study, we have for the first time demonstrated that IL-11 pretreatment is a promising method
`for preventing or minimizing liver IRI. Indeed, IL-11 treatment improved liver function, atten-
`uated histology damage, impaired proinflammatory cytokine/chemokine programs, and re-
`duced hepatocellular death/apoptosis in IR-stressed livers. However, the IL-11 mediated
`protective role was partially impaired by STAT3 siRNA in vivo and in vitro. These findings
`demonstrate that IL-11 treatment attenuates liver IRI through activating the STAT3
`signaling pathway.
`IL-11 is a hematopoietic IL-6 family cytokine first identified from marrow-derived stromal
`cells. It is a key regulator of hematopoiesis and promotes megakaryocyte maturation [24]. IL-
`11 as well as its receptors is expressed in many tissues and cell types, including macrophages,
`hepatocytes, etc. in liver tissues [25]. Du’s group for the first time reported the protective effects
`of IL-11 in models of cytoablative chemoradiotherapy in 1994. Then, some researchers have
`demonstrated that IL-11 play protective roles in various pathophysiologic states [25]. Different
`from other IL-6 family members, IL-11 administration reduces inflammatory responses in
`chronic inflammatory diseases, lipopolysaccharide-induced sepsis, macrophages inflammation,
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`IL-11 in Liver Ischemia/Reperfusion Injury
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`Fig 7. STAT3 knockdown abolished IL-11 protective effects in vivo. Mice were injected with STAT3 siRNA or NS siRNA 4h prior to ischemia, and
`administered with IL-11 1h prior to ischemia, followed by 6h reperfusion. (A) Western blot-assisted detection of P- STAT3, Cleaved Caspase-3 and β-actin.
`(B) sALT. (C) Histopathalogic analysis of livers harvested 6h after reperfusion. Sham group: Normal hepatic architecture; NS siRNA+IR group: severe
`hepatic lobule distortion, sinusoidal congestion, apparent edema, vacuolization and massive necrosis; NS siRNA+IL-11+IR group: mild vacuolization,
`punctate necrosis and edeman; STAT3 siRNA+IL-11+IR group: severe hepatic lobule distortion, sinusoidal congestion, apparent edema, vacuolization and
`massive necrosis. (D) The severity of liver IRI by Suzuki’s histological grading. Data are expressed as mean±SD (n = 6/group), *P<0.05.
`
`doi:10.1371/journal.pone.0126296.g007
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`nephrotoxic nephritis and T-cell mediated liver injury [9–11]. The present study also demon-
`strated that IL-11 treatment effectively inhibited inflammatory responses of ischemic liver, as
`evidenced by reducing pro-inflammatory cytokines (TNF-α and IL-6) and chemokines (IP-10
`and MCP-1), and repressing MPO activtity. In addition to its anti-inflammatory properties,
`IL-11 treatment has been shown to attenuate necrotic and apoptotic cell death in many organs
`including the heart, intestines and endothelial cells [25]. In fact, apoptosis/necrosis is a key
`mechanism of cell death in liver IRI, which directly indicates the extent of liver damage. In our
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`IL-11 in Liver Ischemia/Reperfusion Injury
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`study, hepatocellular death/necrosis as well as apoptosis was also observed in ischemic liver.
`Our data showed that IL-11 attenuated hepatocellular death/necrosis (ALT, Suzuki’s score) as
`well as apoptosis (TUNEL staining, cleaved-Caspase 3 fragmentation) in ischemic liver after
`IRI. In addition, we also analyzed effects of IL-11 on hepatocellular death/necrosis by LDH re-
`lease level in vitro, which was consistent with in vivo results. Thus, we conclude that IL-11 ad-
`ministration provides liver protection against IRI by reducing inflammation and necrosis/
`apoptosis.
`The molecular mechanisms of IL-11 mediated signaling pathway for an anti-inflammatory
`and anti-necrosis/apoptosis might involve the activation of multiple intercellular pathways.
`After IL-11 binds to the IL-11 receptor, the ligand-receptor complex interacts with a common
`receptor subunit, glycoprotein 130 (gp130), leading to gp130-associated kinase-mediated tyro-
`sine phosphorylation [26]. In vascular endothelial and intestinal epithelial cells, IL-11 protects
`against oxidant induced necrosis and apoptosis via mechanisms involving ERK, MAPK, AKT
`and/or induction of HSP25 [22, 27, 28]. In renal IRI, IL-11 performs renal protection by direct
`induction of sphingosine kinase-1 (SK-1) via nuclear translacation of HIF-1α. In cardiac myo-
`cytes, IL-11 treatment attenuates injury and fibrosis via Janus Kinase-Signal Transducer and
`Activation of Transducer 3 (JAK-STAT3) pathway activation [12, 23, 29]. Kawakami T et al
`have demonstrated rhIL-11 confers significant protection against CCl4-induced hepatic injury
`by virtue of its liver-specific HO-1 induction [30]. In addition, a previous studydemonstrated
`STAT3 activation after Ad-HO-1 treatment improved the hepatocellular function in a mouse
`model of segmental liver warm IRI [31]. In the present study, IL-11 treatment rapidly activated
`STAT3 in hepatocytes in vivo and in vitro, and reduced liver injury after reperfusion. However,
`IL-11 administration shows weak hepatocellularprotective effects in STAT3siRNA transfected
`hepatocytes or STAT3siRNA transfected mice. Therefore, hepatocyte is an important target in
`the action of IL-11, and IL-11-mediated protection of liver IRI is partially dependent on
`STAT3 activation of hepatocytes. Whether HO-1 is involved in this protective procedure will
`be further investigated in the future.
`Evidence exists that the STAT3 signaling pathway transduces stress-activating extracellular
`chemical signals into cellular responses for a number of pathophysiological processes, such as
`immunity, inflammation and apoptosis, and is involved in liver IRI. The function of activated
`STAT3 is controversial; some studies have associated it with survival [31–33], while others
`have related it to cell death [34]. Previous studies have confirmed that STAT3 alterations affect
`Bcl-2 and Bax protein expression and induce inflammation and apoptosis in many types of
`tumor cells [35–37]. In mycosis fungoides tumor cells, some apoptosis-related genes, such as
`Bcl-2 and Bax, have been identified as STAT3 target genes [38]. In our study, STAT3 activation
`reduced hepatocellular necrosis/apoptosis and liver injury induced by IR after IL-11 treatment,
`but STAT3 knockdown restored the hepatocellular necrosis/apoptosis and liver injury in IL-
`11-treated mice. These data indicate that IL-11 treatment reduces hepatocellular necrosis/apo-
`ptosis by STAT3 activation. Lee et al demonstrated that co-activated NF-κB and STAT3 modu-
`late Bax/Bcl-xL expression and promote cell survival in head and neck squamous cell
`carcinoma [38]. In primary cortical neurons and murine models of stroke, the activation of
`STAT3 pathway by secretoneurin has been found to exert neuroprotective effects and induce
`neuronal plasticity after hypoxia and ischemic insult [39]. Using a mouse model of myocardial
`infarction, Obana M et al demonstrated that IL-11 exerted protective effects against myocardial
`ischemic injury through IL-11R-mediated STAT3 activation, antiapoptotic signaling and
`proangiogenic activity [23]. In addition, STAT3 has been demonstrated to have an anti-inflam-
`matory function in many pathophysiological processes [23]. Inflammatory response plays a
`pathogenic role in liver I/R injury, especially innate immune responses involved in cytokines
`and chemokines, including TNF-α, IL-6, IP-10, MCP-1 and so on [40–43]. Ke B et al
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`IL-11 in Liver Ischemia/Reperfusion Injury
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`demonstrated that STAT3 activation repressed TLR4-drived inflammation by activating PI3K/
`Akt signaling during liver IRI [23]. Consistent with the above data, our results also showed that
`IL-11-induced STAT3 signaling inhibited pro-inflammatory cytokines and chemokines.
`In conclusion, our findings demonstrate for the first time IL-11-mediated STAT3 attenuates
`IR-triggered liver injury. IL-11-mediated STAT3 signaling