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Icariin protects against intestinal ischemiaereperfusion injury Feng Zhang, MD,a Yan Hu, MD,b Xiaomei Xu, MD,b Xiaohan Zhai, MD,b Guangzhi Wang, MD,a Shili Ning, MD,a Jihong Yao, MD, PhD,b and Xiaofeng Tian, MD, PhDa,* a b
Department of General Surgery, Second Affiliated Hospital of Dalian Medical University, Dalian, China Department of Pharmacology, Dalian Medical University, Dalian, China
article info
abstract
Article history:
Background: This study investigated the role of Sirtuin 1 (SIRT1)/forkhead box O3 (FOXO3)
Received 10 December 2013
pathway, and a possible protective function for Icariin (ICA), in intestinal ischemiae
Received in revised form
reperfusion (I/R) injury and hypoxiaereoxygenation (H/R) injury.
14 August 2014
Materials and methods: Male SpragueeDawley rats were pretreated with different doses of
Accepted 2 October 2014
ICA (30 and 60 mg/kg) or olive oil as control 1 h before intestinal I/R. Caco-2 cells were
Available online 8 October 2014
pretreated with different concentrations of ICA (25, 50, and 100 mg/mL) and then subjected to H/R-induced injury.
Keywords:
Results: The in vivo results demonstrated that ICA pretreatment significantly improved
Intestinal ischemiaereperfusion
I/R-induced tissue damage and decreased serum tumor necrosis factor a and interleukin-6
SIRT1
levels. Changes of manganese superoxide dismutase, Bcl-2, and Bim were also reversed by
Icariin
ICA, and apoptosis was reduced. Importantly, the protective effects of ICA were positively
FOXO3
associated with SIRT1 activation. Increased SIRT1 expression, as well as decreased acety-
Oxidative stress
lated FOXO3 expression, was observed in Caco-2 cells pretreated with ICA. Additionally,
Apoptosis
the protective effects of ICA were abrogated in the presence of SIRT1 inhibitor nicotinamide. This suggests that ICA exerts a protective effect upon H/R injury through activation of SIRT1/FOXO3 signaling pathway. Accordingly, the SIRT1 activator resveratrol achieved a similar protective effect as ICA on H/R injury, whereas cellular damage resulting from H/R was exacerbated by SIRT1 knockdown and nicotinamide. Conclusions: SIRT1, activated by ICA, protects intestinal epithelial cells from I/R injury by inducing FOXO3 deacetylation both in vivo and in vitro These findings suggest that the SIRT1/FOXO3 pathway can be a target for therapeutic approaches intended to minimize injury resulting from intestinal dysfunction. ª 2015 Elsevier Inc. All rights reserved.
1.
Introduction
Intestinal ischemiaereperfusion (I/R) injury is a pathophysiological process typically associated with intestinal and mesenteric vascular dysfunction, which can also occur as a
result of surgery, organ transplantation, and trauma. Intestinal I/R is the initiating event in systemic inflammatory response syndrome and multiple organ dysfunction syndrome, associated with significant morbidity and mortality [1,2]. Thus, it is important to understand the mechanisms that
* Corresponding author. Department of General Surgery, Second Affiliated Hospital of Dalian Medical University, Dalian 116023, China. Tel.: þ86 0411 86110025; fax: þ86 0411 86110010. E-mail address: [email protected] (X. Tian). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.10.004
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mediate the cellular response to injury, and exploit these to mitigate damage to tissue and to the organism. Sirtuin 1 (SIRT1) is a member of the highly conserved nicotinamide adenine dinucleotide-dependent class III histone deacetylases [3,4], which is involved in multiple cellular processes, including DNA damage response, cell cycle regulation, apoptosis, and cellular longevity [5]. Recently, SIRT1 has been shown to play an important role in preventing oxidative damage resulting from myocardial hypoxiae reoxygenation (H/R), acute kidney injury, and hepatic stress [6e10]. During myocardial I/R injury, SIRT1 messenger RNA (mRNA) and protein levels decreased significantly; moreover, mice deficient in cardiac-specific SIRT1 exhibited a greater myocardial infarct size than wild-type mice after I/R [11], indicating that SIRT1 may have protective effects against I/R injury. The forkhead box O (FOXO) family of transcription factors is involved in cell-cycle regulation, DNA repair, and oxidative stress resistance and is also known to have antiapoptotic function [12,13]. FOXO3 has been shown to promote resistance to oxidative stress in cells under hypoxic and/or ischemic conditions, mainly by activating the expression of its target gene superoxide dismutase, which is a scavenger of oxygen free radicals [14]. FOXO3 is also involved in the regulation of apoptosis [15]. Recent reports indicate that SIRT1 can directly modulate FOXO3 deacetylation and activities under conditions of cellular stress [16,17]. However, the function of FOXO3 in intestinal I/R injury, and its relationship to SIRT1, has yet to be determined. Icariin (ICA), a flavonoid extracted from the traditional Chinese herb Epimedium brevicornum Maxim, is known to have anti-inflammatory, antioxidative, and antiapoptotic properties [18e20]. Recently, a neuroprotective function has been observed for ICA on injury induced by oxygen and glucose deprivation, and this effect was associated with an upregulation of SIRT1 [21e23]. We hypothesized that ICA could protect against oxidative stress and apoptosis induced by intestinal I/R, and these beneficial effects may be associated with SIRT1/FOXO3 signaling activation. To test this hypothesis, we determined whether ICA exerts protective effects in a rat model of intestinal I/R injury and in Caco-2 cells H/R injury and examined SIRT1/FOXO3 signaling pathway variation both in vivo and in vitro.
2.
Materials and methods
2.1.
Drugs and reagents
ICA (98% pure), purchased from Shanghai Winherb Medical Science Co, Ltd (Shanghai, China), was dissolved in olive oil (3 and 6 mg/mL) and gavaged before intestinal I/R (at the dose of 10 mL/kg). The rats in the sham and I/R groups were treated with an equal volume of olive oil. The dose of ICA administration was determined from a previous study with modification from our preliminary experiments [23,24]. In cell culture experiments, ICA was dissolved in 0.1% dimethyl sulfoxide at three concentrations (25, 50, and 100 mg/mL), and the cells were treated with ICA for 6 h
before exposure to H/R environment. An equal volume of 0.1% dimethyl sulfoxide was applied as the control. Nicotinamide (NAM, 98% pure) was purchased from Shanghai Source Leaf Biological Technology Co, Ltd (Shanghai, China) and was dissolved in saline.
2.2.
Animals and experimental groups
Male SpragueeDawley rats weighing 180e220 g were obtained from the Animal Center of Dalian Medical University (Dalian, China), housed under specific pathogen-free conditions and provided with standard laboratory chow and water. The rats were fasted overnight with free access to water before operation. The rats were divided into five experimental groups randomly, with eight rats in each of the following: (1) shamoperated group (sham); (2) intestinal I/R group (I/R); (3) sham þ ICA (60 mg/kg), the rats were pretreated with ICA at a dose of 60 mg/kg intragastrically for three consecutive days, and then surgery was performed as that in the sham group; (4) I/R þ ICA (30 mg/kg), the rats were pretreated with ICA at a dose of 30 mg/kg for 3 d, and then surgery was performed as that in the I/R group; and (5) I/R þ ICA (60 mg/kg), the rats were pretreated with ICA at a dose of 60 mg/kg, and then surgery was performed as that in the I/R group. The intestinal I/R model was established according to previous standardized procedures with modification from preliminary experiments [25]. The rats in the sham group underwent surgical preparation including isolation of the superior mesenteric artery without occlusion. The rats in the I/R group were subjected to 1 h intestinal ischemia and 2 h reperfusion after the superior mesenteric artery was isolated and occluded by an atraumatic microvascular clamp. All animals were euthanized at the end of the reperfusion, and tissues and blood samples were harvested for analysis. All procedures were conducted according to the institutional animal care guidelines and were approved by the institutional ethics committee.
2.3.
Intestine morphologic assessment
The isolated intestine was fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, sliced into 4-mm sections, and stained with hematoxylin-eosin according to standard procedures. Pathologic score of the intestine damage was evaluated according to previous reports [26].
2.4. Measurement of serum tumor necrosis factor-a and interleukin-6 by enzyme-linked immunosorbent assay The blood samples were harvested from the abdominal aorta and allowed to coagulate for 30 min at room temperature. Serum was isolated after centrifugation at 2500 rpm for 15 min. The levels of serum tumor necrosis factor-a (TNF-a) and interleukin (IL)-6 were measured with enzymelinked immunosorbent assay kits (ENGTON bio-engineering Co, Ltd, Shanghai, China), according to the manufacturer’s protocols.
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Fig. 1 e ICA pretreatment protects intestinal tissue from I/R injury and decreases I/R-induced acute systematic inflammation. (A) Pathologic alterations of intestinal tissue in each group. (B) Normalized histopathologic properties of intestine. (C) Serum TNF-a level in each group. (D) Serum IL-6 level in each group. All data were presented as the mean ± standard deviation, n [ 8. **P < 0.01 versus control group; ##P < 0.01 versus I/R group. (Color version of the figure is available online.)
2.5. Detection of intestinal mucosal cellular apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay (Roche, Mannheim, Germany) was performed according to the manufacturer’s protocols. Intestine samples were fixed with 4% paraformaldehyde in phosphate-buffered saline, pH 7.4 for 48 h, embedded in paraffin, and sliced into 4-mm sections, which were treated with 20 mg/mL proteinase K and then incubated in a nucleotide mixture containing fluorescein-12-dUTP and terminal deoxynucleotidyl transferase. Positive controls were pretreated with 1 U/mL DNAse, and negative controls were incubated without terminal deoxynucleotidyl transferase. The number of apoptotic cells in each section was calculated by counting the number of TUNEL-positive apoptotic cells in 10e12 40 fields per condition from at least three to five independent samples per group.
2.6.
Immunoblots and immunoprecipitations
Immunoblots were performed using tissue and cell lysates. Briefly, protein extracts from each sample were separated electrophoretically by 10%e15% sodium dodecyl sulfate polyacrylamide gel electropheresis (Bio-Rad, Hercules, CA) and transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA). Nonspecific-binding sites were blocked at 37 C for 2 h with 5% skim milk, and then the membranes were incubated with the following primary antibodies: SIRT1, FOXO3, manganese superoxide dismutase (MnSOD) (all from Abcam Ltd, Cambridge, United Kingdom), Bcl-2 (Beyotime Institute of Biotechnology, Shanghai, China), and b-actin (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 C and were subsequently incubated with biotinylated secondary antibodies for 2 h at 37 C. The bands were visualized by using enhanced chemiluminescence-plus reagents (Beyotime Institute of Biotechnology). Emitted light was documented using a BioSpectrum-410 multispectral
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Fig. 2 e ICA attenuates intestinal oxidative stress and apoptosis induced by I/R. (A) Western blotting analysis for protein levels of MnSOD, Bcl-2 and, Bim in each group. (B) Intestinal apoptosis by TUNEL in each group. All data were presented as the mean ± standard deviation, n [ 3. **P < 0.01 versus control group; #P < 0.05, ##P < 0.01 versus I/R group. (Color version of the figure is available online.)
imaging system (UVP, Inc, Upland, CA) and analyzed using Gel-Pro Analyzer Version 4.0 (Media Cybernetics, Rockville, MD). For total proteins, b-actin was used as a loading control. Immunoprecipitations were performed with the protein A þ G Agarose Kit (Beyotime Institute of Biotechnology), according to the manufacturer’s protocols.
TCATAA-30 ; b-actin sense: 50 -CCAGCACAATGAAGATCAA GA -30 , antisense: 50 -AGAAAGGGTGTAACGCAACTAA-30 . All primers were synthesized by TaKaRa. PCR products were electrophoresed using 1.5% agarose gels and stained with ethidium bromide. A BioSpectrum-410 (UVP, Inc, Upland, CA) multispectral imaging system was used to analyze the intensity of electrophoresis bands.
2.7. Isolation of total RNA and reverse transcriptionpolymerase chain reaction
2.8.
The tissue, which was used for reverse transcriptionpolymerase chain reaction analysis, was soaked in the RNA stabilizing agent. Total RNA was extracted from rat intestine or cell sample using RNAiso Plus (TaKaRa, Dalian, China), according to the manufacturer’s instructions. Reverse transcription into complementary DNA was performed using a TaKaRa RNA polymerase chain reaction (PCR) Kit (AMV) version 3.0 (TaKaRa) for PCR analysis. Primers for rats are shown as follows: SIRT1 sense: 50 -TTGGCACCGATCCT CGAAC-30 , antisense: 50 -CCCAGCTCCAGTCAGAACTAT-30 ; b-actin sense: 50 -CCAGCACAATGAAGATCAAGA-30 , antisense: 50 -AGAAAGGGTGTAACGCAACTAA-30 . Primers for Caco-2 cell are shown as follows: SIRT1 sense: 50 -GCCTCACATGCAA GCTCTAGTGAC-30 , antisense: 50 -TTCGAGGATCTGTGCCAA
Here, we used human colorectal adenocarcinoma cell line Caco-2 exposed to H/R as a model, which is a common model for mimicking organ I/R injury and may, at least partly, reflect the pathophysiology of I/R in vivo [27,28]. The Caco-2 cells were cultured in Dulbecco’s modification of Eagle’s medium supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY), 1% nonessential amino acids, and 1% glutamine in a humidified atmosphere of air with 5% CO2 at 37 C. On confluency, the cells were split using trypsin. The culture medium was then replaced with serum-free Dulbecco’s modification of Eagle’s medium, and the cells were incubated for 24 h before the experimental treatment. To simulate intestinal I/R in vitro, cellular H/R conditions were created by incubating cells in a
Cell culture and H/R model
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Fig. 3 e ICA upregulates SIRT1 expression during intestinal I/R injury in vivo. (A) Western blotting analysis for protein levels of SIRT1 in each group. (B) reverse transcription-polymerase chain reaction analysis for mRNA expression of SIRT1 in each group. (C) Western blotting analysis for protein levels of FOXO3 in nuclear and cytoplasm of each group. All data were presented as the mean ± standard deviation, n [ 3. **P < 0.01 versus control group; #P < 0.05, ##P < 0.01 versus I/R group.
microaerophilic system (Thermo Scientific, Waltham, MA) at 5% CO2 and 1% O2 balanced with 94% N2 for 12 h. Then the cells were cultured in normoxic conditions (95% air and 5% CO2 at 37 C) for 6 h reoxygenation.
(Thermo, Vantaa, Finland). Cell viability was defined relative to untreated control.
2.9.
Caco-2 cells were plated on 35 mm dishes at a density of 1 105 cells per dish. When 50%e60% confluence was reached, for SIRT1 small interfering RNA (siRNA) transfection, the cells were transfected with 100 nmol/L or nontargeting control siRNA complexed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA), according to manufacturer’s instructions. The sense sequences of SIRT1 siRNA was as follows: 5- CCCUGUAAAGCUUUC AGAAdtdt-3; antisense: 5-UUCUGAAAGCUUUACAGGGdtdt-3 (Genepharma, Shanghai, China). After transfection with siRNA for 42 h, the cells were changed to fresh culture medium containing 50 mg/mL ICA for 6 h, and protein expression was examined using Western blot analysis.
MTT assay for cytotoxicity
Cell viability and survival was determined using the MTT assay. 1 105 cells were plated in 96-well microtiter plate and treated with different concentrations of the compounds for 6 h. After treatment, the cultures were incubated with MTT solution (5 mg/mL) for 4 h at 37 C, allowing viable cells to reduce the yellow tetrazolium salt into dark blue formazan crystals. Then, the medium was discarded, and formazan crystals were dissolved with the addition of 100 mL of dimethyl sulfoxide. Absorbance at 570 nm was measured using an enzyme-linked immunosorbent assay microplate reader
2.10.
Small interfering RNA transfection
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Fig. 4 e ICA promotes survival of Caco-2 cells under H/R conditions in vitro. (A) Caco-2 cells were exposed to hypoxic environment for 3, 6, 12, or 24 h and then reoxygenated for 6 h. The viable cells were determined using the MTT assay. (B) Caco-2 cells were pretreated with ICA (25, 50, and 100 mg/mL) for 6 h and then exposed to hypoxia 12 h followed by reoxygenation for 6 h; viable cells were determined using the MTT assay. All data were presented as the mean ± standard deviation, n [ 8. *P < 0.05, **P < 0.01 versus control group; ##P < 0.01 versus H/R group.
2.11. Measurement of intracellular reactive oxygen species (ROS) and cellular apoptosis assay after H/R Cellular reactive oxygen species (ROS) were detected using the fluorescent probe DCFH-DA from Molecular Probes (Invitrogen), according to the protocols described in our previous study [29]. Cell apoptosis was assessed by flow cytometry using Annexin V-FITC/PI Apoptosis Detection Kit from Invitrogen, according to the manufacturer’s instruction.
respectively. These results indicate that ICA exerts a protective function against intestinal I/R injury and systemic inflammation.
3.2. Protective effects of ICA against I/R-induced oxidative stress and apoptosis in vivo
All data are expressed as mean standard deviation. Oneway analysis of variance was used to determine significant differences between the groups. The Student-Newman-Keuls and/or least significant difference test was performed to compare all pair means. A value of P < 0.05 was chosen to indicate statistical significance. All statistical analyses were carried out using the SPSS 16.0 Statistical Software Package (SPSS Inc, Chicago, IL).
To investigate the extent of oxidative stress and apoptosis induced by I/R injury, the expression of the antioxidative stress factor MnSOD, the antiapoptotic factor Bcl-2, and proapoptotic factor Bim was examined in intestinal tissue. MnSOD and Bcl-2 expression decreased, whereas Bim expression increased in the I/R group compared with control animals; however, the opposite was observed in I/R þ ICA animals compared with those that were not treated with ICA before injury (Fig. 2A). Furthermore, TUNEL staining showed that ICA pretreatment also prevented the apoptosis induced by I/R injury (Fig. 2B). These results indicate that ICA protects intestinal tissue from I/R-induced oxidative injury and apoptosis.
3.
3.3. ICA-induced expression of SIRT1 and FOXO3 translocation after I/R injury in vivo
2.12.
Statistical analysis
Results
3.1. Protective effects of ICA after intestinal I/R injury and systemic inflammation To determine whether ICA exerts protective effects, intestinal tissue was examined for histopathologic features, and serum TNF-a and IL-6 levels were measured after I/R injury. Intestine tissues were clearly damaged, with mild swelling edema and villi irregularities (Fig. 1A and B). In accordance with the observed morphologic changes, TNF-a and IL-6 levels were increased in I/R animals compared with the control group (Fig. 1C and D). Pretreatment with ICA resulted in an improvement in the integrity of the tissue. And TNF-a level decreased by 10% (P < 0.01) with ICA 30 mg/kg and by 34% (P < 0.01) with ICA 60 mg/kg, whereas IL-6 decreased by 14% (P < 0.01) and 26% (P < 0.01),
We next examined the possible role of SIRT1 activation in the protective function exerted by ICA. SIRT1 protein expression was significantly reduced in I/R animals compared with controls (Fig. 3A). ICA pretreatment resulted in a significant increase in SIRT1 protein and mRNA levels that was dosedependent (Fig. 3A and B). On intestinal I/R injury, nuclear FOXO3 expression was significantly enhanced compared with control animals, whereas cytoplasmic FOXO3 expression was reduced (Fig. 3C). ICA administration caused a decrease in nuclear FOXO3 accumulation, and a concomitant increase in cytoplasmic FOXO3 distribution, after I/R injury (Fig. 3C). Collectively, these data suggest that the protective effects of ICA are mediated through an upregulation of SIRT1 expression and the subsequent activation of the SIRT1/FOXO3 signaling pathway.
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Fig. 5 e SIRT1 expression is activated by ICA in cultured Caco-2 cells. (A) (B) ICA upregulates SIRT1 protein and mRNA expression in a dose-dependent manner, respectively. (C) (D) ICA upregulates SIRT1 protein and mRNA expression in a time-dependent manner. All data were presented as the mean ± standard deviation, n [ 3. *P < 0.05, **P < 0.01 versus control group.
3.4. ICA-induced expression of SIRT1 under conditions of H/R in vitro
tion induced the expressions of SIRT1 mRNA and protein in Caco-2 cells in a dose- and time-dependent manner (Fig. 5).
To further explore the mechanism underlying the protective effects of ICA, an H/R model was established using Caco-2 cells (Fig. 4A). ICA pretreatment effectively suppressed the apoptosis triggered by H/R in a dose-dependent manner (Fig. 4B). Consistent with the in vivo results, ICA administra-
3.5. Protective effects of ICA against H/R-induced oxidative injury and apoptosis in vitro To examine whether the antioxidant and antiapoptotic activities of ICA were mediated by SIRT1, Caco-2 cells were
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Fig. 6 e SIRT1 activation protects Caco-2 cells against H/R-induced oxidative stress and apoptosis. (A) ROS determination in each group. (B) Apoptosis detection of Caco-2 cells using flow cytometry. All data were presented as the mean ± standard deviation, n [ 3. **P < 0.01 versus control group; ##P < 0.01 versus H/R group; &&P < 0.01 versus H/R D ICA group. CON, Control. (Color version of the figure is available online.)
treated with ICA only, or ICA and the SIRT1 inhibitor NAM for 6 h before exposure to H/R conditions. Elevated levels of cellular ROS were observed after H/R; this effect was abolished, and was accompanied by SIRT1 upregulation, when ICA was administered before H/R. ICA pretreatment also diminished the apoptosis induced by H/R in Caco-2 cells. However, the antioxidant and antiapoptotic effects of ICA were abrogated in the presence of NAM (Fig. 6A and B). These results indicate that SIRT1 mediates the protective effects of ICA against H/R-induced oxidative stress and apoptosis.
3.6. Deacetylation of FOXO3 by ICA-induced SIRT1 activation in Caco-2 cells after H/R To determine whether ICA-induced activation of SIRT1 results in increased deacetylation of the SIRT1 substrate FOXO3, the
interaction between SIRT1 and FOXO3 was examined in Caco2 cells stimulated with ICA. The levels of acetylated FOXO3 increased in response to H/R injury, as determined by immunoprecipitation (Fig. 7A); however, this effect was reversed by ICA pretreatment. In addition, in the presence of NAM, the expression of acetylated FOXO3 remained at a high level. Immunofluorescence double-staining showed colocalization of SIRT1 and FOXO3 in the nucleus, indicating a possible physical interaction between these two proteins. SIRT1 fluorescence was clearly decreased, which was associated with an increase in FOXO3 fluorescence under H/R conditions compared with control cells (Fig. 7B). However, ICA-pretreated cells showed a increased nuclear distribution in SIRT1, but a decreased nuclear colocalization with FOXO3 (Fig. 7B). This effect of ICA was attenuated by NAM. These results suggest that SIRT1 activation by ICA stimulates the
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Fig. 7 e Interaction of SIRT1 and FOXO3 in response to H/R. (A) Western blotting analysis for protein levels of SIRT1 in Caco-2 cells pretreated with ICA or NAM during H/R. Immunoprecipitation analysis for acetylation of FOXO3 in Caco-2 cells pretreated with ICA or NAM during H/R. (B) Immunofluorescence experiment for localization of SIRT1 and FOXO3 in each group. All data were presented as the mean ± standard deviation, n [ 3. *P < 0.05, **P < 0.01 versus control group; ## P < 0.01 versus H/R group; &&P < 0.01 versus H/R D ICA group. CON, Control. (Color version of the figure is available online.)
deacetylation of FOXO3 after H/R, and could potentially mediate the protective effects of ICA.
3.7. H/R-induced expression of oxidative stress and apoptotic factors mediated by SIRT1/FOXO3 signaling in vitro To determine whether ICA-activated SIRT1/FOXO3 signaling induced the expression of oxidative stress and apoptosisrelated factors, the expression of MnSOD, Bcl-2, and Bim was examined after H/R. Caco-2 cells subjected to H/R had
decreased levels of SIRT1 but increased levels of acetylated FOXO3 compared with control cells (Fig. 8A). Pretreatment with ICA, or with the SIRT1 activator resveratrol [30], increased SIRT1 expression and decreased the level of acetylated FOXO3 relative to H/R cells; moreover, the H/R-induced downregulation of MnSOD and Bcl-2 was enhanced, and the H/R-induced upregulation of Bim was suppressed (Fig. 8A). These results confirm that the activation of SIRT1 contributes to the protection against H/R-induced oxidative stress injury and apoptosis, which may be accomplished through the regulation of FOXO3 deacetylation.
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Fig. 8 e SIRT1/FOXO3 pathway is essential for preventing H/R-induced oxidative stress and apoptosis in vitro. (A) Western blotting analysis for protein levels of SIRT1, acetylated FOXO3, MnSOD, Bcl-2, and Bim in Caco-2 cells pretreated with ICA or RES during H/R. (B) Western blotting analysis for protein levels of SIRT1, acetylated FOXO3, MnSOD, Bcl-2, and Bim in Caco-2 cells transfected with a SIRT1 siRNA or NAM under H/R condition. CON, Control; RES, resveratrol.
In addition, siRNA knockdown of SIRT1, or inhibition of SIRT1 by NAM, led to an increase in acetylated FOXO3 expression, in conjunction with increased Bim and decreased MnSOD and Bcl-2 expression (Fig. 8B). Taken together, these findings indicate that preventing FOXO3 deacetylation via SIRT1 inhibition exacerbated H/R-induced injury in Caco-2 cells by causing the upregulation of a proapoptotic factor and the downregulation of antioxidative and antiapoptotic proteins.
4.
Discussion
The mechanisms of intestinal I/R injury have not been completely defined and many researchers, including us, have made efforts on finding an ideal therapy for intestinal I/R [31,32]. In our present study, it was found that ICA protects intestinal I/R injury, and that the protective effects of ICA are mediated by the SIRT1/FOXO3 signaling pathway. These finding may be a potential therapeutic approach to abrogate intestinal damage during I/R. Previous studies have reported the neuroprotective effect of ICA after cerebral I/R injury and ensuing cognitive dysfunction, which was attributed to the antioxidative function of ICA [24,33]. In the model of intestinal I/R injury used in the present study, ICA pretreatment mitigated the histologic damage to intestinal tissue caused by I/R and attenuated the heightened serum levels of inflammatory factors. These findings indicate that ICA exerts protective effects against systemic inflammation, oxidative stress, and cellular apoptosis triggered by I/R injury. The neuroprotective effects of ICA during oxygen and glucose deprivation, or H2O2-induced neurotoxicity, are dependent on the activation of SIRT1 [21,22]. The SIRT1 signaling pathway is known to mitigate injury in various experimental models such as cardiac, renal, and cerebral I/R
[11,34,35]. It was therefore hypothesized that SIRT1 activation, induced by ICA, has a similar protective function in intestinal I/R. Consistent with a previous report [11], downregulation of SIRT1 mRNA and protein was observed after intestinal I/R or H/R in Caco-2 cells, an effect that was abolished by ICA pretreatment (Figs. 3,5). This implies that, like resveratrol, ICA is a naturally occurring activator of SIRT1. The data also showed that ICA may exert its protective function through the upregulation of SIRT1 expression. The ICA-induced increase in SIRT1 expression was negatively correlated with the levels of oxidative stress biomarkers (e.g., ROS) and apoptosis in Caco-2 cells under H/R conditions (Fig. 6), whereas these effects were suppressed in the presence of NAM, implicating SIRT1 as the mediator of ICA function. Recent studies have shown that cardiac-specific FOXO1 and FOXO3 deficiency results in an increase in the myocardial infarct area, accompanied by a decrease in levels of antioxidants and antiapoptotic molecules after acute I/R injury [36]. In mammalian cells, FOXO proteins shuttle to the nucleus and are deacetylated by SIRT1 in response to oxidative stress. This enables FOXO3 to induce cell cycle arrest, heightens resistance to oxidative stress, and reduces FOXO3-dependent apoptosis [16,17,37]. This implies that the regulation of FOXO3 by SIRT1 plays an important role in minimizing the damage resulting from I/R. Indeed, the in vitro experiments showed that levels of acetylated FOXO3 increased in response to H/R (Fig. 7), and that ICA pretreatment promoted the deacetylation of FOXO3 concomitant with SIRT1 activation under these conditions. To determine whether the deacetylation of FOXO3 was dependent on ICA-induced activation of SIRT1, siRNA was used to knock down SIRT1, which prevented the deacetylation of FOXO3 even with ICA pretreatment (Fig. 8). These results indicate that SIRT1 is required for FOXO3 deacetylation, and that this is dependent, at least in part, on ICA-inducted upregulation of SIRT1 expression. Although several studies have established the relationship between
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SIRT1 and FOXO family proteins [11,15e17], the present investigation is the first to demonstrate that SIRT1/FOXO3 signaling provides a cellular mechanism for the protection against intestinal I/R injury. FOXO proteins participate in several signaling pathways that mediate cell-cycle regulation, DNA repair, and resistance to oxidative stress and apoptosis [12,13]. MnSOD is one of the many target genes activated by FOXO [38e41]. Several studies have proposed a link between SIRT1 and the regulation of the MnSOD antioxidant pathway by FOXO3 [42,43]. Consistent with these reports, in this study, parallel decreases in MnSOD and SIRT1 expressions were observed under conditions I/R or H/R (Figs. 2,3), accompanied by increases in acetylated FOXO3 levels (Fig. 7) and ROS accumulation (Fig. 6). Based on these results, a plausible mechanism for the protective effects exerted by ICA would involve the ICA-induced activation of SIRT1, followed by FOXO3 deacetylation, and the activation of MnSOD expression, which would strengthen cellular antioxidant capability and promote survival. Mechanisms that govern apoptosis are closely linked to those that mediate the cellular response to oxidative stress. The Bcl-2 family, which has both proapoptotic and antiapoptotic members (e.g., Bim and Bcl-2, respectively), is critical for the regulation of cell survival. SIRT1 was shown to deacetylate and repress the activity of FOXO3, leading to the downregulation of the forkhead-dependent apoptotic factor Bim [44]. FOXO3/FKHR-L1 specifically activates Bim but not Bcl-2 [45]. In the present study, Bim expression increased as a result of I/R injury, possibly because the reduced expression of SIRT1 prevented the deacetylation of FOXO3. Bcl-2 expression was also reduced on intestinal I/R injury, which could be because of the fact that Bcl-2 expression is regulated by SIRT1 [46,47]. Hence, a balance between the opposing functions of Bcl-2 and Bim, both of which are regulated by SIRT1/FOXO3 signaling, is important for the survival of intestinal epithelial cells during I/R or H/R. However, our study did not include the longer reperfusion time to demonstrate duration of ICA protection, which might be more important in clinical studies and we will add this part in further study. Also the study did not determine the dosedependent effect of ICA and did not use SIRT1 knock-out rats, which plays a crucial role in investigating mechanisms in vivo.
5.
Conclusions
In conclusion, this study provides evidence that ICA-induced SIRT1/FOXO3 signaling is a key mechanism mediating protection against oxidative stress and apoptosis resulting from intestinal I/R injury. These results offer a mechanism by which cell survival is promoted by SIRT1/FOXO3 signaling and downstream processes that activate the antioxidant pathway and suppress proapoptotic factors.
Acknowledgment This work was supported by the grants of National Natural Science Foundation of China (No. 81372037), Specialized
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Research Fund for the Doctoral Program of Higher Education of China (No. 20122105110001), and Science foundation of Liaoning Province of China (No. 2012225003). Authors’ contributions: F.Z. and J.Y. wrote the article. J.Y. and X.T. designed the experiments. F.Z., Y.H., X.X., and X.Z. performed the experiments. G.W. and S.N. collected and analyzed the data. J.Y. and X.T. revised the article and obtained fundings.
Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in this article.
references
[1] Berlanga J, Prats P, Remirez D, et al. Prophylactic use of epidermal growth factor reduces ischemia/reperfusion intestinal damage. Am J Pathol 2002;161:373. [2] Pope MR, Bukovnik U, Tomich JM, et al. Small beta2glycoprotein I peptides protect from intestinal ischemia reperfusion injury. J Immunol 2012;189:5047. [3] Cheng HL, Mostoslavsky R, Saito S, et al. Developmental defects and p53 hyperacetylation in Sir2 homolog(SIRTI)deficient mice. Dev Biol 2002;100:10794. [4] McBurney MW, Yang X, Jardine K, et al. The mammalian SIR2alpha protein has a role in embryogenesis and gametogenesis. Mol Cell Biol 2003;23:38. [5] Marcia CH, Leonard PG. Mammalian sirloins-emerging roles in physiology aging, and calorie restriction. Gen Dev 2008; 20:2913. [6] Hsu CP, Oka S, Shao D, et al. Nicotinamide phosphoribosyltransferase regulates cell survival through NADþ synthesis in cardiac myocytes. Circ Res 2009; 105:481. [7] Rane S, He M, Sayed D, et al. Downregulation of miR-199a derepresses hypoxia-inducible factor-1alpha and sirtuin 1 and recapitulates hypoxia preconditioning in cardiac myocytes. Circ Res 2009;104:879. [8] Kim DH, Jung YJ, Lee JE, et al. SIRT1 activation by resveratrol ameliorates cisplatin-induced renal injury through deacetylation of p53. Am J Physiol Ren Physiol 2011;301:F427. [9] He W, Wang Y, Zhang MZ, et al. Sirt1 activation protects the mouse renal medulla from oxidative injury. J Clin Invest 2010;120:1056. [10] Wang RH, Kim HS, Xiao CY, et al. Hepatic Sirt1 deficiency in mice impairs mTorc2/Akt signaling and results in hyperglycemia, oxidative damage, and insulin resistance. J Clin Invest 2011;121:4477. [11] Hsu CP, Zhai P, Yamamoto T, et al. Silent information regulator 1 protects the heart from ischemia/reperfusion. Circulation 2010;122:2170. [12] Maiese K, Chong ZZ, Shang YC. OutFOXOing disease and disability: the therapeutic potential of targeting FoxO proteins. Trends Mol Med 2008;14:219. [13] Maiese K, Chong ZZ, Shang YC, et al. A “FOXO” in sight: targeting Foxo proteins from conception to cancer. Med Res Rev 2009;29:395. [14] Picone P, Giacomazza D, Vetri V, et al. Insulin-activated Akt rescues Ab oxidative stress-induced cell death by orchestrating molecular trafficking. Aging Cell 2011;10:832. [15] Gilley J, Coffer PJ, Ham J. FOXO transcription factors directly activate Bim gene expression and promote apoptosis in sympathetic neurons. J Cell Biol 2003;162:613.
138
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 4 ( 2 0 1 5 ) 1 2 7 e1 3 8
[16] Brunet A, Sweeney LB, Sturgill JF, et al. Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science 2004;303:2011. [17] Hasegawa K, Wakino S, Yoshioka K, et al. Sirt1 protects against oxidative stress-induced renal tubular cell apoptosis by the bidirectional regulation of catalase expression. Biochem Biophys Res Commun 2008;372:51. [18] Liu ZQ, Luo XY, Sun YX, et al. The antioxidative effect of icariin in human erythrocytes against free-radical-induced haemolysis. J Pharm Pharmacol 2004;56:1557. [19] Liu ZQ. Icariin: a special antioxidant to protect linoleic acid against free-radical-induced peroxidation in micelles. J Phys Chem A 2006;110:6372. [20] Zhao F, Tang YZ, Liu ZQ. Protective effect of icariin on DNA against radical-induced oxidative damage. J Pharm Pharmacol 2007;59:1729. [21] Wang L, Zhang L, Chen ZB. Icariin enhances neuronal survival after oxygen and glucose deprivation by increasing SIRT1. Eur J Pharmacol 2009;609:40. [22] Zhang L, Huang S, Chen Y, et al. Icariin inhibits hydrogen peroxide-mediated cytotoxicity by up-regulating sirtuin type 1-dependent catalase and peroxiredoxin. Basic Clin Pharmacol Toxicol 2010;107:899. [23] Zhu HR, Wang ZY, Zhu XL, et al. Icariin protects against brain injury by enhancing SIRT1-dependent PGC-1a expression in experimental stroke. Neuropharmacology 2010;59:70. [24] Li L, Zhou QX, Shi JS. Protective effects of icariin on neurons injured by cerebral ischemia/reperfusion. Chin Med J (engl) 2005;118:1637. [25] Megison SM, Horton JW, Chao H, et al. A new model for intestinal ischemia in the rat. J Surg Res 1990;49:168. [26] Chiu CJ, McArdle AH, Brown R, et al. Intestinal mucosal lesion in low-flow states. I. A morphological, hemodynamic, and metabolic reappraisal. Arch Surg 1970;101:478. [27] Terui K, Enosawa S, Haga S, et al. Stat3 confers resistance against hypoxia/reoxygenation-induced oxidative injury in hepatocytes through upregulation of Mn-SOD. J Hepatol 2004;41:957. [28] Sato Y, Itagaki S, Kurokawa T, et al. In vitro and in vivo antioxidant properties of chlorogenic acid and caffeic acid. Int J Pharm 2011;403:136. [29] Zhai X, Lin M, Zhang F, et al. Dietary flavonoid genistein induces Nrf2 and phase II detoxification gene expression via ERKs and PKC pathways and protects against oxidative stress in Caco-2 cells. Mol Nutr Food Res 2013;57:249. [30] Huang J, Gan Q, Han L, et al. SIRT1 overexpression antagonizes cellular senescence with activated ERK/S6k1 signaling in human diploid fibroblasts. PLoS One 2008; 3:e1710. [31] SH1 Wen, Ling YH, Li Y, et al. Ischemic postconditioning during reperfusion attenuates oxidative stress and intestinal mucosal apoptosis induced by intestinal ischemia/ reperfusion via aldose reductase. Surgery 2013;153:555. [32] Yucel AF, Kanter M, Pergel A, et al. The role of curcumin on intestinal oxidative stress, cell proliferation and apoptosis
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
after ischemia/reperfusion injury in rats. J Mol Histol 2011; 42:579. Zheng M, Qu L, Lou Y. Effects of icariin combined with Panax notoginseng saponins on ischemia reperfusioninduced cognitive impairments related with oxidative stress and CA1 of hippocampal neurons in rat. Phytother Res 2008;22:597. Lempiainen J, Finckenberg P, Levijoki J, et al. AMPK activator AICAR ameliorates ischaemia reperfusion injury in the rat kidney. Br J Pharmacol 2012;166:1905. Wang P, Xu TY, Guan YF, et al. Nicotinamide phosphoribosyltransferase protects against ischemic stroke through SIRT1-dependent adenosine monophosphateactivated kinase pathway. Ann Neurol 2011;69:360. Sengupta A, Molkentin JD, Paik JH, et al. FOXO transcription factors promote cardiomyocyte survival upon induction of oxidative stress. J Biol Chem 2011;286:7468. Giannakou ME, Partridge L. The interaction between FOXO and SIRT1: tipping the balance towards survival. Trends Cell Biol 2004;14:408. Kops GJ, Dansen TB, Polderman PE, et al. Forkhead transcription factor FOXO3 protects quiescent cells from oxidative stress. Nature 2002;419:316. Chung YW, Kim HK, Kim IY, et al. Dual function of protein kinase C (PKC) in 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced manganese superoxide dismutase (MnSOD) expression. J Biol Chem 2011;286:29681. Guo J, Gertsberg Z, Ozgen N, et al. p66Shc links alpha1adrenergic receptors to a reactive oxygen species-dependent AKT-FOXO3 phosphorylation pathway in cardiomyocytes. Circ Res 2009;104:660. Sundaresan NR, Gupta M, Kim G, et al. Sirt3 blocks the cardiac hypertrophic response by augmenting FOXO3dependent antioxidant defense mechanisms in mice. J Clin Invest 2009;119:2758. Calvert JW, Jha S, Gundewar S, et al. Hydrogen sulfide mediates cardioprotection through Nrf2 signaling. Circ Res 2009;105:365. Tanaka J, Qiang L, Banks AS, et al. Foxo1 links hyperglycemia to LDL oxidation and endothelial nitric oxide synthase dysfunction in vascular endothelial cells. Diabetes 2009; 58:2344. Motta MC, Divecha N, Lemieux M, et al. Mammalian SIRT1 represses Forkhead transcription factors. Cell 2004; 116:551. Dijkers PF, Medema RH, Lammers JW, et al. Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the Forkhead transcription factor FKHR-L1. Curr Biol 2000; 10:1201. Takayama K, Ishida K, Matsushita T, et al. SIRT1 regulation of apoptosis of human chondrocytes. Arthritis Rheum 2009; 60:2731. Mikawa K, Nishina K, Takao Y, et al. ONO-1714, a nitric oxide synthase inhibitor, attenuates endotoxin-induced acute lung injury in rabbits. Anesth Analg 2003;97:1751.
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Icariin protects against intestinal ischemiaereperfusion injury Feng Zhang, MD,a Yan Hu, MD,b Xiaomei Xu, MD,b Xiaohan Zhai, MD,b Guangzhi Wang, MD,a Shili Ning, MD,a Jihong Yao, MD, PhD,b and Xiaofeng Tian, MD, PhDa,* a b
Department of General Surgery, Second Affiliated Hospital of Dalian Medical University, Dalian, China Department of Pharmacology, Dalian Medical University, Dalian, China
article info
abstract
Article history:
Background: This study investigated the role of Sirtuin 1 (SIRT1)/forkhead box O3 (FOXO3)
Received 10 December 2013
pathway, and a possible protective function for Icariin (ICA), in intestinal ischemiae
Received in revised form
reperfusion (I/R) injury and hypoxiaereoxygenation (H/R) injury.
14 August 2014
Materials and methods: Male SpragueeDawley rats were pretreated with different doses of
Accepted 2 October 2014
ICA (30 and 60 mg/kg) or olive oil as control 1 h before intestinal I/R. Caco-2 cells were
Available online 8 October 2014
pretreated with different concentrations of ICA (25, 50, and 100 mg/mL) and then subjected to H/R-induced injury.
Keywords:
Results: The in vivo results demonstrated that ICA pretreatment significantly improved
Intestinal ischemiaereperfusion
I/R-induced tissue damage and decreased serum tumor necrosis factor a and interleukin-6
SIRT1
levels. Changes of manganese superoxide dismutase, Bcl-2, and Bim were also reversed by
Icariin
ICA, and apoptosis was reduced. Importantly, the protective effects of ICA were positively
FOXO3
associated with SIRT1 activation. Increased SIRT1 expression, as well as decreased acety-
Oxidative stress
lated FOXO3 expression, was observed in Caco-2 cells pretreated with ICA. Additionally,
Apoptosis
the protective effects of ICA were abrogated in the presence of SIRT1 inhibitor nicotinamide. This suggests that ICA exerts a protective effect upon H/R injury through activation of SIRT1/FOXO3 signaling pathway. Accordingly, the SIRT1 activator resveratrol achieved a similar protective effect as ICA on H/R injury, whereas cellular damage resulting from H/R was exacerbated by SIRT1 knockdown and nicotinamide. Conclusions: SIRT1, activated by ICA, protects intestinal epithelial cells from I/R injury by inducing FOXO3 deacetylation both in vivo and in vitro These findings suggest that the SIRT1/FOXO3 pathway can be a target for therapeutic approaches intended to minimize injury resulting from intestinal dysfunction. ª 2015 Elsevier Inc. All rights reserved.
1.
Introduction
Intestinal ischemiaereperfusion (I/R) injury is a pathophysiological process typically associated with intestinal and mesenteric vascular dysfunction, which can also occur as a
result of surgery, organ transplantation, and trauma. Intestinal I/R is the initiating event in systemic inflammatory response syndrome and multiple organ dysfunction syndrome, associated with significant morbidity and mortality [1,2]. Thus, it is important to understand the mechanisms that
* Corresponding author. Department of General Surgery, Second Affiliated Hospital of Dalian Medical University, Dalian 116023, China. Tel.: þ86 0411 86110025; fax: þ86 0411 86110010. E-mail address: [email protected] (X. Tian). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.10.004
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mediate the cellular response to injury, and exploit these to mitigate damage to tissue and to the organism. Sirtuin 1 (SIRT1) is a member of the highly conserved nicotinamide adenine dinucleotide-dependent class III histone deacetylases [3,4], which is involved in multiple cellular processes, including DNA damage response, cell cycle regulation, apoptosis, and cellular longevity [5]. Recently, SIRT1 has been shown to play an important role in preventing oxidative damage resulting from myocardial hypoxiae reoxygenation (H/R), acute kidney injury, and hepatic stress [6e10]. During myocardial I/R injury, SIRT1 messenger RNA (mRNA) and protein levels decreased significantly; moreover, mice deficient in cardiac-specific SIRT1 exhibited a greater myocardial infarct size than wild-type mice after I/R [11], indicating that SIRT1 may have protective effects against I/R injury. The forkhead box O (FOXO) family of transcription factors is involved in cell-cycle regulation, DNA repair, and oxidative stress resistance and is also known to have antiapoptotic function [12,13]. FOXO3 has been shown to promote resistance to oxidative stress in cells under hypoxic and/or ischemic conditions, mainly by activating the expression of its target gene superoxide dismutase, which is a scavenger of oxygen free radicals [14]. FOXO3 is also involved in the regulation of apoptosis [15]. Recent reports indicate that SIRT1 can directly modulate FOXO3 deacetylation and activities under conditions of cellular stress [16,17]. However, the function of FOXO3 in intestinal I/R injury, and its relationship to SIRT1, has yet to be determined. Icariin (ICA), a flavonoid extracted from the traditional Chinese herb Epimedium brevicornum Maxim, is known to have anti-inflammatory, antioxidative, and antiapoptotic properties [18e20]. Recently, a neuroprotective function has been observed for ICA on injury induced by oxygen and glucose deprivation, and this effect was associated with an upregulation of SIRT1 [21e23]. We hypothesized that ICA could protect against oxidative stress and apoptosis induced by intestinal I/R, and these beneficial effects may be associated with SIRT1/FOXO3 signaling activation. To test this hypothesis, we determined whether ICA exerts protective effects in a rat model of intestinal I/R injury and in Caco-2 cells H/R injury and examined SIRT1/FOXO3 signaling pathway variation both in vivo and in vitro.
2.
Materials and methods
2.1.
Drugs and reagents
ICA (98% pure), purchased from Shanghai Winherb Medical Science Co, Ltd (Shanghai, China), was dissolved in olive oil (3 and 6 mg/mL) and gavaged before intestinal I/R (at the dose of 10 mL/kg). The rats in the sham and I/R groups were treated with an equal volume of olive oil. The dose of ICA administration was determined from a previous study with modification from our preliminary experiments [23,24]. In cell culture experiments, ICA was dissolved in 0.1% dimethyl sulfoxide at three concentrations (25, 50, and 100 mg/mL), and the cells were treated with ICA for 6 h
before exposure to H/R environment. An equal volume of 0.1% dimethyl sulfoxide was applied as the control. Nicotinamide (NAM, 98% pure) was purchased from Shanghai Source Leaf Biological Technology Co, Ltd (Shanghai, China) and was dissolved in saline.
2.2.
Animals and experimental groups
Male SpragueeDawley rats weighing 180e220 g were obtained from the Animal Center of Dalian Medical University (Dalian, China), housed under specific pathogen-free conditions and provided with standard laboratory chow and water. The rats were fasted overnight with free access to water before operation. The rats were divided into five experimental groups randomly, with eight rats in each of the following: (1) shamoperated group (sham); (2) intestinal I/R group (I/R); (3) sham þ ICA (60 mg/kg), the rats were pretreated with ICA at a dose of 60 mg/kg intragastrically for three consecutive days, and then surgery was performed as that in the sham group; (4) I/R þ ICA (30 mg/kg), the rats were pretreated with ICA at a dose of 30 mg/kg for 3 d, and then surgery was performed as that in the I/R group; and (5) I/R þ ICA (60 mg/kg), the rats were pretreated with ICA at a dose of 60 mg/kg, and then surgery was performed as that in the I/R group. The intestinal I/R model was established according to previous standardized procedures with modification from preliminary experiments [25]. The rats in the sham group underwent surgical preparation including isolation of the superior mesenteric artery without occlusion. The rats in the I/R group were subjected to 1 h intestinal ischemia and 2 h reperfusion after the superior mesenteric artery was isolated and occluded by an atraumatic microvascular clamp. All animals were euthanized at the end of the reperfusion, and tissues and blood samples were harvested for analysis. All procedures were conducted according to the institutional animal care guidelines and were approved by the institutional ethics committee.
2.3.
Intestine morphologic assessment
The isolated intestine was fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, sliced into 4-mm sections, and stained with hematoxylin-eosin according to standard procedures. Pathologic score of the intestine damage was evaluated according to previous reports [26].
2.4. Measurement of serum tumor necrosis factor-a and interleukin-6 by enzyme-linked immunosorbent assay The blood samples were harvested from the abdominal aorta and allowed to coagulate for 30 min at room temperature. Serum was isolated after centrifugation at 2500 rpm for 15 min. The levels of serum tumor necrosis factor-a (TNF-a) and interleukin (IL)-6 were measured with enzymelinked immunosorbent assay kits (ENGTON bio-engineering Co, Ltd, Shanghai, China), according to the manufacturer’s protocols.
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Fig. 1 e ICA pretreatment protects intestinal tissue from I/R injury and decreases I/R-induced acute systematic inflammation. (A) Pathologic alterations of intestinal tissue in each group. (B) Normalized histopathologic properties of intestine. (C) Serum TNF-a level in each group. (D) Serum IL-6 level in each group. All data were presented as the mean ± standard deviation, n [ 8. **P < 0.01 versus control group; ##P < 0.01 versus I/R group. (Color version of the figure is available online.)
2.5. Detection of intestinal mucosal cellular apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay (Roche, Mannheim, Germany) was performed according to the manufacturer’s protocols. Intestine samples were fixed with 4% paraformaldehyde in phosphate-buffered saline, pH 7.4 for 48 h, embedded in paraffin, and sliced into 4-mm sections, which were treated with 20 mg/mL proteinase K and then incubated in a nucleotide mixture containing fluorescein-12-dUTP and terminal deoxynucleotidyl transferase. Positive controls were pretreated with 1 U/mL DNAse, and negative controls were incubated without terminal deoxynucleotidyl transferase. The number of apoptotic cells in each section was calculated by counting the number of TUNEL-positive apoptotic cells in 10e12 40 fields per condition from at least three to five independent samples per group.
2.6.
Immunoblots and immunoprecipitations
Immunoblots were performed using tissue and cell lysates. Briefly, protein extracts from each sample were separated electrophoretically by 10%e15% sodium dodecyl sulfate polyacrylamide gel electropheresis (Bio-Rad, Hercules, CA) and transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA). Nonspecific-binding sites were blocked at 37 C for 2 h with 5% skim milk, and then the membranes were incubated with the following primary antibodies: SIRT1, FOXO3, manganese superoxide dismutase (MnSOD) (all from Abcam Ltd, Cambridge, United Kingdom), Bcl-2 (Beyotime Institute of Biotechnology, Shanghai, China), and b-actin (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 C and were subsequently incubated with biotinylated secondary antibodies for 2 h at 37 C. The bands were visualized by using enhanced chemiluminescence-plus reagents (Beyotime Institute of Biotechnology). Emitted light was documented using a BioSpectrum-410 multispectral
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Fig. 2 e ICA attenuates intestinal oxidative stress and apoptosis induced by I/R. (A) Western blotting analysis for protein levels of MnSOD, Bcl-2 and, Bim in each group. (B) Intestinal apoptosis by TUNEL in each group. All data were presented as the mean ± standard deviation, n [ 3. **P < 0.01 versus control group; #P < 0.05, ##P < 0.01 versus I/R group. (Color version of the figure is available online.)
imaging system (UVP, Inc, Upland, CA) and analyzed using Gel-Pro Analyzer Version 4.0 (Media Cybernetics, Rockville, MD). For total proteins, b-actin was used as a loading control. Immunoprecipitations were performed with the protein A þ G Agarose Kit (Beyotime Institute of Biotechnology), according to the manufacturer’s protocols.
TCATAA-30 ; b-actin sense: 50 -CCAGCACAATGAAGATCAA GA -30 , antisense: 50 -AGAAAGGGTGTAACGCAACTAA-30 . All primers were synthesized by TaKaRa. PCR products were electrophoresed using 1.5% agarose gels and stained with ethidium bromide. A BioSpectrum-410 (UVP, Inc, Upland, CA) multispectral imaging system was used to analyze the intensity of electrophoresis bands.
2.7. Isolation of total RNA and reverse transcriptionpolymerase chain reaction
2.8.
The tissue, which was used for reverse transcriptionpolymerase chain reaction analysis, was soaked in the RNA stabilizing agent. Total RNA was extracted from rat intestine or cell sample using RNAiso Plus (TaKaRa, Dalian, China), according to the manufacturer’s instructions. Reverse transcription into complementary DNA was performed using a TaKaRa RNA polymerase chain reaction (PCR) Kit (AMV) version 3.0 (TaKaRa) for PCR analysis. Primers for rats are shown as follows: SIRT1 sense: 50 -TTGGCACCGATCCT CGAAC-30 , antisense: 50 -CCCAGCTCCAGTCAGAACTAT-30 ; b-actin sense: 50 -CCAGCACAATGAAGATCAAGA-30 , antisense: 50 -AGAAAGGGTGTAACGCAACTAA-30 . Primers for Caco-2 cell are shown as follows: SIRT1 sense: 50 -GCCTCACATGCAA GCTCTAGTGAC-30 , antisense: 50 -TTCGAGGATCTGTGCCAA
Here, we used human colorectal adenocarcinoma cell line Caco-2 exposed to H/R as a model, which is a common model for mimicking organ I/R injury and may, at least partly, reflect the pathophysiology of I/R in vivo [27,28]. The Caco-2 cells were cultured in Dulbecco’s modification of Eagle’s medium supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY), 1% nonessential amino acids, and 1% glutamine in a humidified atmosphere of air with 5% CO2 at 37 C. On confluency, the cells were split using trypsin. The culture medium was then replaced with serum-free Dulbecco’s modification of Eagle’s medium, and the cells were incubated for 24 h before the experimental treatment. To simulate intestinal I/R in vitro, cellular H/R conditions were created by incubating cells in a
Cell culture and H/R model
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Fig. 3 e ICA upregulates SIRT1 expression during intestinal I/R injury in vivo. (A) Western blotting analysis for protein levels of SIRT1 in each group. (B) reverse transcription-polymerase chain reaction analysis for mRNA expression of SIRT1 in each group. (C) Western blotting analysis for protein levels of FOXO3 in nuclear and cytoplasm of each group. All data were presented as the mean ± standard deviation, n [ 3. **P < 0.01 versus control group; #P < 0.05, ##P < 0.01 versus I/R group.
microaerophilic system (Thermo Scientific, Waltham, MA) at 5% CO2 and 1% O2 balanced with 94% N2 for 12 h. Then the cells were cultured in normoxic conditions (95% air and 5% CO2 at 37 C) for 6 h reoxygenation.
(Thermo, Vantaa, Finland). Cell viability was defined relative to untreated control.
2.9.
Caco-2 cells were plated on 35 mm dishes at a density of 1 105 cells per dish. When 50%e60% confluence was reached, for SIRT1 small interfering RNA (siRNA) transfection, the cells were transfected with 100 nmol/L or nontargeting control siRNA complexed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA), according to manufacturer’s instructions. The sense sequences of SIRT1 siRNA was as follows: 5- CCCUGUAAAGCUUUC AGAAdtdt-3; antisense: 5-UUCUGAAAGCUUUACAGGGdtdt-3 (Genepharma, Shanghai, China). After transfection with siRNA for 42 h, the cells were changed to fresh culture medium containing 50 mg/mL ICA for 6 h, and protein expression was examined using Western blot analysis.
MTT assay for cytotoxicity
Cell viability and survival was determined using the MTT assay. 1 105 cells were plated in 96-well microtiter plate and treated with different concentrations of the compounds for 6 h. After treatment, the cultures were incubated with MTT solution (5 mg/mL) for 4 h at 37 C, allowing viable cells to reduce the yellow tetrazolium salt into dark blue formazan crystals. Then, the medium was discarded, and formazan crystals were dissolved with the addition of 100 mL of dimethyl sulfoxide. Absorbance at 570 nm was measured using an enzyme-linked immunosorbent assay microplate reader
2.10.
Small interfering RNA transfection
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Fig. 4 e ICA promotes survival of Caco-2 cells under H/R conditions in vitro. (A) Caco-2 cells were exposed to hypoxic environment for 3, 6, 12, or 24 h and then reoxygenated for 6 h. The viable cells were determined using the MTT assay. (B) Caco-2 cells were pretreated with ICA (25, 50, and 100 mg/mL) for 6 h and then exposed to hypoxia 12 h followed by reoxygenation for 6 h; viable cells were determined using the MTT assay. All data were presented as the mean ± standard deviation, n [ 8. *P < 0.05, **P < 0.01 versus control group; ##P < 0.01 versus H/R group.
2.11. Measurement of intracellular reactive oxygen species (ROS) and cellular apoptosis assay after H/R Cellular reactive oxygen species (ROS) were detected using the fluorescent probe DCFH-DA from Molecular Probes (Invitrogen), according to the protocols described in our previous study [29]. Cell apoptosis was assessed by flow cytometry using Annexin V-FITC/PI Apoptosis Detection Kit from Invitrogen, according to the manufacturer’s instruction.
respectively. These results indicate that ICA exerts a protective function against intestinal I/R injury and systemic inflammation.
3.2. Protective effects of ICA against I/R-induced oxidative stress and apoptosis in vivo
All data are expressed as mean standard deviation. Oneway analysis of variance was used to determine significant differences between the groups. The Student-Newman-Keuls and/or least significant difference test was performed to compare all pair means. A value of P < 0.05 was chosen to indicate statistical significance. All statistical analyses were carried out using the SPSS 16.0 Statistical Software Package (SPSS Inc, Chicago, IL).
To investigate the extent of oxidative stress and apoptosis induced by I/R injury, the expression of the antioxidative stress factor MnSOD, the antiapoptotic factor Bcl-2, and proapoptotic factor Bim was examined in intestinal tissue. MnSOD and Bcl-2 expression decreased, whereas Bim expression increased in the I/R group compared with control animals; however, the opposite was observed in I/R þ ICA animals compared with those that were not treated with ICA before injury (Fig. 2A). Furthermore, TUNEL staining showed that ICA pretreatment also prevented the apoptosis induced by I/R injury (Fig. 2B). These results indicate that ICA protects intestinal tissue from I/R-induced oxidative injury and apoptosis.
3.
3.3. ICA-induced expression of SIRT1 and FOXO3 translocation after I/R injury in vivo
2.12.
Statistical analysis
Results
3.1. Protective effects of ICA after intestinal I/R injury and systemic inflammation To determine whether ICA exerts protective effects, intestinal tissue was examined for histopathologic features, and serum TNF-a and IL-6 levels were measured after I/R injury. Intestine tissues were clearly damaged, with mild swelling edema and villi irregularities (Fig. 1A and B). In accordance with the observed morphologic changes, TNF-a and IL-6 levels were increased in I/R animals compared with the control group (Fig. 1C and D). Pretreatment with ICA resulted in an improvement in the integrity of the tissue. And TNF-a level decreased by 10% (P < 0.01) with ICA 30 mg/kg and by 34% (P < 0.01) with ICA 60 mg/kg, whereas IL-6 decreased by 14% (P < 0.01) and 26% (P < 0.01),
We next examined the possible role of SIRT1 activation in the protective function exerted by ICA. SIRT1 protein expression was significantly reduced in I/R animals compared with controls (Fig. 3A). ICA pretreatment resulted in a significant increase in SIRT1 protein and mRNA levels that was dosedependent (Fig. 3A and B). On intestinal I/R injury, nuclear FOXO3 expression was significantly enhanced compared with control animals, whereas cytoplasmic FOXO3 expression was reduced (Fig. 3C). ICA administration caused a decrease in nuclear FOXO3 accumulation, and a concomitant increase in cytoplasmic FOXO3 distribution, after I/R injury (Fig. 3C). Collectively, these data suggest that the protective effects of ICA are mediated through an upregulation of SIRT1 expression and the subsequent activation of the SIRT1/FOXO3 signaling pathway.
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Fig. 5 e SIRT1 expression is activated by ICA in cultured Caco-2 cells. (A) (B) ICA upregulates SIRT1 protein and mRNA expression in a dose-dependent manner, respectively. (C) (D) ICA upregulates SIRT1 protein and mRNA expression in a time-dependent manner. All data were presented as the mean ± standard deviation, n [ 3. *P < 0.05, **P < 0.01 versus control group.
3.4. ICA-induced expression of SIRT1 under conditions of H/R in vitro
tion induced the expressions of SIRT1 mRNA and protein in Caco-2 cells in a dose- and time-dependent manner (Fig. 5).
To further explore the mechanism underlying the protective effects of ICA, an H/R model was established using Caco-2 cells (Fig. 4A). ICA pretreatment effectively suppressed the apoptosis triggered by H/R in a dose-dependent manner (Fig. 4B). Consistent with the in vivo results, ICA administra-
3.5. Protective effects of ICA against H/R-induced oxidative injury and apoptosis in vitro To examine whether the antioxidant and antiapoptotic activities of ICA were mediated by SIRT1, Caco-2 cells were
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Fig. 6 e SIRT1 activation protects Caco-2 cells against H/R-induced oxidative stress and apoptosis. (A) ROS determination in each group. (B) Apoptosis detection of Caco-2 cells using flow cytometry. All data were presented as the mean ± standard deviation, n [ 3. **P < 0.01 versus control group; ##P < 0.01 versus H/R group; &&P < 0.01 versus H/R D ICA group. CON, Control. (Color version of the figure is available online.)
treated with ICA only, or ICA and the SIRT1 inhibitor NAM for 6 h before exposure to H/R conditions. Elevated levels of cellular ROS were observed after H/R; this effect was abolished, and was accompanied by SIRT1 upregulation, when ICA was administered before H/R. ICA pretreatment also diminished the apoptosis induced by H/R in Caco-2 cells. However, the antioxidant and antiapoptotic effects of ICA were abrogated in the presence of NAM (Fig. 6A and B). These results indicate that SIRT1 mediates the protective effects of ICA against H/R-induced oxidative stress and apoptosis.
3.6. Deacetylation of FOXO3 by ICA-induced SIRT1 activation in Caco-2 cells after H/R To determine whether ICA-induced activation of SIRT1 results in increased deacetylation of the SIRT1 substrate FOXO3, the
interaction between SIRT1 and FOXO3 was examined in Caco2 cells stimulated with ICA. The levels of acetylated FOXO3 increased in response to H/R injury, as determined by immunoprecipitation (Fig. 7A); however, this effect was reversed by ICA pretreatment. In addition, in the presence of NAM, the expression of acetylated FOXO3 remained at a high level. Immunofluorescence double-staining showed colocalization of SIRT1 and FOXO3 in the nucleus, indicating a possible physical interaction between these two proteins. SIRT1 fluorescence was clearly decreased, which was associated with an increase in FOXO3 fluorescence under H/R conditions compared with control cells (Fig. 7B). However, ICA-pretreated cells showed a increased nuclear distribution in SIRT1, but a decreased nuclear colocalization with FOXO3 (Fig. 7B). This effect of ICA was attenuated by NAM. These results suggest that SIRT1 activation by ICA stimulates the
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Fig. 7 e Interaction of SIRT1 and FOXO3 in response to H/R. (A) Western blotting analysis for protein levels of SIRT1 in Caco-2 cells pretreated with ICA or NAM during H/R. Immunoprecipitation analysis for acetylation of FOXO3 in Caco-2 cells pretreated with ICA or NAM during H/R. (B) Immunofluorescence experiment for localization of SIRT1 and FOXO3 in each group. All data were presented as the mean ± standard deviation, n [ 3. *P < 0.05, **P < 0.01 versus control group; ## P < 0.01 versus H/R group; &&P < 0.01 versus H/R D ICA group. CON, Control. (Color version of the figure is available online.)
deacetylation of FOXO3 after H/R, and could potentially mediate the protective effects of ICA.
3.7. H/R-induced expression of oxidative stress and apoptotic factors mediated by SIRT1/FOXO3 signaling in vitro To determine whether ICA-activated SIRT1/FOXO3 signaling induced the expression of oxidative stress and apoptosisrelated factors, the expression of MnSOD, Bcl-2, and Bim was examined after H/R. Caco-2 cells subjected to H/R had
decreased levels of SIRT1 but increased levels of acetylated FOXO3 compared with control cells (Fig. 8A). Pretreatment with ICA, or with the SIRT1 activator resveratrol [30], increased SIRT1 expression and decreased the level of acetylated FOXO3 relative to H/R cells; moreover, the H/R-induced downregulation of MnSOD and Bcl-2 was enhanced, and the H/R-induced upregulation of Bim was suppressed (Fig. 8A). These results confirm that the activation of SIRT1 contributes to the protection against H/R-induced oxidative stress injury and apoptosis, which may be accomplished through the regulation of FOXO3 deacetylation.
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Fig. 8 e SIRT1/FOXO3 pathway is essential for preventing H/R-induced oxidative stress and apoptosis in vitro. (A) Western blotting analysis for protein levels of SIRT1, acetylated FOXO3, MnSOD, Bcl-2, and Bim in Caco-2 cells pretreated with ICA or RES during H/R. (B) Western blotting analysis for protein levels of SIRT1, acetylated FOXO3, MnSOD, Bcl-2, and Bim in Caco-2 cells transfected with a SIRT1 siRNA or NAM under H/R condition. CON, Control; RES, resveratrol.
In addition, siRNA knockdown of SIRT1, or inhibition of SIRT1 by NAM, led to an increase in acetylated FOXO3 expression, in conjunction with increased Bim and decreased MnSOD and Bcl-2 expression (Fig. 8B). Taken together, these findings indicate that preventing FOXO3 deacetylation via SIRT1 inhibition exacerbated H/R-induced injury in Caco-2 cells by causing the upregulation of a proapoptotic factor and the downregulation of antioxidative and antiapoptotic proteins.
4.
Discussion
The mechanisms of intestinal I/R injury have not been completely defined and many researchers, including us, have made efforts on finding an ideal therapy for intestinal I/R [31,32]. In our present study, it was found that ICA protects intestinal I/R injury, and that the protective effects of ICA are mediated by the SIRT1/FOXO3 signaling pathway. These finding may be a potential therapeutic approach to abrogate intestinal damage during I/R. Previous studies have reported the neuroprotective effect of ICA after cerebral I/R injury and ensuing cognitive dysfunction, which was attributed to the antioxidative function of ICA [24,33]. In the model of intestinal I/R injury used in the present study, ICA pretreatment mitigated the histologic damage to intestinal tissue caused by I/R and attenuated the heightened serum levels of inflammatory factors. These findings indicate that ICA exerts protective effects against systemic inflammation, oxidative stress, and cellular apoptosis triggered by I/R injury. The neuroprotective effects of ICA during oxygen and glucose deprivation, or H2O2-induced neurotoxicity, are dependent on the activation of SIRT1 [21,22]. The SIRT1 signaling pathway is known to mitigate injury in various experimental models such as cardiac, renal, and cerebral I/R
[11,34,35]. It was therefore hypothesized that SIRT1 activation, induced by ICA, has a similar protective function in intestinal I/R. Consistent with a previous report [11], downregulation of SIRT1 mRNA and protein was observed after intestinal I/R or H/R in Caco-2 cells, an effect that was abolished by ICA pretreatment (Figs. 3,5). This implies that, like resveratrol, ICA is a naturally occurring activator of SIRT1. The data also showed that ICA may exert its protective function through the upregulation of SIRT1 expression. The ICA-induced increase in SIRT1 expression was negatively correlated with the levels of oxidative stress biomarkers (e.g., ROS) and apoptosis in Caco-2 cells under H/R conditions (Fig. 6), whereas these effects were suppressed in the presence of NAM, implicating SIRT1 as the mediator of ICA function. Recent studies have shown that cardiac-specific FOXO1 and FOXO3 deficiency results in an increase in the myocardial infarct area, accompanied by a decrease in levels of antioxidants and antiapoptotic molecules after acute I/R injury [36]. In mammalian cells, FOXO proteins shuttle to the nucleus and are deacetylated by SIRT1 in response to oxidative stress. This enables FOXO3 to induce cell cycle arrest, heightens resistance to oxidative stress, and reduces FOXO3-dependent apoptosis [16,17,37]. This implies that the regulation of FOXO3 by SIRT1 plays an important role in minimizing the damage resulting from I/R. Indeed, the in vitro experiments showed that levels of acetylated FOXO3 increased in response to H/R (Fig. 7), and that ICA pretreatment promoted the deacetylation of FOXO3 concomitant with SIRT1 activation under these conditions. To determine whether the deacetylation of FOXO3 was dependent on ICA-induced activation of SIRT1, siRNA was used to knock down SIRT1, which prevented the deacetylation of FOXO3 even with ICA pretreatment (Fig. 8). These results indicate that SIRT1 is required for FOXO3 deacetylation, and that this is dependent, at least in part, on ICA-inducted upregulation of SIRT1 expression. Although several studies have established the relationship between
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SIRT1 and FOXO family proteins [11,15e17], the present investigation is the first to demonstrate that SIRT1/FOXO3 signaling provides a cellular mechanism for the protection against intestinal I/R injury. FOXO proteins participate in several signaling pathways that mediate cell-cycle regulation, DNA repair, and resistance to oxidative stress and apoptosis [12,13]. MnSOD is one of the many target genes activated by FOXO [38e41]. Several studies have proposed a link between SIRT1 and the regulation of the MnSOD antioxidant pathway by FOXO3 [42,43]. Consistent with these reports, in this study, parallel decreases in MnSOD and SIRT1 expressions were observed under conditions I/R or H/R (Figs. 2,3), accompanied by increases in acetylated FOXO3 levels (Fig. 7) and ROS accumulation (Fig. 6). Based on these results, a plausible mechanism for the protective effects exerted by ICA would involve the ICA-induced activation of SIRT1, followed by FOXO3 deacetylation, and the activation of MnSOD expression, which would strengthen cellular antioxidant capability and promote survival. Mechanisms that govern apoptosis are closely linked to those that mediate the cellular response to oxidative stress. The Bcl-2 family, which has both proapoptotic and antiapoptotic members (e.g., Bim and Bcl-2, respectively), is critical for the regulation of cell survival. SIRT1 was shown to deacetylate and repress the activity of FOXO3, leading to the downregulation of the forkhead-dependent apoptotic factor Bim [44]. FOXO3/FKHR-L1 specifically activates Bim but not Bcl-2 [45]. In the present study, Bim expression increased as a result of I/R injury, possibly because the reduced expression of SIRT1 prevented the deacetylation of FOXO3. Bcl-2 expression was also reduced on intestinal I/R injury, which could be because of the fact that Bcl-2 expression is regulated by SIRT1 [46,47]. Hence, a balance between the opposing functions of Bcl-2 and Bim, both of which are regulated by SIRT1/FOXO3 signaling, is important for the survival of intestinal epithelial cells during I/R or H/R. However, our study did not include the longer reperfusion time to demonstrate duration of ICA protection, which might be more important in clinical studies and we will add this part in further study. Also the study did not determine the dosedependent effect of ICA and did not use SIRT1 knock-out rats, which plays a crucial role in investigating mechanisms in vivo.
5.
Conclusions
In conclusion, this study provides evidence that ICA-induced SIRT1/FOXO3 signaling is a key mechanism mediating protection against oxidative stress and apoptosis resulting from intestinal I/R injury. These results offer a mechanism by which cell survival is promoted by SIRT1/FOXO3 signaling and downstream processes that activate the antioxidant pathway and suppress proapoptotic factors.
Acknowledgment This work was supported by the grants of National Natural Science Foundation of China (No. 81372037), Specialized
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Research Fund for the Doctoral Program of Higher Education of China (No. 20122105110001), and Science foundation of Liaoning Province of China (No. 2012225003). Authors’ contributions: F.Z. and J.Y. wrote the article. J.Y. and X.T. designed the experiments. F.Z., Y.H., X.X., and X.Z. performed the experiments. G.W. and S.N. collected and analyzed the data. J.Y. and X.T. revised the article and obtained fundings.
Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in this article.
references
[1] Berlanga J, Prats P, Remirez D, et al. Prophylactic use of epidermal growth factor reduces ischemia/reperfusion intestinal damage. Am J Pathol 2002;161:373. [2] Pope MR, Bukovnik U, Tomich JM, et al. Small beta2glycoprotein I peptides protect from intestinal ischemia reperfusion injury. J Immunol 2012;189:5047. [3] Cheng HL, Mostoslavsky R, Saito S, et al. Developmental defects and p53 hyperacetylation in Sir2 homolog(SIRTI)deficient mice. Dev Biol 2002;100:10794. [4] McBurney MW, Yang X, Jardine K, et al. The mammalian SIR2alpha protein has a role in embryogenesis and gametogenesis. Mol Cell Biol 2003;23:38. [5] Marcia CH, Leonard PG. Mammalian sirloins-emerging roles in physiology aging, and calorie restriction. Gen Dev 2008; 20:2913. [6] Hsu CP, Oka S, Shao D, et al. Nicotinamide phosphoribosyltransferase regulates cell survival through NADþ synthesis in cardiac myocytes. Circ Res 2009; 105:481. [7] Rane S, He M, Sayed D, et al. Downregulation of miR-199a derepresses hypoxia-inducible factor-1alpha and sirtuin 1 and recapitulates hypoxia preconditioning in cardiac myocytes. Circ Res 2009;104:879. [8] Kim DH, Jung YJ, Lee JE, et al. SIRT1 activation by resveratrol ameliorates cisplatin-induced renal injury through deacetylation of p53. Am J Physiol Ren Physiol 2011;301:F427. [9] He W, Wang Y, Zhang MZ, et al. Sirt1 activation protects the mouse renal medulla from oxidative injury. J Clin Invest 2010;120:1056. [10] Wang RH, Kim HS, Xiao CY, et al. Hepatic Sirt1 deficiency in mice impairs mTorc2/Akt signaling and results in hyperglycemia, oxidative damage, and insulin resistance. J Clin Invest 2011;121:4477. [11] Hsu CP, Zhai P, Yamamoto T, et al. Silent information regulator 1 protects the heart from ischemia/reperfusion. Circulation 2010;122:2170. [12] Maiese K, Chong ZZ, Shang YC. OutFOXOing disease and disability: the therapeutic potential of targeting FoxO proteins. Trends Mol Med 2008;14:219. [13] Maiese K, Chong ZZ, Shang YC, et al. A “FOXO” in sight: targeting Foxo proteins from conception to cancer. Med Res Rev 2009;29:395. [14] Picone P, Giacomazza D, Vetri V, et al. Insulin-activated Akt rescues Ab oxidative stress-induced cell death by orchestrating molecular trafficking. Aging Cell 2011;10:832. [15] Gilley J, Coffer PJ, Ham J. FOXO transcription factors directly activate Bim gene expression and promote apoptosis in sympathetic neurons. J Cell Biol 2003;162:613.
138
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 4 ( 2 0 1 5 ) 1 2 7 e1 3 8
[16] Brunet A, Sweeney LB, Sturgill JF, et al. Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science 2004;303:2011. [17] Hasegawa K, Wakino S, Yoshioka K, et al. Sirt1 protects against oxidative stress-induced renal tubular cell apoptosis by the bidirectional regulation of catalase expression. Biochem Biophys Res Commun 2008;372:51. [18] Liu ZQ, Luo XY, Sun YX, et al. The antioxidative effect of icariin in human erythrocytes against free-radical-induced haemolysis. J Pharm Pharmacol 2004;56:1557. [19] Liu ZQ. Icariin: a special antioxidant to protect linoleic acid against free-radical-induced peroxidation in micelles. J Phys Chem A 2006;110:6372. [20] Zhao F, Tang YZ, Liu ZQ. Protective effect of icariin on DNA against radical-induced oxidative damage. J Pharm Pharmacol 2007;59:1729. [21] Wang L, Zhang L, Chen ZB. Icariin enhances neuronal survival after oxygen and glucose deprivation by increasing SIRT1. Eur J Pharmacol 2009;609:40. [22] Zhang L, Huang S, Chen Y, et al. Icariin inhibits hydrogen peroxide-mediated cytotoxicity by up-regulating sirtuin type 1-dependent catalase and peroxiredoxin. Basic Clin Pharmacol Toxicol 2010;107:899. [23] Zhu HR, Wang ZY, Zhu XL, et al. Icariin protects against brain injury by enhancing SIRT1-dependent PGC-1a expression in experimental stroke. Neuropharmacology 2010;59:70. [24] Li L, Zhou QX, Shi JS. Protective effects of icariin on neurons injured by cerebral ischemia/reperfusion. Chin Med J (engl) 2005;118:1637. [25] Megison SM, Horton JW, Chao H, et al. A new model for intestinal ischemia in the rat. J Surg Res 1990;49:168. [26] Chiu CJ, McArdle AH, Brown R, et al. Intestinal mucosal lesion in low-flow states. I. A morphological, hemodynamic, and metabolic reappraisal. Arch Surg 1970;101:478. [27] Terui K, Enosawa S, Haga S, et al. Stat3 confers resistance against hypoxia/reoxygenation-induced oxidative injury in hepatocytes through upregulation of Mn-SOD. J Hepatol 2004;41:957. [28] Sato Y, Itagaki S, Kurokawa T, et al. In vitro and in vivo antioxidant properties of chlorogenic acid and caffeic acid. Int J Pharm 2011;403:136. [29] Zhai X, Lin M, Zhang F, et al. Dietary flavonoid genistein induces Nrf2 and phase II detoxification gene expression via ERKs and PKC pathways and protects against oxidative stress in Caco-2 cells. Mol Nutr Food Res 2013;57:249. [30] Huang J, Gan Q, Han L, et al. SIRT1 overexpression antagonizes cellular senescence with activated ERK/S6k1 signaling in human diploid fibroblasts. PLoS One 2008; 3:e1710. [31] SH1 Wen, Ling YH, Li Y, et al. Ischemic postconditioning during reperfusion attenuates oxidative stress and intestinal mucosal apoptosis induced by intestinal ischemia/ reperfusion via aldose reductase. Surgery 2013;153:555. [32] Yucel AF, Kanter M, Pergel A, et al. The role of curcumin on intestinal oxidative stress, cell proliferation and apoptosis
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
after ischemia/reperfusion injury in rats. J Mol Histol 2011; 42:579. Zheng M, Qu L, Lou Y. Effects of icariin combined with Panax notoginseng saponins on ischemia reperfusioninduced cognitive impairments related with oxidative stress and CA1 of hippocampal neurons in rat. Phytother Res 2008;22:597. Lempiainen J, Finckenberg P, Levijoki J, et al. AMPK activator AICAR ameliorates ischaemia reperfusion injury in the rat kidney. Br J Pharmacol 2012;166:1905. Wang P, Xu TY, Guan YF, et al. Nicotinamide phosphoribosyltransferase protects against ischemic stroke through SIRT1-dependent adenosine monophosphateactivated kinase pathway. Ann Neurol 2011;69:360. Sengupta A, Molkentin JD, Paik JH, et al. FOXO transcription factors promote cardiomyocyte survival upon induction of oxidative stress. J Biol Chem 2011;286:7468. Giannakou ME, Partridge L. The interaction between FOXO and SIRT1: tipping the balance towards survival. Trends Cell Biol 2004;14:408. Kops GJ, Dansen TB, Polderman PE, et al. Forkhead transcription factor FOXO3 protects quiescent cells from oxidative stress. Nature 2002;419:316. Chung YW, Kim HK, Kim IY, et al. Dual function of protein kinase C (PKC) in 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced manganese superoxide dismutase (MnSOD) expression. J Biol Chem 2011;286:29681. Guo J, Gertsberg Z, Ozgen N, et al. p66Shc links alpha1adrenergic receptors to a reactive oxygen species-dependent AKT-FOXO3 phosphorylation pathway in cardiomyocytes. Circ Res 2009;104:660. Sundaresan NR, Gupta M, Kim G, et al. Sirt3 blocks the cardiac hypertrophic response by augmenting FOXO3dependent antioxidant defense mechanisms in mice. J Clin Invest 2009;119:2758. Calvert JW, Jha S, Gundewar S, et al. Hydrogen sulfide mediates cardioprotection through Nrf2 signaling. Circ Res 2009;105:365. Tanaka J, Qiang L, Banks AS, et al. Foxo1 links hyperglycemia to LDL oxidation and endothelial nitric oxide synthase dysfunction in vascular endothelial cells. Diabetes 2009; 58:2344. Motta MC, Divecha N, Lemieux M, et al. Mammalian SIRT1 represses Forkhead transcription factors. Cell 2004; 116:551. Dijkers PF, Medema RH, Lammers JW, et al. Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the Forkhead transcription factor FKHR-L1. Curr Biol 2000; 10:1201. Takayama K, Ishida K, Matsushita T, et al. SIRT1 regulation of apoptosis of human chondrocytes. Arthritis Rheum 2009; 60:2731. Mikawa K, Nishina K, Takao Y, et al. ONO-1714, a nitric oxide synthase inhibitor, attenuates endotoxin-induced acute lung injury in rabbits. Anesth Analg 2003;97:1751.
