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Original Basic Science—Liver

Agmatine Attenuates Liver Ischemia Reperfusion Injury by Activating Wnt/β-catenin Signaling in Mice

Han, Zhenyi MD1,2,3,4; Li, Yakun MD1,2,3,4; Yang, Bo PhD1,2,3,4; Tan, Rumeng PhD1,2,3,4; Wang, Meixi MD1,2,3,4; Zhang, Bo PhD1,2,3,4; Dai, Chen PhD1,2,3,4; Wei, Lai PhD1,2,3,4; Chen, Dong PhD1,2,3,4; Chen, Zhishui PhD1,2,3,4

Author Information
doi: 10.1097/TP.0000000000003161

Abstract

INTRODUCTION

Liver ischemia reperfusion injury (LIRI) is one of the major causes of liver injury in clinical practice. It often occurs during liver resection, liver transplantation, and shock and is related to the primary graft dysfunction1 and biliary complications after liver transplantation.2 In these clinical processes, transient ischemia of the liver causes hypoxia-induced hepatocyte death. However, when the blood supply is resumed, large amounts of inflammatory cells, inflammatory mediators, reactive oxygen species, and reactive nitrogen species accumulate in the liver, which destroy the cell homeostasis and induce severe inflammatory responses, apoptosis, and necrosis, ultimately leading to further cell death.3 Although the mechanisms of LIRI have been widely investigated, clinical treatment of LIRI is still limited to intraoperative prevention and postoperative supportive treatment, with no effective targeted treatment measures for LIRI.

Agmatine (AGM) is an endogenous polyamine, synthesized from L-arginine via a decarboxylation reaction involving arginine decarboxylase on the mitochondrial membrane of cells,4 and it is found in bacteria, plants, insects, invertebrates, and mammals.5,6 In mammals, AGM is widely distributed in the brain, stomach, liver, and other organs.7 It is usually present at low concentrations in the body under physiological conditions, but its level increase during growth and development or after stimulation.8 AGM is widely considered to be an endogenous agonist of the imidazoline receptor and the α2 adrenergic receptor, acting as a neurotransmitter.4 It is also an antagonist of the NMDA receptor.9 In addition, AGM is an endogenous regulator of the synthesis of NO,10 it reduces NO production under pathological conditions, thus regulating vasomotor and anti-inflammatory effects.11,12 A recent study also reported that AGM was central to the antiaging effects of metformin.13 Previous studies have shown that AGM exerts protective effects against ischemia-reperfusion injury of various tissues and organs, such as the brain,14 kidney,15 and heart.16 However, its protective effects against LIRI have not yet been reported.

The Wnt-mediated signaling pathway includes the canonical Wnt signaling pathway and the noncanonical Wnt signaling pathway,17 and has been proved to play an important regulatory role in cell proliferation, differentiation, development, and death.18 The most studied Wnt pathway is the canonical Wnt signaling pathway, namely the Wnt/β-catenin pathway, which functions by regulating the amount of the transcriptional coactivator β-catenin. The Wnt/β-catenin pathway has been extensively studied and is reported to be involved in the regulation of liver development,19 nutrient metabolism,20 tumorigenesis,21 and oxidative stress in hepatocytes.22 Using results of previous studies,23,24 we made a brief schematic diagram to illustrate the role of Wnt/β-catenin signaling in LIRI (Figure 1). In the model of LIRI, activating the Wnt/β-catenin pathway can promote hepatocyte proliferation, alleviate inflammation, and reduce apoptosis, while inhibiting this pathway may aggravate liver damage.25,26 These findings suggest that the Wnt/β-catenin signaling pathway may be a therapeutic target for alleviating LIRI.

FIGURE 1.
FIGURE 1.:
Schematic illustration of the Wnt/β-catenin signaling in the regulation of LIRI. When the Wnt/β-catenin signaling was activated during LIRI, β-catenin would not be phosphorylated by the destruction complex (Axin, APC and GSK3β) and then it translocated into the nucleus. After this, the β-catenin binded with TCF4 and activated the expression of its target genes to promote cell proliferation; it could also depress the PTEN activity, which promoted the PI3K/Akt signaling to reduce apoptosis and inflammatory responses; additionally, the β-catenin could directly inhibit NF-κB activation, thus inhibiting a series of inflammatory responses. APC, adenomatous polyposis coli; LIRI, liver ischemia reperfusion injury; TCF4, T-cell factor-4; PTEN, phosphatase and tensin homolog deleted on chromosome 10; PI3K, phosphoinositide 3-kinase; NF-κB, nuclear factor kappa B.

In this study, we used a mouse model of partial hepatic ischemia reperfusion injury and a hepatocyte model of cobalt chloride (CoCl2)-induced hypoxia to investigate whether AGM can improve LIRI and explore whether the protective effects of AGM on LIRI are mediated via the regulation of the Wnt/β-catenin pathway.

MATERIALS AND METHODS

Animals

Male C57BL/6J mice (6–8 wk old) were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China) and housed in the animal facility of the Institute of Transplantation of the Tongji Hospital Affiliated to the Tongji Medical College, Huazhong University of Science and Technology. All animal experiments were approved by the Institutional Animal Care and Use Committee at the Tongji Medical College, Huazhong University of Science and Technology, and all procedures were performed in accordance with the relevant guidelines and regulations of the Chinese Council on Animal Care.

Partial LIRI Model

Male C57BL/6J mice (weighing 20–22g) were used in this experiment. The partial LIRI model was prepared as previously described.27 The sham-operated mice underwent the same operative procedure without clamping the blood vessels. After 6 hours of reperfusion, the mice were sacrificed for the collection of blood and liver tissue samples.

The mice belonging to the LIRI + AGM group were intraperitoneally injected with AGM (100 mg/kg, Sigma-Aldrich, A7127) 10 minutes prior hepatic ischemia and at the same time as the reperfusion (with the same dose of AGM).

The mice belonging to inhibitor-treatment groups were intraperitoneally injected with the Wnt/β-catenin signaling pathway inhibitor XAV-939 (20 mg/kg, Selleck, S1180,) or ICG-001 (10 mg/kg, MCE, HY-14428). XAV-939 and ICG-001 were dissolved in 4% DMSO and corn oil.

Serum Biochemistry Assay

The blood samples were centrifuged at 8000 rpm for 5 minutes at room temperature. The levels of serum ALT and AST were measured using a standard autoanalyzer (ANTECH Diagnostics, Los Angeles, CA).

Cell Culture and Hepatocyte Hypoxia Model

Murine hepatocyte cell line AML12 (ATCC®CRL-2254TM) was used in this experiment. The cells were cultured in Dulbecco’s modified Eagle’s medium/F-12 (DMEM/F-12, Hyclone) supplemented with 10% fetal bovine serum (Gibco), ITS Liquid Media Supplement (Sigma-Aldrich, I3146), and 40 ng/mL dexamethasone (Sigma-Aldrich, D4902). The AML12 cells were maintained in a humidified incubator at 37°C under 5% CO2 conditions.

Hypoxic environment was simulated in vitro by using the hypoxia-inducing chemical CoCl2 (Sigma-Aldrich, C8661). Cells were cultured until they attained 60%–70% confluence, and then cultured in FBS-free DMEM/F-12 with 300 μM CoCl2 for 24 hours. For cells in the CoCl2 + AGM group, 0.5 mmol/L AGM was added at the same time with CoCl2.

Cells in the inhibitor-treatment group were cultured with XAV-939 or ICG-001 both at a final concentration of 10 μM.

PATHOLOGICAL ANALYSIS

Hematoxylin and Eosin Staining

After reperfusion for 6 hours, liver samples were procured and fixed with 4% paraformaldehyde for paraffin embedding. The embedded tissues were sectioned at a thickness of 5 μm and stained with hematoxylin and eosin. The level of liver necrosis was evaluated using Suzuki’s score.27

Terminal Deoxynucleotidyl Transferase-mediated Deoxyuridine Triphosphate Nick-end Labeling Assay

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay was performed using the in situ Cell Death Detection kit (Roche, 11684817910) following the manufacturer’s instructions. The liver tissue sections were subjected to immunohistochemical staining via the TUNEL assay.

Immunohistochemical Staining for Myeloperoxidase (MPO) in Liver Tissues

MPO staining of the liver sections was performed according to the manufacturer’s guidelines for immunohistochemical assays. We examined and evaluated the stained samples under a light microscope.

Cell Viability Assay

Cell viability was detected using the CCK-8 kit (Dojindo). The cells were plated in 96-well plates at a density of 5000 cells/well and incubated for 24 hours. They were then subjected to hypoxia treatment as described above; next, 10 μL of CCK8 solution was added to each well, followed by incubation for 100 minutes at 37°C. Finally, the absorbance values of the samples were measured at a wavelength of 450 nm using a microplate reader.

Flow Cytometry Analysis for Cell Apoptosis

After the hypoxia treatment as described above, AML12 cells were collected to assess apoptosis using an Annexin V-FITC and propidium iodide staining kit according to the manufacturer’s instructions (Multi Sciences, Hangzhou, China). The percentages of apoptotic cells were quantified via flow cytometry analysis, which was performed on a FACSCalibur instrument (BD Biosciences). The FlowJo software was used for data analysis.

Quantitative Real-time Polymerase Chain Reaction

Total mRNA was isolated from the liver tissues using the TRIzol reagent (Takara, Shiga, Japan) and reverse-transcribed into cDNA using a high-capacity cDNA reverse transcription kit (TAKARA, Shiga, Japan) according to the manufacturer’s instructions. The expression levels of the target genes were quantified by quantitative real-time polymerase chain reaction (qRT-PCR) analysis, which was performed using the SYBR Green kit (Trans Gene). The data were analyzed using the StepOne Software (Thermo Fisher Scientific), and the relative gene expression was calculated using the 2−(ΔΔCT) method, with β-actin as the reference gene. The mouse primer sequences are listed in the supplementary material (Table S1, SDC, http://links.lww.com/TP/B877).

Western Blotting Analysis

The total protein from liver homogenates or cell lysates was extracted using IP lysis buffer (Beyotime, Shanghai, China) containing protease inhibitors. The protein concentrations were quantified with the BCA Protein Assay Kit (Beyotime, Shanghai, China) using a Microplate Reader (BioTek). The protein samples were separated by electrophoresis on 10% or 12% sodium dodecyl sulfate polyacrylamide gels; the resultant bands were transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% skim milk and then incubated overnight at 4°C with the primary antibodies. Antibodies against Bax (2772), Bcl-2 (3498), cleaved-caspase3 (9664), and Cyclin D1 (2978) were purchased from Cell Signaling Technology. Antibodies against β-catenin (ab32572) and c-Myc (ab32072) were purchased from Abcam. Antibodies against β-actin (60008-1-Ig) and Wnt1 (27935-1-AP) were purchased from Proteintech. On the next day, the polyvinylidene fluoride membranes were incubated with horseradish peroxidase-conjugated antimouse or antirabbit antibodies (Proteintech) for 1 hour at 37°C. The proteins were subjected to chemiluminescent detection using ECL reagents (Beyotime, Shanghai, China). All results were analyzed using the Image-Pro Plus software (Media Cybernetics).

Statistical Analysis

All experiments were conducted in triplicate and repeated independently. Statistical analysis was performed using the Prism software (GraphPad). Results were expressed as the mean ± SEM. Comparisons were evaluated using the unpaired t test between 2 groups and 1-way ANOVA for multiple groups followed by a Turkey post hoc test. P < 0.05 were considered statistically significant.

RESULTS

AGM Protects the Liver From Ischemia Reperfusion Injury

To investigate whether AGM had a protective effect on LIRI in mice, a liver ischemia reperfusion mouse model was successfully established using wild-type mice. The mice were randomly divided into the following 3 groups: sham, LIRI, and LIRI + AGM. In accordance with previous studies,28,29 mice of the LIRI + AGM group were intraperitoneally administered with AGM at a dose of 100 mg/kg. Based on our preliminary studies, there were no significant effects when AGM was injected for once (Figure S1, SDC, http://links.lww.com/TP/B877). Then we chose to use AGM 10 minutes before ischemia and at the same time as the reperfusion. Liver tissues and blood samples were obtained 6 hours after reperfusion. Liver damage in response to LIRI was evaluated based on the levels of the serum aminotransferases ALT and AST; tissue necrosis was also observed in the liver, and Suzuki’s criteria were applied to grade the necrotic content (on a scale from 0 to 4).

Levels of ALT and AST in the mice from the LIRI group increased significantly compared with those from the sham group, while AGM dramatically reduced them (Figure 2A). Similarly, severe hepatic injury was observed in mice from the LIRI group, which was evident from the increased necrotic area and the Suzuki’s score of their liver tissues. Contrastingly, the degree of hepatic injury was significantly reduced in mice from the LIRI + AGM group (Figure 2B and C). These observations suggest that AGM protects the liver from LIRI.

FIGURE 2.
FIGURE 2.:
Agmatine exhibits protective effects against LIRI. A, After 6 h of reperfusion, the serum ALT and AST levels in the mice from the sham, LIRI, and LIRI + AGM groups were measured. B, Representative images for the histological analysis of the HE-stained liver tissues at ×200 magnification. Scale bar: 100 μm. C, The extent of liver injury was graded using the Suzuki’s scores. The data represent the mean ± SEM (n = 4–6 mice per group). *** P < 0.001 vs the sham group; ## P < 0.01 vs the LIRI group. AGM, agmatine; HE, hematoxylin and eosin; LIRI, liver ischemia reperfusion injury; SEM, standard error of the mean.

AGM Ameliorates Inflammatory Responses in the Liver During LIRI

Severe sterile inflammation is an important characteristic of LIRI. Additionally, a large number of proinflammatory cytokines are produced during ischemia reperfusion, further aggravating the inflammatory damage. We used qRT-PCR to examine the expression of TNF-α and IL-1β in the liver tissues of mice. As shown in Figure 3A and B, the expression levels of TNF-α and IL-1β in the LIRI group increased significantly compared with the sham group, whereas the expression levels of TNF-α and IL-1β in the LIRI + AGM group were lower than those in the LIRI group. We also performed MPO staining assay to detect neutrophil infiltration. As shown in Figure 3C and D, in samples from mice in the LIRI+AGM group, neutrophil infiltration was notably suppressed, compared with samples from mice in the LIRI group. These results indicate that AGM alleviates the inflammatory responses during LIRI.

FIGURE 3.
FIGURE 3.:
Agmatine decreases the inflammation caused by LIRI. The mRNA levels of TNF-α (A) and IL-1β (B) were measured by RT-PCR. C, Representative images of the immunohistochemical staining (MPO staining) of the liver sections prepared from the tissues of mice in the sham, LIRI, and LIRI + AGM groups at ×400 magnification. Scale bar: 50 μm. D, MPO-positive cells were quantified. The data represent the mean ± SEM (n = 4–6 mice per group). ** P < 0.01 and *** P < 0.001 vs the sham group; ## P < 0.01 vs the LIRI group. AGM, agmatine; LIRI, liver ischemia reperfusion injury; MPO, myeloperoxidase; RT-PCR, real-time polymerase chain reaction; SEM, standard error of the mean.

AGM Alleviates LIRI-induced Cell Apoptosis

Severe inflammatory responses resulting from ischemia reperfusion may cause cell damage and death. To examine whether AGM had a protective effect against LIRI-induced cell damage, liver tissue sections were subjected to the TUNEL assay. As shown in Figure 4A, the number of TUNEL-positive cells increased significantly in the LIRI group, compared with that of the sham group; however, after the AGM treatment, the number of TUNEL-positive cells decreased. Moreover, LIRI increased the expression of the procell death protein cleaved-caspase3 and decreased the expression of the antiapoptosis protein Bcl-2 in the liver tissues, the same being dramatically reversed after AGM treatment (Figure 4B). Collectively, these results indicate that AGM reduces the degree of cell death resulting from LIRI.

FIGURE 4.
FIGURE 4.:
Agmatine alleviates liver tissue apoptosis during LIRI. A, Representative images of the immunohistochemical staining (TUNEL assay) of the liver sections prepared from the tissues of mice in the sham, LIRI, and LIRI + AGM groups. Scale bar: 100 μm. TUNEL-positive cells were quantified from 5 randomly selected fields at ×200 magnification. B, Western blotting analysis for quantifying the Bcl-2 and cleaved-caspase3 levels after 6 h of reperfusion. β-actin served as the loading control. The experiments were repeated at least thrice. The data represent the mean ± SEM (n = 4–6 mice per group). ** P < 0.01 and *** P < 0.001 vs the sham group; ## P < 0.01 vs the LIRI group. AGM, agmatine; LIRI, liver ischemia reperfusion injury; SEM, standard error of the mean; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling.

AGM Protects AML12 Cells From CoCl2-induced Hypoxic Injury

Since CoCl2 can be used as a hypoxia-simulating agent, we treated AML12 cells with CoCl2 to establish a hypoxia model in vitro. To determine the proper experimental conditions, we treated AML12 cells with different concentrations of CoCl2 for 24 hours and then subjected these cells to CCK-8 assay. As shown in Figure 5A, the viability of AML12 cells decreased significantly when they were treated with CoCl2 for 24 hours. When the cell survival rate was around 50%, the corresponding concentrations of CoCl2 could be selected for the following experiments and here we chose 300 µmol/L. To estimate the protective effects of AGM against cell hypoxia, the AML12 cells were treated with 300 μM CoCl2 and various concentrations of AGM (0.1–5 mmol/L) for 24 hours. As shown in Figure 5B, when treated with 0.5 mmol/L AGM, the viability of the AML12 cells increased significantly, compared with that for cells in the CoCl2 group.

FIGURE 5.
FIGURE 5.:
Agmatine protects AML12 cells from CoCl2-induced hypoxic injury. The AML12 cells were subjected to the CCK-8 assay to determine the appropriate concentration of CoCl2 (A) and detect the effective concentration of AGM (B). C, The protein levels of Bcl-2, Bax, and cleaved-caspase3 were detected using Western blotting analysis. β-actin served as the loading control. D, Apoptosis was also determined using Annexin V/PI staining. The experiments were repeated at least thrice. The data represent the mean ± SEM (n = 3 samples per group). * P < 0.05, ** P < 0.01, and *** P < 0.001 vs the normal group; # P < 0.05 and ## P < 0.01 vs the CoCl2 group. AGM, agmatine; CoCl2, cobalt chloride; PI, propidium iodide; SEM, standard error of the mean.

Western blotting analysis demonstrated that the expression of the procell death proteins Bax and cleaved-caspase 3 was upregulated in the CoCl2 group, and the expression of the antiapoptosis protein Bcl-2 was downregulated. In contrast, the AGM group showed lower expression levels of Bax and cleaved-caspase3 and a higher expression level of Bcl-2, compared with the CoCl2 group (Figure 5C).

Furthermore, to detect whether AGM could protect AML12 cells from hypoxic injury-induced apoptosis, flow cytometry was performed. After the cells were stained with Annexin V/propidium iodide, they were analyzed using flow cytometry. As shown in Figure 5D, the apoptosis rate (Q2 + Q3) of cells from the CoCl2 group increased significantly compared with that of the normal group, while the CoCl2 + AGM group had a lower apoptosis rate than the CoCl2 group. The above results indicate that AGM effectively protects the AML12 cells from the damage caused by CoCl2-induced hypoxia.

AGM Activates the Wnt/β-catenin Signaling Pathway During LIRI

The obvious protective effects of AGM against LIRI-induced liver damage led us to investigate the mechanisms underlying these effects. Previous studies have reported that the Wnt/β-catenin signaling pathway plays an important role in the regulation of both the inflammatory responses and cell apoptosis during LIRI.22,25 Therefore, we analyzed several key molecules of Wnt/β-catenin signaling. Western blotting analysis demonstrated that the expression of Wnt1, β-catenin, c-Myc, and Cyclin D1 in liver tissues (Figure 6A and B) and AML12 cells (Figure 6C) decreased after LIRI or hypoxia treatment; however, AGM treatment increased the expression of these key proteins. These results indicate that there is a correlation between AGM and the Wnt/β-catenin signaling during LIRI.

FIGURE 6.
FIGURE 6.:
Agmatine activates the Wnt/β-catenin signaling pathway during LIRI. Western blotting analysis of β-catenin (A), Wnt1, c-Myc and Cyclin D1 (B), and the band density ratio relative to β-actin in vivo (n = 4–6 mice per group). ** P < 0.01 vs the sham group; # P < 0.05 and ## P < 0.01 vs the LIRI group. C, Western blotting analysis of β-catenin, Wnt1, c-Myc, and Cyclin D1and the band density ratio relative to β-actin in vitro (n = 3 samples per group). * P < 0.05 and ** P < 0.01 vs the normal group; # P < 0.05 vs the CoCl2 group. The experiments were repeated at least thrice. The data represent the mean ± SEM. AGM, agmatine; CoCl2, cobalt chloride; LIRI, liver ischemia reperfusion injury; SEM, standard error of the mean.

Blocking Wnt/β-catenin Signaling Weakens the Protective Effects of AGM Against LIRI

Based on the results obtained, we speculated that AGM exhibited its protective effects against LIRI by activating the Wnt/β-catenin signaling. To further verify our speculation, we chose 2 kinds of Wnt/β-catenin signaling inhibitors, XAV-939 and ICG-001. XAV-939 selectively inhibited Wnt/β-catenin-mediated transcription by inhibiting tankyrase and ICG-001 was a selective antagonist of the interaction between β-catenin and TCF4.

In experiments in vivo, mice from the LIRI + AGM + XAV-939 group were treated with XAV-939 (20 mg/kg, i.p.) 36 hours before modeling (Figure S2, SDC, http://links.lww.com/TP/B877) and mice from the LIRI + AGM + ICG-001 group were treated with ICG-001 (10 mg/kg, i.p.) 48 hours before modeling (Figure S3, SDC, http://links.lww.com/TP/B877). The mice were then subjected to partial LIRI model, and AGM was administered as described earlier. Liver tissues and blood samples were obtained 6 hours after reperfusion. As shown in Figure 7A, the serum ALT and AST levels were significantly increased in the LIRI + AGM + XAV-939 group and LIRI + AGM + ICG-001 group compared with the LIRI + AGM group. Hematoxylin and eosin staining of liver tissue samples showed that the liver necrosis area and Suzuki’s score of LIRI + AGM + XAV-939 group and LIRI + AGM + ICG-001 group were higher than those of the LIRI + AGM group (Figure 7B). These results demonstrated that the protective effects of AGM on liver damage diminished when the Wnt/β-catenin signaling pathway was blocked.

FIGURE 7.
FIGURE 7.:
Blocking the Wnt/β-catenin pathway weakens the protective effects of AGM on LIRI. A, Serum ALT and AST levels in mice from the sham, LIRI, LIRI + vehicle, LIRI + AGM, LIRI + AGM + XAV-939, and LIRI + AGM + ICG-001 groups. B, Representative images for the histological analysis of the HE-stained liver tissues at ×200 magnification. Scale bar: 100 μm. The extent of liver injury was graded using the Suzuki’s scores. C, Representative images of MPO staining of liver sections at ×400 magnification. Scale bar: 50 μm. MPO-positive cells were quantified. D, The mRNA levels of TNF-α and IL-1β of each group were measured by RT-PCR. E, Representative images of TUNEL assay of liver sections in each group. Scale bar: 100 μm. TUNEL-positive cells were quantified from 5 randomly selected fields at ×200 magnification. F, Western blotting analysis for quantifying the β-catenin, Bcl-2, and cleaved-caspase3 levels. β-actin served as the loading control. The experiments were repeated at least thrice. The data represent the mean ± SEM (n = 4–6 mice per group). ** P < 0.01 and *** P < 0.001 vs the sham group; # P < 0.05 and # # P < 0.01 vs the LIRI group; & P < 0.05 and && P < 0.01 vs the LIRI + AGM group. Vehicle: 4% DMSO + corn oil. AGM, agmatine; HE, hematoxylin and eosin; LIRI, liver ischemia reperfusion injury; MPO, myeloperoxidase; SEM, standard error of the mean; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling.

We also used MPO staining and qRT-PCR to detect the level of inflammatory injuries. As shown in Figure 7C, the degrees of neutrophil infiltration in LIRI + AGM + XAV-939 group and LIRI + AGM + ICG-001 group were more severe than that in LIRI + AGM group; qRT-PCR also showed consistent results, the mRNA levels of TNF-α and IL-1β were significantly increased in LIRI + AGM + XAV-939 group and LIRI + AGM + ICG-001 group (Figure 7D). These results indicated that blocking the Wnt/β-catenin pathway diminished the curative ability of AGM on liver tissue inflammation.

Finally, we examined the degrees of apoptosis in each group. TUNEL staining of liver tissue sections showed that the number of TUNEL positive cells in the LIRI + AGM + XAV-939 group and the LIRI + AGM + ICG-001 group were significantly higher than that in the LIRI + AGM group (Figure 7E). Moreover, the detection results of apoptosis-related proteins were also consistent. As shown in Figure 7F, when compared with the LIRI + AGM group, the expression of Bcl-2 in the LIRI + AGM + XAV-939 group and the LIRI + AGM + ICG-001 group decreased, while cleaved-caspase3 levels were significantly elevated. Combining these data, we conclude that when Wnt/β-catenin signaling is inhibited, the protective effects of AGM on liver injuries, inflammatory response and apoptosis are attenuated.

Inhibitors of Wnt/β-catenin Pathway Weaken Protective Effects of AGM on CoCl2-induced Hypoxic Injury

In experiments in vitro, we treated AML12 cells with XAV-939 or ICG-001. We determined the optimal concentrations of these 2 inhibitors by detecting the protein expression levels of β-catenin following treatment of the cells with different concentrations of XAV-939 or ICG-001. As shown in Figure S4 (SDC, http://links.lww.com/TP/B877), when the concentration of XAV-939 was 10 µmol/L, the expression of β-catenin was notably reduced. Similarly, we determined 10 µmol/L as the optimum concentration of ICG-001 (Figure S5, SDC, http://links.lww.com/TP/B877).

Six treatment groups were established as follows: normal, DMSO, CoCl2, CoCl2 + AGM, CoCl2 + AGM + XAV-939, and CoCl2 + AGM + ICG-001. We performed the CCK-8 assay to measure the cell viability in each group. After treatment with XAV-939 or ICG-001, the cell survival rate decreased significantly compared with that of the CoCl2 + AGM group (Figure 8A). We also used flow cytometry to detect the apoptosis rate of cells from each group. As shown in Figure 8B and C, cells from the CoCl2 + AGM + XAV-939 and CoCl2 + AGM + ICG-001 groups had higher apoptosis rates than those from the CoCl2 + AGM group. In addition, as shown in Figure 8D and E, the protein levels of cleaved-caspase3 in the CoCl2 + AGM + XAV-939 and CoCl2 + AGM + ICG-001 group were higher than those in CoCl2 + AGM group, while the expression of Bcl-2 was decreased. All these results demonstrated that the protective effects of AGM were inhibited after the Wnt/β-catenin signaling pathway was blocked, thus confirming that AGM exhibited protective effects against LIRI by activating the Wnt/β-catenin pathway.

FIGURE 8.
FIGURE 8.:
Inhibitors of Wnt/β-catenin pathway weaken the protective effects of AGM on CoCl2-induced hypoxic injury. A, Cell viability rate in normal, DMSO, CoCl2, CoCl2 + AGM, CoCl2 + AGM + XAV-939, and CoCl2 + AGM + ICG-001 groups were detected through CCK-8 assay. B and C, Representative images of flow cytometry analysis stained with Annexin V/PI. D and E, Western blotting analysis and the relative band density ratio of β-catenin, Bcl-2, and cleaved-caspase3 in cells from the normal, DMSO, CoCl2, CoCl2 + AGM, CoCl2 + AGM + XAV-939, and CoCl2 + AGM + ICG-001 groups, respectively. β-Actin served as the loading control. These experiments were repeated at least thrice. The data represent the mean ± SEM (n = 3 samples per group). ** P < 0.01 and *** P < 0.001 vs the normal group; # P < 0.05 and ## P < 0.01 vs the CoCl2 group; & P < 0.05 and && P < 0.01 vs the CoCl2 + AGM group. AGM, agmatine; CoCl2, cobalt chloride; PI, propidium iodide; SEM, standard error of the mean.

DISCUSSION

LIRI is common after liver transplantation and other hepatic surgeries and it seriously affects the prognosis of patients. The pathological process of LIRI includes liver tissue damage, inflammatory responses, and hepatocyte apoptosis.30 Although there have been many studies on hepatic ischemia reperfusion injury, its mechanism has not yet been clearly explained, and there is still a lack of effective treatments for LIRI in clinical practice. In our study, we demonstrated that AGM could improve LIRI-induced liver damage and hepatocyte apoptosis.

Recently, it has been found that AGM not only enhances morphine-induced analgesia31 and antidepression32 activities but also plays an important protective role against ischemia reperfusion injury of various organs.15,16,29 In this experiment, we discovered the protective effects of AGM against hepatic ischemia reperfusion injury. The results showed that following ischemia reperfusion injury, the serum ALT and AST levels and the pathological damage observed in the liver tissues of mice increased substantially, but AGM significantly reduced these levels. It is currently believed that the excessive release of inflammatory factors and cell death play important roles in the pathogenesis of ischemia reperfusion injury.33 Another study has reported that AGM decreases the production of TNF-α and IL-1β,34 which is consistent with the results of our study. We also found that the levels of TNF-α and IL-1β and the neutrophil infiltration (indicated by MPO staining) in the LIRI + AGM group were significantly lower than those in the LIRI group, suggesting that AGM alleviated the LIRI-induced inflammatory responses. Previous researches have shown that AGM could reduce NO production, but we found no difference in NO synthesis among groups (data not shown). It has been found that AGM inhibits cell apoptosis following hyperoxia-induced acute lung injury.35 In our study, the TUNEL staining and Western blotting analysis showed that AGM significantly diminished LIRI-induced apoptosis. We detected the survival rate and apoptosis level of cells in each group in vitro, and these results also validated the protective effects of AGM. In conclusion, our results suggested that AGM significantly ameliorated the liver damage caused by LIRI both in vivo and in vitro.

While exploring the possible mechanisms underlying these effects of AGM, we found that the expression level of β-catenin, the most critical coactivator in the canonical Wnt pathway, was significantly decreased in the liver tissues of mice from the LIRI group. Additionally, other proteins involved in Wnt/β-catenin signaling, such as Wnt1, c-Myc, Cyclin D1, showed the same trends as β-catenin. This was consistent with the previous studies, which had reported that under hypoxic conditions, the transcription of genes present downstream of the Wnt/β-catenin pathway was inhibited.36 However, AGM administration increased the levels of these proteins. These results suggested that AGM could be related to the Wnt/β-catenin pathway during LIRI.

The Wnt/β-catenin pathway is a complex pathway that is highly conserved during the evolution of organisms; it plays crucial roles in cell growth, proliferation, and differentiation and in the maintenance of cell homeostasis.37 In recent years, an increasing number of studies have shown that the Wnt/β-catenin pathway is associated with ischemia reperfusion injury.38,39 In LIRI, activation of the Wnt/β-catenin pathway may attenuate oxidative stress damage in the liver by regulating the cellular redox balance,26 reduce liver inflammation by regulating the innate and adaptive immunity of the body,23 and also reduce apoptosis and the expression of apoptosis-related proteins.40 To further validate the correlation between AGM and the Wnt/β-catenin pathway during LIRI, we blocked the Wnt/β-catenin pathway using 2 inhibitors and the results showed the protective effects of AGM were reduced both in vivo and in vitro. Combined with these experimental data, we can conclude that AGM plays a protective role against liver damages by activating the Wnt/β-catenin signaling during LIRI.

However, our study has a few limitations. First, in the cell experiments, we used CoCl2 to induce hypoxia rather than the hypoxic incubator, because we found that using CoCl2 provided more stability in our experimental conditions; several previous studies have also adopted this method.26,41 Second, we have verified that AGM can protect the liver from LIRI-induced damage by activating the Wnt/β-catenin pathway, but we have not elucidated the specific mechanism through which AGM regulates the Wnt/β-catenin signaling. Studies have reported several ways to activate Wnt/β-catenin signaling during ischemia reperfusion injuries. Tenascin-C could protect against renal ischemia reperfusion injury by recruiting the Wnt ligands to activate Wnt/β-catenin signaling.42 Another study demonstrated that miR-1246 activated the Wnt/β-catenin signaling by inhibiting GSK3β in hepatic ischemia reperfusion injury.43 However, little studies have reported about how AGM regulates the Wnt/β-catenin signaling. We will attempt to address this query in our future studies.

In conclusion, this study demonstrates that AGM reduces the inflammatory response and apoptosis during LIRI by activating the Wnt/β-catenin pathway, thereby preventing liver damages. This study is the first to discover that AGM can protect the liver from ischemia reperfusion injury and prove that AGM could regulate the Wnt/β-catenin pathway during LIRI. Despite the shortcomings in our study, we believe it will inspire new ideas toward the exploration of effective drugs for the treatment and prevention of hepatic ischemia reperfusion injury.

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