Introduction
Fulminant hepatic failure (FHF) is a severe clinical syndrome that is characteristic of hepatic cell injury resulting from a variety of hepatic disease processes. FHF leads to hepatic encephalopathy, severe coagulopathy, jaundice, hydroperitoneum and high rates of patient mortality. Several mechanisms are responsible for FHF, including viral infection, drug or toxin intake and metabolic disorders (1). However, the pathophysiology of FHF remains poorly understood and further investigations are required. No therapy is currently available except liver transplantation, which is limited due to a shortage of donors, the rapid progression of FHF and the expense of the surgical procedure (2). Effective prophylactic or therapeutic interventions are urgently required to improve the prognosis of patients with FHF.
D-galactosamine (D-GalN) and lipopolysaccharide (LPS)-induced hepatic failure in mice is a universally used animal model that resembles acute hepatic failure in the clinic (3). D-GalN has been found to markedly strengthen the susceptibility of mice to the lethal effects of LPS (4). Upon stimulation with LPS, liver macrophages secrete various proinflammatory cytokines, leading to hepatic necrosis, decreased levels of antioxidant enzymes and the scavenging of free radicals (5,6). Therefore, decreasing the generation of proinflammatory mediators and oxidative substances may be an effective strategy for the prevention or treatment of FHF.
Produced by interstitial fibroblasts in the kidney and in the fetal liver, erythropoietin (EPO) is an endogenous hormone that acts as a potent stimulator of bone marrow activity and red blood cell production (7). Although EPO is primarily synthesized in the kidney, it has been recognized that EPO and EPO receptors (EPORs) are expressed by other tissues and organs, including the brain, heart, kidney and liver (8–10). Furthermore, studies have revealed that EPO exerts antiapoptotic (11,12), antioxidative (13–15) and anti-inflammatory (16) effects. Activation of the EPOR activates various intracellular pathways, including the mitogen-activated protein kinase, c-Jun N-terminal kinase, nuclear factor κ-light-chain-enhancer of activated B cell and phosphatidylinositol 3-kinase (PI3K)/Akt signaling cascades (17,18). In the present study, it was hypothesized that EPO may protect liver tissues against D-GalN and LPS-induced liver failure in mice. This study aimed to investigate this hypothesis.
Materials and methods
Materials
D-GalN and LPS were purchased from Sigma-Aldrich (St. Louis, MO, USA). Detection kits for alanine aminotransferase (ALT), aspartate aminotransferase (AST), superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
Experimental animals, groups and treatment
Six- to eight-week-old BALB/c mice weighing between 18 and 22 g were obtained from the Experimental Animal Center of Xi’an Jiaotong University College of Medicine (Xi’an, China). All animals were acclimated to the laboratory environment for one week prior to the conduction of experimental procedures. The mice were allowed free access to drinking water and food and were maintained at 22±2°C in an automatic 12-h light/dark cycle with 50% humidity. The study was approved by the Ethics Committee of Xi’an Jiaotong University and performed in accordance with the Practice Guidelines for Laboratory Animals of China.
The mice were randomly divided into the following five groups (n=8/group): Normal, D-GalN/LPS, and 1,000, 3,000 and 10,000 U/kg EPO. The dosages were selected based on previous investigations (9,17) and a preliminary study. The EPO groups were administered EPO intraperitoneally once per day for 3 days. One hour after the final administration, mice in the normal group were injected with the same volume of sterile saline. The mice in the other group were simultaneously intraperitoneally injected with 700 mg/kg D-GalN and 10 μg/kg LPS. The mice were fasted for one night (12 h) prior to D-GalN and LPS administration. Eight hours after D-GalN and LPS injection, all mice were sacrificed under deep ether anesthesia. Blood samples were taken from the retro-orbital plexus of the mice. Serum was separated using centrifugation at 1,368 × g for 10 min, then stored at −80°C for analysis of the serum levels of ALT and AST. Liver tissue samples were divided into two sections. One was immersed in 10% phosphate-buffered formalin for morphological analysis and the other was stored at −80°C for the analysis of biochemical indicators of the liver.
Hepatic function tests
Serum AST and ALT levels were determined to assess liver function using AST and ALT commercial kits (Nanjing Jiancheng Bioengineering Institute).
MDA, SOD and GSH-Px assays
Liver tissue was washed with cold physiological saline and the connective tissue was removed. The liver tissue was then dried using filter paper and weighed. Tissue homogenate was prepared using cold physiological saline at a ratio of 1/9 (w/v), followed by centrifugation at 1,596 × g for 10 min. The supernatant was then collected and the concentration of MDA, and the activities of SOD and GSH-Px were measured using the corresponding kits according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute).
Histopathological analysis
Liver tissue was fixed in 10% neutral-buffered formalin and embedded in paraffin. The samples were sliced into 5-μm sections and the slides were stained with hematoxylin and eosin.
RNA isolation and quantitative polymerase chain reaction (qPCR) analysis
Total RNA was isolated from mouse liver tissues using the TriPure RNA isolation reagent (Roche, Basel, Switzerland) and 2 μg RNA was reverse transcribed using the PrimeScript™ RT Master Mix (Perfect Real Time) (Takara Bio, Inc., Tokyo, Japan). qPCR analysis was performed using SYBR® Premix Ex Taq™ II (Perfect Real Time) (Takara Bio, Inc.). PCR reactions were performed in 96-well plates in an iQ5 Real-Time PCR Detection system (Bio-Rad Laboratories, Hercules, CA, USA).
All of the primers and probes for qPCR were obtained from Takara Bio, Inc. Murine EPOR was amplified using the primers EPOR-F (5′-GCT CCG GGA TGG ACT TCA ACT A-3′) and EPOR-R (5′-CTG GTG CAG GCT ACA TGA CTT TC-3′); murine PI3K was amplified using the primers PIK3R1-F (5′-GCT CCT GGA AGC CAT TGA GAA-3′) and PIK3R1-R (5′-CGT CGA TCA TCT CCA AGT CCA C-3′); and murine GAPDH was amplified using the primers GAPDH-F (5′-TGT GTC CGT CGT GGA TCT GA-3′) and GAPDH-R (5′-TTG CTG TTG AAG TCG CAG GAG-3′). GAPDH served as an endogenous control and the results were normalized to GAPDH. The efficiency of qPCR for the target genes and the endogenous control was approximately equal. −ΔCT expresses the difference between the number of cycles (CT) of the target genes and the endogenous control. Results are expressed as 2−ΔΔCT and show the fold increase in gene expression compared with the control group. Bio-Rad iQ5 software (Bio-Rad Laboratories) was used to perform the data analysis and generate the standard curve.
Statistical analysis
All data are presented as the mean ± standard error of the mean. The results were evaluated using one-way analysis of variance and Tukey’s multiple comparison tests. A value of P<0.05 was considered to indicate a statistically significant difference.
Results
Effect of EPO on serum levels of ALT and AST in a D-GalN/LPS-induced model of FHF
ALT and AST are two important indicators of liver function. As shown in Fig. 1, ALT and AST levels were observed to be significantly elevated in the mice in the D-GalN/LPS group compared with those in the normal group (P<0.01), indicating severe liver injury in the D-GalN/LPS mice. Pretreatment with various doses of EPO was found to reduce the increases in ALT and AST by varying degrees. In the mice in the 10,000 U/kg EPO group, serum AST and ALT activities were significantly reduced compared with activities in the D-GalN/LPS group (P<0.01).
Effect of EPO on MDA, SOD and GSH-Px levels
As indicated in Fig. 2, 8 h after D-GalN/LPS administration, the concentration of MDA, a marker of free radical-induced injury, was determined. The activities of the antioxidant enzymes SOD and GSH-Px were also analyzed. Liver tissue MDA levels were increased significantly in the D-GalN/LPS group compared with those in the normal group (P<0.01). However, the activities of SOD and GSH-Px were markedly reduced in the mice in the D-GalN/LPS group compared with those in the normal group (P<0.01). The elevation in MDA was significantly attenuated with 3,000 and 10,000 U/kg EPO compared with the D-GalN/LPS group (P<0.01). Furthermore, EPO pretreatment was found to enhance the activities of SOD and GSH-Px.
EPO alleviates liver injury in D-GalN/LPS-induced FHF mice
In the normal group, the hematoxylin and eosin-stained liver sections exhibited an integrated architecture of hepatic lobules and normal cell structure without necrosis (Fig. 3). However, injection with D-GalN/LPS was found to induce destruction of the liver structure, severe hepatocyte necrosis, hemorrhage and inflammatory cell infiltration. Liver tissue damage was ameliorated in the mice pretreated with EPO, compared with those in the D-GalN/LPS group.
Effects of EPO on the expression of EPOR and PI3K
In order to further investigate the mechanism underlying the effect of EPO on FHF, the hepatic expression of EPOR and PI3K was assessed in the different groups. The expression of EPOR and PI3K was calculated relative to that of the endogenous control, GAPDH. As shown in Fig. 4, the expression of EPOR and PI3K in the mice in the D-GalN/LPS group was significantly decreased compared with that in the normal group (P<0.05). However, pretreatment with EPO was found to significantly increase the mRNA expression of EPOR and PI3K mRNA in comparison with the D-GalN/LPS group (P<0.05).
Discussion
D-GalN/LPS-treated mouse models are frequently used to study the pathogenesis of liver injury (19–21). D-GalN is a specific hepatotoxin that selectively depletes uridine nucleotides, ultimately inhibiting macromolecule synthesis in hepatocytes, which results in abnormalities in the structure and function of hepatic cells. In addition, D-GalN may act synergistically with LPS. Activated neutrophils, which accumulate around damaged liver cells, undergo bursts of respiration and degranulation, releasing oxygen free radicals and leading to lipid peroxidation. Oxygen free radicals and lipid peroxidation target liver parenchyma and vascular endothelial cells, resulting in cell damage or death (22,23). In the present study, intraperitoneal injection of D-GalN/LPS in mice resulted in severe hepatic injury, which was associated with elevated serum activity of AST and ALT. Histopathological analysis also showed that D-GalN/LPS induced severe necrosis in hepatocytes, hemorrhage and inflammatory cell infiltration. Reactive oxygen species have a major role in the initiation of D-GalN/LPS-induced liver injury (24,25), which is consistent with the findings of the present study. MDA, a marker of lipid peroxidation, was observed to be present in significantly higher levels in the liver tissue of mice in the D-GalN/LPS group compared with mice in the normal group, and the activity of SOD and GSH-Px, antioxidant markers, was significantly lower.
The present study showed that, in the mice pretreated with EPO, the serum activity of AST and ALT was decreased and the degree of liver tissue damage was ameliorated, compared with mice in the D-GalN/LPS group. This suggests that EPO has a strong hepatoprotective effect on D-GalN/LPS-induced liver injury. EPO has been shown to exert antioxidative effects against hypoxic-ischemic brain injury (26) and chemical toxins (27). In the present study, intraperitoneal administration of EPO decreased the level of MDA and increased the activity of antioxidant enzymes, including SOD and GSH-Px, in the liver tissue compared with the D-GalN/LPS group. These findings are in accordance with those of other in vivo studies (28–30) and the histopathological observations.
EPO has been proposed to be a tissue-protective hormone with pleiotropic potential. EPO exerts protective effects against tissue destruction surrounding sites of injury by signaling through a nonhemopoietic receptor (31). In addition, when exerting its extra-hematopoietic effects, EPO has been shown to bind to heterodimeric EPOR, which consists of EPOR and the β common receptor (32). One of the most important signaling pathways is the PI3K/Akt pathway. The PI3K/Akt signaling pathway is an important pathway for survival, and protects the body against various stresses. The activation of the PI3K/Akt pathway regulates cell apoptosis, survival and proliferation through the regulation of its downstream factors. Studies have shown that this pathway has a positive protective effect in liver ischemia-reperfusion injury (33–35). Furthermore, another study found that EPO has a protective effect against injury in proximal tubule cells and cardiac myocytes by activating EPOR, which leads to activation of PI3K and ultimately Akt (36). Thus, it was of interest to examine the effect of EPO pretreatment on the PI3K/Akt pathway in D-GalN/LPS-induced acute liver failure in a murine model. The present study showed that EPOR and PI3K gene expression was significantly decreased in D-GalN/LPS mice, while pretreatment with EPO significantly increased EPOR and PI3K mRNA expression in a dose-dependent manner. Therefore, EPO may exert its hepatoprotective effects through upregulation of EPOR and PI3K.
In conclusion, to the best of our knowledge, the results of the present study are the first to demonstrate a protective effect of exogenous EPO against D-GalN/LPS-induced hepatic injury. In addition, the mechanism underlying the protective effect of EPO has been shown to be associated with antioxidant properties and the upregulation of EPOR and PI3K expression. These findings may contribute to the development of novel agents for the treatment of FHF. However, further investigations are required to determine the precise protective mechanism.