Inhibition of RKIP aggravates thioacetamide-induced acute liver failure in mice.

Accumulating evidence has indicated that Raf kinase inhibitor protein (RKIP) is involved in several intracellular signaling pathways; its abnormal expression is associated with tumor progression and metastasis in several human neoplasms. However, the role of RKIP in acute liver injury has remained elusive. In the present study, acute liver failure was induced by thioacetamide in mice, and locostatin was used to interfere with RKIP expression. It was found that RKIP expression was significantly inhibited by locostatin. Down-regulation of RKIP expression resulted in severe liver injury and extensive release of alanine aminotransferase and aspartate aminotransferase. In addition, reduced RKIP expression significantly enhanced the levels of reactive oxygen species and the content of pro-inflammatory factors such as tumor necrosis factor-α as well as interleukin-6 and -1β, and decreased the levels of nuclear factor E2-related factor-2 and heme oxygenase-1. Furthermore, down-regulation of RKIP promoted the activation of the nuclear factor-κB and extracellular signal-regulated kinase signaling pathways. In conclusion, the present study indicates an inverse correlation between RKIP level and the degree of hepatic injury, that is, a decrease in RKIP expression may exacerbate acute liver failure.

Introduction

Acute liver injury usually arises from variety of reasons such as viral infection, autoimmune disorders, ischemia and xenobiotics, and results in severe clinical problems such as hepatic encephalopathy, severe infection and multiple organ failure. Hepatic inflammation is the hallmark of liver injury, fibrosis, cirrhosis and even cancer (1). Thus, inhibition of oxidative stress and inflammation is effective in the prevention and treatment of liver injury. It has been reported that mitogen-activated protein kinases (MAPKs) mediate the signaling cascades leading to the expression of pro-inflammatory genes (2). Furthermore, nuclear factor κB (NF-κB) directly regulates inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and inducible nitric oxide synthase (iNOS), promoting the inflammatory response (3). Therefore, the MAPK and NF-κB pathways have been considered as effective targets for therapeutics against various inflammatory diseases.

Raf kinase inhibitor protein (RKIP), the extracellular signal-regulated kinase (ERK)/MAPK pathway inhibitor (4), has been reported to have a vital role in cell proliferation, apoptosis and metastasis in numerous tumor cell types (5,6). However, the role of RKIP in acute liver injury has remained to be fully elucidated. In the present study, acute liver failure was induced in mice by thioacetamide (TAA) and locostatin was used to interfere with RKIP expression. The biological role of RKIP on the severity of liver injury was assessed.

Materials and methods

Materials

Locostatin, an RKIP-specific inhibitor (7), was obtained from MerckKGaA (Darmstadt, Germany). TAA was purchased from Sigma-Aldrich (Merck KGaA). Alanine aminotransferase (ALT; 20160105) and aspartate aminotransferase (AST; 20160329) were purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). TNF-α ELISA kit (KGEHC103a-1) was purchased from NanjingKeygen Biological Technology (Nanjing, China). ELISA kits for IL-6 (2015116250) and IL-1β (201510730) were obtained from Beijing Huaying Biological Technology (Beijing, China).

TAA-induced acute liver failure in mice and drug administration

A total of 60 male ICR mice (6 weeks in age), weighing 18–22 g, were obtained from the Experimental Animal Center of Guangxi Medical University (Guangxi, China) and the animal experiment was approved by the Ethical Committee of Guangxi Medical University (Guangxi, China). The animals were housed under controlled conditions at 25±2°C, a relative humidity of 60±10%, room air changes 12–18 times/h and a 12-h light/dark cycle. Food and water were available ad libitum.

Acute hepatic injury was induced by TAA as previously described (8). In brief, 60 mice were divided into 4 groups (15 animals/group): Normal control, locostatin control, TAA model and TAA plus locostatin (TL) groups. The mice in the locostatin control and TL groups were administered locostatin (0.5 mg/kg) intraperitoneally, while the animals in the normal and TAA model groups received an equivalent volume of normal saline once a day for 7 days. At the end of the pre-treatment, the animals in the TAA model and TL groups were injected intraperitoneally with 300 mg/kg TAA once a day for 2 days, whereas the mice in the normal and locostatin control groups were injected with an equivalent volume of saline. At 24 h after the last TAA administration, all of the animals were sacrificed, and blood and liver samples were collected. A proportion of each of the livers was fixed in formalin, while the others were stored at −80°C for further experiments.

Immunohistochemical (IHC) analysis of hepatic RKIP

The livers were fixed in formalin, embedded in paraffin and sectioned into 5-µm slices. The localization and expression of RKIP was then observed by IHC staining according to the protocol of a previous study (9).

Histological examination of liver tissues

Liver tissues were fixed in 10% formalin, embedded in paraffin and sectioned at 5-µm thickness. The liver pathology was observed by hematoxylin-eosin staining as described previously (10,11).

Determination of ALT and AST activities

Serum AST and ALT activities were measured using the commercial kits according to the manufacturer's instructions.

Measurement of reactive oxygen species (ROS) generation in liver tissues

Dichlorofluorescein diacetate (DCFH-DA; Molecular Probes; Thermo Fisher Scientific, Inc., Waltham, MA, USA) reacts with ROS to produce the highly fluorescent compound dichlorofluorescein (DCF), which is an indicator to reflect the level of ROS. In the present study, the fluorescent product DCF was measured using a spectrofluorimeter with excitation at 484 nm and emission at 530 nm as previously described (10). DCFH-DA in the absence of homogenates was used to determine background fluorescence.

Determination of inflammatory cytokines in serum

Serum TNF-α, IL-6 and IL-1β content was determined using respective ELISA kits according to the manufacturer's instructions.

Assessment of NF-κB activity

Nuclear protein was isolated from liver tissues by using a Nuclear Extraction kit (ActiveMotif, Carlsbad, CA, USA). The activity of NF-κB-p65 was then detected using an NF-κB/p65 ActivELISA kit (Imgenex, San Diego, CA, USA) as previously described (11).

Western blot analysis

Total hepatic proteins were extracted from liver tissues using radioimmunoprecipitation assay buffer containing a protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). The protein concentration of the tissue homogenate was determined according to our previous study (11) with bovine serum albumin (BSA) as a standard. Protein (60 µg) from liver homogenates was loaded per lane on 10% polyacrylamide gels and electrophoresed. Proteins were transferred to nitrocellulose membranes and the membrane was blocked overnight in 4% non-fat dry milk in PBS with 0.2% Tween-20 at 4°C and then incubated with the primary antibodies including RKIP, ERK, phosphorylated (p)-ERK, p38, p-p38, c-Jun N-terminal kinase (JNK), p-JNK, NF-κB-p65 (p65), p-p65, inhibitor of NF-κB α (IκBα), p-IκBα, nuclear factor E2-related factor-2 (Nrf2), heme oxygenase-1 (HO-1) and GAPDH (1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight. The membranes were subsequently washed 3 times in Tris-buffered saline with Tween 20 for 15 min each and incubated with 1:5,000 dilution of alkaline phosphatase conjugated goat anti-mouse IgG (Calbiochem; San Diego, CA, USA) for 1 h at 37°C. The protein was visualized with an enhanced chemiluminescence western blotting detection kit (GE Healthcare; Chicago, IL, USA). The membranes were finally exposed to X-ray film for 1 min. The relative expression of various proteins was quantified by densitometric scanning with ImageJ 1.38 Software (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis

All group values are expressed as the mean ± standard deviation. Data were evaluated using SPSS 11.5 for Windows (SPSS Inc., Chicago, IL, USA). Differences between groups were tested for statistical significance using one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

Results

Hepatic RKIP expression

IHC staining revealed that RKIP was primarily localized in the cytoplasm of hepatic cells. The RKIP-positive cells were significantly decreased in the TL group compared with those in the TAA model group (Fig. 1A). Similarly, western blot analysis demonstrated that locostatin treatment of TAA-induced mice led to a significant decrease in RKIP expression (Fig. 1B).

Figure 1.

Locostatin enhances the reduction of RKIP in liver tissues of mice with acute liver injury. (A) Immunohistochemical staining (magnification, ×400). The arrows indicate RKIP-positive cells (dark brown). (B) Hepatic RKIP expression was assessed by western blot analysis. Lanes: 1, Normal control; 2, locostatin control; 3, TAA model; 4, TL group. Values are expressed as the mean ± standard deviation (n=15); *P<0.05 vs. normal control group; #P<0.05 vs. TAA model group. TAA, thioacetamide; RKIP, Raf kinase inhibitor protein; TL, TAA+ locostatin.

Inhibition of RKIP aggravates liver injury

The histological examination revealed that the hepatic tissues in the normal control and locostatin control groups had a normal structure, and the hepatic cells were neatly arranged (Fig. 2A1 and A2), while the liver tissues from the TAA model group were edematous with fatty degeneration, and lobular architecture was destroyed or had disappeared (Fig. 2A3). Of note, compared with the TAA model group, locostatin treatment led to more severe damage, such as steatosis and hepatic lesions (Fig. 2A4).

Figure 2.

Inhibition of Raf kinase inhibitor protein by locostatin aggravates TAA-induced acute liver injury. (A) Hepatic histological changes were examined by hematoxylin and eosin staining (magnification, ×200). (B) AST and ALT activities were detected by using commercially available kits. Values are expressed as the mean ± standard deviation (n=15); *P<0.05 vs. normal control group; #P<0.05 vs. TAA model group. TAA, thioacetamide; TL, TAA+ locostatin; AST, aspartate aminotransferase; ALT, alanine amiontransferase.

Serum ALT and AST are the important indicators of liver injury. The activities of the two enzymes were significantly increased in the TAA model group, which were further enhanced by treatment with locostatin (Fig. 2B). These results indicated that inhibition of RKIP expression aggravates liver injury.

Inhibition of RKIP promotes ROS generation and pro-inflammatory cytokine production in mice with acute liver injury

Increasing ROS levels indicate the production of free radicals, leading to oxidative stress, which is crucial in acute hepatic disorders. As presented in Fig. 3, the levels of ROS, TNF-α, IL-6 and IL-1β in the TL group were higher than those of the TAA model group, suggesting that inhibition of RKIP aggravated hepatic injury largely due to oxidative stress and inflammatory response.

Figure 3.

Inhibition of Raf kinase inhibitor protein by locostatin increases the production of ROS in the liver and TNF-α, IL-6 and IL-1β in the sera of mice with acute liver injury. (A) ROS production in liver tissues was detected using the DCFH-diacetate method. (B) The production of TNF-α, IL-6 and IL-1β in liver tissues was detected by commercially available kits. Values are expressed as the mean ± standard deviation (n=15); *P<0.05 vs. normal control group; #P<0.05 vs. TAA model group. TAA, thioacetamide; TL, TAA+ locostatin; ROS, reactive oxygen species; TNF, tumor necrosis factor; IL, interleukin; DCFH, dichloro-dihydro-fluorescein.

Inhibition of RKIP decreases Nrf2 and HO-1 levels in mice with acute liver injury

As displayed in Fig. 4, the expression of Nrf2 and HO-1 was markedly lowered in the TAA model group compared with the normal control group. Furthermore, locostatin treatment resulted in a significant decrease in the levels of Nrf2 and HO-1 compared with those in the TAA model group, suggesting that the inhibition of RKIP aggravates oxidative stress in acute liver injury, at least in part, by suppressing Nrf2 and HO-1 levels.

Figure 4.

Inhibition of Raf kinase inhibitor protein by locostatin inhibits Nrf2 and HO-1 expression in the livers of mice with acute liver injury. The expression of (A) Nrf2 protein and (B) HO-1 protein was detected by western blot analysis. Lanes: 1, Normal control; 2, locostatin control; 3, TAA model; 4, TL group. Values are expressed as the mean ± standard deviation (n=15); *P<0.05 vs. normal control group; #P<0.05 vs. TAA model group. TAA, thioacetamide; TL, TAA+ locostatin; Nrf2, nuclear factor E2-related factor-2; HO-1, heme oxygenase-1.

Inhibition of RKIP increases NF-κB activation in mice with acute liver injury

To fully understand the role of RKIP in the inflammatory response, the NF-κB activity in liver tissues was determined by ELISA. As presented in Fig. 5A, compared with the TAA model group, the NF-κB activity in the TL group was significantly elevated. In addition, western blot analysis revealed that in the TAA model group, IκBα and p65 phosphorylation was significantly increased compared with that in the normal control group, which was significantly amplified by locostatin treatment (Fig. 5B and C). These results suggested that inhibition of RKIP enhances NF-κB pathway activation during acute liver injury.

Figure 5.

Inhibition of Raf kinase inhibitor protein by locostatin induces NF-κB activation in the livers of mice with acute liver injury. (A) The activity of NF-κB in liver tissues was detected using an ELISA kit. The phosphorylation of (B) IκBα and (C) NF-κB-p65 (p65) was detected by using western blot analysis. Lanes: 1, Normal control; 2, locostatin control; 3, TAA model; 4, TL group. Values are expressed as the mean ± standard deviation (n=15); *P<0.05 vs. normal control group; #P<0.05 vs. TAA model group. TAA, thioacetamide; TL, TAA+ locostatin; p-NF-κB, phosphorylated nuclear factor κB; IκBα, inhibitor of NF-κB α.

Inhibition of RKIP induces ERK/MAPK pathway activation in mice with acute liver injury

During the inflammatory response, the ERK/MAPK signaling pathway is activated, thereby promoting the expression of numerous pro-inflammatory genes. As presented in Fig. 6, compared with that in the TAA group, locostatin treatment significantly increased the phosphorylation of ERK, p38 and JNK in liver tissues, which indicated that inhibition of RKIP enhanced the activation of the ERK/MAPK pathway during acute liver injury.

Figure 6.

Inhibition of Raf kinase inhibitor protein by locostatin enhances ERK/mitogen-activated protein kinase pathway activation in the livers of mice with acute liver injury. The phosphorylation of (A) ERK, (B) p38 and (C) JNK was detected by western blot. Lanes: 1, Normal control; 2, locostatin control; 3, TAA model; 4, TL group. Values are expressed as the mean ± standard deviation (n=15); *P<0.05 vs. normal control group; #P<0.05 vs. TAA model group. TAA, thioacetamide; TL, TAA+ locostatin; p-ERK, phosphorylated extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase.

Discussion

RKIP is a specific ERK/MAPK pathway inhibitor (12,13), which plays a vital role in cell proliferation, apoptosis and metastasis of tumor cells (5,6). However, its explicit function in acute liver injury has remained to be fully elucidated. In the present study, a mouse model of TAA-induced acute hepatic failure was generated and locostatin was administered to interfere with RKIP expression. The results demonstrated that RKIP expression was significantly inhibited by locostatin administration. TAA treatment induced severe histological changes in the liver and significantly increased the activities of serum ALT and AST. Of note, locostatin enhanced the severity of histological damage and led to a more significant increase in the activity of the two enzymes compared with those in the TAA model group. These results indicated that inhibition of RKIP aggravates TAA-induced acute liver injury.

Numerous studies have reported that TAA-induced liver injury is characterized by increased oxidative stress and impairment of the anti-oxidant defense. Elevated ROS are an important indicator of oxidative stress and their generation contributed to the accumulation of lipid oxidation products, leading to cell necrosis and liver injury (14). Inflammation is another important pathological mechanism propagating TAA-induced liver injury (15). A significant amount of pro-inflammatory mediators is released during the inflammatory process. Pro-inflammatory cytokines such as IL-6, IL-1β and TNF-α are known to be crucial in inflammatory processes and hepatic damage. The results of the present study demonstrated that locostatin significantly increased the levels of ROS, IL-6, IL-1β and TNF-α compared with those in the TAA model group, suggesting that inhibition of RKIP aggravates liver injury partly by promoting oxidative stress and inflammatory response.

Nrf2 is one of the important redox-sensitive transcription factors (16). Under oxidative stress conditions, Nrf2 is dissociated from Kelch-like ECH-associated protein1 and translocates into the nucleus to promote the expression of numerous anti-oxidant defense genes (17), thereby protecting the liver from oxidative insult (18). In addition, HO-1 is a stress protein induced in response to a variety of oxidative challenges and has a protective role in liver injury (19). In the present study, locostatin administration notably decreased the expression of Nrf2 and HO-1 compared with that in the TAA model group. This finding suggested that inhibition of RKIP exacerbates oxidative injury, at least in part, by inhibiting Nrf2 and HO-1, thereby destroying the cellular balance between oxidants and anti-oxidants.

In order to explore the underlying mechanisms of the roles of RKIP in the inflammatory response, the present study further assessed the NF-κB pathway. Activation of NF-κB has a central role in inflammation through its ability to induce the transcription of pro-inflammatory genes (20). The rapid phosphorylation of IκBα and its subsequent degradation following exposure of cells to external stimuli, such as carcinogens, inflammatory cytokines and reactive oxygen species, leads to increased nuclear translocation and DNA binding of NF-κB. The results of the present study demonstrated that the phosphorylation of IκBα and p65 in the TL group were higher than those in the TAA model group, which suggested that inhibition of RKIP enhanced NF-κB pathway activation.

MAPKs are serine-threonine protein kinases that have important roles in signal transduction from the cell surface to the nucleus (21). The MAPK pathway is known to be influenced not only by receptor ligand interactions, but also by different stressors placed on the cell. One type of stress that induces potential activation of the MAPK pathway is the oxidative stress caused by ROS. In general, increased ROS production in a cell leads to the activation of the major MAPK family proteins (ERK, JNK or p38), which further enhances the production of certain pro-inflammatory cytokines (22). Persistent activation of the MAPK signaling pathway has been revealed to increase the development of human inflammatory diseases due to the induction of iNOS expression (23). It has been reported that RKIP directly interacts with Raf-1 and MAPK kinase (MEK) and disrupts the Raf-1/MEK interaction, thereby preventing MEK activation and its downstream targets (24). Over-expression of RKIP suppressed MAPK signaling, while down-regulation of RKIP had the opposite effect (25). In the present study, treatment with locostatin significantly increased the phosphorylation of JNK, p38 and ERK in liver tissues. This result indicated that inhibition of RKIP promotes ROS generation and oxidative stress in mice with liver failure, partly through enhancing the MAPK pathway and subsequently exacerbating the inflammatory response.

In conclusion, the present study indicated that inhibition of RKIP may be a factor that aggravates acute liver injury, which may provide a possible target for the prevention and treatment of liver failure in the future. However, to verify the potential therapeutic target, further studies are required to be performed; for instance, it should be investigated whether over-expression of RKIP alleviates liver failure.