Nobiletin attenuates lipopolysaccharide/D‑galactosamine‑induced liver injury in mice by activating the Nrf2 antioxidant pathway and subsequently inhibiting NF‑κB‑mediated cytokine production

Introduction

Acute liver failure (ALF) is a severe condition, which is associated with a high rate of mortality resulting from excessive hepatocyte death in a short time (1). At present, there are limited effective preventative and therapeutic strategies for the treatment of this disease, short of liver transplantation (2,3). Therefore, there is an urgent need to develop novel drugs to control this condition. Lipopolysaccharide (LPS)- and D-galactosamine (GalN)-induced acute liver injury in mice is a well-established animal model that may accurately represent clinical symptoms in humans (4). It is widely used to investigate the underlying mechanisms and potential therapies for ALF. GalN is a specific hepatotoxic agent that inhibits RNA and protein synthesis in hepatocytes (4). LPS induces the production of inflammatory cytokines, resulting in subsequent liver tissue injury (5). Furthermore, LPS induces Kupffer cell activation via the Toll-like receptor 4 signaling pathway, and subsequently activates nuclear factor (NF)-κB and initiates the release of inflammatory cytokines, including interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α (6,7).

Nobiletin is an O-methylated flavonoid present in citrus peels, with an empirical formula of C21H22O8 and a molecular weight of 402.39, which has been reported to possess anti-inflammatory and antioxidant properties (8–10). Previous studies have reported that nobiletin may protect against cisplatin-induced acute kidney injury by preserving renal function and restoring antioxidant status (11). Furthermore, a Citrus aurantium extract, which is rich in nobiletin and tangeretin, has been demonstrated to inhibit ethanol-induced liver injury in mice via modulation of AMP-activated protein kinase and NF-E2-related factor 2 (Nrf2)-related signals (12). In addition, nobiletin and tangeretin have been reported to suppress LPS-induced osteoclast formation and bone resorption in an experimental model of periodontitis (13). However, the effects of nobiletin on LPS/GalN-induced liver injury remain unclear. The present study aimed to determine the effects of nobiletin on LPS/GalN-induced liver injury in mice.

Materials and methods

Animals

Male C57BL/6 mice (weight, 18–22 g; age, 6 weeks) were purchased from the Center of Experimental Animals of Chongqing Medical University (Chongqing, China). The mice were housed in an environmentally controlled room (temperature, 24±1°C; humidity, 40–80%) under a 12-h dark/light cycle with free access to food and water. All animal experimentation described in the present study was performed according to the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The study was approved by the committee on the Ethics of Animal Experiments of Chongqing Medical University.

Experimental design

A total of 50 mice were randomly divided into five groups: PBS control group, LPS/GalN group and nobiletin (50, 100 and 200 mg/kg) + LPS/GalN groups. To induce acute liver injury, the mice were intraperitoneally injected with LPS (100 µg/kg; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) and GalN (700 mg/kg; Sigma-Aldrich; Merck Millipore). A total of 50, 100 or 200 mg/kg nobiletin (92600; Sigma-Aldrich; Merck Millipore) was administered intraperitoneally 2 h prior to LPS/GalN injection. The mice were sacrificed under anesthesia with sodium pentobarbital (40 mg/kg, intraperitoneally). Blood and liver tissues were subsequently collected for analysis.

Serum hepatic enzymes

Following LPS and GalN injection, all groups were fasted for 24 h, and blood samples were collected from the eyeballs. Each blood sample was incubated for 30 min at room temperature, and was then centrifuged at 1,200 × g for 10 min at 4°C. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were analyzed using an automatic serum analyzer (7600–120; Hitachi High-Technologies Corporation, Tokyo, Japan).

ELISA assay

Liver tissue samples were homogenized in ice-cold PBS (Wuhan Boster Biological Technology Co., Ltd., Wuhan, China) and were centrifuged at 1,200 × g for 20 min at 4°C. Supernatants were collected and TNF-α, IL-1β and IL-6 expression was analyzed using commercially available ELISA kits (cat. nos. EK0394, EK0411 and EK0527; Wuhan Boster Biological Technology Co., Ltd.) according to the manufacturer's protocols. The absorbance was measured at 450 nm using a microplate reader (Bio-Tek ELx800; Bio-Tek Instruments, Inc., Winooski, VT, USA).

Histological analysis

Liver tissues collected from the mice were fixed with 10% formalin (10D15B, Wuhan Boster Biological Technology Co., Ltd.) for 24 h at room temperature, embedded in paraffin wax and cut into 6-µm sections. For hematoxylin and eosin staining, the slides bearing tissue sections were stained with hematoxylin (Beyotime Institute of Biotechnology, Haimen, China) for 5 min and 1% eosin solution (Beyotime Institute of Biotechnology) for 30 sec at room temperature. Following hematoxylin and eosin staining, the slides were observed for conventional morphological evaluation under a light microscope (Eclipse TE2000-U; Nikon Corporation, Tokyo, Japan) and images were captured at ×400 magnification.

Western blot analysis

A total of 24 h after LPS/GalN injection, liver tissues were harvested and homogenized in radioimmunoprecipitation assay lysis buffer (P0013D; Beyotime Institute of Biotechnology) to prepare whole protein extracts. Tissue lysates were centrifuged at 12,000 × g for 10 min at 4°C. A nuclear/cytoplasmic protein extraction kit (P0028; Beyotime Institute of Biotechnology) was used to extract nuclear proteins in accordance with the manufacturer's protocol. Protein concentrations were quantified by bicinchoninic acid assay (Beyotime Institute of Biotechnology) and aliquots of 50 µg per sample of liver tissue homogenates or nucleus fractionation were electrophoresed. Subsequently, proteins were separated by 10% SDS-PAGE and were transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked for 1 h at 37°C using bovine serum albumin (Beyotime Institute of Biotechnology) Each membrane was incubated separately with the following rabbit primary antibodies: Anti-inducible nitric oxide synthase (iNOS) (sc-8310; 1:600), anti-cyclooxygenase (COX)-2 (sc-7951; 1:600), anti-NF-κB (sc-372; 1:500), anti-phosphorylated (p)-inhibitor of NF-κB (IκB) (sc-101713; 1:500), anti-IκB (sc-371, 1:500), anti-Nrf2 (sc-722; 1:500), anti-heme oxygenase (HO)-1 (sc-10789; 1:500), anti-GADPH (sc-25778; 1:1,000) and anti-TATA box binding protein (sc-33736; 1:1,000) (all Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Subsequently, the membranes were incubated with horseradish peroxidase-linked goat anti-rabbit immunoglobulin G (1:10,000; cat. no. 7074; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature. Protein bands were visualized using a western blotting detection system (ChemiDoc™ XRS+; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and analyzed using a densitometry system (Quantity One v4.6.2; Bio-Rad Laboratories, Inc.) according to the manufacturer's protocol.

Statistical analysis

An independent sample t-test was used to determine the statistical differences between two groups. Multiple group comparisons were tested using one-way analysis of variance, followed by Tukey post hoc test and a 95% confidence interval (P<0.05) was accepted as indicative of significance. All statistical analyses were performed using SPSS 12.0 statistical software (SPSS, Inc., Chicago, IL, USA). All experiments were repeated three times and data are presented as the mean + standard error.

Results

Nobiletin inhibits LPS/GalN-induced serum ALT and AST levels

To assess the protective effects of nobiletin on LPS/GalN-induced liver injury in mice, serum ALT and AST levels were detected 24 h after LPS/GalN treatment. As shown in Fig. 1, ALT and AST levels were significantly increased after LPS/GalN treatment. However, the increased levels were attenuated following administration of nobiletin (50, 100 and 200 mg/kg).

Figure 1. Effects of nobiletin on serum (A) ALT and (B) AST levels. Data are presented as the mean ± standard error of the mean (n=10/group). #P<0.01 vs. control group, *P<0.05 and **P<0.01 vs. LPS/GalN group. ALT, alanine aminotransferase; AST, aspartate aminotransferase; LPS, lipopolysaccharide; GalN, D-galactosamine.

LPS/GalN-induced histopathological changes are reduced by nobiletin treatment

The histopathological state of the liver tissues was observed under a light microscope. As shown in Fig. 2, liver sections from the normal control group exhibited normal lobular architecture and cellular structure. Liver sections from the LPS/GalN group exhibited marked pathological alterations, including extensive areas of portal inflammation, cellular necrosis, and inflammatory cell infiltration. However, these pathological alterations were markedly attenuated in the nobiletin-treated groups.

Figure 2. Effects of nobiletin on histopathological changes in liver tissues. Representative histological changes in liver tissues obtained from mice of various groups. (A) Control group, (B) LPS/GalN group, (C) LPS/GalN + nobiletin (50 mg/kg) group, (D) LPS/GalN + nobiletin (100 mg/kg) group, (E) LPS/GalN + nobiletin (200 mg/kg) group (hematoxylin and eosin staining, magnification 400x). LPS, lipopolysaccharide; GalN, D-galactosamine.

Nobiletin inhibits hepatic IL-1β, IL-6 and TNF-α production

To determine the anti-inflammatory effects of nobiletin, hepatic IL-1β, IL-6 and TNF-α production was detected using ELISA kits. As presented in Fig. 3, IL-1β, IL-6 and TNF-α levels were significantly increased in the LPS/GalN group compared with in the control group. Nobiletin treatment (50, 100 and 200 mg/kg) significantly suppressed LPS/GalN-induced hepatic IL-1β, IL-6 and TNF-α levels in a dose-dependent manner.

Figure 3. Effects of nobiletin on hepatic (A) IL-1β, (B) IL-6 and (C) TNF-α levels. Data are presented as the mean ± standard error of the mean (n=10/group). #P<0.01 vs. control group, *P<0.05 and **P<0.01 vs. LPS/GalN group. IL, interleukin, TNF-α, tumor necrosis factor-α; LPS, lipopolysaccharide; GalN, D-galactosamine.

Nobiletin inhibits hepatic iNOS and COX-2 expression

Hepatic iNOS and COX-2 expression levels were detected 24 h after LPS/GalN treatment by western blot analysis. The results indicated that iNOS and COX-2 levels were significantly increased in the LPS/GalN group compared with in the control group. Treatment with nobiletin (50, 100 and 200 mg/kg) dose-dependently reduced hepatic iNOS and COX-2 levels (Fig. 4).

Figure 4. Effects of nobiletin on hepatic iNOS and COX-2 expression. Data are presented as the mean ± standard error of the mean (n=10/group). #P<0.01 vs. control group, **P<0.01 vs. LPS/GalN group. iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; LPS, lipopolysaccharide; GalN, D-galactosamine.

Nobiletin inhibits NF-κB activation

NF-κB has been reported to have a dominant role in the inflammatory response (14). In the present study, the effects of nobiletin on the degradation of IκBα and the activation of NF-κB were detected by western blot analysis. The results revealed that LPS/GalN exposure induced phosphorylation and degradation of IκBα, and NF-κB p65 translocation into the nucleus; however, these effects were attenuated following nobiletin treatment (Fig. 5).

Figure 5. Nobiletin inhibits LPS/GalN-induced (A) IκBα phosphorylation and (B) NF-κB activation, as determined by western blotting. Data are presented as the mean ± standard error of the mean (n=10/group). #P<0.01 vs. control group, **P<0.01 vs. LPS/GalN group. IκBα, inhibitor of NF-κB α; NF-κB, nuclear factor-κB; LPS, lipopolysaccharide; GalN, D-galactosamine.

Nobiletin promotes the expression of Nrf2 and HO-1

Nrf2 is considered an important transcription factor in regulation of the antioxidant response (15). In the present study, the effects of nobiletin were detected on Nrf2 and HO-1 expression by western blot analysis. As shown in Fig. 6, the expression levels of Nrf2 in the nucleus and HO-1 in the cytoplasm were significantly increased 24 h after LPS/GalN treatment. Notably, these increases in Nrf2 and HO-1 expression were augmented by nobiletin.

Figure 6. Nobiletin augmented the expression of Nrf2 and HO-1. Data are presented as the mean ± standard error of the mean (n=10/group). #P<0.01 vs. control group, *P<0.05 and **P<0.01 vs. LPS/GalN group. HO-1, heme oxygenase-1; Nrf2, NF-E2-related factor 2; TBP, Tata box binding protein; LPS, lipopolysaccharide; GalN, D-galactosamine.

Discussion

The present study demonstrated that nobiletin exerts a protective effect on LPS/GalN-induced acute liver injury in mice. The results indicated that nobiletin treatment significantly attenuated hepatic pathological damage, hepatic enzyme release, proinflammatory cytokine production and inflammatory cell infiltration. Furthermore, the results revealed that nobiletin inhibits NF-κB activation, and upregulates the expression levels of Nrf2 and HO-1. Therefore, the present study suggests that nobiletin may be considered a promising therapeutic reagent for the treatment of acute liver injury.

ALT and AST are two transaminase enzymes that are important for the synthesis of amino acids. ALT is predominantly present in the liver, whereas AST is also present in other organs and tissues. Significantly elevated levels of ALT and AST are often associated with hepatic ailments, such as viral hepatitis, liver damage and bile duct problems; and are therefore commonly used for assessing the progression of liver disease (16). Treatment with LPS/GalN can increase hepatocyte membrane permeability and induce hepatocyte necrosis, which may increase the levels of AST and ALT. In the present study, serum ALT and AST levels, and histological analysis of liver tissues, were used to assess the protective effects of nobiletin on LPS/GalN-induced acute liver injury. The results indicated that nobiletin significantly decreased serum ALT and AST levels. Furthermore, histological analysis demonstrated that nobiletin attenuated cellular swelling and necrosis in the liver, and inhibited inflammatory cell infiltration. These results suggested that nobiletin may exert a protective effect on LPS/GalN-induced hepatic injury.

Inflammation is an important pathological mechanism responsible for propagating LPS/GalN-induced liver injury (17). Previous studies have revealed that LPS may activate Kupffer cells, which can mediate the hepatic inflammatory process by producing TNF-α, IL-1β and IL-6, as well as other proinflammatory cytokines (18,19). TNF-α is an important inflammatory mediators involved in LPS/GalN-induced liver injury, which can potentially induce apoptosis of hepatocytes, subsequently leading to organ failure (20–22). In addition, TNF-α may initiate the inflammatory cascade and induce the production of other cytokines, including IL-1β and IL-6 (23). Previous studies have reported that inhibition of TNF-α synthesis inhibits cytokine production and attenuates liver injury (24). In addition, iNOS and COX-2 are important proinflammatory enzymes involved in the development of liver injury (25,26). Elevated iNOS and COX-2 levels are often observed in LPS/GalN-induced liver injury in mice (27). In the present study, nobiletin significantly inhibited LPS/GalN-induced increases in hepatic TNF-α, IL-1β, IL-6, COX-2 and iNOS levels.

NF-κB is an upstream regulator of various genes associated with the inflammatory response (28). Once activated by LPS, NF-κB p65 dissociates from its inhibitory protein IκBα and translocates from the cytoplasm to the nucleus, where it may induce transcription of inflammatory mediators, such as TNF-α, COX-2 and iNOS (29,30). Therefore, the present study assessed the effects of nobiletin on the NF-κB signaling pathway. The results indicated that LPS/GalN induced the phosphorylation and degradation of IκBα and the nuclear translocation of NF-κB p65; however, these effects were attenuated by nobiletin.

Nrf2 is an important transcription factor involved in regulating the oxidative stress response (31,32). It regulates the transcription of antioxidant and detoxification genes, such as HO-1 (33,34). In the present study, the expression levels of Nrf2 in the nucleus and HO-1 in the cytoplasm were significantly increased in the liver tissue of LPS/GalN-treated mice. Notably, these increases were augmented by nobiletin. These results suggested that nobiletin may alleviate LPS/GalN-induced oxidative damage by stimulating the oxidative defense system. Furthermore, several studies have reported that there is an interaction between the Nrf2 and NF-κB signaling pathways (35,36). Nrf2 can negatively regulate the NF-κB signaling pathway and ameliorate NF-κB-related inflammatory response and oxidative injury in mice (37). Therefore, the inhibition of NF-κB observed in the liver tissue may be due to activation of Nrf2 by nobiletin treatment.

In conclusion, the present study demonstrated that treatment with nobiletin reduces LPS/GalN-induced proinflammatory cytokine production and attenuates liver injury in a mouse model. This protective mechanism may be dependent on the inhibition of NF-κB activation and the activation of Nrf2. These findings support the potential use of nobiletin as a therapeutic agent for the prevention of acute liver injury.

References