International Immunopharmacology 21 (2014) 406–411
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Protective effects of hesperidin on concanavalin A-induced hepatic injury in mice Gang Li a,1, Mao-jian Chen a,1, Chao Wang a, Hao Nie a, Wen-jian Huang a, Ting-dong Yuan a, Ting Sun a, Ke-gang Shu a, Chang-fu Wang b, Quan Gong a,⁎, Shao-qian Tang c,⁎⁎ a b c
Department of Immunology, School of Medical, Yangtze University, Jingzhou 434023, PR China Clinical Laboratory Department, Jingzhou Center Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jingzhou 434020, PR China Department of Gastroenterology, Jingzhou Center Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jingzhou 434020, PR China
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Article history: Received 11 December 2013 Received in revised form 30 April 2014 Accepted 15 May 2014 Available online 24 May 2014 Keywords: Hepatic injury Hesperidin Concanavalin A HMGB1
a b s t r a c t Hesperidin (HDN) is a citrus bioflavonoid, which widely exists in many plants. Previous researches have proved that HDN has several functions such as anti-oxidant, anti-tumor, anti-inflammatory, immune regulation and so on. In the present study, we explored the protective effects of HDN on concanavalin A (Con A)-induced hepatic injury. Acute hepatic injury model was established successfully by intravenous administration of Con A (15 mg/kg) in male C57BL/6 mice, and HDN was pretreated for 10 days before Con A challenge. It was found that the hepatic injury was notably improved in HDN pretreated mice. Furthermore, hepatic oxidative stress and the production of proinflammatory cytokines including TNF-α and IFN-γ were decreased by HDN pretreatment. More importantly, compared with Con A-treated mice, the expression and releasing of HMGB1 and T-cell activation were markedly reduced in HDN pretreated mice. Thus, these results suggest that HDN protects mice from Con A-induced hepatic injury by suppressing hepatocyte oxidative stress, producing cytokines, expressing and releasing HMGB1 and activating T cells. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Acute hepatic failure, viral hepatitis, alcoholic liver disease and autoimmune hepatitis have become a major health problem worldwide [1,2], as many can progress to chronic fibrosis, cirrhosis and even hepatocellular carcinoma [3]. T cells play an important role in the pathogenesis of these liver diseases, for activation of T cells can instigate liver injury directly via the cytotoxicity against hepatocytes. As a potent T-cell mitogen, Con A has been widely used to induce hepatitis in animals [4], and Con Ainduced hepatitis (CIH) is a well-established murine model that resembles viral hepatitis, autoimmune hepatitis and other immune-mediated liver diseases in humans. CIH is characterized by elevated plasma levels of various cytokines including TNF-α, IFN-γ, IL-4, IL-6 and IL-18 that secreted by activation of CD4+T cells and NKT cells [5–7]. Among several proinflammatory cytokines involved, TNF-α and IFN-γ play a critical role in the development of hepatocyte apoptosis and necrosis [8]. High mobility group box 1 protein (HMGB1) is a highly conserved non-histone nuclear DNA-binding protein [9]. It plays an important role in organizing nuclear architecture, DNA replication, DNA repair and transcriptional regulation of gene expression. HMGB1 is composed of 215 amino acids with two box structures (A-box at N-terminal and ⁎ Corresponding author. Tel./fax: +86 716 8062733. ⁎⁎ Corresponding author. Tel./fax: +86 716 8497225. E-mail addresses: [email protected] (Q. Gong), [email protected] (S. Tang). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.intimp.2014.05.018 1567-5769/© 2014 Elsevier B.V. All rights reserved.
B-box at central) as well as a C-terminal acidic tail [10]. The B-box domain has the properties of proinflammatory, while the A-box has no the effect of proinflammatory but can attenuate inflammatory cascade [11,12]. HMGB1 can be passively released from damaged or necrotic cells [10]; in addition, it can actively secreted by immune cells, such as dendritic cells (DCs), macrophages and natural killer (NK) cells under the stimulation of TNF-α, IL-1 and IFN-γ and oxidative stress [13]. Multiple receptors such as Toll-like receptors (TLR2 and TLR4) and receptor for advanced glycation end products (RAGE) can interact with extracellular HMGB1 [14]. They have been implicated in many diseases including cancer, sepsis, atherosclerosis, stroke and rheumatoid arthritis [15]. Recently, we demonstrated that HMGB1 could promote Con A-induced liver damage [16], and blocking it could protect mice from T-cell-mediated hepatitis [17]. The flavonoid HDN, a family of polyphenolic compounds, exists in several vegetables and fruits such as orange and grapes, especially in pericarp and membranous of citrus fruits. It has shown the pharmacology properties of anti-oxidant, anti-inflammatory, anti-tumor, antifungal, anti-viral and immune regulation [18–20]. Previous study has shown that HDN treatment protects against carbon tetrachloride (CCL4)-induced hepatic injury [21]. However, there is little information available about whether HDN could protect mice from Con A-induced liver injury. The aim of this study is to test the hypothesis that HDN could inhibit the expression and releasing of HMGB1 and thus improve the liver injury.
G. Li et al. / International Immunopharmacology 21 (2014) 406–411
2. Materials and methods 2.1. Reagent HDN, Con A and carboxyl methyl cellulose (CMC) were obtained from Sigma Aldrich (St. Louis, USA). Alanine transferase (ALT), aspartate transferase (AST), total superoxide dismutase (T-SOD), malondialdehyde (MDA) and Coomassie brilliant blue protein assay kits were purchased from Nanjing Jiancheng Biological Product. The cytokine enzymelinked immunosorbent assay (ELISA) kits of TNF-α and IFN-γ were purchased from R&D Systems (Minneapolis, MN, USA). HMGB1 ELISA kit was purchased from Elab (Elabscience, Wuhan, China). RNA simple Total RNA kit was purchased from TIANGEN (TIANGEN Biotech, China). RNA PCR kit was obtained from Takara (Takara Biotechnology, China). All the antibodies for FACS analysis were obtained from BD Pharmingen (San Diego, CA, USA) 2.2. Animals Six- to eight-week-old male C57BL/6 mice (20–25 g) were purchased from animal center of Wuhan University (Wuhan, China). The mice were raised in a laboratory with standard food and water and were exposed to a 12-h light/12-h dark cycles. The room temperature was 25 ± 1 °C, and humidity was 50 ± 5%. All of the animal studies were approved by the institutional animal care and use committee (IACUC) at the Yangtze University. 2.3. Study design HDN was dissolved in 0.5% CMC. Con A was dissolved in pyrogenfree normal saline solution (NSS) at a concentration of 2 mg/ml and then injected via tail vein with a single dose of 15 mg/kg body weight to induce liver injury as described [3]. Mice were randomly divided into three groups: Normal control group was injected with NSS via tail vein alone; model group was injected with Con A and solvent CMC treatment; and HDN (1000 mg/kg body weight) pretreatment group was pretreated with HDN for 10 days before Con A challenge. All the mice was sacrificed by cervical decapitation at the pathognomonic points after Con A challenge. The blood samples were collected in tubes without heparin and serum was separated by centrifugation to detect ALT, AST, TNF-α, IFN-γ and HMGB1. The liver was dissected and liver homogenate (1% or 10%, w/v) was prepared for biochemical assays. 2.4. Serum and tissue biochemical analysis 2.4.1. Activities of hepatic marker enzymes Hepatocyte injury was evaluated by determination of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. ALT and AST activities were measured according to the standard procedures using an automated chemistry analyzer (Olympus AU Japan). 2.4.2. Determination of lipid peroxidation in the liver tissue Lipid peroxidation in the liver tissue was assayed by determination of malondialdehyde (MDA) according to the method of Wills (1966). Liver tissue homogenate (1% or 10%, w/v) was prepared in normal saline containing protease inhibitor, and the homogenates were then centrifuged at 4000 rpm (4 °C) for 20 min to collect supernatants for determination of MDA and SOD concentrations. MDA was estimated by measuring thiobarbituric acid (TBA) using an MDA assay kit according to the manufacturers' instructions and expressed as nmol/mg protein. SOD activity was detected through nitroblue tetrazolium coloration by SOD assay kit according to the manufacture's instruction and expressed as U/mg protein. Coomassie brilliant blue protein assay kit was used for the measurement of liver tissue protein.
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2.4.3. Enzyme-linked immunosorbent assay (ELISA) Serum protein levels of TNF-α, IFN-γ and HMGB1 were estimated by ELISA according to the manufactures' instructions. 2.4.4. Histopathology Liver tissues were fixed in formalin and embedded in paraffin. Paraffin sections were stained with hematoxylin and eosin (H&E) for morphological evaluation under light microscope. 2.5. Immunohistochemistry The formalin fixed and paraffin-embedded liver sections were dewaxed and then rehydrated by a series of graded alcohols. In order to block endogenous peroxidase, 3% hydrogen peroxide was used to incubate slider for 15 min. The non-specific proteins were blocked with 10% goat serum for 30 min. For HMGB1 staining, specimens were incubated overnight with rabbit monoclonal antibody to HMGB1 (ab79823, Abcam, UK, 1:1000) at 4 °C, followed by 30 min incubation with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Zhongshan Golden Bridge Biotechnology CO; LTD, China). The sections were incubated with diamino-Benzidine (DAB) at last as a chromogenic substrate and counterstained with hematoxylin. 2.6. Reverse transcriptase polymerase chain reaction (RT-PCR) Total RNA was isolated from liver homogenates using RNA simple Total RNA kit at the indicated time points followed the manufacturer's instructions. After RNA was extracted, the reaction of reverse transcription from total RNA to cDNA was performed. RNA PCR kit was used for the polymerase chain reaction (Takara Biotechnology, China). The specific primer sequences involved were as follows: for β-action (F) 5′-CTG TCC CTG TAT GCC TCT G-3′, (R) 5′-CAT CGT ACT CCT GCT TGC T-3′; for HMGB1 (F) 5′-GAT GGG CAA AGG AGA TCC TAA G-3′, (R) 5′-TCA CTT TTT TGT CTC CCC TTT GGG-3′. 2.7. Flow cytometric analysis 2.7.1. T-cell activation in vivo Livers were harvested 6 h and 16 h after Con A injection. Liver MNCs were isolated by 40% percoll (10 × PBS 0.12 ml, percoll 1.08 ml, PBS 1.8 ml) and 70% percoll (10 × PBS 0.14 ml, percoll 1.26 ml and PBS 0.6 ml). T cells were stained with Percp-cy7-conjugated anti-mouse CD3 and PE-conjugated anti-mouse CD69 mAb and analyzed by BD FACS-Calibur. 2.7.2. T-cell activation in vitro Spleen lymphocytes were extracted from normal mice. The cells were divided into three groups: normal control group, HDN pretreatment group and Con A group. Cells were pretreated with 50 μM HDN [22] and cultured at a density of 2 × 106 cells/ml in RPMI-1640 contain 10% heat-inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 0.05 mM 2-mercaptoethanol for 3 h. Then, 5 μg/ml Con A were added and continue cultured for 12 h. Finally, cells were washed and stained with Percp-cy7-conjugated anti-mouse CD3 and PE-conjugated antimouse CD69 mAb and analyzed by BD FACS-Calibur. 2.8. Statistical analysis The results were presented as mean ± SD. Statistical significance of differences between groups was determined by one-way analysis of variance (ANOVA) or Student's t test. All data analyses were performed using SPSS v17.0 statistical analysis software. P values less than 0.05 were considered as statistically significant.
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3. Results 3.1. HDN pretreatment prevents mice from Con A-induced hepatic injury To explore the effects of HDN on Con A-induced liver injury, HDN was administrated to mice daily for 10 days, and the last treatment was performed 2 h prior to Con A injection. Serum ALT and AST were detected to determine the liver injury 16 h after Con A administration. As shown in Fig. 1A, compared with NSS control, serum ALT and AST levels were significantly increased in mice after Con A challenge. As expected, HDN pretreatment significantly attenuated Con A-induced elevation of serum ALT and AST. Furthermore, hepatic histopathological analyses were used to define the extent of liver inflammatory infiltration and necrosis by light microscopy. As shown in Fig. 1B, severe inflammatory infiltration and extensive necrosis were observed in Con A injection group. In contrast, mice pretreated with HDN showed minor liver damage. Altogether, HDN pretreatment significantly protected mice from Con A-induced hepatic injury.
3.2. HDN pretreatment prevents Con A-induced oxidative stress in liver To determine the effects of HDN on Con A-induced oxidative stress, the concentrations of MDA and SOD in liver were determined 16 h after Con A injection. As shown in Fig. 2A, hepatic MDA content was markedly elevated in Con A treatment mice. On the contrary, HDN treatment significantly alleviated Con A-induced increase of MDA. In addition, the SOD activity was obviously decreased after Con A injection, but this reduction was significantly improved by the pretreatment with HDN (Fig. 2B). These data suggest that HDN pretreatment reduces Con A-induced hepatic oxidative stress.
Fig. 1. Hesperidin treatment attenuates Con A-induced liver injury. (A) Mice were treated with NSS, CMC and HDN (1000 mg/kg) before Con A administration. The levels of serum ALT and AST were detected at 16 h after Con A injection. Data were expressed as mean ± SD. ⁎⁎P b 0.01 significantly different as compared with NSS. ##P b 0.01 vs CMC/Con A (n = 6–8). (B) Hematoxylin and eosin staining of liver specimens. Original magnification: ×100 and ×400. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
Fig. 2. Hesperidin pretreatment reduces Con A-caused oxidative stress in mice. Liver homogenate were analyzed at 16 h after Con A administration. (A) The activity of MDA and (B) the activity of SOD were measured. Data were expressed as mean ± SD. ⁎⁎P b 0.01 vs NSS. ##P b 0.01 vs CMC/Con A, #P b 0.05 vs CMC/Con A (n = 6–8).
3.3. HDN pretreatment inhibits cytokines production in Con A-treated mice A number of studies have proved that TNF-α and IFN-γ play a pivotal role in Con A-induced hepatitis [8]. To investigate whether HDN influences these cytokine productions, the levels of TNF-α, IFN-γ in serum were measured at 2 h, 6 h and 16 h after Con A treatment by ELISA. As is shown in Fig. 3, the levels of IFN-γ and TNF-α were dramatically increased in Con A-treated mice. As expected, the elevation of serum IFN-γ concentrations were significantly suppressed by HDN at 2 h and
Fig. 3. Hesperidin pretreatment inhibits TNF-α and IFN-γ production in Con A-treated mice. Serum samples were collected at 2 h, 6 h and 16 h after Con A challenge. The production of TNF-α and IFN-γ was measured. All the data were expressed as mean ± SD. (A) The production of TNF-α and (B) the production of IFN-γ were down-regulated. ⁎⁎P b 0.01 significantly different as compared with NSS. ##P b 0.01 significantly different as compared with CMC/Con A. #P b 0.05 vs CMC/Con A (n = 6–8).
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6 h, and the elevation of TNF-α were also significantly suppressed by HDN at 2 h and 6 h after Con A injection. Of note, as an early proinflammatory cytokine, TNF-α was undetectable in serum after 16 h of Con A induction (data not shown). These data suggest that HDN pretreatment suppresses TNF-α and IFN-γ secretion in CIH. 3.4. HMGB1 expression and releasing is inhibited by HDN in Con A-induced hepatitis
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(Fig. 5A). Interestingly, T-cell activation percentage at 16 h did not show significant difference between Con A and HDN treatment groups (data not shown). In vitro, after 12 h of Con A stimulation, CD69+ T cells were increased in Con A treatment group (Con A vs NSS: 83.39 ± 2.16 vs 11.23 ± 1.14, P b 0.01), but it markedly decreased in HDN treatment group (HDN vs Con A: 64.59 ± 5.8 vs 83.39 ± 2.16, P b 0.01) (Fig. 5B). 4. Discussion
Based on the above observations, we further explored the possible mechanisms by which HDN attenuates liver injury induced by Con A. Our previous study demonstrated that HMGB1 is an important inflammatory mediator in the initiation and progress of Con A-induced liver injury [16]. We, therefore, investigated the effect of HDN on the expression and releasing of HMGB1 by RT-PCR, ELISA and immunohistochemical analyses. As shown in Fig. 4B, HMGB1 was only stained in the nucleus of hepatocytes in normal liver tissue. In contrast, HMGB1 was also present in the cytoplasm other than the nucleus in damaged cells at 16 h after Con A injection, which indicates that HMGB1 is passively released from damaged cells after Con A challenged. As expected, mice pretreated with HDN showed a significant reduction of HMGB1. Furthermore, the hepatic mRNA expression of HMGB1 was examined at 4 h after Con A challenge. As shown in Fig. 4A, HMGB1 mRNA expression was significantly up-regulated at 4 h in Con A-treated mice and was obviously down-regulated in HDN pretreated mice. Besides, serum HMGB1 concentration were elevated in Con A-treated mice in 6 h and 16 h but markedly suppressed by HDN pretreatment in 16 h as shown in Fig. 4C. These data suggest that HDN pretreatment may inhibit HMGB1 releasing and expression and thus attenuates Con A-induced hepatic injury. 3.5. HDN pretreatment inhibits T-cell activation in Con A-treated mice T cells were pivotal for Con A-mediated liver injury. To explore the effect of HDN on the activation of T cells, we analyzed the percentage of CD69+ T cells in vivo and in vitro. It was found that T-cell activation ratio was elevated at 6 h after Con A treatment in vivo (Con A vs NSS: 90.21 ± 8.36 vs 7.18 ± 2.15, P b 0.01), but this elevation was reversed by HDN at 6 h (HDN vs Con A: 70.05 ± 6.85 vs 90.21 ± 8.36, P b 0.05)
Phytochemicals extracted from natural resources, such as vegetables, fruits and herbs, have come into the spotlight as potential treatment for inflammatory diseases. HDN, a flavanone glycoside in citrus fruits, exerts anti-inflammatory, antioxidant and immune regulation actions. A recent study showed that HDN protected against carbon tetrachloride (CCL4)-induced hepatic injury by increasing GSH, SOD and CAT levels [21]. In the present study, for the first time, we explored the effect of HDN on liver injury induced by Con A and the possible mechanisms in mice. Our data demonstrated that HDN treatment attenuated Con A-induced elevation of serum ALT and AST, hepatocyte necrosis, suggesting that HDN was able to protect mice from immunemediated liver injury in CIH. HDN also markedly reduced hepatic oxidative stress and inflammatory cytokines such as TNF-α and IFN-γ production initiated by Con A. Furthermore, we demonstrated that HDN significantly suppressed the expression and releasing of HMGB1 in Con A-induced hepatic injury, and this might explain some of the mechanisms whereby HDN elicits its benefits. Con A-mediated hepatic injury model is characterized by a variety of activation of T cells and releasing of a large amount of cytokines such as TNF-α, IFN-γ, IL-4, IL-6, IL-12 and IL-18. Of those cytokines, TNF-α and IFN-γ are considered to play a critical role in the initiation of hepatocyte damage. TNF-α, mainly secreted by activated Kupffer cells, is essential in the early stage of CIH to lead to hepatocyte necrosis and apoptosis, anti-TNF-α antibody or TNF-α inhibitor treatment markedly attenuated Con A-induced liver injury [23]; TNFR1- and TNFR2-deficient mice failed to develop liver injury upon Con A injection. HDN also can modulate serum levels of adipose tissue TNF-α and resistin mRNA expression in type 2 diabetic rats [24]. IFN-γ is mainly secreted by Th1 cells, and it can induce expression of pro-apoptotic genes and also directly induce
Fig. 4. Hesperidin pretreatment down-regulates Con A-induced hepatic HMGB1 expression and releasing. (A) Liver samples were collected at 4 h after Con A injection to determine the mRNA level of HMGB1 by RT-PCR. (B) Liver samples were collected at 16 h after Con A injection for immunohistochemical staining of HMGB1 (original magnification: ×400). (C) Serum samples were collected at 6 h and 16 h after Con A challenge. The production of HMGB1 was measured. Data were expressed as mean ± SD. ⁎⁎P b 0.01 significantly different as compared with NSS. ##P b 0.01 vs CMC/Con A. #P b 0.05 vs CMC/Con A (n = 6–8). (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
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Fig. 5. Hesperidin pretreatment down-regulated the activation of T cells in Con A-induced hepatic injury. (A, C) Hepatic MNCs were isolated and analyzed by FACS at 6 h in vivo. The activation of T cells was elevated in Con A-treated mice at 6 h, but this elevation was reversed by HDN. (B, D) Spleen T cells were extracted and analyzed by FACS after 12 h of Con A stimulation in vitro. CD69+ T cells were increased in Con A treatment group, but it markedly decreased in HDN treatment group. Data were expressed as mean ± SD. ⁎⁎P b 0.01 significantly different as compared with NSS. ##P b 0.01 vs CMC/Con A. #P b 0.05 vs CMC/Con A (n = 6–8). (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
cellular apoptosis in an IRF-1-dependent manner. Mice pretreated with anti-IFN-γ antibody or IFN-γ knockout mice do not develop hepatitis after exposed to Con A. HDN significantly inhibited Th2 cytokine (IL-4) and IFN-γ production in a mouse model of allergic asthma [25]. These studies suggest that the inhibition of TNF-α and IFN-γ tended to alleviate liver injury. In the current study, we found that TNF-α expressed in the early stage of Con A-induced hepatitis, which is consistent with previous study. The level of IFN-γ increased upon Con A stimulation and reached its peak in plasma about 6 h after Con A treatment. As expected, HDN pretreated group significantly decreased the production of IFN-γ and TNF-α. These data suggest that HDN pretreatment significantly repressed the production of TNF-α and IFN-γ to exert protection on hepatocyte in CIH mice. In addition to its nuclear role, extracellular HMGB1 is considered as a critical mediator of innate immune response to tissue infection and injury. Extracellular HMGB1 has been implicated as a putative danger signal involved in the pathogenesis of a variety of infectious and noninfectious inflammatory conditions such as sepsis [10], acute lung injury [26], ischemia/reperfusion (I/R) injury, trauma and cancer. HMGB1 antagonists including neutralization antibody and recombinant A-box confer protection against tissue damage in HMGB1-mediated diseases. We also found that HMGB1 is a vital inflammation mediator in response to Con A-induced hepatic damage [27]. The administration of exogenous HMGB1 worsens Con A caused hepatitis, while the blockage of HMGB1 protected mice from T-cell-mediated hepatitis [17]. In our experiment, we demonstrated that HDN inhibited the expression of HMGB1 mRNA at 4 h in comparison to Con A treatment mice. Furthermore, HDN pretreatment also suppressed HMGB1 transfer from nucleus to cytoplasm, suggesting that HDN pretreatment may inhibit HMGB1 releasing and expression and thus protects mice from Con A-induced hepatic injury. However, the mechanisms of HDN on blocking the expression and releasing of HMGB1 are needed to be further elucidated. It has been well demonstrated that Con A exposure rapidly activates T cells in vivo, and excessive T-cell activation are associated with Con A-induced liver injury [28]. Therefore, preventing the activation of T cells may protect immune-mediated liver injury. In the present study, we found that HDN pretreatment markedly inhibited T-cell activation induced by Con A, which could be one of the mechanisms underlying the protective effects of HDN. Further studies are needed to clarify the precise mechanisms by which HDN pretreatment modulates Con A-induced T-cell activation.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Protective effects of hesperidin on concanavalin A-induced hepatic injury in mice Gang Li a,1, Mao-jian Chen a,1, Chao Wang a, Hao Nie a, Wen-jian Huang a, Ting-dong Yuan a, Ting Sun a, Ke-gang Shu a, Chang-fu Wang b, Quan Gong a,⁎, Shao-qian Tang c,⁎⁎ a b c
Department of Immunology, School of Medical, Yangtze University, Jingzhou 434023, PR China Clinical Laboratory Department, Jingzhou Center Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jingzhou 434020, PR China Department of Gastroenterology, Jingzhou Center Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jingzhou 434020, PR China
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Article history: Received 11 December 2013 Received in revised form 30 April 2014 Accepted 15 May 2014 Available online 24 May 2014 Keywords: Hepatic injury Hesperidin Concanavalin A HMGB1
a b s t r a c t Hesperidin (HDN) is a citrus bioflavonoid, which widely exists in many plants. Previous researches have proved that HDN has several functions such as anti-oxidant, anti-tumor, anti-inflammatory, immune regulation and so on. In the present study, we explored the protective effects of HDN on concanavalin A (Con A)-induced hepatic injury. Acute hepatic injury model was established successfully by intravenous administration of Con A (15 mg/kg) in male C57BL/6 mice, and HDN was pretreated for 10 days before Con A challenge. It was found that the hepatic injury was notably improved in HDN pretreated mice. Furthermore, hepatic oxidative stress and the production of proinflammatory cytokines including TNF-α and IFN-γ were decreased by HDN pretreatment. More importantly, compared with Con A-treated mice, the expression and releasing of HMGB1 and T-cell activation were markedly reduced in HDN pretreated mice. Thus, these results suggest that HDN protects mice from Con A-induced hepatic injury by suppressing hepatocyte oxidative stress, producing cytokines, expressing and releasing HMGB1 and activating T cells. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Acute hepatic failure, viral hepatitis, alcoholic liver disease and autoimmune hepatitis have become a major health problem worldwide [1,2], as many can progress to chronic fibrosis, cirrhosis and even hepatocellular carcinoma [3]. T cells play an important role in the pathogenesis of these liver diseases, for activation of T cells can instigate liver injury directly via the cytotoxicity against hepatocytes. As a potent T-cell mitogen, Con A has been widely used to induce hepatitis in animals [4], and Con Ainduced hepatitis (CIH) is a well-established murine model that resembles viral hepatitis, autoimmune hepatitis and other immune-mediated liver diseases in humans. CIH is characterized by elevated plasma levels of various cytokines including TNF-α, IFN-γ, IL-4, IL-6 and IL-18 that secreted by activation of CD4+T cells and NKT cells [5–7]. Among several proinflammatory cytokines involved, TNF-α and IFN-γ play a critical role in the development of hepatocyte apoptosis and necrosis [8]. High mobility group box 1 protein (HMGB1) is a highly conserved non-histone nuclear DNA-binding protein [9]. It plays an important role in organizing nuclear architecture, DNA replication, DNA repair and transcriptional regulation of gene expression. HMGB1 is composed of 215 amino acids with two box structures (A-box at N-terminal and ⁎ Corresponding author. Tel./fax: +86 716 8062733. ⁎⁎ Corresponding author. Tel./fax: +86 716 8497225. E-mail addresses: [email protected] (Q. Gong), [email protected] (S. Tang). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.intimp.2014.05.018 1567-5769/© 2014 Elsevier B.V. All rights reserved.
B-box at central) as well as a C-terminal acidic tail [10]. The B-box domain has the properties of proinflammatory, while the A-box has no the effect of proinflammatory but can attenuate inflammatory cascade [11,12]. HMGB1 can be passively released from damaged or necrotic cells [10]; in addition, it can actively secreted by immune cells, such as dendritic cells (DCs), macrophages and natural killer (NK) cells under the stimulation of TNF-α, IL-1 and IFN-γ and oxidative stress [13]. Multiple receptors such as Toll-like receptors (TLR2 and TLR4) and receptor for advanced glycation end products (RAGE) can interact with extracellular HMGB1 [14]. They have been implicated in many diseases including cancer, sepsis, atherosclerosis, stroke and rheumatoid arthritis [15]. Recently, we demonstrated that HMGB1 could promote Con A-induced liver damage [16], and blocking it could protect mice from T-cell-mediated hepatitis [17]. The flavonoid HDN, a family of polyphenolic compounds, exists in several vegetables and fruits such as orange and grapes, especially in pericarp and membranous of citrus fruits. It has shown the pharmacology properties of anti-oxidant, anti-inflammatory, anti-tumor, antifungal, anti-viral and immune regulation [18–20]. Previous study has shown that HDN treatment protects against carbon tetrachloride (CCL4)-induced hepatic injury [21]. However, there is little information available about whether HDN could protect mice from Con A-induced liver injury. The aim of this study is to test the hypothesis that HDN could inhibit the expression and releasing of HMGB1 and thus improve the liver injury.
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2. Materials and methods 2.1. Reagent HDN, Con A and carboxyl methyl cellulose (CMC) were obtained from Sigma Aldrich (St. Louis, USA). Alanine transferase (ALT), aspartate transferase (AST), total superoxide dismutase (T-SOD), malondialdehyde (MDA) and Coomassie brilliant blue protein assay kits were purchased from Nanjing Jiancheng Biological Product. The cytokine enzymelinked immunosorbent assay (ELISA) kits of TNF-α and IFN-γ were purchased from R&D Systems (Minneapolis, MN, USA). HMGB1 ELISA kit was purchased from Elab (Elabscience, Wuhan, China). RNA simple Total RNA kit was purchased from TIANGEN (TIANGEN Biotech, China). RNA PCR kit was obtained from Takara (Takara Biotechnology, China). All the antibodies for FACS analysis were obtained from BD Pharmingen (San Diego, CA, USA) 2.2. Animals Six- to eight-week-old male C57BL/6 mice (20–25 g) were purchased from animal center of Wuhan University (Wuhan, China). The mice were raised in a laboratory with standard food and water and were exposed to a 12-h light/12-h dark cycles. The room temperature was 25 ± 1 °C, and humidity was 50 ± 5%. All of the animal studies were approved by the institutional animal care and use committee (IACUC) at the Yangtze University. 2.3. Study design HDN was dissolved in 0.5% CMC. Con A was dissolved in pyrogenfree normal saline solution (NSS) at a concentration of 2 mg/ml and then injected via tail vein with a single dose of 15 mg/kg body weight to induce liver injury as described [3]. Mice were randomly divided into three groups: Normal control group was injected with NSS via tail vein alone; model group was injected with Con A and solvent CMC treatment; and HDN (1000 mg/kg body weight) pretreatment group was pretreated with HDN for 10 days before Con A challenge. All the mice was sacrificed by cervical decapitation at the pathognomonic points after Con A challenge. The blood samples were collected in tubes without heparin and serum was separated by centrifugation to detect ALT, AST, TNF-α, IFN-γ and HMGB1. The liver was dissected and liver homogenate (1% or 10%, w/v) was prepared for biochemical assays. 2.4. Serum and tissue biochemical analysis 2.4.1. Activities of hepatic marker enzymes Hepatocyte injury was evaluated by determination of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. ALT and AST activities were measured according to the standard procedures using an automated chemistry analyzer (Olympus AU Japan). 2.4.2. Determination of lipid peroxidation in the liver tissue Lipid peroxidation in the liver tissue was assayed by determination of malondialdehyde (MDA) according to the method of Wills (1966). Liver tissue homogenate (1% or 10%, w/v) was prepared in normal saline containing protease inhibitor, and the homogenates were then centrifuged at 4000 rpm (4 °C) for 20 min to collect supernatants for determination of MDA and SOD concentrations. MDA was estimated by measuring thiobarbituric acid (TBA) using an MDA assay kit according to the manufacturers' instructions and expressed as nmol/mg protein. SOD activity was detected through nitroblue tetrazolium coloration by SOD assay kit according to the manufacture's instruction and expressed as U/mg protein. Coomassie brilliant blue protein assay kit was used for the measurement of liver tissue protein.
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2.4.3. Enzyme-linked immunosorbent assay (ELISA) Serum protein levels of TNF-α, IFN-γ and HMGB1 were estimated by ELISA according to the manufactures' instructions. 2.4.4. Histopathology Liver tissues were fixed in formalin and embedded in paraffin. Paraffin sections were stained with hematoxylin and eosin (H&E) for morphological evaluation under light microscope. 2.5. Immunohistochemistry The formalin fixed and paraffin-embedded liver sections were dewaxed and then rehydrated by a series of graded alcohols. In order to block endogenous peroxidase, 3% hydrogen peroxide was used to incubate slider for 15 min. The non-specific proteins were blocked with 10% goat serum for 30 min. For HMGB1 staining, specimens were incubated overnight with rabbit monoclonal antibody to HMGB1 (ab79823, Abcam, UK, 1:1000) at 4 °C, followed by 30 min incubation with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Zhongshan Golden Bridge Biotechnology CO; LTD, China). The sections were incubated with diamino-Benzidine (DAB) at last as a chromogenic substrate and counterstained with hematoxylin. 2.6. Reverse transcriptase polymerase chain reaction (RT-PCR) Total RNA was isolated from liver homogenates using RNA simple Total RNA kit at the indicated time points followed the manufacturer's instructions. After RNA was extracted, the reaction of reverse transcription from total RNA to cDNA was performed. RNA PCR kit was used for the polymerase chain reaction (Takara Biotechnology, China). The specific primer sequences involved were as follows: for β-action (F) 5′-CTG TCC CTG TAT GCC TCT G-3′, (R) 5′-CAT CGT ACT CCT GCT TGC T-3′; for HMGB1 (F) 5′-GAT GGG CAA AGG AGA TCC TAA G-3′, (R) 5′-TCA CTT TTT TGT CTC CCC TTT GGG-3′. 2.7. Flow cytometric analysis 2.7.1. T-cell activation in vivo Livers were harvested 6 h and 16 h after Con A injection. Liver MNCs were isolated by 40% percoll (10 × PBS 0.12 ml, percoll 1.08 ml, PBS 1.8 ml) and 70% percoll (10 × PBS 0.14 ml, percoll 1.26 ml and PBS 0.6 ml). T cells were stained with Percp-cy7-conjugated anti-mouse CD3 and PE-conjugated anti-mouse CD69 mAb and analyzed by BD FACS-Calibur. 2.7.2. T-cell activation in vitro Spleen lymphocytes were extracted from normal mice. The cells were divided into three groups: normal control group, HDN pretreatment group and Con A group. Cells were pretreated with 50 μM HDN [22] and cultured at a density of 2 × 106 cells/ml in RPMI-1640 contain 10% heat-inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 0.05 mM 2-mercaptoethanol for 3 h. Then, 5 μg/ml Con A were added and continue cultured for 12 h. Finally, cells were washed and stained with Percp-cy7-conjugated anti-mouse CD3 and PE-conjugated antimouse CD69 mAb and analyzed by BD FACS-Calibur. 2.8. Statistical analysis The results were presented as mean ± SD. Statistical significance of differences between groups was determined by one-way analysis of variance (ANOVA) or Student's t test. All data analyses were performed using SPSS v17.0 statistical analysis software. P values less than 0.05 were considered as statistically significant.
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3. Results 3.1. HDN pretreatment prevents mice from Con A-induced hepatic injury To explore the effects of HDN on Con A-induced liver injury, HDN was administrated to mice daily for 10 days, and the last treatment was performed 2 h prior to Con A injection. Serum ALT and AST were detected to determine the liver injury 16 h after Con A administration. As shown in Fig. 1A, compared with NSS control, serum ALT and AST levels were significantly increased in mice after Con A challenge. As expected, HDN pretreatment significantly attenuated Con A-induced elevation of serum ALT and AST. Furthermore, hepatic histopathological analyses were used to define the extent of liver inflammatory infiltration and necrosis by light microscopy. As shown in Fig. 1B, severe inflammatory infiltration and extensive necrosis were observed in Con A injection group. In contrast, mice pretreated with HDN showed minor liver damage. Altogether, HDN pretreatment significantly protected mice from Con A-induced hepatic injury.
3.2. HDN pretreatment prevents Con A-induced oxidative stress in liver To determine the effects of HDN on Con A-induced oxidative stress, the concentrations of MDA and SOD in liver were determined 16 h after Con A injection. As shown in Fig. 2A, hepatic MDA content was markedly elevated in Con A treatment mice. On the contrary, HDN treatment significantly alleviated Con A-induced increase of MDA. In addition, the SOD activity was obviously decreased after Con A injection, but this reduction was significantly improved by the pretreatment with HDN (Fig. 2B). These data suggest that HDN pretreatment reduces Con A-induced hepatic oxidative stress.
Fig. 1. Hesperidin treatment attenuates Con A-induced liver injury. (A) Mice were treated with NSS, CMC and HDN (1000 mg/kg) before Con A administration. The levels of serum ALT and AST were detected at 16 h after Con A injection. Data were expressed as mean ± SD. ⁎⁎P b 0.01 significantly different as compared with NSS. ##P b 0.01 vs CMC/Con A (n = 6–8). (B) Hematoxylin and eosin staining of liver specimens. Original magnification: ×100 and ×400. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
Fig. 2. Hesperidin pretreatment reduces Con A-caused oxidative stress in mice. Liver homogenate were analyzed at 16 h after Con A administration. (A) The activity of MDA and (B) the activity of SOD were measured. Data were expressed as mean ± SD. ⁎⁎P b 0.01 vs NSS. ##P b 0.01 vs CMC/Con A, #P b 0.05 vs CMC/Con A (n = 6–8).
3.3. HDN pretreatment inhibits cytokines production in Con A-treated mice A number of studies have proved that TNF-α and IFN-γ play a pivotal role in Con A-induced hepatitis [8]. To investigate whether HDN influences these cytokine productions, the levels of TNF-α, IFN-γ in serum were measured at 2 h, 6 h and 16 h after Con A treatment by ELISA. As is shown in Fig. 3, the levels of IFN-γ and TNF-α were dramatically increased in Con A-treated mice. As expected, the elevation of serum IFN-γ concentrations were significantly suppressed by HDN at 2 h and
Fig. 3. Hesperidin pretreatment inhibits TNF-α and IFN-γ production in Con A-treated mice. Serum samples were collected at 2 h, 6 h and 16 h after Con A challenge. The production of TNF-α and IFN-γ was measured. All the data were expressed as mean ± SD. (A) The production of TNF-α and (B) the production of IFN-γ were down-regulated. ⁎⁎P b 0.01 significantly different as compared with NSS. ##P b 0.01 significantly different as compared with CMC/Con A. #P b 0.05 vs CMC/Con A (n = 6–8).
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6 h, and the elevation of TNF-α were also significantly suppressed by HDN at 2 h and 6 h after Con A injection. Of note, as an early proinflammatory cytokine, TNF-α was undetectable in serum after 16 h of Con A induction (data not shown). These data suggest that HDN pretreatment suppresses TNF-α and IFN-γ secretion in CIH. 3.4. HMGB1 expression and releasing is inhibited by HDN in Con A-induced hepatitis
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(Fig. 5A). Interestingly, T-cell activation percentage at 16 h did not show significant difference between Con A and HDN treatment groups (data not shown). In vitro, after 12 h of Con A stimulation, CD69+ T cells were increased in Con A treatment group (Con A vs NSS: 83.39 ± 2.16 vs 11.23 ± 1.14, P b 0.01), but it markedly decreased in HDN treatment group (HDN vs Con A: 64.59 ± 5.8 vs 83.39 ± 2.16, P b 0.01) (Fig. 5B). 4. Discussion
Based on the above observations, we further explored the possible mechanisms by which HDN attenuates liver injury induced by Con A. Our previous study demonstrated that HMGB1 is an important inflammatory mediator in the initiation and progress of Con A-induced liver injury [16]. We, therefore, investigated the effect of HDN on the expression and releasing of HMGB1 by RT-PCR, ELISA and immunohistochemical analyses. As shown in Fig. 4B, HMGB1 was only stained in the nucleus of hepatocytes in normal liver tissue. In contrast, HMGB1 was also present in the cytoplasm other than the nucleus in damaged cells at 16 h after Con A injection, which indicates that HMGB1 is passively released from damaged cells after Con A challenged. As expected, mice pretreated with HDN showed a significant reduction of HMGB1. Furthermore, the hepatic mRNA expression of HMGB1 was examined at 4 h after Con A challenge. As shown in Fig. 4A, HMGB1 mRNA expression was significantly up-regulated at 4 h in Con A-treated mice and was obviously down-regulated in HDN pretreated mice. Besides, serum HMGB1 concentration were elevated in Con A-treated mice in 6 h and 16 h but markedly suppressed by HDN pretreatment in 16 h as shown in Fig. 4C. These data suggest that HDN pretreatment may inhibit HMGB1 releasing and expression and thus attenuates Con A-induced hepatic injury. 3.5. HDN pretreatment inhibits T-cell activation in Con A-treated mice T cells were pivotal for Con A-mediated liver injury. To explore the effect of HDN on the activation of T cells, we analyzed the percentage of CD69+ T cells in vivo and in vitro. It was found that T-cell activation ratio was elevated at 6 h after Con A treatment in vivo (Con A vs NSS: 90.21 ± 8.36 vs 7.18 ± 2.15, P b 0.01), but this elevation was reversed by HDN at 6 h (HDN vs Con A: 70.05 ± 6.85 vs 90.21 ± 8.36, P b 0.05)
Phytochemicals extracted from natural resources, such as vegetables, fruits and herbs, have come into the spotlight as potential treatment for inflammatory diseases. HDN, a flavanone glycoside in citrus fruits, exerts anti-inflammatory, antioxidant and immune regulation actions. A recent study showed that HDN protected against carbon tetrachloride (CCL4)-induced hepatic injury by increasing GSH, SOD and CAT levels [21]. In the present study, for the first time, we explored the effect of HDN on liver injury induced by Con A and the possible mechanisms in mice. Our data demonstrated that HDN treatment attenuated Con A-induced elevation of serum ALT and AST, hepatocyte necrosis, suggesting that HDN was able to protect mice from immunemediated liver injury in CIH. HDN also markedly reduced hepatic oxidative stress and inflammatory cytokines such as TNF-α and IFN-γ production initiated by Con A. Furthermore, we demonstrated that HDN significantly suppressed the expression and releasing of HMGB1 in Con A-induced hepatic injury, and this might explain some of the mechanisms whereby HDN elicits its benefits. Con A-mediated hepatic injury model is characterized by a variety of activation of T cells and releasing of a large amount of cytokines such as TNF-α, IFN-γ, IL-4, IL-6, IL-12 and IL-18. Of those cytokines, TNF-α and IFN-γ are considered to play a critical role in the initiation of hepatocyte damage. TNF-α, mainly secreted by activated Kupffer cells, is essential in the early stage of CIH to lead to hepatocyte necrosis and apoptosis, anti-TNF-α antibody or TNF-α inhibitor treatment markedly attenuated Con A-induced liver injury [23]; TNFR1- and TNFR2-deficient mice failed to develop liver injury upon Con A injection. HDN also can modulate serum levels of adipose tissue TNF-α and resistin mRNA expression in type 2 diabetic rats [24]. IFN-γ is mainly secreted by Th1 cells, and it can induce expression of pro-apoptotic genes and also directly induce
Fig. 4. Hesperidin pretreatment down-regulates Con A-induced hepatic HMGB1 expression and releasing. (A) Liver samples were collected at 4 h after Con A injection to determine the mRNA level of HMGB1 by RT-PCR. (B) Liver samples were collected at 16 h after Con A injection for immunohistochemical staining of HMGB1 (original magnification: ×400). (C) Serum samples were collected at 6 h and 16 h after Con A challenge. The production of HMGB1 was measured. Data were expressed as mean ± SD. ⁎⁎P b 0.01 significantly different as compared with NSS. ##P b 0.01 vs CMC/Con A. #P b 0.05 vs CMC/Con A (n = 6–8). (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
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Fig. 5. Hesperidin pretreatment down-regulated the activation of T cells in Con A-induced hepatic injury. (A, C) Hepatic MNCs were isolated and analyzed by FACS at 6 h in vivo. The activation of T cells was elevated in Con A-treated mice at 6 h, but this elevation was reversed by HDN. (B, D) Spleen T cells were extracted and analyzed by FACS after 12 h of Con A stimulation in vitro. CD69+ T cells were increased in Con A treatment group, but it markedly decreased in HDN treatment group. Data were expressed as mean ± SD. ⁎⁎P b 0.01 significantly different as compared with NSS. ##P b 0.01 vs CMC/Con A. #P b 0.05 vs CMC/Con A (n = 6–8). (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
cellular apoptosis in an IRF-1-dependent manner. Mice pretreated with anti-IFN-γ antibody or IFN-γ knockout mice do not develop hepatitis after exposed to Con A. HDN significantly inhibited Th2 cytokine (IL-4) and IFN-γ production in a mouse model of allergic asthma [25]. These studies suggest that the inhibition of TNF-α and IFN-γ tended to alleviate liver injury. In the current study, we found that TNF-α expressed in the early stage of Con A-induced hepatitis, which is consistent with previous study. The level of IFN-γ increased upon Con A stimulation and reached its peak in plasma about 6 h after Con A treatment. As expected, HDN pretreated group significantly decreased the production of IFN-γ and TNF-α. These data suggest that HDN pretreatment significantly repressed the production of TNF-α and IFN-γ to exert protection on hepatocyte in CIH mice. In addition to its nuclear role, extracellular HMGB1 is considered as a critical mediator of innate immune response to tissue infection and injury. Extracellular HMGB1 has been implicated as a putative danger signal involved in the pathogenesis of a variety of infectious and noninfectious inflammatory conditions such as sepsis [10], acute lung injury [26], ischemia/reperfusion (I/R) injury, trauma and cancer. HMGB1 antagonists including neutralization antibody and recombinant A-box confer protection against tissue damage in HMGB1-mediated diseases. We also found that HMGB1 is a vital inflammation mediator in response to Con A-induced hepatic damage [27]. The administration of exogenous HMGB1 worsens Con A caused hepatitis, while the blockage of HMGB1 protected mice from T-cell-mediated hepatitis [17]. In our experiment, we demonstrated that HDN inhibited the expression of HMGB1 mRNA at 4 h in comparison to Con A treatment mice. Furthermore, HDN pretreatment also suppressed HMGB1 transfer from nucleus to cytoplasm, suggesting that HDN pretreatment may inhibit HMGB1 releasing and expression and thus protects mice from Con A-induced hepatic injury. However, the mechanisms of HDN on blocking the expression and releasing of HMGB1 are needed to be further elucidated. It has been well demonstrated that Con A exposure rapidly activates T cells in vivo, and excessive T-cell activation are associated with Con A-induced liver injury [28]. Therefore, preventing the activation of T cells may protect immune-mediated liver injury. In the present study, we found that HDN pretreatment markedly inhibited T-cell activation induced by Con A, which could be one of the mechanisms underlying the protective effects of HDN. Further studies are needed to clarify the precise mechanisms by which HDN pretreatment modulates Con A-induced T-cell activation.
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