International Immunopharmacology 24 (2015) 42–49

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Paeoniflorin protects against concanavalin A-induced hepatitis in mice☆ Mingsheng Chen a,1, Lijun Cao a,1,⁎, Yijun Luo a,1, Xiaofeng Feng b,1, Lu Sun a, Min Wen c, Shaobin Peng c a b c

Department of Anesthesiology, No. 113 Hospital of People's Liberation Army, Ningbo 315040, China Department of Gastroenterology, No. 113 Hospital of People's Liberation Army, Ningbo 315040, China Department of Stomatology, No. 113 Hospital of People's Liberation Army, Ningbo 315040, China

a r t i c l e

i n f o

Article history: Received 28 August 2014 Received in revised form 29 October 2014 Accepted 10 November 2014 Available online 20 November 2014 Keywords: Paeoniflorin Concanavalin A Hepatitis Lymphocyte TLR4 NF-κB

a b s t r a c t Paeoniflorin (PF) is one of the main effective components of the total glucosides of peony, which has been reported to have anti-inflammatory ability. However, the effects of paeoniflorin on concanavalin A (Con A)-induced hepatitis have not been carefully examined. The aim of this study was to investigate the protective effect of paeoniflorin and elucidate potential mechanisms of paeoniflorin on Con A-induced hepatitis. C57BL/6 mice were divided randomly into the following four experimental groups: PBS group, PF group, Con A group, and Con A + PF group. Mice received paeoniflorin (50 mg/kg) by tail vein before Con A intravenous administration. We found that paeoniflorin pretreatment can significantly reduce the elevated plasma aminotransferase levels and liver necrosis in Con A-induced hepatitis. Also, paeoniflorin pretreatment suppressed the secretion of proinflammatory cytokines (TNF-α, INF-γ, IL-6), compared with Con A group. Meanwhile, paeoniflorin pretreatment decreased CD4+, CD8+ and NKT cell infiltration in the liver. Besides, we observed that paeoniflorin pretreatment can decrease the expression level of Toll-like receptor (TLR) 4 mRNA or protein in liver tissues. Further results showed that paeoniflorin pretreatment was capable of suppressing the activation of the NF-κB pathway by inhibiting IκBα kinase and p65 phosphorylation in Con A-induced liver injury. These results suggest that paeoniflorin pretreatment protects mice against Con A-induced liver injury via inhibition of several inflammatory mediators and, at least in part, by suppressing CD4+, CD8+ and NKT cell infiltration in liver. The beneficial effect of paeoniflorin may be related to the downregulation of TLR4 expression and the inhibition of NF-κB activation. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Liver diseases are still a major public health problem worldwide because of significant morbidity and mortality. The most common causes of liver damage in humans are infections with hepatitis B or C viruses, and autoimmune hepatitis [1–3]. It was estimated that one percent of people infected with hepatitis viruses develop fulminant hepatitis without intervention [4]. Concanavalin A-induced hepatitis in mice is a well established model that closely mimics the pathogenic mechanisms and pathological changes with viral and autoimmune hepatitis in human. In this model, inflammatory cells, such as T cells [5–7], and a variety of

☆ Authors' contributions: Mingsheng Chen, Yijun Luo, Xiaofeng Feng, and Lu Sun performed the experiments and interpreted the results of experiments; Mingsheng Chen, Yijun Luo, Xiaofeng Feng, and Min Wen analyzed the data; Mingsheng Chen, Yijun Luo, Xiaofeng Feng, Lu Sun, Min Wen, and Shaobin Peng contributed reagents, materials and analysis tools; Mingsheng Chen, Yijun Luo, Xiaofeng Feng drafted the manuscript; Lijun Cao designed this study, supervised the data collection and revised this article. All authors have read and approved the final manuscript. ⁎ Corresponding author. Tel./fax: +86 574 27754305, +86 15888579036 (mobile). E-mail addresses: [email protected] (M. Chen), [email protected] (L. Cao), [email protected] (Y. Luo), [email protected] (X. Feng), [email protected] (L. Sun), [email protected] (M. Wen), [email protected] (S. Peng). 1 Contributed equally to this article.

http://dx.doi.org/10.1016/j.intimp.2014.11.006 1567-5769/© 2014 Elsevier B.V. All rights reserved.

hepatotoxic cytokines, i.e., tumor necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-6, play a key role in hepatocyte damage [8–11]. Previous studies indicated that many therapeutic agents that abrogate liver injury might transect with the Toll-like receptor (TLR) 4 signaling pathway [12–14]. Moreover, some studies showed that TLR4 was critically involved in the pathogenesis of Con A-induced liver damage [15–17]. It has been reported that Con A also upregulated NF-κB expression in liver [18], and there was increasing evidence shown that Con A-induced liver injury was significantly attenuated via inhibiting NF-κB activation [19–21]. Therefore, inhibition of the expression of TLR4 and suppressing the activation of NF-κB pathway may well represent therapeutic targets for T cell-mediated hepatitis. Paeoniflorin (PF) is a monoterpene glycoside isolated from the roots of Paeonia lactiflora Pall. which have been used for more than 1500 years in traditional Chinese medicine. Previous investigations of paeoniflorin exhibited many pharmacological effects such as anti-inflammation [22], anti-hyperglycemia [23], and neuroprotective effects [24]. Moreover, paeoniflorin has been proven to be effective in the treatment of many diseases in animal models, such as collagen-induced arthritis and acute lung injury [25,26]. Jiang et al. demonstrated that paeoniflorin inhibited systemic inflammation and improved survival in experimental sepsis via inhibiting the NF-κB activation [27]. But to our knowledge,

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it is unclear that whether paeoniflorin also provides a protective effect on Con A-induced hepatitis in mice. Based on these studies, it is tempting to speculate that paeoniflorin might play an important role in alleviation of Con A-induced hepatitis. Therefore, the aim of this study was to investigate the effect of paeoniflorin and elucidate potential mechanisms of paeoniflorin on Con A-induced hepatitis. 2. Materials and methods 2.1. Reagents and mice Paeoniflorin (CAS Number: 23180-57-6, purity N 98%, molecular formula: C23H28O11, molecular weight: 480.46) was purchased from Sigma-Aldrich Corporation. Con A was provided by Solarbio Corporation (Beijing, China). All of the other chemicals and reagents were standard commercially available biochemical quality. Deionized water was purified with a Milli-Q purification system and was used to prepare all solutions. Six- to eight-week-old male C57BL/6 mice weighing between 20 and 25 g were obtained from the animal center of the Animal Experimentation Center of No. 113 Hospital of People's Liberation Army (Ningbo, China). The animals were kept in an environmentally controlled room (23 ± 2 °C, 55 ± 10% humidity) with a 12-h light and -dark cycle and allowed free access to food and water. All experiments were performed in accordance with the guidelines of Institutional Animal Ethics Committee of No. 113 Hospital of People's Liberation Army (Permit Number: 20120919-56). 2.2. Animal treatment Paeoniflorin and Con A were dissolved in pathogen-free phosphatebuffered saline (PBS). Mice were administrated intravenously with paeoniflorin (50 mg/kg, 100 μl) or PBS (100 μl) as a control. The mice were injected intravenously with Con A (15 mg/kg, 100 μl). This dosage was proved effective by previous studies in mice [28]. Mice were randomly divided into four groups: PBS group, PF group, Con A group, and Con A + PF group. In PBS group mice, only the carrier solution (PBS) was injected. PF group mice were administrated paeoniflorin, without Con A treatment. Con A group mice were injected with Con A, without paeoniflorin treatment. Con A + PF group was a treatment group, in which mice were injected with paeoniflorin 3 h prior to Con A administration. The blood and liver tissue were harvested 12 h after Con A administration. 2.3. Measurement of serum aminotransferase and cytokine secretion The blood was obtained 12 h after Con A administration. The blood was collected by heart puncture after sevoflurane anesthesia. Plasma was separated after centrifugation at 300 ×g for 5 min. Activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured spectrophotometrically using an automatic biochemical analyzer (Hitachi Auto Analyzer 7170, Japan). The concentrations of TNF-α, IFN-γ and IL-6 were detected by using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions (R&D system, USA). The thresholds of ELISA for TNF-α, IFN-γ and IL-6 were 0.36–7.21 pg/ml, 12.5–400 pg/ml, and 1.3–1.8 pg/ml, respectively. 2.4. Histopathological analysis Liver samples were harvested 12 h after Con A administration. Tissue sections (thickness, 5 μm) were stained with hematoxylin and eosin (H&E) and were examined for liver damage by light microscopy. All sections were graded blindly by three pathologists under light microscopy according to the following criteria: 0, none; 1, individual cell necrosis;

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2, ≤ 30% lobular necrosis; 3, ≤ 60% lobular necrosis; 4, N60% lobular necrosis [29]. 2.5. Liver mononuclear cell preparation Liver samples were harvested 12 h after Con A administration. In brief, liver tissues were infiltrated in collagenase type II (Life Technologies, USA) for 30 min. After flushing the liver with cold PBS, the livers were crushed through a stainless mesh (size 60, Sigma, St. Louis, MO) and suspended in RPMI 1640 medium (Gibco, BRL). Cell suspensions in PBS were centrifuged at 500 g for 5 min for removal of debris and placed through a nylon mesh presoaked in PBS. The supernatants containing hepatic mononuclear cells (MNCs) were collected and washed once with PBS, and then the cells were re-suspended in 40% Percoll (Sigma). The cell suspension was gently overlaid on top of 70% Percoll and centrifuged for 30 min at 800 g. MNCs were collected from the interphase, and washed twice in PBS. Approximately 1 × 107 cells/ mouse liver was recovered. 2.6. Isolation of total RNA and detection of mRNA by reverse transcription Liver samples were collected 12 h after Con A administration. Total RNA was isolated from the homogenate of the liver with Trizol reagent according to the protocol provided by the manufacturer. cDNA was synthesized from 2 μg of total RNA using PrimeScript™1st Strand cDNA Synthesis Kit (TaKaRa Biotechnology, China). The murine primer sequences are shown as follows: TNF-α (forward, F) 5′-GGGCTACAGGCTTGTCACTCG-3′ and (reverse, R) 5′-ACTCCAGGCGGTGCCTATGTC-3′, IFN-γ (F) 5′-CCTCAAACTTGGCAATACTCA-3′ and (R) 5′-CTCAAGTG GCATAGATGTGGA-3′, IL-6 (F) 5′-AGTTGCCTTCTTGGGACTGA-3′ and (R) 5′-TCCACGATTTCC CAGAGAAC-3′, TLR4 (F) 5′-GGTGTGAAATTGAGACAATTGAAAAC-3′ and (R) 5′-GTTT CCTGTCAGTACCAAGGTTGA-3′, NF-κB (F) 5′-AGAAGGCTGGGGTCAATCTT-3′ and (R) 5′-CTCAGGCT TTGTAGCCAAGG-3′, GAPDH (F) 5′-AGAGTGGGAGTTGCTGTTG-3′ and (R) 5′-GCCTTCCG TGTTCCTACC-3′. Total RNA was treated with DNase I to eliminate genomic DNA contamination, followed by synthesis of the first-strand using reverse transcription system. Reverse transcription was carried out as follows: 42 °C for 60 min, 70 °C for 10 min and first-strand cDNA was stored at −20 °C. Real-time PCR was performed in a 20 μl of reaction solution containing SYBR Premix Ex Taq, primers, and cDNAs. The cycles for PCR were as follows: 95 °C for 2 min, 40 cycles of 95 °C for 15 s, 58 °C for 20 s, and 72 °C for 20 s. Melting curves were determined by heat-denaturing PCR products over a 35 °C temperature gradient at 0.5 °C/s from 65 to 99.5 °C. GAPDH was used as an internal control [30]. The relative amount of mRNA was determined using the ΔΔCT technique as described previously [31]. The levels of mRNA were expressed as fold changes after normalization to GAPDH. 2.7. Western-blotting analysis of NF-κB and TLR4 Livers were carefully excised and homogenized into lysis buffer (Thermo, USA) to yield a homogenate. After centrifugation, protein concentration was determined by BCA protein assay kit (Thermo, USA) with bovine serum albumin as a standard. Equal amounts of protein extracts separated discontinuously on 10% SDS-PAGE and transferred onto PVDF membranes. After blockade of nonspecific binding sites, membranes were incubated for 2 h at room temperature with various antibodies against IκBα, phospho-IκBα, NF-κB p65, phospho-NF-κB p65

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and TLR4 (Cell Signal Technology, USA) for 2 h at room temperature. Protein bands were demonstrated by ECL detection system (Thermo USA). Signals were densitometrically assessed and normalized to the β-actin signals. 2.8. Flow cytometry analysis of CD4+, CD8+ and NKT cells Single-cell suspensions of the liver were obtained 12 h after the Con A-induced hepatitis was set up. Cells were then stained with fluorescence-labeled antibody (anti-CD4 APC, code: 17-0041-82; clone: GK1.5, eBioscience USA; anti-CD8 PE, code: 11-0081-85; clone: 53-6.7, eBioscience USA; anti-NKT1.1 PE, code: 12-5941, clone: PK136, eBioscience USA; anti-CD3 APC, code: 17-0031, clone: 145-2C11, eBioscience USA). The percentage of infiltrating CD4+, CD8+ and NKT cells in the liver was analyzed by flow cytometry (Miltenyi, Germany). 2.9. Statistical analysis Data reported are the mean ± standard deviation. All statistical analyses were performed using Prism 5.0 (Graph Pad Software, USA). All comparisons among groups were performed by one-way analysis of variance (ANOVA). For multi-group analysis, intergroup comparisons were performed by Dunn's test. Significance was accepted at P b 0.05. 3. Results 3.1. Paeoniflorin pretreatment attenuates Con A-induced hepatitis Plasma ALT and AST levels increased 12 h after intravenous Con A administration. Pretreatment with paeoniflorin markedly attenuated the plasma levels of ALT and AST compared to Con A group (P b 0.01) (Fig. 1), indicating that paeoniflorin has a protective effect on Con A-induced hepatitis. To further confirm the protective effect of paeoniflorin on liver injury, we performed hematoxylin and eosin staining of the liver. After Con A administration intravenously, massive hepatocyte necrosis in Con A group was observed. However, in paeoniflorinpretreated group, the area and extent of necrosis were diminished (Fig. 2a). Besides, the pathological scores showed that liver injury in Con A group was significantly higher than that in paeoniflorin pretreatment group (P b 0.01) (Fig. 2b). These results suggest that paeoniflorin attenuates liver injury induced by Con A intravenous injection via the tail. 3.2. Paeoniflorin pretreatment inhibited the release of proinflammatory cytokines Proinflammatory cytokines play pivotal roles in Con A-induced hepatitis. As shown in Fig. 3, the increased plasma levels of TNF-α, INF-γ, and IL-6 in response to Con A were prevented by pretreatment with the paeoniflorin (P b 0.05). Furthermore, the intrahepatic expression levels of TNF-α, IFN-γ and IL-6 mRNA were significantly decreased in paeoniflorin pretreated mice (P b 0.05) (Fig. 4). Thus, the prevention

by paeoniflorin of Con A-induced liver injury is also associated with the inhibition of proinflammatory cytokine release. 3.3. Effect of paeoniflorin on liver infiltrating CD4+, CD8+ and NKT cells Accumulating evidence demonstrated that T cells play a critical role in Con A-induced liver injury [5–7]. In our study, we calculated the percentage of CD4+ and CD8+ T cells in the liver by flow cytometry. As expected, compared with those from Con A group mice, the percentage of infiltrating CD4+ and CD8+ cells were significantly decreased in livers of Con A + PF group mice (17.4% vs. 6.37%, 13.3% vs. 6.56%, respectively) (P b 0.01) (Fig. 5d–e). In this study, we compared the percentage of NKT cells in the liver among the four groups without any statistical significance. But we found that the absolute amounts of infiltrating of NKT cells in the liver increased in the livers of Con A group mice, compared to Con A + PF group mice (14.56 × 105 vs. 8.89 × 105) (P b 0.01) (Fig. 5f). Pretreatment with paeoniflorin significantly reduces CD4+, CD8+ and NKT cells recruited into the liver. 3.4. Influence of paeoniflorin on Con A-induced liver TLR4 mRNA and protein We tested the expression of TLR4 protein by western blot in the liver. As shown in Fig. 6a, the expression of TLR4 protein was reduced by paeoniflorin pretreated compared with Con A group. Additionally, we also examined intrahepatic mRNA expression of TLR4 at 12 h after Con A injection. We found that the intrahepatic TLR4 mRNA levels increased upon Con A injection at 12 h, and the expression of these TLR4 genes in paeoniflorin pretreated group was also reduced significantly compared with Con A group (P b 0.05) (Fig. 6b). 3.5. Effect of paeoniflorin on NF-κB pathway in Con A-induced hepatitis Since the transcription factor NF-κB plays an important role in Con A-induced hepatitis and is associated with inflammatory response, we therefore wondered whether the effect of paeoniflorin on cytokine might be due to its influence on NF-κB activation in vivo. In our study, we found that liver expression of NF-κB mRNA was significantly enhanced after ConA injection, and paeoniflorin pretreatment inhibited the expression of NF-κB mRNA compared with that in the Con A administration group (P b 0.05) (Fig. 7a). Furthermore, we tested the levels of phosphorylation of IκBα and p65 in cytoplasm by Western blot analysis. As shown in Fig. 7b, paeoniflorin pretreatment inhibited IκBα and p65 phosphorylation markedly compared with Con A group. 4. Discussion Autoimmune and viral hepatitis represent a significant health issue, as they can progress into cirrhosis and no effective therapies exist up to date. Peony is one of a number of well-known herbs in China. Several clinical observations reported in China indicated that peony, which

Fig. 1. Effect of paeoniflorin on serum ALT and AST levels. Mice were treated with PBS, paeoniflorin 3 h before Con A administrated. Twelve hours after Con A injection, serum were collected by heart puncture. Administration of paeoniflorin led to a significant decrease of serum AST and ALT levels in Con A + PF group mice, treated with paeoniflorin before Con A administration, compared with Con A group mice that did not receive paeoniflorin. In the absence of Con A, administration of paeoniflorin did not induce a significant change in serum AST or ALT levels (n = 7 mice/group); **P b 0.01 vs. PBS group, ##P b 0.01 vs. Con A group.

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Fig. 2. Histopathological changes of mice. The liver tissue sections were stained with H&E. (a) Histological sections of livers from Con A + PF group mice revealed markedly attenuated damage compared with sections of Con A group livers, in which massive hepatocyte necrosis was present (n = 7 mice/group). Original magnification ×100. (b) The severity of liver injury was scored as described in the Materials and Methods section. **P b 0.01 vs. PBS group, ##P b 0.01 vs. Con A group.

contains more than 90% of paeoniflorin, was a safe and effective medicine in the treatment of various inflammatory and autoimmune diseases [32]. Recently, several researches indicated that paeoniflorin was an effective medicine in the treatment of various diseases [22–27]. In light of these observations, we investigated the therapeutic potential of paeoniflorin in a hepatitis model induced by Con A. In the current study, we showed that paeoniflorin pretreatment 3 h before Con A challenge protected the mice from Con A-induced hepatitis. According to the results of our study, paeoniflorin pretreatment significantly decreased the serum level of aminotransferase and inhibited the severity of hepatic necrosis in Con A-treated mice (Figs. 1, 2). The potential beneficial mechanisms of this effect involve inhibiting inflammatory responses, which led us to investigate the effect of paeoniflorin on proinflammatory cytokine expression in mouse. A number of studies have shown that proinflammatory cytokines were the main factors which were responsible for pathogenesis of Con A-induced hepatitis [8–11]. Previous studies have shown that antibodies against TNF-α or IFN-γ can attenuate Con A-induced liver injury in mice [33,34]. Similar findings have demonstrated that IFN−/−TNF−/− mice did not develop liver injury upon Con A stimulation [10]. Cao et al.'s experiments showed that paeoniflorin inhibited LPS-stimulated tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β release [35]. In addition, Wang et al. reported that paeoniflorin exerted its protection of allergic contact dermatitis in mice via inhibiting inflammatory responses [22]. Similar to the previous studies, we found that paeoniflorin pretreatment significantly diminished TNF-α and IFN-γ levels in the plasma in Con A-treated mice. At the same time, compared with the Con A group, paeoniflorin markedly reduced the expression of TNF-α and IFN-γ mRNA in the liver. Moreover, Liang et al. found that IL-6 also played an important role in liver injury [36]. In our study, we also observed a significant reduction in the IL-6 level in plasma as well as mRNA expression in the liver with treatment by paeoniflorin (Figs. 3, 4). Our data suggested that paeoniflorin exerts its hepatoprotective

effect through inhibition of proinflammatory cytokine production in this model. During the development of inflammatory Con A-induced injury, lymphocyte, including T lymphocyte, activated natural killer T (NKT) cells, macrophages, dendritic cells (DCs) and Kupffer cells, recruitment increased significantly and these infiltrating lymphocytes in intrahepatic will determine the severity of liver injury [5–7,37,38]. Among these immune cells, CD4+ T cells and NKT cells were the predominant T lymphocytes recruited to the liver following Con A injection [20,39]. Previous research reported that mice treated with anti-CD4 monoclonal antibodies did not develop hepatitis following Con A injection [7]. Additionally, Mizuhara et al. found that mice pretreated with FK506 (a T cellspecific immunosuppressive drug) before Con A challenge failed to develop disease [40]. In our previous report, we demonstrated that CD4+ and CD8+ T cell recruitment increased significantly in the liver in this model [28]. Moreover, the study conducted by Takeda et al. found that NKT cells were depleted by continuous observation (0 h, 2 h, 4 h, 8 h, 16 h, 20 h) after Con A injection and indicated that they acted as predominant effector cells in Con A-induced hepatitis and might play a crucial role in the Con A-induced hepatitis [41]. However, the number of NKT cell was significantly higher after Con A administration after 12 h, compared with control group [42]. In this study, we found that the percentage of CD4+, CD8+ and the number of NKT cells in the liver increased markedly after Con A injection without paeoniflorin pretreatment at 12 h, compared with control group without Con A-treatment. Our study data showed that paeoniflorin pretreatment was associated with a remarkable decrease of CD4+, CD8+ and NKT cells (Fig. 5d–f). These findings suggested that paeoniflorin protects against Con A-induced hepatitis, at least in part, by suppressing CD4+, CD8+ and NKT cell infiltration in the liver. In our study, we only demonstrated that paeoniflorin was able to decrease the intrahepatic lymphocyte (CD4+, CD8+ and NKT cells) infiltration. However, we did not explore why paeoniflorin could decrease

Fig. 3. Effect of paeoniflorin on serum levels of cytokine in Con A-induced liver injury. Mice were treated with PBS, paeoniflorin 3 h before Con A administrated. Twelve hours after Con A injection, serum was collected by heart puncture. The concentrations of TNF-α, IFN-γ and IL-6 were detected by using ELISA kits according to the manufacturer's instructions. Serum TNF-α, IFN-γ and IL-6 were significantly lower in Con A + PF group mice, compared with Con A group animals (n = 7 mice/group). **P b 0.01 vs. PBS group, ##P b 0.01 vs. Con A group.

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Fig. 4. Influence of paeoniflorin on intrahepatic cytokine levels in Con A-induced liver injury. The levels of mRNA expression in the liver were examined by quantitative real time PCR at 12 h after injection of Con A to PBS- or paeoniflorin-pretreated mice. TNF-α, IFN-γ and IL-6 mRNA levels were normalized with GAPDH expression in each sample. Paeoniflorin alleviated intrahepatic mRNA expression of these cytokines (n = 7 mice/group). **P b 0.01 vs. PBS group, ##P b 0.01 vs. Con A group.

the lymphocytes in the present study, which may include antiproliferative of T lymphocytes, inhibition of chemokine or adhesion molecule production and so on. Further investigation of this question

will help better understand the molecular mechanisms of immunomodulating action of paeoniflorin in the treatment of autoimmune and viral hepatitis. Moreover, increasing studies have shown that the activation

Fig. 5. The effect of paeoniflorin on T lymphocyte infiltrated liver. At 12 h after Con A challenge, hepatic lymphocytes were isolated from each group mice, and stained with APC- or PEconjugated mAbs and analyzed by flow cytometry. Shown are (a) CD4+ T cells (using anti-CD4 Ab) (b) CD8+ T cells (using anti-CD8 Ab) (c) NKT cells (using anti-NK1.1 plus anti-CD3 Ab). (a–c) Representative images from four animals per group are shown. (d–e) Shown is the percentage of CD4+ and CD8+ T cells. The percentage of CD4+ and CD8+ T cells in the liver increased significantly following Con A administration without paeoniflorin pretreatment, compared with Con A + PF group. (f) The absolute number of infiltrated NKT cells was calculated by multiplying the total number of hepatic MNCs with percentage of NKT cells. Administration of paeoniflorin led to a marked decrease in the intrahepatic NKT lymphocyte number in Con A + PF group animals, compared with Con A group animals. Paeoniflorin pretreatment reduced liver-infiltrating CD4+, CD8+ and NKT cells after Con A administration (n = 7 mice/group). **P b 0.01 vs. PBS group, ##P b 0.01 vs. Con A group. Results are representative of three independent experiments.

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Fig. 6. Effect of paeoniflorin on Con A-induced liver TLR4 expression. (a) Liver tissues harvested 12 h after Con A administration. Hepatic TLR4 protein levels were analyzed by western blot with antibody against TLR4. The β-actin was used as an internal control. Paeoniflorin pretreatment followed by Con A administration attenuated TLR4 protein expression in the liver. (b) The levels of mRNA were determined at 12 h after Con A injection by quantitative real time PCR. TLR4 mRNA levels were normalized with GAPDH expression in each sample. Paeoniflorin pretreatment alleviated intrahepatic mRNA expression of TLR4 (n = 7 mice/group). **P b 0.01 vs. PBS group, ##P b 0.01 vs. Con A group.

of macrophages and DCs also played pivotal roles in the development of this model [37,38] and further studies were needed to clarify the effects of paeoniflorin in these cell types. Toll-like receptor (TLR) 4, which is a pattern recognition receptor (PRR) and is widely expressed on Kupffer cells, hepatocytes, hepatic stellate cells, biliary epithelial cells, sinusoidal endothelial cells, and hepatic dendritic cells (DCs) in the liver [43,44], has been reported to play an important role in the regulation of inflammation [45]. Some researches have shown that TLR4 expression was upregulated in a mouse model of Con A-induced hepatic injury as compared with the normal liver [15–17]. It has been reported that TLR4 knockout mice exposed to dextran sulfate sodium or alcohol developed significantly

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less inflammation in the liver [46,47]. A recent work by Xu et al. demonstrated that cytokine production was attenuated and liver injury was prevented in the absence of a functional TLR4 pathway in mice [44]. In our study, TLR4 was upregulated in the Con A group; however, the upregulation of TLR4 was significantly reversed by paeoniflorin pretreatment (Fig. 6). Given that TLR4 plays an important role in regulating inflammation, we speculated that paeoniflorin exerts its hepatoprotective effect, at least in part, via inhibition of the expression of TLR4 in this model. Supporting our data, some study reported that paeoniflorin abrogates DSS-induced colitis via decreasing the expression of TLR4 [48]. However, what the endogenous ligands are and how these ligands activate TLR4 in this model are still elusive. Hence, it suggested that the regulation of the Con A-induced liver injury via TLR4 must be very complex. Previous papers reported that several other receptors are involved in the development of Con A-induced liver injury, including the Fas/Fas ligand system, vitamin D receptor, TLR2, TLR3 and TLR9 [16,49–51]. Moreover, Hung et al. demonstrated that paeoniflorin exerted pharmacologic effect through the Fas/Fas ligand system [52]. In addition, a recent work by Lin reported that paeoniflorin served as a LXRα agonist to exert cholesterol-lowering, antidiabetic, and neuroprotective effects through LXRα pathway [53]. These studies suggested that multitargets may be involved in protective effects of paeoniflorin in this model. Therefore, arduous works should be implemented to clarify the precise mechanism of protective effect of paeoniflorin in this model. To further elucidate the possible causes to paeoniflorin protective effect of anti-inflammatory in Con A-induced liver injury model, we therefore examined the effects of paeoniflorin on intrahepatic NF-κB level in the model. Nuclear factor κB (NF-κB) is a nuclear transcription factor that regulates the expression of a large number of genes that are critical for the regulation of viral replication, tumorigenesis, inflammation, and various autoimmune diseases. Emerging evidence suggested that blockade of NF-κB activation can ameliorate Con A-induced liver injury and reduce the secretion of inflammatory molecules [19–21]. Several previous studies have demonstrated that paeoniflorin exhibited its impacts by regulating NF-κB signaling pathway. For example, Zhou et al. showed that paeoniflorin was able to alleviate acute lung injury through downregulation of the activation of NF-κB pathway in lung tissues [26]. Guo et al. reported that paeoniflorin protected against ischemia-induced brain damages in rats via inhibiting NF-κB pathway [54]. Here, our study also revealed that paeoniflorin pretreatment was capable of suppressing IκBα kinase and p65 phosphorylation in Con Ainduced liver injury (Fig. 7), which suggested that inhibiting NF-κB activation by paeoniflorin may contribute to the protective effects

Fig. 7. The effect of paeoniflorin pretreatment on NF-κB mRNA and protein expression. (a) The levels of mRNA were determined at 12 h after Con A injection by quantitative real time PCR. NF-κB mRNA levels were normalized with GAPDH expression in each sample. Paeoniflorin pretreatment 3 h before Con A administration attenuated intrahepatic NF-κB mRNA expression (n = 7 mice/group). **P b 0.01 vs. PBS group, ##P b 0.01 vs. Con A group. (b) At 12 h after Con A challenge, liver tissues were harvested. Hepatic NF-κB protein levels were analyzed by western blot with antibodies against IκBα, phospho-IκBα, NF-κB p65, and phospho-NF-κB p65. The β-actin was used as an internal control. Paeoniflorin pretreatment followed by Con A administration attenuated IκBα and p65 phosphorylation.

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against Con A-induced liver injury. Consequently, we speculated that the mechanism of paeoniflorin exerting the protective effects against Con A-induced liver injury by suppression of inflammatory responses might be related to the downregulation of TLR4 expression and inhibition of NF-κB signaling pathway. In this study, only one dose of paeoniflorin was used in the study, which may ignore its dose dependent effect. Our study is limited by the lack of investigation of the other pharmacological actions of paeoniflorin, such as antioxidant, which may participate in Con Ainduced hepatitis. Further studies are required to clarify the mechanism underlying the pharmacological action of paeoniflorin in this model of Con A-induced hepatitis. TLR4 and NF-κB are general signaling pathways in modulating the effect of paeoniflorin on Con A-induced liver injury model, which may not explain the mechanism clearly. Further studies are required to elucidate their specific signaling pathways in this model. 5. Conclusion In conclusion, our research indicated that paeoniflorin exerts a hepatoprotective effect on T cell-mediated hepatitis via inhibiting inflammatory response and, at least partly, by suppressing CD4+, CD8+ and NKT cell infiltration in the liver. The NF-κB and TLR4 signaling pathways may be involved in the protective effects of paeoniflorin, which may account for reduction of increased pro-inflammatory cytokine levels and thereby prevent liver injury. These results may give insight into the further evaluation of paeoniflorin as a therapeutic agent or potential adjunct to T cell mediated liver disease therapy. Abbreviations Con A ALT AST TLR NF-κB

Concanavalin A Alanine aminotransferase Aspartate aminotransferase Toll-like receptor Nuclear factor κB

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