Inflammation ( # 2015) DOI: 10.1007/s10753-014-0108-7

Trichostatin A Protects Against Experimental Acute-on-Chronic Liver Failure in Rats Through Regulating the Acetylation of Nuclear Factor-κB Qian Zhang,1 Fan Yang,1 Xun Li,1 Lu-wen Wang,1 Xiao-gang Chu,1 Hong Zhang,2 and Zuo-jiong Gong1,3

Abstract—Histone deacetylase inhibitors (HDACi) were recently shown to suppress inflammatory responses in experimental models of autoimmune and inflammatory diseases. In this study, the protective effects of Trichostatin A (TSA), an HDACi, on experimental acute-on-chronic liver failure (ACLF) in rat were explored. An ACLF model was established in rats, and animals were randomly divided into control, model, and TSA-treated groups. The rats in TSA-treated group received TSA (2 mg/kg) at 2 h before induction of ACLF. Samples were obtained at 24 h after ACLF induction. We found that the rats in model group showed severe damage to liver tissue at 24 h after ACLF induction. TSA improved liver injury effectively. Serum tumor necrosis factor-alpha (TNF-α), interferon-γ (IFN-γ), interleukin (IL)10, and IL-18 levels were significantly increased in model group compared with control group, but TSA reduced serum TNF-α, IFN-γ, IL-10, and IL-18 levels effectively compared with model group. In addition, TSA reduced the total HDAC activity, promoted the acetylation of histone, and decreased the expressions of class I HDAC in liver tissue. TSA also increased the acetylation levels and decreased phosphorylation levels in NF-κB p65. The median survival time of the rats was significantly prolonged in TSA-treated group. To conclude, TSA can inhibit the release of multiple inflammatory cytokines, prolong the survival time, and protect against ACLF in rats. The mechanisms were probably through enhancing the acetylation levels of non-histones rather than histone. KEY WORDS: Trichostatin A; inflammation; histone deacetylase; acute-on-chronic liver failure; nuclear factor-κB.

INTRODUCTION Acute-on-chronic liver failure (ACLF) is a severe lifethreatening condition, which is the most common type of liver failure with a high fatality in Asia, especially in China [1]. The main pathological characteristic of ACLF is massive or submassive hepatocellular necrosis accompanied with the inflammatory cell infiltration on the basis of chronic liver injury. Although the pathogenesis of ACLF is not entirely clear, the intestinal endotoxemia and the release of inflammatory cytokines are proved to involve 1

Department of Infectious Diseases, Renmin Hospital of Wuhan University, Wuhan, 430060, China 2 Department of Pharmaceutical, Renmin Hospital of Wuhan University, Wuhan, 430060, China 3 To whom correspondence should be addressed at Department of Infectious Diseases, Renmin Hospital of Wuhan University, Wuhan, 430060, China. E-mail: [email protected]

in the progress of the disease [1–3]. Several studies have demonstrated that the rising levels of inflammatory cytokines in patients with liver cirrhosis are correlated to the emergence of clinical complications [4–9]. In contrast with the clinical picture of acute liver failure (ALF), ACLF shares many similarities with severe sepsis or systemic inflammatory response syndrome (SIRS) with multiple organ failure [10–12]. During the development of ACLF, inflammatory genes, which drive the host’s immune response, are regulated closely at multiple checkpoints, including signaling pathways and transcription factors [13, 14]. Recently, particular attention has been paid to the unique role of chromatin in transcriptional regulation [15]. Dynamic equilibrium of chromatin remodeling is crucial for transcriptional activation and gene expression. One of the best studied epigenetic modifications is histone acetylation [16, 17], which is regulated by histone acetyltransferases (HATs)

0360-3997/15/0000-0001/0 # 2015 Springer Science+Business Media New York

Zhang, Yang, Li, Wang, Chu, Zhang, and Gong and histone deacetylases (HDACs). HATs activate transcription by enhancing nucleosome relaxation. There are 18 members of the HDAC family that have been identified [18]. The class I (HDACs1, 2, 3, and 8) and class II (HDACs4, 5, 6, 7, 9, 10, and 11) isoforms are Zn-dependent, whereas class III HDACs (Sirtuins 127) are NAD+dependent. Trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) have a hydroxamate structure and inhibit Zn-dependent HDAC subtypes broadly [18, 19]. It is confirmed that HDACs can stabilize nucleosome structures and suppress transcription [20]. Therefore, theoretically, TSA as an HDAC inhibitor (HDACi) can relax nucleosome structures and promote transcription. It is reported that HDACi, such as TSA, SAHA, and VPA, was able to decrease hemorrhage-associated lethality in hemorrhagic shock [21–23], suppress expression of proinflammatory cytokines, and improve survival in a mouse model of septic shock [24–26]. However, it is also reported that the reduction of HDAC3 activity by exposure of human macrophages to smoke resulted in enhanced inflammatory cytokine production [27]. Whether HDACi could protect against ACLF effectively is not sure yet. In the present study, we established an ACLF rat model and explored whether TSA as an HDACi could prevent the release of proinflammatory cytokines and had protective effects on ACLF.

MATERIALS AND METHODS Experimental Animals Specific pathogen free (SPF) Wistar rats, weighing 120 to 160 g, were purchased from the Experimental Animal Center of Hubei Province. All rats had been acclimated to the research laboratory for 5 days before experiments and maintained in a light-controlled room (12 h light and dark cycle) at an ambient temperature of 25 °C with free access to water and standard chow. The rats were given humanistic concern in accordance with the animal laboratory guidelines.

multipoint with 0.5 mL above solutions containing 4 mg HSA at a total of four times (14-day intervals between the first and second time; 14-day intervals between the second and third time; 10-day intervals between the third and fourth time). After sensitization by HSA, the rats were injected 4 mg HSA into tail vein twice a week for 6 weeks. The chronic liver injury in rats was firstly induced by HSA. Secondly, the rats were administrated by intraperitoneal injection with D-Gal (purity of 98 %, Sigma-Aldrich Co., USA) at a dose of 400 mg/kg combined with LPS (purity of 99 %, Sigma-Aldrich Co., USA) at a dose of 100 μg/kg, which induce acute liver failure on the basis of chronic liver injury. The normal rats were administrated with physiological saline (i.e., the vehicle solution of HSA, D-gal, and LPS) at the same time points as did in the model rats, which served as a control. Experimental Design Forty-five animals were randomly divided into control group (n = 15), model group (n = 15), TSA-treated group (n=15). The time of combined administration of D-Gal and LPS was accepted as baseline (time point 0). The rats in TSA treatment groups were injected intravenously with TSA at a dose of 2 mg/kg at 2 h before induction of ACLF. The rats in control group and model group were injected intravenously with the same volume of normal saline solution. Then, five rats in each group were randomly sacrificed for blood and liver samples at 24 h time point after induction of ACLF. The remaining rats in each group were observed for survival duration after induction of ACLF in a week. Determinations of Histopathology For routine histology, inflation-fixed livers were harvested, fixed, dehydrated, paraffin embedded, and sliced into 4-μm-thick sections. After deparaffinization, slides were stained with hematoxylin and eosin (HE) using standard methods. The pathological changes evaluated under light microscope.

Establishment of Animal Model As described previously [28], the ACLF rat models were induced by human serum albumin (HSA), D-galactosamine (D-Gal), and lipopolysaccharide (LPS). In brief, HSA (Octapharma GmbH, Austria) was diluted to a concentration of 8 g/L with physiological saline and emulsified with an equal amount of incomplete Freund’s adjuvant. The rats were injected subcutaneously at

Determinations of Blood Samples The contents of cytokines (TNF-α, IFN-γ, IL-10, and IL-18) in blood samples were detected by using ELISA kit (eBoscience, California, USA) according to the instructions’ of the manufacturer. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) were measured by routine biochemical

Trichostatin A Protects Against Experimental Acute-on-Chronic Liver Failure in Rats methods using a Hitachi Automatic Analyzer (Hitachi, Inc. Japan). Determinations of Total HDAC Activity of the Liver Tissue The activity of HDAC was measured using a commercial HDAC activity assay kit as described by the instructions. In brief, total extract was prepared from freshly harvested liver tissue. Fifty micrograms of total extract was incubated with HDAC assay substrate and incubated at 30 °C for 1 h. After the addition of reagent, samples were kept at room temperature for 15 min, and readings were taken with a fluorescent microplate reader at an excitation of 360 nm and emission of 450 nm. A standard curve was performed according to the manufacturer’s protocol. Western Blot of Class I HDACs, Histone, and NF-κB p65 in Liver Tissue Fifty microgram protein extracts from 100 mg liver tissue samples were subjected to 12 % SDS-PAGE and then transferred to a PVDF membrane (Millipore, Germany). The membrane was incubated with different primary antibodies (anti-HDAC, histone, and NF-κB p65 antibody from Cell Signaling Technology Co. USA) and then with a secondary antibody (LICOR Co., USA) and finally detected by Odyssey infrared imaging system (LICOR Co., USA). Membranes were also probed for β-actin (Santa Cruz Co., USA) as additional loading controls. Statistical Analysis All data were expressed as means±SD. Differences among groups were assessed using Student’s t test. SPSS 13.0 software was used for statistical analysis. P value less than 0.05 was considered to be statistically significant.

decrease of inflammatory cell infiltration, alleviation of hepatocyte swelling, and less cell necrosis. As shown in Fig. 1d–f, serum ALT, AST, and TBIL increased significantly in model group (5475.04 ± 431.8 U/L, 2816.53 ± 342.43 U/L, and 98.74 ± 18.56 μmol/L, respectively) as compared with normal group (72.42 ± 8.26 U/L, 54.27 ± 7.15 U/L, and 3.68 ± 0.57 μmol/L, respectively). However, serum ALT, AST, and TBIL were markedly suppressed in TSA-treated group (849.25 ± 90.44 U/L, 285.65 ± 39.55 U/L, and 28.65 ± 5.66 μmol/L, respectively) as compared with model group. TSA Decreased Serum Levels of TNF-α, INF-γ, IL-10, and IL-18 As shown in Fig. 2, the serum levels of TNF-α, IFNγ, IL-10, and IL-18 were increased significantly in the model group (1315.75±168.70, 735.75±114.14, 993.26 ±212.42, and 1099.52±218.56 pg/mL, respectively) compared with those in control group (162.96±62.15, 66.96 ±26.21, 68.46±27.81, and 69.93±25.12 pg/mL, respectively) (P<0.05). However, the serum levels of TNF-α, INF-γ, IL-10, and IL-18 were significantly reduced in TSA-treated group compared with those in model group (417.11 ± 97.52, 221.01 ± 95.38, 197.21 ± 91.52, and 267.78±78.66 pg/mL, respectively) (P<0.05). TSA Reduced the Total HDAC Activity of the Liver Tissue As shown in Fig. 3, compared with the control group, the total HDAC activity of liver tissue in model group increased significantly. However, HDAC activity was reduced obviously in TSA-treated group compared with model group (40.3±1.45 vs. 56.7±4.24 OD/mg, P<0.05). TSA Promoted the Acetylation of Histone

RESULTS TSA Improved Liver Histology and Liver Function The representative images of liver tissue stained with HE for each group were shown in Fig. 1. In control group (Fig. 1a), hepatocytes were arrayed radiatively around the central vein. Neither degeneration nor necrosis was presented. However, massive or submassive necrosis could be observed in model group (Fig. 1b). In TSA-treated group (Fig. 1c), liver histology was significantly improved as compared with the model group. There was an obvious

As shown in Fig. 4, compared with control group, acetylation levels of H3 and H4 were both increased in model group (P<0.05). Moreover, compared with model group, the acetylation levels of H3 and H4 were increased even further in TSA-treated group (P<0.05). TSA Decreased the Expressions of Class I HDACs As shown in Fig. 5, compared with control group, the expression levels of HDAC1, HDAC2, and HDAC3 were increased significantly (P<0.05). However, the expression levels of HDAC1, HDAC2, and HDAC3 were all

Zhang, Yang, Li, Wang, Chu, Zhang, and Gong

Fig. 1. TSA improved liver histology and liver function of rats with ACLF. a Control group, b model group, c TSA-treated group, d ALT, e AST, f TBIL. Rats in the different group were killed at 24 h time point. Livers were harvested and stained with hematoxylin and eosin. The pathological changes were evaluated under light microscope (a1, b1, and c1, ×100), (a2, b2, and c2, ×200). Compared with the control group,*P < 0.05. Compared with the model group, #P < 0.05.

decreased in TSA-treated group compared with model group (P<0.05).

increased in the model group but decreased in the TSAtreated group compared with control group (P<0.05).

TSA Enhanced the Acetylation of NF-κB p65

TSA Treatment Prolonged the Survival Time

As shown in the Fig. 6, the acetylation level of NF-κB p65 was decreased obviously in model group compared with that in control group (P<0.05), while it increased significantly in TSA-treated group (P<0.05). However, there were different changes in phosphorylation level of NF-κB p65 compared with acetylation level of NF-κB p65. The expressions of phosphorylated NF-κB p65 were

Kaplan–Meier survival curves (Fig. 7) showed that the ACLF rats all died within 4 days without TSA treatment, and their median survival time was 48 h. TSA treatment significantly prolonged the median survival time of the ACLF rats to 120 h. By log-rank test, there was a significant difference among the curves (χ2 = 15.91, P < 0.0001).

Trichostatin A Protects Against Experimental Acute-on-Chronic Liver Failure in Rats

Fig. 2. TSA decreased serum levels of TNF-α, INF-γ, IL-10, and IL-18 in rats with ACLF. Control: control group, model: model group, TSA: TSA-treated group. Rats in the different group were killed at 24 h time point. Blood was harvested, and TNF-α, INF-γ, IL-10, and IL-18 were examined using ELISA kits. Data expressed as means ± SD. Compared with the control group, *P < 0.05. Compared with the model group, #P < 0.05.

DISCUSSION Acetylation is emerging as key post-translational modifications of histones that maintain the structure of chromatin and regulate gene transcription [29]. The

Fig. 3. TSA reduced the total HDAC activity of the liver tissue. Control: control group, model: model group, TSA: TSA-treated group. Rats in the different group were killed at 24 h time point. Total extract of liver was prepared for HDAC activity assay. Readings were performed using a fluorescent microplate reader at an excitation of 360 nm and emission of 450 nm. Compared with the control group, *P < 0.05. Compared with the model group, #P < 0.05.

acetylation status of chromatin is controlled by the antagonistic activities of HATs and HDACs. HATs transfer acetyl groups from acetyl-coenzyme A to the amino group of lysines in the amino-terminal region of histones. HDAC is a group of enzymes that remove acetyl groups from histones and epigenetically modulate the expression of various genes [30]. Although HDAC inhibitors are used as anti-tumor reagents in clinic, their protective effects on inflammatory diseases are becoming increasingly recognized [19, 31]. The most widely studied potent inhibitor of mammalian HDAC is TSA, which selectively inhibits the classes I and II mammalian HDACs [32], thereby regulating the acetylation of histones and non-histone proteins. The results of recent studies showed that TSA can alleviate inflammation [33, 34]. In the present study, TSA was used as HDAC inhibitors to explore its protective effect on experimental ACLF in rats. It was found that TSA could alleviate liver injury effectively in ACLF, which suggested a crucial pathophysiological significance of HDAC in the liver injury in ACLF. ACLF is not only the presence of organ failure with a high mortality rate but also the presence of the severe systemic inflammation [35]. In order to evaluate the

Zhang, Yang, Li, Wang, Chu, Zhang, and Gong

Fig. 4. TSA promoted the acetylation of histone (H3 and H4). Control: control group, model: model group, TSA: TSA-treated group. Rats in the different group were killed at 24 h time point. Total extract of liver was prepared for Western blot. a Hepatic acetylation of H3 and H4 levels were detected by Western blot, and β-actin was probed as a loading control. b Relative expressions of AH3 and AH4. The relative protein expression levels are represented as the ratio of the band density of AH3 and AH4 to H3 and H4. Compared with the control group, *P < 0.05. Compared with the model group, #P < 0.05.

Fig. 5. TSA decreased the expressions of class I HDACs. Control: control group, model: model group, TSA: TSA-treated group. Rats in the different group were killed at 24 h time point. Total extract of liver was prepared for Western blot. a Hepatic class I HDACs levels were detected by Western blot, and β-actin was probed as a loading control. b Relative expressions of HDAC1, HDAC2, and HDAC3. The relative protein expression levels are represented as the ratio of the band density of HDAC1, HDAC2, and HDAC3 bands to β-actin. Compared with the control group, *P < 0.05. Compared with the model group, # P < 0.05.

Trichostatin A Protects Against Experimental Acute-on-Chronic Liver Failure in Rats

Fig. 6. TSA enhanced the acetylation of NF-κB p65. Control: control group, model: model group, TSA: TSA-treated group. Rats in the different group were killed at 24 h time point. Total extract of liver was prepared for Western blot. a Acetylation and phosphorylation of NF-κB p65 levels were detected by Western blot, and β-actin was probed as a loading control. b Relative expressions of acetylation and phosphorylation of NF-κB p65. The relative protein expression levels are represented as the ratio of the band density of acetylation and phosphorylation of NF-κB p65 to the band density of NF-κB p65. Compared with the control group, *P < 0.05. Compared with the model group, #P < 0.05.

systemic inflammatory status, the serum levels of TNF-α, IFN-γ, IL-10, and IL-18 were determined in this study. TNF-α, IFN-γ, IL-10, and IL-18 are pivotal inflammatory cytokines, which are greatly upregulated in the patients with ACLF [28, 36, 37]. In the present study, the serum levels of TNF-α, IFN-γ, IL-10, and IL-18 were increased significantly in the model group

Fig. 7. TSA treatment prolonged the survival time of the rats with ACLF. Control: control group, model: model group, TSA: TSA-treated group. The ACLF rats all died within 4 days without TSA treatment, and their median survival time was 48 h. TSA treatment significantly prolonged the median survival time of the ACLF rats to 120 h. By log-rank test, there was a significant difference among the curves (χ2 = 15.91, P < 0.0001).

compared with those in control group but were significantly reduced in TSA-treated group compared with those in model group. The results suggested that pretreatment with TSA could decrease the circulating levels of inflammatory factors significantly and suppress the inflammatory response in ACLF. Previous studies in experimental inflammatory animal models for asthma, colitis, arthritis, and LPS-induced septic shock were also confirmed the inhibitory effects of TSA on inflammatory response [23, 38–40]. Therefore, the anti-inflammatory effect of TSA might be attributed to its ability to regulate the expression of inflammation-related genes. The administration of HDAC inhibitors usually results in enhanced acetylation of histone. This alteration might be associated with the opening of chromatinic structure, which facilitates gene transcription [41]. In our study, the acetylation levels of H3 and H4 were measured for exploring the effects of TSA on the acetylation of histone. The results showed that the acetylation levels of H3 and H4 were both increased in model group compared with control group. Moreover, the acetylation levels of H3 and H4 were further increased in TSA-treated group compared with model group. These results are consistent with previous

Zhang, Yang, Li, Wang, Chu, Zhang, and Gong similar studies [42, 43]. Except for the acetylation levels of H3 and H4, the expressions of class I HDACs were also detected. The result showed that expressions of HDAC1, HDAC2, and HDAC3 were all increased in the model group but were all decreased in the TSA-treated group. These results were consistent with the changes of HDAC activity detected in the liver tissue in this study. It was suggested that HDACs may promote the inflammatory response, while the HDAC inhibitors could downregulate the expressions of HDACs and play anti-inflammatory effects. It was recently reported that HDAC inhibitors could also enhance the transcription of TNF-α mRNA, but it reduced the protein level of TNF-α in the hemorrhaged leukocytes after a LPS “second hit” [44]. Moreover, HDAC inhibitors were reported to have anti-inflammatory effects both in vitro and in vivo [45]. Therefore, the anti-inflammatory function of HDAC inhibitors seemed to be associated with their effect on the chromatin. Previous study has proved that HATs and HDACs target not only histone but also non-histone. In a human cell study, it was found that there were more than 1750 kinds of protein that can be modified by acetylation on lysine residue [46]. Lysine residues of proteins can be modified by methylation, ubiquitination, acetylation, and ADP glycosylation [47]. Therefore, the function of non-histone proteins can be regulated by acetylation, which probably contributes to the antiinflammatory activity. Some groups have reported that mitogen-activated protein kinase phosphatase-1 (MKP1) is acetylated on lysine residue, which increases its phosphatase activity and interrupts mitogen-activated protein kinase signaling [48]. In addition, some scholars found that nuclear factor-κB (NF-κB) was also acetylated at various lysine residues. These acetylation modifications have been reported to both positively and negatively regulate the subcellular location, DNAbinding affinity as well as the transcriptional activity of NF-κB [49, 50]. In our study, we had detected the expressions of acetylation and phosphorylation of NFκB p65. It was found that acetylation of NF-κB p65 was deceased in model group and increased in the TSA-treated group, while the phosphorylation of NFκB p65 was increased in model group and decreased in TSA-treated group. These results suggested that TSA influenced the NF-κB p65 signal pathway and the balance of acetylation and phosphorylation of NF-κB p65. The acetylized modification might occur in both histone and non-histone proteins, which offer multiple

regulatory points to control gene expression in the process of inflammation. TSA inhibits inflammatory responses probably through regulating non-histone acetylation. In conclusion, the results of this study suggested that HDAC inhibitor TSA could inhibit the release of multiple inflammatory cytokines, prolong the survival time, and protect against experimental ACLF in rats. Moreover, it is suggested that the inhibitory effect of TSA on the inflammation may mainly through enhancing the acetylation levels of non-histones rather than histone. Although the exact molecular events and cell signaling in the course of TSA inhibiting inflammatory response remains to be further studied, the acetylized modification represents a novel antiinflammatory strategy through regulating the expressions of inflammatory genes.

ACKNOWLEDGMENTS This study was supported by a grant from the National Natural Science Foundation of China (No. 81371789).

Conflict of Interests. The authors have declared that no competing interests exist.

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