Inflammation

, Volume 36, Issue 6, pp 1453–1459

Valproic Acid Attenuates Lipopolysaccharide-Induced Acute Lung Injury in Mice

Authors

  • Mu-huo Ji
    • Department of Anesthesiology, Jinling Hospital, School of MedicineNanjing University
  • Guo-min Li
    • Department of Anesthesiology and Intensive Care, Jintan HospitalJiangsu University
  • Min Jia
    • Department of Anesthesiology, Jinling Hospital, School of MedicineNanjing University
  • Si-hai Zhu
    • Department of Anesthesiology, Jinling Hospital, School of MedicineNanjing University
  • Da-peng Gao
    • Department of Anesthesiology, Jinling Hospital, School of MedicineNanjing University
  • Yun-xia Fan
    • Department of Anesthesiology and Intensive Care, Jintan HospitalJiangsu University
  • Jing Wu
    • Department of Anesthesiology, Jinling Hospital, School of MedicineNanjing University
    • Department of Anesthesiology, Jinling Hospital, School of MedicineNanjing University
Article

DOI: 10.1007/s10753-013-9686-z

Cite this article as:
Ji, M., Li, G., Jia, M. et al. Inflammation (2013) 36: 1453. doi:10.1007/s10753-013-9686-z

Abstract

Valproic acid (VPA) has been shown to exert anti-inflammatory and antioxidant effects in a range of diseases including septic shock. However, the effects of VPA on lipopolysaccharide (LPS)-induced acute lung injury (ALI) remains not well understood. We found that VPA pretreatment attenuated the LPS-induced ALI, as evidenced by the reduced histological scores, myeloperoxidase activity, and wet-to-dry weight ratio in the lung tissues. This was accompanied by the downregulated nuclear factor kappa B (NF-κB) p65, nitric oxide, and inducible nitric oxide synthase in the lung tissues and the decreased levels of tumor necrosis factor alpha and interleukin-1β in the bronchoalveolar lavage fluid. Furthermore, VPA reduced the nuclear histone deacetylase (HDAC)3 expression whereas increased the cytoplasmic HDAC3 expression. Our results suggested that VPA attenuates the LPS-induced ALI via inhibiting the NF-κB activation probably through a mechanism depending on HDAC3 redistribution.

KEY WORDS

sepsispro-inflammatory cytokineHDACsacute lung injury

INTRODUCTION

Acute lung injury (ALI) is a leading cause of respiratory dysfunction, which affects a diverse array of medical patients in the intensive care unit throughout the world [1]. Sepsis is the most frequent cause of ALI, leading to increased permeability pulmonary edema, enhanced polymorphonuclear neutrophil sequestration, and respiratory failure [13]. Although the pathogenesis is not well understood, overproduction of pro-inflammatory cytokines has been shown to play a pivotal role in the development of ALI [2, 3].

Chromatin modification is a key mechanism responsible for the regulation of gene transcription, including various inflammation-related genes [4]. Histone acetylation is one of the most widely studied histone modifications, which is controlled by the balance between histone acetyl transferase (HAT) and histone deacetylases (HDACs) [5]. Accumulating evidence suggests that HDAC inhibitors regulate inflammatory gene expression, as indicated by the findings that pan-HDAC inhibitors exert potent anti-inflammatory effects in animal models of inflammatory diseases [59]. Valproic acid (VPA) is an approved anticonvulsant and mood-stabilizing drug with a long history of clinical use, which also belongs to the class I HDAC inhibitors. However, the role of VPA in sepsis-induced ALI is not well elucidated.

Therefore, we aimed to investigate whether VPA can improve lipopolysaccharide (LPS)-induced ALI in a mouse model. In addition, the possible mechanisms of protection were also explored in the present study.

MATERIALS AND METHODS

Animals

The present study was approved by the Animal Care and Use Committee of Jinling Hospital, Nanjing University. Male C57BL/6 mice weighing 25–30 g were purchased from the Animal Center of Jinling Hospital. Animals involved in this experiment were treated in accordance with the Guide for Care and Use of Laboratory Animals of National Institutes of Health. Animals were housed in standard conditions and maintained in a 12:12-h light/dark cycle with food and water ad libitum.

Animal Model of ALI

Mice were anesthetized with 1 % sodium pentobarbital in saline (40 mg/kg, intraperitoneally; Sigma Chemical Co., St. Louis, MO, USA). The trachea was exposed using a neck incision as previously described [10]. ALI was induced by an intratracheal instillation of LPS from Escherichia coli serotype 055:B5 (Sigma Chemical, St. Louis, MO) at the dose of 5 mg/kg. After recovery from anesthesia, mice were returned to their cages and allowed to food and water ad libitum.

Experimental Protocol

Mice were randomly allocated into one of the following three groups (n = 8 for each group): (1) sham group, animals received an intratracheal injection of sterile saline (10 ml/kg); (2) LPS group, animals received an intratracheal injection of LPS (10 mg/kg diluted in 10 ml/kg saline); and (3) LPS + VPA group, 30 min before LPS instillation, animals received intraperitoneally an injection of VPA (200 mg/kg diluted in 10 ml/kg saline). The doses of VPA were selected on the basis of one previous study and our preliminary experiment [8]. At 6 h after LPS or saline instillation, bronchoalveolar lavage fluid (BALF) and lung tissues were collected for the determination of TNF-α, IL-1β, and IL-6 concentrations and other analyses, respectively.

Histological Analysis

A pathologist blinded to the group assignment analyzed the samples and determined the levels of lung injury. The severity of microscopic injury was graded from 0 (normal) to 4 (severe) based on the following categories: neutrophil infiltration, interstitial edema, hemorrhage, and hyaline membrane. The sum of all scores was combined to calculate a composite score as described previously [11].

Enzyme-Linked Immunosorbent Assay

The concentrations of inflammatory cytokines in BALF were quantified using specific ELISA kits for mice in accordance with the manufacturers’ instructions (TNF-α from Diaclone Research, Besanson Cedex, France; IL-1β from R&D Systems, Minneapolis, MN, USA; and IL-6 from Biosource Europe SA, Nivelles, Belgium).

Western Blotting

The lung tissue samples collected at 6 h after LPS instillation were homogenized, and cytoplasmic and nuclear proteins were extracted separately using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's instructions. Nuclear protein extracts were used to detect the HDAC1, HDAC2, HDAC3, HDAC8, and nuclear factor kappa B (NF-κB) p65 subunit. Cytoplasmic extracts were used to detect HDAC3, inducible nitric oxide synthase (iNOS), and β-actin. Western blotting was performed as previously described [11] using the following primary antibodies: HDAC1, HDAC2, HDAC3, HDAC8, NF-κB p65, and iNOS from Santa Cruz Biotechnology and β-actin from Cell Signaling Technology. We used the NIH Image J software (National Institutes of Health, Bethesda, MD, USA) to quantify the protein band concentrations.

Myeloperoxidase Activity and Nitric Oxide Concentration

The myeloperoxidase (MPO; Jiancheng Bioengineering Institute, Nanjing, China) activity and nitric oxide (NO; Jiancheng Bioengineering Institute, Nanjing, China) content in lung tissues were determined as previously described [11].

Wet-to-Dry Weight Ratio

The lung weight-to-dry (W/D) ratio was calculated as a parameter of lung edema. At the end of the experiment, animals were sacrificed with an overdose of sodium pentobarbital of 60 mg/kg. The lungs were removed, weighed, and then dried in an oven at 80 °C for 48 h to obtain lung W/D ratio.

Statistical Analysis

Data are expressed as the mean ± standard error of the mean (SEM). Statistical significance was determined by analysis of variance followed by Bonferroni tests for post hoc comparisons. P value of <0.05 was regarded as statistically significant.

RESULTS

No animal died in the sham group during the observation period. However, one animal died in the LPS group as well as in the LPS + VPA group.

Histopathologic Changes in the Lung Tissues

The lung tissues of mice subjected to LPS administration illustrated an increase in alveolar wall thickness that was caused by the increased neutrophil infiltration and alveolar congestion, which was not observed in the sham group. However, the degree of the lung tissue lesion was significantly attenuated in the LPS + VPA group, as evidenced by less alveolar septal thickening, neutrophil infiltration, and alveolar congestion (P = 0.008) (Fig. 1).
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Fig. 1

Histological changes in the lung tissues 6 h after an intratracheal instillation of LPS (hematoxylin–eosin, ×400). VPA pretreatment significantly decreased histological scores when compared with LPS group (P < 0.05). Data are expressed as mean ± SEM; *P < 0.05.

MPO Activity and Water Content in the Lung Tissues

The MPO activity and the W/D ratio in the lung tissues were significantly increased after LPS administration, whereas VPA pretreatment decreased the MPO activity and the W/D ratio significantly (P = 0.047 and P = 0.013, respectively) (Fig. 2).
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Fig. 2

Pulmonary levels of myeloperoxidase (MPO) activity and wet-to-dry ratio. VPA pretreatment significantly decreased pulmonary MPO and W/D when compared with LPS group (P < 0.05). Data are expressed as mean ± SEM; *P < 0.05.

HDAC1, HDAC2, HDAC3, and HDAC8 Levels in the Lung Tissues

No significant difference was observed with regard to HDAC1, HDAC2, and HDAC8 levels in the lung tissues among the three groups (P > 0.05) (Fig. 3). The nuclear HDAC3 level in the lung tissues was significantly decreased, while the cytoplasmic HDAC3 level was increased after LPS stimulation. However, VPA pretreatment increased the nuclear HDAC3 level and reduced the cytoplasmic HDAC3 level significantly (P = 0.003 and P = 0.004, respectively) (Fig. 4).
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Fig. 3

Changes of histone deacetylase (HDAC)1, HDAC2, and HDAC8 expressions in the lung tissues. No difference was observed in HDAC1, HDAC2, and HDAC8 expression in the lung tissues among the three groups (P > 0.05). Data are expressed as mean ± SEM.

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Fig. 4

Changes of HDAC3 expression in the lung tissues. Nuclear HDAC3 level in the lung tissues was significantly reduced, while cytoplasmic HDAC3 expression was increased after LPS stimulation. However, VPA pretreatment increased nuclear HDAC3 expression and reduced cytoplasmic HDAC3 levels (P < 0.05). Data are expressed as mean ± SEM; *P < 0.05.

Effects of VPA Pretreatment on NF-κB p65 and iNOS Activities in the Lung Tissues

LPS induced the increased activities of NF-κB p65 and iNOS that were abolished by VPA pretreatment (P = 0.016 and P = 0.028, respectively) (Fig. 5).
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Fig. 5

Changes of NF-κB p65 and iNOS expressions in the lung tissues. LPS induced increased levels of NF-κB p65 and iNOS expressions, which were suppressed by VPA pretreatment (P < 0.05). Data are expressed as mean ± SEM; *P < 0.05.

Concentrations of TNF-α, IL-6, IL-1β, and NO in the BALF

The concentrations of TNF-α, IL-1β, and NO in the BALF were significantly increased, which were inhibited by VPA pretreatment (P = 0.011, P = 0.001, and P = 0.043, respectively) However, no difference was observed in the IL-6 concentration among groups (P = 1.000) (Fig. 6).
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Fig. 6

Changes of TNF-α, IL-6, IL-1β in the BALF, and NO levels in the lung tissues. The concentrations of TNF-α and IL-1β in BALF and NO levels in the lung were significantly increased, which was reduced by pretreatment of VPA (P < 0.05). Data are expressed as mean ± SEM; *P < 0.05.

DISCUSSION

In this study, we found that VPA attenuates lung inflammation of LPS-induced ALI. These results are consistent with previous studies and may thus extend the protective potential of HDAC3 inhibitor to sepsis-induced ALI.

The cytokines (TNF-α, IL-1β, and IL-6) secreted by alveolar macrophages in response to bacterial endotoxin play a key role in neutrophil recruitment to the lung and contribute to significant tissue damage [12, 13]. In addition, LPS stimulates iNOS expression and NO overproduction in diverse pulmonary cells including macrophage, which may induce tissue damage by enhancing the production of other inflammatory mediators [11]. The balance between HDACs and HAT is known to play an important role in regulating gene transcription through remodeling of chromatin [4]. The family of HDACs consists of 17 isoforms grouped into four classes: class I (HDAC1, HDAC2, HDAC3, and HDAC8), class II (HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10), class III (sirtuin 1–7), and class IV (HDAC11) [46]. VPA is a broad-acting HDAC inhibitor, which mainly targets class I enzymes including HDAC1, HDAC2, HDAC3, and HDAC8. Previous studies suggest that class I HDAC inhibitors suppress the production of pro-inflammatory cytokines in a range of diseases including sepsis [8, 14]. However, the individual HDACs responsible for these effects remain unclear.

HDAC3 has been identified as a key mediator in the activation of the inflammatory gene expression in macrophages [15]. In contrast to the predominant nuclear localization of HDAC1, HDAC2, and HDAC8, HDAC3 has both a nuclear import and export signal and thus may shuttle between the cytoplasm and nucleus [7]. Furthermore, HDAC3 differs from other class I HDACs in that HDAC3 exists as a component of the nuclear receptor corepressor/silencing mediator for retinoid and thyroid hormone receptor corepressor complex [15, 16]. The most widely appreciated function of this complex is the constitutive repression (via histone deacetylation) of genes bound by unliganded nuclear hormone receptors [17]. Consistently, we showed that HDAC3 expression reduced in the nucleus but increased in the cytoplasm, implying translocation of HDAC3 after LPS stimulation. However, VPA treatment reversed HDAC3 expression pattern, a phenomenon might be attributed to HDAC3 translocation from the nucleus to the cytoplasm rather than direct inhibition.

NF-κB pathway has been implicated to play a key role in the transcription of most pro-inflammatory molecules, including cytokines, adhesion molecules, as well as iNOS [18]. It has been suggested that HDAC1 and HDAC3 can interact directly with the p65 subunit of NF-κB to exert a corepressor function in the nucleus [19]. On the other hand, cytoplasmic HDAC3 is responsible for deacetylating NF-κB, a key process in its association with the inhibitor of NF-κB alpha prior to nuclear export [20]. Thus, reduction in HDAC3 expression in the nucleus combined with elevated cytoplasmic HDAC3 levels might contribute to an increased synthesis of NF-κB-driven inflammatory cytokines. However, this phenomenon was partially reversed by VPA pretreatment. Our data are in agreement with a recent work demonstrating that HDAC3 is a positive regulator of IL-1-induced gene expression [21]. Consistent with the attenuated lung lesions, we detected the decreased leukocyte infiltration on histological examination and the lower pulmonary MPO activities and W/D ratio in mice treated with VPA, suggesting a protective effect of VPA in ALI, which is related to the attenuation of neutrophil influx into the lung tissue.

There are some limitations that have to be acknowledged in the present study. The observation period was limited to 6 h, which was insufficient to evaluate the long-term effects of VPA on sepsis-induced ALI. Furthermore, using more specific HDAC3 inhibitor other than VPA may be more effective in elucidating the role of HDAC3 in inflammatory diseases such as ALI.

In conclusion, our study suggests that VPA pretreatment attenuates inflammation, decreases neutrophil infiltration in the lungs, and provides an alternative therapeutic target to limit sepsis-induced ALI. The mechanism of the salutary effect seems to be related to the suppression of NF-κB activation through a mechanism depending on HDAC3 redistribution.

Acknowledgments

This work was supported by the Natural Science Foundation of Jiangsu Province (BK2012778).

Conflict of Interest

The authors have no potential conflicts of interest to disclose.

Copyright information

© Springer Science+Business Media New York 2013