Valproic Acid Attenuates Lipopolysaccharide-Induced Acute Lung Injury in Mice
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- Ji, M., Li, G., Jia, M. et al. Inflammation (2013) 36: 1453. doi:10.1007/s10753-013-9686-z
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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 WORDSsepsispro-inflammatory cytokineHDACsacute lung injury
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 . Sepsis is the most frequent cause of ALI, leading to increased permeability pulmonary edema, enhanced polymorphonuclear neutrophil sequestration, and respiratory failure [1–3]. 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 . 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) . 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 [5–9]. 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
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 . 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.
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 . 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.
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 .
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).
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  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 .
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.
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.
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
MPO Activity and Water Content in the Lung Tissues
HDAC1, HDAC2, HDAC3, and HDAC8 Levels in the Lung Tissues
Effects of VPA Pretreatment on NF-κB p65 and iNOS Activities in the Lung Tissues
Concentrations of TNF-α, IL-6, IL-1β, and NO in the BALF
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 . The balance between HDACs and HAT is known to play an important role in regulating gene transcription through remodeling of chromatin . 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) [4–6]. 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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.
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.