NEMO-Binding Domain Peptide Attenuates Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting the NF-κB Signaling Pathway

Hindawi Publishing Corporation Mediators of Inflammation Volume 2016, Article ID 7349603, 11 pages http://dx.doi.org/10.1155/2016/7349603 Research Article NEMO-Binding Domain Peptide Attenuates Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting the NF-𝜅B Signaling Pathway Jianhua Huang,1,2 Li Li,1 Weifeng Yuan,1 Linxin Zheng,1 Zhenhui Guo,3,4 and Wenjie Huang1,4 1 Department of Respiratory Medicine, General Hospital of Guangzhou Military Command of PLA, Guangzhou, Guangdong, China Department of Pulmonary Medicine, Chenzhou No. 1 People’s Hospital, Chenzhou, Hunan, China 3 Department of Medical Intensive Care Unit, General Hospital of Guangzhou Military Command of PLA, Guangzhou, Guangdong, China 4 Guangdong Provincial Key Laboratory of Geriatric Infection and Organ Function Support, Guangzhou, Guangdong, China 2 Correspondence should be addressed to Zhenhui Guo; and Wenjie Huang; Received 11 July 2016; Revised 5 September 2016; Accepted 15 September 2016 Academic Editor: Yutong Zhao Copyright © 2016 Jianhua Huang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The aim of the present study is to investigate the protective effects and relevant mechanisms exerted by NEMO-binding domain peptide (NBD) against lipopolysaccharide- (LPS-) induced acute lung injury (ALI) in mice. The ALI model was induced by intratracheally administered atomized LPS (5 mg/kg) to BABL/c mice. Half an hour before LPS administration, we treated the mice with increasing concentrations of intratracheally administered NBD or saline aerosol. Two hours after LPS administration, each group of mice was sacrificed. We observed that NBD pretreatment significantly attenuated LPS-induced lung histopathological injury in a dose-dependent manner. Western blotting established that NBD pretreatment obviously attenuated LPS-induced I𝜅B-𝛼 and NF-𝜅Bp65 activation and NOX1, NOX2, and NOX4 overexpression. Furthermore, NBD pretreatment increased SOD and TAOC activity and decreased MDA levels in lung tissue. In addition, NBD also inhibited TNF-𝛼 and IL-1𝛽 secretion in BALF after LPS challenge. In conclusion, NBD protects against LPS-induced ALI in mice. 1. Introduction Acute lung injury (ALI) is caused by microbial infection, sepsis, trauma, and ischemia and reperfusion, leading to epithelial integrity disruption, neutrophil accumulation, noncardiogenic pulmonary edema, severe hypoxemia, and intense pulmonary inflammatory responses. The acute respiratory distress syndrome (ARDS) is a more severe form of ALI. Both ALI and ARDS are major causes of acute respiratory failure and leading causes of morbidity and mortality in critically ill patients [1, 2]. In recent years, rapid advances in supportive care, such as mechanical ventilation, have been achieved. However, several data analyses have shown that the mortality rate associated with ALI- or ARDS-induced acute respiratory failure is still high at approximately 40% [3–5]. The pathogenesis of ALI/ARDS is characterized by polymorphonuclear cells (PMNs) infiltration into the lungs, which may cause interstitial edema. In addition, the alveoli develop fibrin leakage, resulting in increases in the levels of macrophage-derived cytokines, chemokines, and other proinflammatory mediators in the lungs [6]. The results of previous studies indicate that many specific therapies have not proven beneficial with respect to managing ALI/ARDS [7]. Therefore, investigating the mechanisms underlying ALI/ARDS is necessary, as such investigations may contribute to the development of novel effective treatments for ALI/ARDS. ALI research relies mainly on animal models. The intratracheal lipopolysaccharide (LPS) administration model is the most commonly used clinically relevant severe lung injury model for studying the pathophysiologic mechanisms underlying ALI, as it simulates the human disease [8]. LPS 2 are components of gram-negative bacterial walls and play an important role in ALI by inducing PMNs infiltration into injured lung tissue, mimicking clinical ALI progression. TNF-𝛼 and keratinocyte-derived chemokines are secreted during this process and recruit intravascular PMNs into the alveolar spaces [9]. These activated PMNs generate superoxide anions (O2 − ) and release proteases via respiratory bursts and degranulation [10]. This excessive inflammatory response induces significant lipid peroxidation and antioxidant enzyme activity alterations, thereby disrupting lung endothelial integrity [11]. It is accepted that NF-𝜅B, a critical transcriptional factor, plays an important role in the pathogenesis of ALI/ARDS [12]. A variety of experimental techniques have demonstrated that NF-𝜅B exists in both the cytoplasm and the nucleus. NF𝜅B activation induces its translocation from the cytoplasm to the nucleus. NF-𝜅B is activated by LPS and some cytokines, such as TNF-𝛼 and IL-1𝛽. These cytokines initiate a cascade of events leading to I𝜅B phosphorylation by I𝜅B kinase (IKK), which triggers I𝜅B degradation by the ubiquitin– proteasome pathway. I𝜅B, an inhibitory protein, binds to P65 and P50, two NF-𝜅B subunits, under normal conditions. I𝜅B degradation removes a nuclear localization signal from NF-𝜅B, resulting in its uncoiling and translocation into the nucleus. This uncoiling is thought to activate the transcription of cytokines and other proinflammatory mediators [13, 14]. IKK comprises three subunits, IKK𝛼, IKK𝛽, and IKK𝛾, which are also collectively known as NEMO (NF-𝜅B essential modulator). IKK𝛾 has no catalytic domain and plays a critical role in biology only when being a part of the IKK complex [15]. The NH2-terminus of NEMO associates with a hexapeptide sequence (Leu-Asp-Trp-Ser-Trp-Leu) within the COOH terminus of IKK𝛼 and IKK𝛽 termed the NEMObinding domain (NBD). Previous studies have shown that LPS induces the NF-𝜅B activation required for NBD activity. NBD disrupts the association between NEMO and IKK𝛽 and blocks LPS-induced NF-𝜅B activation in cells, which ameliorates the inflammatory response and oxidative stress in distinct animal models to some extent [16, 17]. The results of previous studies indicate that understanding the mechanisms underlying the protective effects of NBD may facilitate the development of therapies that are effective against ALI. Therefore, the aim of the current study was to elucidate the mechanisms underlying the protective effects exerted by NBD against LPS-induced ALI. 2. Materials and Methods 2.1. Chemicals and Reagents. LPS (from Escherichia coli 055: B5) was purchased from Sigma-Aldrich, St. Louis, MO, USA. NBD and N-NBD (negative control) were obtained from MERCK (NBD amino acid sequence: H-Asp-Arg-Gln-IleLys-IIe-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-ThrAla-Leu-Asp-Trp-Ser-Trp-Leu-Gln-Thr-Glu-OH; N-NBD amino acid sequence: H2N-Asp-Arg-Gln-Ile-Lys-IIe-TrpPhe-Gl (...truncated)