Improvement of ventilation-induced lung injury in a rodent model by inhibition of inhibitory κB kinase.

ORIGINAL ARTICLE

Improvement of ventilation-induced lung injury in a rodent model by inhibition of inhibitory JB kinase Yu-Sheng Shu, MD, Wei Tao, MS, Qian-Bing Miao, MD, Ya-Bing Zhu, MD, and Yi-Feng Yang, MD, Jiangsu, China BACKGROUND: Inhibition of nuclear factor JB (NF-JB) activation is a well-know strategy to ameliorate ventilation-induced lung injury (VILI). Inhibitory JB kinase (IKK) plays a key role in the regulation of NF-JB activation. In this study, we determined whether inhibition of IKK by an IKK inhibitor exerts lung protection in a rat model of VILI. METHODS: Anesthetized and mechanically ventilated Sprague-Dawley rats were randomly assigned to a standard (tidal volume, 8 mL/kg) or hightidal volume (tidal volume, 25 mL/kg) ventilation group. An IKK inhibitor (IKK 16) or vehicle was administrated 1 hour before the induction of VILI. All groups were ventilated and observed for 5 hours. RESULTS: High-pressure ventilation caused activation of NF-JB, increased pulmonary inflammatory mediator levels, lung edema, and impairment of gas exchange. The IKK inhibitor treatment significantly reduced these changes and increased interleukin 10 levels, heme oxygenase 1 activity, protein kinase B (Akt) phosphorylation levels, and nuclear amounts of nuclear factor E2Yrelated factor 2 protein. CONCLUSION: IKK may be a therapeutic target for VILI. An IKK inhibitor, IKK 16, can dampen VILI in rats. The beneficial effect of the IKK 16 may be mediated through the inhibition of NF-JB pathway and up-regulation of nuclear factor E2Yrelated factor 2Yregulated heme oxygenase 1 through the activation of the phosphatidylinositol 3 kinase/Akt. (J Trauma Acute Care Surg. 2014;76: 1417Y1424. Copyright * 2014 by Lippincott Williams & Wilkins) KEY WORDS: Inhibitor of IJB kinase; nuclear factor JB; ventilation-induced lung injury; heme oxygenase 1.

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echanical ventilation (MV) is a lifesaving strategy for severe lung diseases, such as acute lung injury (ALI)/ adult respiratory distress syndrome.1 However, misusing MV may also cause ventilation-induced lung injury (VILI), a specific type of ALI.1,2 Nuclear factor JYlight-chain-enhancer of activated B cells (NF-JB), a well-known transcription factor, regulates the expression of inflammation mediators, which may cause inflammatory cell activation and infiltration in VILI.3 A growing body of evidence suggests that suppression of the NF-JB pathway by non-specific NF-JB inhibitors can dampen lung injury.3,4 NF-JB sequestered in cytoplasm in an unstimulated state through interaction with its inhibitory proteins, Inhibitory JB (IJB). Phosphorylation of IJB results in its ubiquitination and degradation, while NF-JB is activated and translocated from the cytoplasm to the nucleus.5 Overexpression of the IJB using an adeno-associated virus vectors reduces early pneumonia-induced lung injury in a rat model.6 IJB kinase (IKK) plays a key role in regulating ubiquitination and degradation of IJB.5 Lung inflammation in a lipopolysaccharide/ peptidoglycan or cecal ligation and puncture challenged sepsis is attenuated by an IKK inhibitor effectively.7 As the activation

Submitted: October 14, 2013, Revised: January 11, 2014, Accepted: January 13, 2014. From the Department of Cardiothoracic Surgery (Y.-S.S., Y.-F.Y.), the Second Xiangya Hospital of Central South University, Changsha, Hunan; Department of Cardiothoracic Surgery (Y.-S.S., Q.-B.M.), Subei People’s Hospital of Jiangsu Province, Yangzhou, Jiangsu; Department of Surgery (W.T.), Yangzhou East Hospital, Yangzhou, Jiangsu; and Department of Cardiothoracic Surgery (Y.-B.Z.), Hangzhou First People’s Hospital, Hangzhou, Zhejiang, China. Address for reprints: Yu-Sheng Shu, MD, Department of Cardiothoracic Surgery, Subei People’s Hospital of Jiangsu Province, Yangzhou 225001, Jiangsu, China; email: [email protected]; Yi-Feng Yang, Department of Cardiothoracic Surgery, the Second Xiangya Hospital of Central South University, 139 Renmin Rd, Changsha 410011, Hunan, China; email: [email protected]. DOI: 10.1097/TA.0000000000000229

of NF-JB pathway plays a key role in the pathogenesis of VILI,8,9 the IKK could be a reasonable therapeutic target for it. A previous study has shown that endotoxin-induced ALI can be reduced by an IKK inhibitor (BMS-345541).10 IKK 16 is a novel specific IKK inhibitor11 and has shown its beneficial effect on sepsis.7 However, it remains unknown whether the IKK 16 has any protective effect on VILI, a noninfectious caused lung injury. In this study, we used a rat model of VILI to investigate whether the IKK 16 has any beneficial effect on it and, if so, to elucidate its potential mechanism.

MATERIALS AND METHODS Animals and Experimental Protocols All experiments were performed according to the guidelines for the care and use of animals as established by the Animal Ethics Committee of Jiangsu Province. Sixty male SpragueDawley rats (200Y220 g, Yangzhou, Jiangsu, China) were used in these studies. Rats were anaesthetized by intraperitoneal injection of carbrital (40 mg/kg) 1 hour after IKK 16 (30 mg/kg) or vehicle (5 mL/kg 10% DMSO) intravenous injection (IV). Additional doses were administrated when necessary to keep the animals completely anaesthetized. Anaesthetized animals were then placed on a servo-controlled heated table under a heating pad to maintain normal body temperature, and a tracheotomy was performed by midline incision followed by an insertion of an endotracheal tube. The animals were randomly assigned to one of four groups as follows: (1) sham + vehicle group where rats received a standard tidal volume ventilation protocol12 (tidal volume, 8 mL/kg; 2 cm H2O of positive end-expiratory pressure; respiratory rate, 50 breaths/min; fraction of inspired O2 [FIO2], 21%) and vehicle IV 1 hour before MV; (2) sham + IKK 16 group where rats received a standard tidal volume ventilation protocol

J Trauma Acute Care Surg Volume 76, Number 6

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and IKK 16 (30 mg/kg) IVas described elsewhere11 1 hour before MV; (3) high-tidal volume ventilation (HVT) + vehicle group where rats received an HVT protocol13 (tidal volume, 25 mL/kg; 0 cm H2O of positive end-expiratory pressure; respiratory rate, 20 breaths/min; FIO2, 21%) to induce VILI and vehicle IV 1 hour before MV; and (4) HVT + IKK 16 group where rats received an HVT protocol and IKK 16 (30 mg/kg) IV11 1 hour before MV. IKK 16 was prepared as 30 mg in 5 mL 10% DMSO before administration. All groups were ventilated and observed for 5 hours. A sample of blood was collected with a heparinized syringe from the abdominal aortic artery and immediately measured with a blood gas analyzer. Then, the animals were exsanguinated through the vena cava. A steel cannula was inserted into the right primary bronchi and secured with a silk suture, and then bronchoalveolar lavage (BAL) was performed. After that, the right lung was excised, rinsed of blood, then was homogenized in phosphatebuffered saline on ice to make the 10% pulmonary homogenate and stored at j70-C for further analysis.

Lung Water Content Determination and Histologic Examination The upper left lung was weighed before being dried in an oven at 80-C for 72 hours, and then, the dried lung was weighed again to calculate the pulmonary wet-dry (W/D) ratio. The lung sections were stained with hematoxylin and eosin and examined with light microscopy. The Murakami technique14 was used to grade the degree of lung injury, which is based on the following histologic features: edema, congestion, infiltration of inflammatory cells, and hemorrhage. Each feature was graded as 0, absent and appears normal; 1, light; 2, moderate; 3, strong; and 4, intense. A total score was calculated for each animal.

BAL Methods and Albumin Concentration Assay BAL was performed through a tracheal cannula with saline. In each rat examined, approximately 90% of BAL fluid (BALF) was recovered and immediately centrifuged at 1,000 G for 10 minutes. The supernatant was stored at j70-C for further study. Albumin concentration in cell-free BALF was measured by using enzyme-linked immunosorbent assay (ELISA) kits (Sigma Chemicals, St. Louis, MO) according to the manufacturers’ manual.

Measurements of BALF Inflammatory Mediator Levels The levels of monocyte chemoattractant protein 1 (MCP-1) (PharMingen, San Diego, CA), tumor necrosis factor > (TNF->),

interleukin 6 (IL-6), and IL-10 in BALF were determined by using ELISA kits (R&D Systems, Inc., Minneapolis, MN) according to the manufacturers’ manual.

Measurement of IKKA Activity The IKKA activity in lung homogenates was determined with the GST-IJB> and [F-32P]ATP, the substrates of IKKA, as described elsewhere.15 The quantity of phosphorylated IJB> was measured by SDS-PAGE and autoradiography.15

Western Blot Analysis of IJB>, Protein Kinase B, and Nuclear Factor E2YRelated Factor 2 Cytoplasmic extracts of lung homogenates were prepared for Western blot analysis of nuclear factor E2Yrelated factor 2 (Nrf 2), IJB>, and protein kinase B (Akt) expression using a cytosol extraction kit (BioVision, Inc., Mountain View, CA). Extracted proteins were subjected to SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were incubated with specific antibodies overnight at 4-C. The blots were then washed in TBST five times for 10 minutes. Blots were incubated with horseradish peroxidaseYlinked antiYrabbit IgG (Cell Signaling Technology, Danvers, MA) for 1 hour at room temperature and then washed five times in TBST for 10 minutes. A chemiluminescent peroxidase substrate (ECL; GE Healthcare Bio-Sciences, Piscataway, NJ) was applied according to the manufacturer’s instructions, and the membranes were exposed briefly to x-ray film. The band densities were determined with ImageJ 1.46 software (National Institutes of Health, Bethesda, MD). Phosphorylated IJB> or Akt and IJB> or Akt antibodies were purchased from Cell Signaling Technology. The antibody for Nrf 2 and Lamin B were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

NF-JB Binding Assay Nuclear extracts of lung homogenates were prepared with a nuclear extract kit (Active Motif North America, Carlsbad, CA). The DNA-binding activity of NF-JB p65 was determined using an ELISA-based NF-JB p65 transcription factor assay kit (Active Motif North America) according to the manufacturers’ manual.

Measurements of Heme Oxygenase 1 Activity and Lipid Hydroperoxide Assay The activity of heme oxygenase 1 (HO-1) in lung tissues was determined using specific ELISA kits (R&D Systems, Inc.), according to the manufacturer’s instructions.

TABLE 1. Comparison of the W/D Ratio, MCP-1, and HO-1 Activity (Mean T SEM) in a Standard (Sham) or HVT Group Treated With Vehicle or an IJB Kinase Inhibitor (IKK 16) Group Sham + vehicle Sham + IKK 16 HVT + vehicle HVT + IKK 16

W/D Ratio 3.07 T 0.14 3.12 T 0.11* 6.33 T 0.32** 4.95 T 0.24**†

MCP-1, ng/mL 0.62 T 0.60 T 12.54 T 5.52 T

0.01 0.01* 0.03** 0.08**†

HO-1 Activity, pmol/mg Protein/h 18.50 T 0.39 19.43 T 0.31* 49.02 T 0.24** 114.19 T 0.71**†

*p 9 0.05 versus the sham + vehicle group. **p G 0.05 versus the sham + vehicle group. †p G 0.05 versus the HVT + vehicle group.

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The Student-Newman-Keuls method was used for comparison between groups. Kruskal-Wallis one-way ANOVA on ranks and the Student-Newman-Keuls method were used for statistical evaluation of the histopathologic scores. p value less than 0.05 was considered to be statistically significant.

RESULTS IKK 16 Attenuates VILI In the present study, VILI was characterized by increased lung water content, permeability, lipid hydroperoxide levels, infiltration of neutrophils, and deteriorated pulmonary oxygenation ( p G 0.05, Table 1 and Figs. 1 and 2). These changes were markedly improved by the IKK 16 administration ( p G 0.05, Table 1, and Figs. 1 and 2).

IKK 16 Modulates BALF Inflammatory Mediator Levels In the vehicle-treated rats, HVT caused a dramatic increase in BALF TNF->, IL-6, and MCP-1 levels ( p G 0.05, Fig. 3A and B, Table 1). IKK 16 significantly prevented the rise in BALF levels of IL-6, TNF->, and MCP-1 ( p G 0.05, Fig. 3A and B, Table 1). Meanwhile, the IL-10 production, known as an anti-inflammatory cytokine, was elevated markedly in the HVT + IKK 16 group compared with the HVT + vehicle group ( p G 0.05, Fig. 3C ).

IKK 16 Prevents Pulmonary NF-JB Activation HVT caused a twofold increase in the amount of active NF-JB p65 levels in lung nuclear extracts, compared with rats treated with a standard tidal volume ventilation protocol and vehicle ( p G 0.05, Fig. 5A). IKK 16 significantly attenuated the DNA-binding activity of NF-JB p65 in HVT-treated rats ( p G 0.05, Fig. 5A). The IKKA activity as well as the ratio of phosphorylated IJB> and total IJB> in cytoplasmic extracts of lung homogenates were markedly decreased by the IKK 16 pretreatment compared with rats in the HVT + vehicle group ( p G 0.05, Fig. 4A and B).

Effects of IKK 16 on Pulmonary HO-1 Activity and Akt Phosphorylation

Figure 1. Alterations of albumin concentration in cell-free BALF (A), the PaO2)/FIO2 ratio (B), or lipid hydroperoxide levels (C) in a standard (sham) or HVT group treated with vehicle or an IJB kinase inhibitor (IKK 16). Data are expressed as mean T SEM (n = 8Y15) and compared by one-way ANOVA and the Student-Newman-Keuls method. Hp G 0.05 when compared with the sham + vehicle group. †p G 0.05 when compared with the HVT + vehicle group. ††p 9 0.05 when compared with the sham + vehicle group.

The level of lipid hydroperoxide in lung tissues was measured using a lipid hydroperoxide assay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturers’ manual.

Statistical Analyses All data are presented as mean T SEM. Statistical significance was determined by one-way analysis of variance (ANOVA).

The activity of HO-1 and Akt phosphorylation levels were increased markedly in the HVT + vehicle group compared with the sham groups ( p G 0.05, Table 1, Fig. 5B). IKK 16 pretreatment further elevated HO-1 activity and Akt phosphorylation levels in the HVT + IKK 16 group compared with the HVT + vehicle group ( p G 0.05, Table 1, Fig. 5B).

Effects of IKK 16 on the Translocation of Nrf2 Western blot analysis was used to determine the nuclear amounts of Nrf 2 protein. IKK 16 preconditioning significantly increased the Nrf 2 translocation in comparison with vehicle in the HVT-treated group ( p G 0.05, Fig. 5C).

DISCUSSION Several studies have documented the beneficial effect of IKK inhibitors in experimental animal models of pulmonary inflammation in sepsis-associated multiple-organ dysfunction7 or endotoxin-induced ALI,10 antigen-driven model of asthma,16



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Figure 2. Morphologic alterations of the lungs were determined by photomicrography. A, Photomicrograph of a pulmonary section from a rat 5 hours after standard (sham) tidal volume ventilation and vehicle treatment. B, Photomicrograph of a lung section from a rat 5 hours after standard (sham) tidal volume ventilation and an IJB kinase inhibitor (IKK 16) treatment. C, Photomicrograph of a lung section from a rat 5 hours after HVT and vehicle treatment. D, Photomicrograph of a lung section from a rat 5 hours after HVT and IKK 16 treatment. E, Histopathologic scoring of animals 5 hours after a standard (sham) or HVT and treated with vehicle or IKK 16. Data are expressed as mean T SEM (n =8Y15) and compared by Kruskal-Wallis one-way ANOVA on ranks and the Student-Newman-Keuls method. Hp G 0.05 when compared with the sham + vehicle group. †p G 0.05 when compared with the HVT + vehicle group. ††p 9 0.05 when compared with the sham + vehicle group. Original magnification 400.

as well as lipopolysaccharide aerosol or cigarette smoke exposure.17 This is the first study to investigate the effect of an IKK inhibitor, IKK 16, on VILI. In the present study, we found that the IKK 16 pretreatment led to a remarkable protection against HVTinduced lung injury evidenced by the markedly improved pulmonary alveolar-capillary barrier dysfunction, accumulation of neutrophils, and inflammatory mediator levels. Previous study has shown that the severity of lung injury correlates with sustained NF-JB activation.10 Activation of NF-JB is triggered by the IKK.5 The IKK complex consists of 1420

three subunits as follows: IKK>, IKKA, and IKKF/NEMO (NF-JB essential modulator). The IKK is thought as a vital therapeutic target in numerous animal studies.18Y20 Phosphorylated IJB> is the downstream product of IKK. Increased phosphorylated IJB> levels are accompanied by elevated NF-JB activation.5,21 Inhibition of NF-JB pathway by reducing IJB decrement is capable of attenuating VILI.9 Our results are consistent with previous studies. In the present study, the NF-JB p65 DNA-binding activity, a sensitive indicator for NF-JB activation, and phosphorylated IJB> levels were decreased markedly by the * 2014 Lippincott Williams & Wilkins



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modulated by autophagy, an intracellular proteolytic system, via the activation of the NF-JB pathway.8 NF-JB is an essential transcription factor that regulates the gene expression of various proinflammatory mediators, such as TNF-> and IL-6, which have been shown to play a key role in neutrophil activation and migration into the lung.4,17 IL-6 is known as an important inflammatory indicator.4,23 Increased levels of IL-6 have been consistently shown to correlate with aggravated lung injury.4,22 IL-6 deletion using an IL-6Yspecific inactivating antibody or IL-6 geneYdeficient mice results in ameliorated lung injury following ischemic acute kidney injury or bilateral nephrectomy.23 Moreover, it is suggested that IL-6 could be a reasonable therapeutic target for VILI not only for its antiinflammatory effect.24 IL-6 geneYdeficient mice exhibit a markedly improved pulmonary vascular permeability when compared with a wild-type control in a rodent model of VILI,24 and this protective effect of IL-6 is inflammation independent,24

Figure 3. Alterations of BALF TNF-> (A), IL-6 (B), or IL-10 (C) levels in a standard (sham) or HVT group treated with vehicle or an IJB kinase inhibitor (IKK 16). Data are expressed as mean T SEM (n = 8Y15) and compared by one-way ANOVA and the Student-Newman-Keuls method. Hp G 0.05 when compared with the sham + vehicle group. †p G 0.05 when compared with the HVT + vehicle group. ††p 9 0.05 when compared with the sham + vehicle group.

IKK 16 administration, and these effects were accompanied by a significant dampened lung injury. These results indicate that IKK is an effective therapeutic target for VILI. One of the key steps required for VILI is inflammation.2 An administration of drugs with anti-inflammatory features has shown a decrease in VILI.3,22 Lung inflammation triggered by excessive lung stretch in VILI is thought to be partially

Figure 4. Alterations of the ratio of phosphorylated (P) IJB> and total IJB> (A) or IJB kinase (IKK) A activity (B) in a standard (sham) or HVT group treated with vehicle or an IJB kinase inhibitor (IKK 16). Data are expressed as mean T SEM (n = 8Y15) and compared by one-way ANOVA and the Student-Newman-Keuls method. Hp G 0.05 when compared with the sham + vehicle group. †p G 0.05 when compared with the HVT + vehicle group. ††p 9 0.05 when compared with the sham + vehicle group.



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Figure 5. Alterations of NF-JB p65 DNA-binding activity (A), the ratios of phosphorylated (P) Akt and total Akt (B) or nuclear levels of Nrf2 (C) in a standard (sham) or HVT group treated with vehicle or an IJB kinase inhibitor (IKK 16). Data are expressed as mean T SEM (n = 8Y15) and compared by one-way ANOVA and the Student-Newman-Keuls method. Lamin B was used as a loading control for Nrf2. Hp G 0.05 when compared with the sham + vehicle group. †p G 0.05 when compared with the HVT + vehicle group. ††p 9 0.05 when compared with the sham + vehicle group.

suggesting that IL-6 may also play a vital role on barrier dysfunction in VILI. Our data are consistent with previous studies, the elevated IL-6 production was accompanied by barrier dysfunction and lung edema. IKK 16 pretreatment significantly reduced IL-6 levels and improved pulmonary inflammation and edema. IL-10, a well-known anti-inflammatory mediator, plays a protective effect on alveolar cells.25 Pretreatment of IL-10 is capable of blocking mechanical stretchYinduced inflammatory 1422

cytokine release in fetal mouse lung fibroblasts.26 Our results showed that the IL-10 production was increased in the HVT + vehicle group; this effect may be an endogenous protective mechanism.22 The IKK 16 administration further elevated IL10 levels, and this was accompanied by a decreased IL-6 concentration. It is suggested that a reduced IL-10 production is thought to be mediated via the activation of IL-6 suppressor of cytokine signaling 3 signaling pathway in a fetal Type II epithelial cell exposed to mechanical stretch.27 Our results show that the IKK 16 administration has no effect on the elevation of IL-10 production in sham. We speculate that the reduced IL-6 levels that were triggered by the IKK 16 pretreatment may contribute to the increased IL-10 production in the HVT + IKK 16 group. Alveolar-capillary barrier dysfunction detected by deteriorated BALF protein levels, lung W/D ratio, and pulmonary oxygenation is a sensitive indicator for the assessment of lung injury. Our data showed that the albumin concentration in cellfree BALF, an indicator of pulmonary permeability, and W/D ratio were alleviated significantly in the HVT+ IKK 16 group. These results indicate that the IKK 16 pretreatment can ameliorate lung permeability in VILI. It is suggested that the IKKA activity contributes to a major proportion of total IKK activity.15 In this study, we measured the IKKA activity and its downstream product, the phosphorylated IJB> concentration. According to our data, the highest level of IKKA activity was in the HVT + vehicle group, which was accompanied by the highest phosphorylated IJB> concentration. Although it is suggested that endothelial-specific IKKA gene knockout mice on an atherosclerosis-prone ApoE-null genetic background manifest increased vascular permeability,28 our results suggest that a markedly elevated IKKA activity in VILI may also cause barrier dysfunction. Meanwhile, the IKK could be a double-edged sword. Evidence has shown that the ablation of IKKA in enterocytes prevents systemic inflammatory response induced by intestinal ischemia-reperfusion but triggers severe apoptotic damage to the reperfused intestinal mucosa.29 Although the concise mechanism of HO-1 on VILI is still not well described, accumulating evidence strongly supports the hypothesis that HO-1 possesses protective properties against VILI.30 The expression of HO-1 has been demonstrated to be regulated by Nrf 2Yantioxidant response element (ARE) pathway.31 It is suggest that the phosphatidylinositol 3 kinase (PI3K)/ Akt pathway is essential in regulating Nrf 2-ARE pathway activation.32 Coldewey et al.7 have shown that the IKK 16 can attenuate sepsis via activation of Akt/endothelial nitric oxide synthase survival pathway. Our results shown that the PI3K/ Akt pathway was activated in the HVT + vehicle group; IKK 16 can further elevate phosphorylated Akt level. Moreover, nuclear amounts of Nrf 2 protein were increased markedly in the HVT + IKK 16 group. As the rate-limiting enzyme in heme catabolism, a process that leads to the generation of equimolar amounts of biliverdin, free iron, and carbon monoxide (CO),33 HO-1 is also considered to act as endogenous antioxidative enzymes33 and a regulator of the balance between proinflammatory and antiinflammatory mediators.30 As an adaptive cellular response against oxidative stress, the expression of HO-1 is up-regulated in a rat model of VILI.34 Our data show that the HO-1 activity was elevated in the HVT + vehicle group, which is in agreement with a previous study,34 and the IKK 16 pretreatment can further * 2014 Lippincott Williams & Wilkins



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increase the HO-1 activity in VILI in rats. Studies indicate that HO-1 attenuates oxidative stress through its metabolic products CO and bilirubin.35 HO-1Yderived bilirubin is an efficient scavenger of reactive oxygen species.36 CO inhalation reduces VILI in a rat model via p38 mitogen-activated protein kinase pathway but independent of activator protein 1 and NF-JB,34 and it exerts antioxidative feature via the activation of Nrf 2.37 Up-regulation of HO-1 has shown its vasculoprotective effects via antioxidant and anti-inflammatory pathways.38 In the present study, the markedly increased HO-1 activity was coupled with the reduction of lipid hydroperoxide levels and lung injury.

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CONCLUSION IKK may be a therapeutic target for VILI. An IKK inhibitor, IKK 16, can dampen VILI in rats. The beneficial effect of the IKK 16 may be mediated by the inhibition of NF-JB pathway and up-regulation of Nrf 2-regulated HO-1 through the activation of PI3K/Akt. AUTHORSHIP Y.S.S., W.T., and Y.F.Y. designed the study. W.T. and Q.B.M. contributed to animal work and writing. Q.B.M. and Y.B.Z. contributed to statistical analysis and interpretation, figures and tables editing.

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17. ACKNOWLEDGMENT We thank Mei Cao, Qing Han, Shi-Chun Lu, and Jun Yi for their expert technical assistance and Ying-Ying Zhang for the linguistic advice.

DISCLOSURE The authors declare no conflicts of interest.

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Inhibition of Adenosine Kinase Attenuates Acute Lung Injury.

Osthole prevents intestinal ischemia-reperfusion-induced lung injury in a rodent model.

Rapamycin reverses paraquat-induced acute lung injury in a rat model through inhibition of NFκB activation.

Using a non-invasive assessment of lung injury in a murine model of acute lung injury.

Biodistribution and Efficacy of Targeted Pulmonary Delivery of a Protein Kinase C-δ Inhibitory Peptide: Impact on Indirect Lung Injury.

A rapid lateral fluid percussion injury rodent model of traumatic brain injury and post-traumatic epilepsy.

A preclinical rodent model of acute radiation-induced lung injury after ablative focal irradiation reflecting clinical stereotactic body radiotherapy.

Anti-inflammatory and antioxidant effects of curcumin on acute lung injury in a rodent model of intestinal ischemia reperfusion by inhibiting the pathway of NF-Kb.

Quercetin suppresses lung cancer growth by targeting Aurora B kinase.

Inhibition of acute lung injury by rubriflordilactone in LPS-induced rat model through suppression of inflammatory factor expression.

Protective effect of leflunomide against oxidative intestinal injury in a rodent model of sepsis.

Effect of vasopressin on hippocampal injury in a rodent model of asphyxial cardiopulmonary arrest.

The effects of iloprost on ischemia-reperfusion injury in skeletal muscles in a rodent model.

reperfusion.

Inflammatory cytokine receptor blockade in a rodent model of mild traumatic brain injury.

Anti-EGFR antibody reduces lung nodules by inhibition of EGFR-pathway in a model of lymphangioleiomyomatosis.

Protective effect of pioglitazone on sepsis-induced intestinal injury in a rodent model.

Improvement of immunogenic chemotherapy by STAT3 inhibition.

Amelioration of meconium-induced acute lung injury by parecoxib in a rabbit model.

Increased intestinal protein permeability in a model of lung injury induced by phorbol myristate acetate.

Alterations of lung microbiota in a mouse model of LPS-induced lung injury.

Development of a standardized trauma-related lung injury model.

A biomechanical model of pendelluft induced lung injury.

A new rodent model of cerebral hyperperfusion.