Inflammation ( # 2018) DOI: 10.1007/s10753-018-0758-y

ORIGINAL ARTICLE

Effect of Soluble Epoxide Hydrolase in Hyperoxic Acute Lung Injury in Mice Ping-Song Li,1 Wei Tao,2 Liu-Qing Yang,3,5 and Yu-Sheng Shu4,5

Abstract— Hyperoxic acute lung injury is a serious complication of oxygen therapy that causes high mortality. Inhibition of soluble epoxide hydrolase (sEH) has been reported to have protective effect on lipopolysaccharide-induced acute lung injury (ALI). This study investigates whether sEH plays any role in the pathogenesis of hyperoxic ALI. Wild-type and sEH gene knockout (sEH−/−) mice were exposed to 100% O2 for 72 h to induce hyperoxic ALI. Hyperoxia caused infiltration of inflammatory cells, elevation of interleukin-1β and interleukin-6 levels, and deterioration of alveolar capillary protein leak as well as wet/dry weight ratio in the lung. The hyperoxia-induced pulmonary inflammation and edema were markedly improved in sEH−/− mice. Survival rate was significantly improved in sEH−/− mice compared with that in wild-type mice. Moreover, the levels of epoxyeicosatrienoic acids and heme oxygenase-1 activity were notably elevated in sEH−/− mice compared with those in wild-type mice after exposure to 100% O2 for 72 h. The nucleotide-binding domains and leucine-rich repeat pyrin domains containing 3 (NLRP3) inflammasome activation and caspase-1 activity induced by hyperoxia were inhibited in sEH−/− mice compared with those in wild-type mice. Inhibition of sEH by an inhibitor, AUDA, dampened hyperoxiainduced ALI. sEH plays a vital role in hyperoxic ALI and is a potential therapeutic target for ALI. KEY WORDS: soluble epoxide hydrolase; hyperoxia; acute lung injury; inflammation.

INTRODUCTION Ping-Song Li and Wei Tao contributed equally to this work. 1

Department of Burn and Plastic Surgery, Subei People’s Hospital of Jiangsu Province, Yangzhou, Jiangsu, People’s Republic of China 2 Department of Surgery, Yangzhou Dongfang Hospital, Yangzhou, Jiangsu, People’s Republic of China 3 Department of Anesthesiology, Subei People’s Hospital of Jiangsu Province, Yangzhou, Jiangsu, People’s Republic of China 4 Department of Thoracic Surgery, Subei People’s Hospital of Jiangsu Province, Yangzhou, Jiangsu, People’s Republic of China 5 To whom correspondence should be addressed to Liu-Qing Yang at Department of Anesthesiology, Subei People’s Hospital of Jiangsu Province, Yangzhou, Jiangsu, People’s Republic of China. E-mail: [email protected]; and Yu-Sheng Shu at Department of Thoracic Surgery, Subei People’s Hospital of Jiangsu Province, Yangzhou, Jiangsu, People’s Republic of China. E-mail: [email protected]

Oxygen therapy is a strategy for critical ill patients with severe pulmonary or other diseases. However, increased reactive oxygen species (ROS) production and amplified pulmonary inflammation could be seen in patients with prolonged oxygen therapy or exposure to very high concentrations of oxygen [1]. It has been reported that hyperoxia can cause acute lung injury (ALI) [1]. However, the potential molecular mechanisms of hyperoxia-induced ALI are not well understood. Disruption of inflammation and anti-inflammation balance is an important mechanism contributing to ALI [2–4]. Proinflammatory cytokine interleukin (IL)-1β is one of the most potent inflammatory-initiating cytokines in ALI [5].

0360-3997/18/0000-0001/0 # 2018 Springer Science+Business Media, LLC, part of Springer Nature

Li, Tao, Yang, and Shu The nucleotide-binding domains and leucine-rich repeat pyrin domains containing 3 (NLRP3) inflammasome is the most studied inflammasome that leading to maturation of IL-1β. NLRP3 deficiency is protective for hyperoxiainduced ALI [6]. Recent studies reported that inhibition of soluble epoxide hydrolase (sEH) is a lung protective strategy [7–10]. sEH is a multifunctional protein encoded by the EPHX2 gene which expressed in many tissues including the lung [11, 12]. As an epoxy fatty acids, epoxyeicosatrienoic acids (EETs) are the endogenous substrates of sEH [13]. sEH hydrolyzes EETs which have anti-inflammatory features [14]. In terms of ALI, significantly increased sEH activity was detected in a lipopolysaccharide (LPS)-induced ALI animal model [7]. Lung injury was markedly dampened by using a specific inhibitor of sEH [7]. These results suggest that sEH plays an important effect in the pathogenesis of LPS-induced ALI. It is still unknown that whether sEH plays any role in hyperoxia-induced ALI. Gene deficiency is an effective strategy to elucidate the role of a specific gene. sEH gene deletion is protective for cigarette smokeinduced pulmonary inflammation [15], myocardial and kidney ischemia-reperfusion injury [16, 17], and acute pancreatitis in animal studies [18]. In the present study, we used sEH gene deficiency mice model and sEH inhibitor-treated wild-type mice to test the hypothesis that sEH plays a vital role in hyperoxic ALI.

MATERIALS AND METHODS All studies were performed in accordance with the National Institutes of Health guidelines for the use of experimental animals. The current project was approved by the Ethics Committee of Animal Research of Yangzhou University. Transgenic sEH−/− mice with targeted disruption of EPHX2 gene were generated by Runze Technology Industrial Co., LTD (Changsha, Hunan, China) as described previously [19]. The mice were back-crossed onto a C57BL/6 genetic background for more than ten generations. C57BL/6 wild-type mice were used as control. Male mice aged 6~8 weeks were housed in an airtight plexiglass cage and maintained on a 12-h light/12-h dark cycle at a controlled room temperature and had free access to standard chow and tap water. The animals were allowed to acclimate in the animal facility for 1 week before use. Then, the mice were exposed to hyperoxia or room air as described previously [20]. Seventy-two hours after hyperoxia or air exposure, mice were killed by cervical

dislocation and exsanguinated by cutting the vena cava inferior. We also used a sEH inhibitor, AUDA, to investigate the effect of sEH in hyperoxia-induced ALI. C57BL/6 wild-type mice were treated with AUDA (10 mg/kg) [7] intraperitoneal injection 30 min before hyperoxia exposure, and additional dosage (10 mg/kg) was given per 24 h. Bronchoalveolar Lavage Fluid Collection Bronchoalveolar lavage fluid (BALF) collection was performed on the right lung lobe six times with 5 mL of phosphate-buffered saline. The BALF was centrifuged at 14,000 rpm for 30 min at 4 °C, and the supernatant from the first two washes was pooled and analyzed for total protein and proinflammatory cytokines. Lung Wet/Dry Weight Ratio The wet/dry (W/D) weight ratio was used to estimate lung edema. Briefly, the fresh lung was weighed, dried in an oven at 80 °C for at least 48 h, and then weighed again after drying to calculate the W/D weight ratio. Lung Histology The lungs were perfused via the pulmonary artery with 0.1 mol L −1 PBS followed by 4% buffered paraformaldehyde. The lungs were dissected, postfixed for 24 h, and embedded in paraffin. Five-micrometer sections were deparaffinized and hematoxylin/eosin staining was performed. Histological analysis was initially performed in a blinded fashion. Lung injury scores were performed according to a previous study [21]. Briefly, the following histological features were scored: pulmonary edema, neutrophil infiltration, hyperemia, hemorrhage, and cellular hyperplasia. A score of 0 represented absent damage, l represented mild damage, 2 represented moderate damage, and 3 represented severe damage. Protein Concentration Assay Protein concentrations in BALF were measured using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Proinflammatory Cytokine Measurement The expression levels of interleukin (IL)-1β and IL-6 in BALF were assessed by a commercial enzyme-linked immunosorbent assay (ELISA) kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions.

Soluble Epoxide Hydrolase in Lung Injury EET Measurement The EET levels in lung homogenate samples were assessed by a commercial 14, 15-EET/DHET ELISA kit (Detroit R&D, MI, USA) according to the manufacturers’ manual. Heme Oxygenase-1 and Caspase-1 Activity Assay Heme oxygenase (HO)-1 is an inducible antioxidant enzyme playing an important role in host defense against oxidative injury. The HO-1 activity in lung homogenate samples was assessed as previously reported [22]. The absorbance of the sample was measured by spectrophotometer. The amount of bilirubin formed was calculated from the difference in absorbance at 530 nm. An NADPHfree reaction mixture provided a baseline against which the measured concentrations were compared. The values are expressed as picomoles of bilirubin formed per milligram of protein per hour. Caspase-1 is an aspartate-specific cysteine protease which cleaves IL-1β to generate a mature and active form. Caspase-1 enzymatic activity was measured using a colorimetric assay kit (R&D Systems Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions. Determination of Malondialdehyde Activity The malondialdehyde (MDA), an aldehydic secondary product of lipid peroxidation, is used as a marker of ROS production. MDA activity in lung tissue samples was assessed using an assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. Myeloperoxidase Activity Analysis The myeloperoxidase (MPO) activity is an indicator of neutrophil accumulation. A commercial ELISA kit was used to detect the MPO activity in lung homogenates according to the manufacturers’ instructions (R&D Systems Inc., Minneapolis, MN, USA). Quantitative Real-Time PCR Total RNA in the lung tissue was extracted using a RNA isolation kit (Takara, Japan) and reverse-transcribed using Prime Script™ RT Reagent Kit (Takara). The obtained cDNAs were then amplified with SYBR®Premix Ex Taq™ Kit (Takara) with primer pairs specific to each gene: sEH forward, 5′-TGCCATCCTCACCAACAC-3′ sEH reverse, 5′-ACGGACCCTGGGCTTTAC-3′

β-actin forward, 5′-TGACCGGGTCACCCACAC TGTGCCCATCTA-3′ β-actin reverse, 5′-CTAGAAGCATTTGCGGTG GACGATGGAGGG-3′ NLRP3 forward, 5′-CCACAGTGTAACTTGCAG AAGC-3′ NLRP3 reverse, 5′-GGTGTGTGAAGTTCTGGTTG G-3′ using Bio-Rad IQ5 Real-Time system (BioRad, Hercules, CA). PCR conditions were as follows: initial denaturation at 95 °C for 30 s, denaturation at 95 °C for 30 s, annealing at 60 °C for 10 s, and elongation at 72 °C for 15 s for total 40 cycles. Results were normalized against the RNA level of β-actin (as internal control). Western Blot Analysis The lung tissue was lysed, and the protein concentration was measured by use of the BCA protein assay kit. Protein samples (50 μg) were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and transferred to a nitrocellulose membrane. Proteins were detected by use of primary antibodies against sEH (1:500; Santa Cruz Biotechnology, Santa Cruz, CA) and NLRP3 (1:500; Cell Signaling Technology, Beverley, CA) followed by a HRP-conjugated secondary antibody. The protein bands were visualized by the ECL detection system (Amersham, Arlington Heights, IL), and the densities of the bands were quantified by use of Scion Image software (Scion, Frederick, MD). Proteins of β-actin were used as an internal control (anti-β-actin, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA). Survival Study The survival rate was performed using additional animals (n = 20 in each group). The sEH−/− mice, wildtype mice, and AUDA-treated mice were subjected to continuous 100% O2 exposure and observed at 24-h intervals to assess survival. Observation was continued until the last mouse survived. Statistical Analysis All data are presented as means ± SEM and analyzed using the Statistical Package for the Social Sciences (SPSS) 19.0 software (Chicago, IL, USA). One-way analysis of variance (ANOVA) followed by Dunnett’s test was used to determine significant differences between groups. Pulmonary histopathologic scores were estimated by Wallis one-way analysis of variance on ranks and the Student-

Li, Tao, Yang, and Shu Newman-Keuls method. Survival rates were estimated by the Kaplan-Meier method and compared by log-rank test. Significant level was set at P < 0.05.

RESULTS

Effect of Hyperoxia on sEH and EETs Both mRNA and protein levels of sEH were significantly increased after 72 h of 100% O2 exposure in wildtype mice compared with room air exposure (Fig. 1a). The levels of EETs were significantly reduced in wild-type mice after 72 h of 100% O2 exposure (Fig. 1b). However, the EET levels were markedly increased in sEH −/− mice and AUDA-treated mice exposed to hyperoxia (Fig. 1b).

Effect of sEH on MDA and HO-1 Activity Compared with wild-type mice, the levels of MDA were markedly decreased in sEH −/− mice and AUDAtreated mice (Fig. 1c). The activity of HO-1 was significantly increased after 72 h of 100% O2 exposure in sEH −/− mice and AUDA-treated mice compared with that in wildtype mice (Fig. 1d).

Effect of sEH in Hyperoxia-Induced ALI Compared with wild-type mice, the sEH−/− mice and AUDA-treated mice experienced reduction of neutrophil accumulation in lungs (Fig. 2a), proinflammatory cytokine IL-1β and IL-6 concentrations in BALF (Fig. 2b, c), and attenuation of alveolar capillary protein leak (Fig. 2d) as well as lung water content (Fig. 2e). Hyperoxia-induced histopathological changes were markedly dampened in sEH−/− mice and AUDA-treated mice compared with those in wild-type mice (Figs. 2f and 3a). Effect of sEH on Mortality Our results show that all wild-type mice died within 4– 6 days under hyperoxia exposure (Fig. 3b). In contrast, the sEH−/− mice and AUDA-treated mice were more resistant to hyperoxia; the survival in 100% O2 was significantly enhanced compared with that in wild-type mice (Fig. 3b). Effect of sEH on NLRP3 Activation and Caspase-1 Activity Our results show that hyperoxia exposure significantly elevated NLRP3 both in mRNA and protein levels in wild-type mice (Fig. 3c). However, this hyperoxia-induced elevation of NLRP3 was markedly inhibited in sEH−/− mice and AUDA-treated mice (Fig. 3c).

Fig. 1. Effects of hyperoxia on soluble epoxide hydrolase (sEH) (a) in wild-type mice. Epoxyeicosatrienoic acid (EET) levels (b), malondialdehyde (MDA) activity (c), and heme oxygenase (HO)-1 activity (d) in wild-type or sEH−/− mice exposed to hyperoxia for 72 h. Results represent mean ± SEM (n = 6–9 per group). AUDA, an inhibitor of sEH. *P < 0.05.

Soluble Epoxide Hydrolase in Lung Injury

Fig. 2. Effects of hyperoxia on myeloperoxidase (MPO) activity (a) in lung homogenates, interleukin (IL)-1β (b), IL-6 (c), and protein levels (d) in bronchoalveolar lavage fluid (BALF), lung wet/dry (W/D) ratio (e), and lung histopathological changes (f) in wild-type or soluble epoxide hydrolase (sEH) gene deficiency (sEH−/−) mice exposed to hyperoxia for 72 h. Results represent mean ± SEM (n = 6–9 per group). AUDA, an inhibitor of sEH. Bar 50 μm; * P < 0.05.

The activity of caspase-1 was markedly increased in wild-type mice exposed to 100% O2 for 72 h (Fig. 3d). This hyperoxia-induced caspase-1 activation was significantly inhibited in sEH−/− mice and AUDA-treated mice (Fig. 3d).

DISCUSSION ALI is a life-threatening disease with high mortality [2, 3]. In the present study, hyperoxia-induced mortality was markedly decreased in sEH−/− mice compared with that in wild-type mice. The hyperoxia-induced pulmonary inflammatory cell accumulation, proinflammatory cytokine concentrations, lung water content, and alveolar capillary protein leak were significantly improved in sEH−/− mice and AUDA-treated mice. These results indicated that sEH plays an important role in hyperoxia-induced ALI. sEH has been suggested as a pharmacological target for inflammatory disorder such as ALI [7, 23]. Enhanced activity or expression of sEH has been reported in renal

injury [24], ALI [7], and acute pancreatitis [25]. In the present study, the expression of sEH was significantly enhanced in wild-type mice exposed to hyperoxia for 72 h. However, cisplatin-induced renal injury, cerulein- and arginine-induced acute pancreatitis, and hyperoxia- or LPS-induced ALI were markedly dampened in sEH inhibitor-treated or gene knockout animals [7, 24, 25]. These results suggest that sEH may be a harmful factor for inflammatory disorders. sEH plays an important effect in hydrolyzing EETs. EETs have multifunction such as antiinflammatory and vasoactive properties [14]. EETs influence nuclear factor-κB pathway activation [26]. Previous studies reported that LPS- or cisplatin-induced nuclear factor-κB activation was dampened by inhibitors of sEH or in sEH−/− mice [7, 24]. Nuclear factor-κB regulates the expression of proinflammatory cytokines which play an important role in ALI [27]. IL-1β is one of the most important proinflammatory cytokines which activated and accumulated neutrophils [5]. Infiltration of neutrophils into the lung is one of the key features of ALI. In the present study, hyperoxia exposure significantly increased IL-1β levels and

Li, Tao, Yang, and Shu

Fig. 3. Lung injury scores (a), survival rate (b) (n = 20 per group), nucleotide-binding domains and leucine-rich repeat pyrin domains containing 3 (NLRP3) inflammasome mRNA and protein levels (c), and caspase-1 activity (d) in wild-type or soluble epoxide hydrolase (sEH) gene deficiency (sEH−/−) mice exposed to hyperoxia for 72 h. Results represent mean ± SEM (n = 6–9 per group). AUDA, an inhibitor of sEH. *P < 0.05.

neutrophil infiltration in the lung. However, the hyperoxiainduced upregulation of IL-1β and neutrophil infiltration were markedly inhibited in sEH−/− mice and AUDAtreated mice. This result suggests sEH may be involved in the regulation of IL-1β expression. Nevertheless, a precursor of IL-1β is generated after transcription which has no bio-activity and needed to be modified and converted to an active form [28]. The precursor of IL-1β is processed by caspase-1 which is a component of NLRP3 inflammasome complex [29, 30]. Our results shown that the caspase-1 activity was markedly increased in wild-type mice exposed to hyperoxia, but significantly inhibited in sEH−/− mice and AUDA-treated mice. Inhibition of caspase-1 is associated with dampened pulmonary inflammation. Our result is consistent with previous studies [6, 31]. NLRP3 inflammasome is implicated in hyperoxiainduced ALI [6, 31]. Consistent with previous studies [6, 31], our results show that hyperoxia induced NLRP3 activation in wild-type animals. However, the hyperoxiainduced activation of NLRP3 was markedly inhibited in sEH−/− mice and AUDA-treated mice. This result suggests that sEH is involved in the hyperoxia-induced activation of NLRP3. Previous studies have demonstrated that ROS are implicated in the stimulation of the NLRP3 inflammasome [32]. Previous findings, combined with our data, suggest

that sEH inhibition or gene deletion is associated with reduced ROS production [33]. HO-1 induction is known as an important defense mechanism against oxidative stress during ALI [34, 35]. Evidence has shown that EETs can increase HO-1 expression by inhibiting a negative regulator of HO-1 expression, Bach-1 [36]. sEH inhibition is associated with induction of HO-1 in an animal model of diabetic renal injury [37]. Our results show that the EET level was significantly elevated in sEH−/− mice and AUDA-treated mice, and the HO-1 activity was increased simultaneously. The increased HO-1 activity may reduce ROS production and dampen NLRP3 inflammasome activation.

CONCLUSIONS sEH plays a vital role in hyperoxic ALI and is a potential therapeutic target for ALI. ACKNOWLEDGEMENTS We thank Sun Liu and Xuan Jiang for critical reading of the manuscript, and Qin Xiao and Yue-Xiang Chen for technical assistance.

Soluble Epoxide Hydrolase in Lung Injury Author Contributions. PSL, WT, and YSS conceived and designed the experiments. WT and LQY performed the experiments. WT and LQY analyzed data. WT drafted the manuscript. LQY edited and revised the manuscript. PSL and WT interpreted results of the experiments. PSL, WT, LQY, and YSS approved the final version of the manuscript. COMPLIANCE WITH ETHICAL STANDARDS All studies were performed in accordance with the National Institutes of Health guidelines for the use of experimental animals. The current project was approved by the Ethics Committee of Animal Research of Yangzhou University.

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Conflict of Interest. The authors declare that they have no conflict of interest.

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