Hyperbaric oxygen preconditioning protects the lung against acute pancreatitis induced injury via attenuating inflammation and oxidative stress in a nitric oxide dependent manner

Accepted Manuscript Hyperbaric oxygen preconditioning protects the lung against acute pancreatitis induced injury via attenuating inflammation and oxidative stress in a nitric oxide dependent manner Qihong Yu, Peixi Zhang, Ying Liu, Wenwu Liu, N.A. Yin PII:

S0006-291X(16)31209-8

DOI:

10.1016/j.bbrc.2016.07.087

Reference:

YBBRC 36164

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 16 July 2016 Accepted Date: 20 July 2016

Please cite this article as: Q. Yu, P. Zhang, Y. Liu, W. Liu, N.A. Yin, Hyperbaric oxygen preconditioning protects the lung against acute pancreatitis induced injury via attenuating inflammation and oxidative stress in a nitric oxide dependent manner, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.07.087. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Hyperbaric oxygen preconditioning protects the lung against acute pancreatitis induced injury via attenuating inflammation

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and oxidative stress in a nitric oxide dependent manner

Qihong Yu 1, Peixi Zhang 2, Ying Liu 3, Wenwu Liu 4, Na Yin 5,# 1

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Department of Gastroenterology, Changhai Hospital, the Second Military Medical University, Shanghai, China 2 Department of Cardiothoracic Surgery, the First Hospital of Jining City, No 6, Jiankang Road, Jining City, Shandong, 272011, P. R. China 3 Department of Pathology, Yantaishan Hospital, No 91, Jiefang Road, Zhigang District, Yantai City, Shandong, 264001, P. R. China 4 Department of Diving and Hyperbaric Medicine, the Second Military Medical University, Shanghai, China 5 Department of Anesthesiology & Critical Care Medicine, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China.

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Both Yu Qi-hong and Zhang Peixi contributed to this paper equally.

Author for correspondence: Na Yin

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Department of Anesthesiology & Critical Care Medicine, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China.

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Phone: +86-21-25078999 Fax: +86-21-25078999

E-mail: [email protected]

Liu Wenwu

Department of Diving and Hyperbaric Medicine, the Second Military Medical University, Shanghai, China Phone: +86-21-81871144-809 Fax: +86-21-62352382 E-mail: [email protected]

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Abstract This study aimed to investigate the protective effects of hyperbaric oxygen preconditioning (HBO-PC) on acute pancreatitis AP associated acute lung injury (ALI) and the potential mechanisms. Rats were randomly divided into sham group, AP

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group, HBO-PC+AP group and HBO-PC + L-NAME group. Rats in HBO-PC+AP group received HBO-PC once daily for 3 days, and AP was introduced 24 h after last HBO-PC. In HBO-PC + L-NAME group, L-NAME (40 mg/kg) was intraperitoneally

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injected before each HBO-PC. At 24 h after AP, the blood lipase and amylase activities were measured; the lung and pancreas were harvested for pathological

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examination; the bronchoalveolar lavage fluid was collected for the detection of lactate dehydrogenase (LDH) and proteins; inflammatory factors, superoxide dismutase (SOD) activity and malonaldehyde content were measured in the lung and blood; the Nrf2, SOD-1 and haem oxygenase-1 (HO-1) protein expression was

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measured in the lung. The lung nitric oxide (NO) and NO synthase activity increased significantly after HBO-PC. HBO-PC was able to reduce blood lipase and amylase activities, improve lung and pancreatic pathology, decrease LDH and proteins in

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BALF, inhibit the production of inflammatory factors, reduce malonaldehyde content and increase SOD activity in the lung and blood as well as increase protein expression

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of Nrf2, SOD-1 and HO-1 in the lung. However, L-NAME before HBO-PC significantly attenuated protective effects of HBO-PC. HBO-PC is able to protect the lung against AP induced injury by attenuating inflammation and oxidative stress in the lung via a NO dependent manner.

Key words: acute pancreatitis associated lung injury; inflammation; oxidative stress; hyperbaric oxygen preconditioning; nitric oxide

ACCEPTED MANUSCRIPT Introduction To date, oxygen has been one of the most widely used therapeutic agents [1]. Administration of oxygen in either normobaric or hyperbaric environment is feasible in almost all diseases resulting from tissue hypoxia [2,3]. The beneficial effects of

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oxygen treatment are exerted via numerous mechanisms including the improvements of hemodynamics, energy metabolism, inflammation, delayed cell death and vascular permeability besides the elevated oxygen content in the blood and subsequent

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elevation of oxygen supply to hypoxic tissues [3].

Preconditioning refers to a process by which an organism’s exposure to a

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sublethal stress/stimulus allows it be more resilient against subsequent fatal stimulus. Preconditioning has been introduced as a strategy for the prevention of diseases or alleviation of disease severity [4]. Pre-oxygenation by breathing oxygen at elevated partial pressure (hyperbaric or normobaric hyperoxia) before an event has been

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documented to induce various protective effects that mirror the physiologic and therapeutic applications demonstrated both in extreme environments and in selected clinical applications [5-7]. Hyperbaric oxygen (HBO) has been widely used in the

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therapy of numerous diseases, especially the carbon dioxide toxicity, decompression

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sickness (DCS), problematic wound and stroke [8]. In recent years, HBO is also employed as a strategy for preconditioning, and the effectiveness of HBO preconditioning (HBO-PC) has been confirmed in numerous animal models and clinical trials, which is largely ascribed to its anti-inflammatory, anti-oxidative and anti-apoptotic effects [5-7]. Acute pancreatitis (AP) is thought caused principally by autodigestion of the pancreas, with extravasation of proteolytic enzymes and vasoactive mediators leading to inflammation of contiguous tissues [9]. Entry of these noxious substances into the

ACCEPTED MANUSCRIPT systemic circulation results in multiorgan complications, and lung injury is the most pertinent manifestation of extra-abdominal organ dysfunction in AP [10,11]. Approximately one third of AP patients will develop acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), which account for 60% of all deaths

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within the first week [12]. Various therapeutic endeavors have been proposed for the therapy of AP associated lung injury, but few exhibit effectiveness in the clinical settings and its mortality is still at a high level [13,14].

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HBO has been used in the therapy of pancreatitis [15], but no study has been conducted to investigate the protective effects of HBO-PC on the AP and AP

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associated ALI. On the basis of importance of inflammation and oxidative stress in the pathogenesis of AP associated ALI [10,16], this study was undertaken to investigate whether HBO-PC is able to protect the lung against AP associated injury by alleviating inflammation and oxidative stress and whether the protective effects are

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dependent on nitric oxide (NO). Materials and methods Animals and grouping

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Adults male Sprague–Dawley rats weighing 220-250 g (n=96) were purchased

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from the Experimental Animal Center of the Second Military Medical University. Rats were allowed to accommodate to the environment for 7 days and given ad libitum access to water and food. After surgery, animals were housed independently in cages. The experimental procedures were carried out in accordance with standard guidelines for the care of animals and were approved by the Ethics Committee for Animal Experiments of the Second Military Medical University. Rats were randomly divided into 5 groups: sham group (n=18), HBO-PC group (n=6), AP group (n=24), HBO-PC+AP group (n=24) and HBO-PC+NO inhibitor

ACCEPTED MANUSCRIPT (L-NAME) group (n=24). In sham group, rats received sham surgery; in HBO-PC group, rats received HBO-PC and were sacrificed after the last HBO exposure; in SP group, AP was induced alone; in HBO-PC+AP group, rats received HBO-PC and then AP was induced at 24 h after the last HBO exposure; in HBO-PC+L-NAME group,

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rats were intraperitoneally injected with L-NAME at 40 mg/kg 30 min before each

HBO exposure and other treatments were the same to those in HBO-PC+AP group. HBO preconditioning

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For HBO-PC, pure oxygen was supplied continuously at a pressure of 2

atmosphere absolute (ATA) for 1 h once daily for consecutive 3 days. Compression

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was performed at 1 kg/cm2/min, and decompression was performed at 0.2 kg/cm2/min. No seizures were observed in any animal during any HBO exposure. The animals in sham group were placed in the chamber, which was not pressurized. Chamber temperature was maintained between 22 and 25°C. Accumulation of carbon dioxide

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was prevented by using a small container with calcium carbonate crystals. To minimize the effects of diurnal variation, all exposures were started at 8:00 AM [17]. Establishment of rat AP model

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AP was induced based on a previously described method [18] with minor

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modifications. Rats received food deprivation for 12 h before surgery. After weighing, animals were anesthetized intraperitoneally with 10% chloral hydrate (Shanghai Biochemical Reagent Factory) at 0.4 ml/100 g. Then, they were placed in a supine position, and a midline incision was made at the abdomen. The descending duodenum was identified and exposed, and the trend of bile-pancreatic duct was discerned. The bile duct was clamped at the site of hepatoduodenal ligament close to the porta hepatis, and then 5% sodium taurocholate (Sigma-Aldrich, St. Louis, MO, USA) at 0.1 ml/100 g was retrogradely infused into the distal end of the bile-pancreatic duct. The

ACCEPTED MANUSCRIPT proximal bile duct was temporarily clamped at the porta hepatis with a vascular clamp. Subsequently, the vascular clamp was removed and the duodenal and abdominal wounds were closed. Rats in sham group underwent only a laparotomy and subsequent exposure of the duodenum. Above procedures were conducted under an

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asceptic condition. Detection of serum amylase and lipase activities

At 24 h after surgery, rats were sacrificed and blood was collected from the heart.

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Serum samples were obtained from harvested blood specimens by centrifugation

(2000 ×g for 15 min at 4°C) and the amylase and lipase activities were measured

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through the colorimetric method using commercial kits (Jiancheng Co., Nanjing, China). The activities of amylase or lipase were expressed as units per liter (U/L). Pathological examinations of the lung and pancrease

At 24 h after surgery, rats were sacrificed, and the left lung and pancrease were

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collected and fixed in 4% paraformaldehyde at 4oC. After dehydration in ethanol, tissues were embedded in paraffin and then cut into 4-µm sections. Then, these sections were processed for HE staining.

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Detection of lung NO, NO synthase activity and wet/dry ratio

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After the last HBO expression, rats were sacrificed, and the lungs were collected for the detection of NO and NO synthase (NOS) activity with corresponding kits. NOS catalyzed the formation of NO and L-citrulline from L-arginine and molecular oxygen, and NO reacted with a nucleophile to generate color compounds. The absorbance of NOS at 530 nm was calculated and expressed as U/mg protein. One unit of NOS activity was defined as the production of 1 nmol NO per second per mg protein. The lungs were homogenized and centrifuged at 1000 r/min for 5 min at 4°C. The supernatant was taken for NO assay and total protein determination. NO was

ACCEPTED MANUSCRIPT measured as follows: 10% tissue homogenate (100 µL) was incubated with 200 µL of substrate buffer, 10 µL of reaction accelerator and 100 µL of chromogenic agent at 37°C for 15 min. NO was assayed spectrophotometrically by measuring total nitrate plus nitrite (NO3- plus NO2-) and the stable end products of NO metabolism. The

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results were expressed as µmol/g protein. The detections were conducted according to the manufacturer’s instructions (Nanjing Jiancheng Biotech Co., Ltd).

At 24 h after surgery, rats were sacrificed and the right lung was collected and

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washed in PBS. The water on the lung was removed with filters and then the right

lung was weighed (wet weight; W). After drying in an oven at 60oC for 72 h, the right

reflect the severity of lung edema.

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lung was weighed (dry weight; D). The lung wet/dry ratio (W/D) was calculated to

Bronchoalveolar lavage fluid collection and detection

Bronchoalveolar lavage fluid (BALF) collection was performed with 4 mL of

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phosphate-balanced saline solution (PBS). The collected BALF was centrifuged at 1000 g for 10 min and the supernatant was collected for the detection of lactate dehydrogenase (LDH) activity and proteins. Total protein content in BALF was

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measured by the BCA protein assay reagents (Nanjing Jiancheng Bioengineering

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Institute, China). Under anaerobic conditions, LDH catalyzes lactate acid to generate pyruvate, which then reacts with 2,4-dinitrophenyl hydrazine and forms pyruvate dinitrobenzene hydrazone. The latter compound was quantified with an assay kit (Nanjing Jiancheng Bioengineering Inst., Jiangsu, China) and a spectrophotometer (DU-800, Beckman Coulter Inc., USA) at 440 nm. One unit of LDH activity was defined as the amount that produces 1 µmol of pyruvate after 15 min at 37 °C. LDH activity was expressed as units per gram protein (U/g protein). Detection of lung and blood inflammation related cytokines

ACCEPTED MANUSCRIPT At 24 h after surgery, rats were sacrificed, and the lung and blood were collected for further examinations. In brief, the lung tissues were homogenized, followed by centrifugation at 3000 rpm for 15 min. The supernatant was collected. The blood was centrifuged at 3000 rpm for 15 min, and the serum was harvested. The lung and blood

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contents of tumor necrosis factor-α (TNF-α), monocyte chemotactic protein 1 (MCP-1) and intercellular cell adhesion molecule-1 (ICAM-1) were detected by enzyme-linked immunoassay (ELISA) with corresponding kits (Xitang Biotech Co., Ltd, Shanghai,

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China).

Detection of lung and blood superoxide dismutase activity and malondialdehyde

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content

As a marker of oxidative stress and free oxygen radical-mediated damage, malondialdehyde (MDA) contents of the lung and blood were measured using the MDA Assay Kit (Beyotime, Jiangsu, China) according to the procedure recommended

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by the manufacturer. Briefly, lung tissues were homogenized. After centrifugation, free MDA in the supernatant was converted to a stable carbocyanin dye by the chemical reaction with N-methyl-2-phenylindole. Protein concentration was

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determined by the BCA Protein Assay. MDA levels were normalized against protein

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(µmol/g protein). SOD activity was determined by a colorimetric assay (at 550 nm) based on a xanthine oxidase method (Beyotime, Jiangsu, China). One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of xanthine and xanthine oxidase system reaction in 1 ml enzyme extraction of 1 mg protein. SOD activity was expressed as units per milligram protein (U/mg protein). Detection of anti-oxidase protein expression At 24 h after surgery, the lungs were harvested and total proteins were extracted by using an extraction kit (Nanjing Jiancheng Biotech Co., Ltd). The protein

ACCEPTED MANUSCRIPT concentration was determined with BCA method. Equal amounts of the protein samples were loaded per lane. The primary antibodies were mouse anti-rat polyclonal antibody against SOD-1, rabbit anti-rat polyclonal antibody against HO-1 (1:500), rabbit anti-rat polyclonal antibody against Nrf2 (1:200) and mouse anti-rat monclonal

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antibody against β-actin (1:1000) (Santa Cruz Biotechnology, Inc., USA). Western

blots were performed by means of horseradish peroxidase (HRP)-conjugated IgG and the use of enhanced chemiluminescence detection reagents (Pierce). Bands were

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scanned using a densitometer (model GS-700, Bio-Rad Laboratories), and quantification was performed using Image J software (NIH Image).

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Statistical analysis

Data are expressed as mean ± standard deviation ( X ±SD). Statistical analysis was performed with SPSS version 16.0 (SPSS Inc., Chicago, USA), and comparisons were done with one way analysis of variance (ANOVA) among groups, followed by

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SNK-q test between two groups. A value of P<0.05 was considered statistically significant. Results

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HBO preconditioning increases lung NO content and NOS activity

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The lung NO content was 6.83±2.2 µmol/g protein in control group and 20.97±5.82 µmol/g protein in HBO-PC group, showing significant difference between two groups (P<0.01).

The NOS activity was 5.08±1.30 U/mg protein in control group and 15.13±4.12

U/mg protein in HBO-PC group, showing marked difference between them (P<0.01). HBO preconditioning reduces serum amylase and lipase activities and improves lung and pancreas histology As shown in Figure 1, the blood amylase content was 705±132 U/L in control

ACCEPTED MANUSCRIPT group, but increased significantly in AP group (4765±856 U/L; P<0.01). However, serum amylase content in HBO-PC+AP group (3567±616 U/L) markedly reduced when compared with AP group (P<0.05; Figure 1B). In the presence of L-NAME, HBO-PC failed to reduce serum amylase content (P>0.05 vs AP group; P<0.05 vs

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HBO-PC+AP group). Similar changes were also observed in blood lipase (Figure 1C). In control group, there were no obvious changes in the pancreas of rats. The pancreatic structure was clear, cell morphology was normal, and there was no evident

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interstitial edema, no hemorrhage and no necrosis. In AP group, there were disruption of pancreatic acini and lobular structure, massive hemorrhagic and necrotic areas, and

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infiltration of a large amount of neutrophils. In HBO-PC+AP group, there was swelling of pancreatic acini, hemorrhage and necrosis were observed in a few pancreatic acini, lobular space was enlarged, and there was infiltration of some inflammatory cells (Figure 1A). The pancreatic pathology was deteriorated in

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HBO-PC+L-NAME group as compared to HBO-PC+AP group. In control group, the alveolar structure was normal and clear, there were no infiltration of inflammatory cells in the alveolar wall and interstitium. However, in AP

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group, there were diffuse interstitial edema of the lung, broadening of lung

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interstitium, infiltration of a large amount of neutrophils and mononuclear cells into lung interstitium and alveolar space, and disruption and disordered structure of some alveoli. In HBO-PC+AP group, there were mild lung edema, infiltration of a few inflammatory cells and attenuated thickening of lung interstitium, and the alveolar structure was largely intact (Figure 1A). The lung pathology was deteriorated in HBO-PC+L-NAME group as compared to HBO-PC+AP group. HBO preconditioning inhibits lung edema As shown in Figure 2A, the W/D ratio was 5.65±0.58 in AP group, which was

ACCEPTED MANUSCRIPT significantly higher than in control group (4.12±0.36; P<0.01) and in HBO-PC+AP group (4.64±0.58; P<0.05). This indicates that the lung edema was attenuated in the presence of HBO-PC. However, in the presence of L-NAME, HBO-PC failed to effectively attenuate lung edema secondary to AP (P>0.05 vs AP group; P<0.05 vs

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HBO-PC+AP group).

HBO preconditioning reduces LDH activity and total protein of BALF

LDH activity and total protein content of the BALF may serve as indicators of

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lung injury. In the present study, LDH activity and total protein content in the BALF were measured in rats of different groups. Results showed the lung LDH activity

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(78.69±10.32 U/g protein) and total protein content (83.26±9.38 µg/ml) in AP group, were significantly higher than in sham group (8.12±2.38 U/g protein and 23.67±6.68 µg/ml, respectively). However, after HBO-PC, the lung LDH activity (36.33±8.24 U/g protein) and total protein content (45.43±8.19 µg/ml) reduced markedly as compared

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to those in AP group (P<0.05). The LDH activity and total protein content in HBO-PC+L-NAME group increased significantly as compared to HBO-PC+AP group (P<0.05) (Figure 2B and 2C). Moreover, LDH activity and total protein content

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HBO-PC+L-NAME group were still markedly lower than in AP group (P<0.05). This

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suggests that L-NAME before HBO-PC partially inhibit the protective effects of HBO-PC.

HBO preconditioning reduces inflammation related cytokines, SOD activity and MDA content of the lung and blood Oxidative stress and inflammation play important roles in the pathogenesis of AP related lung injury [10,16]. Thus, in the present study, we further detected the contents of inflammation related factors (MCP-1, ICAM-1 and TNF-α), SOD activity and MDA content of the lung and blood.

ACCEPTED MANUSCRIPT Results showed the contents of MCP-1, ICAM-1 and TNF-α increased significantly in the blood and lung of AP rats (P<0.05 vs sham group), but their contents reduced markedly in rats receiving HBO-PC (P<0.05 vs AP group). These indicate that HBO-PC is able to attenuate inflammation in the lung and blood. In

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addition, the SOD activity of the blood and lung in AP rats reduced significantly as compared to sham group (P<0.05), but it was markedly higher in HBO-PC+AP group than in AP group (P<0.05). The MDA content of the blood and lung in AP group

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increased dramatically when compared with sham group, but it in HBO-PC+AP group was significantly lower than in AP group (P<0.05). These suggest that HBO-PC is

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able to alleviate oxidative stress in the lung and blood. However, the inflammation and oxidative stress deteriorated in the presence of L-NAME during HBO-PC as compared to HBO-PC+AP group (increases in inflammatory factors and MDA content as well as reduction in SOD activity) (Figure 3). Of note, the MCP-1,

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ICAM-1,TNF-α and MDA contents in L-NAME group were still significantly lower than in AP group, but SOD activity in L-NAME group was still markedly higher than

HBO-PC.

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in AP group, suggesting that L-NAME partially inhibits the protective effects of

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HBO preconditioning increases protein expression of anti-oxidases in the lung We further detected the protein expression of major anti-oxidases in the lung by

Western blot assay. Results showed the protein expression of Nrf2, SOD-1 and HO-1 reduced significantly in the lung after AP, suggesting the compromised anti-oxidative capability. However, the anti-oxidative capability increased significantly in the presence of HBO-PC, characterized by increased protein expression of Nrf2, SOD-1 and HO-1 in the lung (P<0.01 vs AP group). Of note, in the presence of L-NAME during HBO-PC, the protein expression of anti-oxidases reduced markedly as

ACCEPTED MANUSCRIPT compared to HBO-PC+AP group (P<0.05) (Figure 4). Of note, the protein expression of Nrf2, SOD-1 and HO-1 in the lung of L-NAME group was still significantly higher than in AP group (P<0.05), suggesting that L-NAME partially inhibits the protective effects of HBO-PC.

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Discussion

Respiratory complications are frequent in AP, and respiratory dysfunction, the

severity of which ranges from mild oxygenation abnormalities to severe ARDS or ALI,

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is most pertinent manifestation of extra-abdominal organ dysfunction in AP [10,11],

which accounts for 60% of all deaths within the first week [12]. In ALI secondary to

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AP, the endothelial and epithelial barrier permeability increases with leakage of a protein-rich exudate into the alveolar space and interstitial tissues, thus compromising oxygenation and gas exchange [10,19]. In available studies, inflammation and oxidative stress are the most extensively studied mechanisms underlying the

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pathogenesis of ALI secondary to AP [10,16]. Inflammatory cells become activated after AP induced ALI, secret a large amount of pro-inflammatory cytokines including TNF-α and simultaneously are recruited at different phases in the presence of

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chemokines such as MCP-1 [10]. Moreover, these cells may produce excess reactive

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oxygen/nitrogen species (ROS/RNS), leading to oxidative/nitrative stress, which is also crucial for the pathogenesis of ALI secondary to AP [16]. HBO-PC has been found to exert protective effects on the brain injury [20], lung

injury [21], heart injury [22], skin flap [23] and liver injury [24] and in other pathological situations. Studies have confirmed that HBO-PC may improve the anti-oxidative, anti-inflammatory and anti-apoptotic capabilities in the body [5] although the underlying mechanisms are complex. In animal models of lung injury, HBO-PC has been confirmed to protect the lung against lipopolysaccharide (LPS)

ACCEPTED MANUSCRIPT induced injury [21], hyperoxia induced injury [25] and high-altitude pulmonary edema [26]. In a clinical study, a single preoperative HBO-PC on the day before surgery was found to reduce the complication rate in pancreaticoduodenectomy,

investigate the protective effects of AP associated with ALI.

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including lung complications [27]. To date, no study has been conducted to

In the present study, retrograde infusion of sodium taurocholate was employed to establish AP animal model. It has been confirmed that this model may (in part) better

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mimic the events preceding the development of biliary AP and simulate severe

necrotizing disease [28]. Our results showed HBO-PC (once daily for consecutive 3

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days) was able to reduce the lung W/D ratio, improve lung and pancreatic histology, decrease inflammation related factors (TNF-α, MCP-1 and ICAM-1) in the lung and blood, attenuate oxidative stress (MDA) of the lung and blood and increase the anti-oxidative capability (SOD) of the lung and blood.

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In a previous study, HBO-PC with our protocol was also confirmed in a rat partial hepatectomy model [29]. In addition, our previous studies found the protective effects of HBO-PC still existed at 24 h after last HBO exposure [30,31]. Thus, HBO

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exposure once daily for consecutive 3 days was employed to this study, and AP was

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induced 24 h after the last HBO exposure. In addition, in our pilot study, intraperitoneal L-NAME had no influence on the pancreatic injury and lung injury in rats with SAP. As mentioned by the Orzelska-Gorka et al, L-NAME is a reversible NOS inhibitor and has the half-life of approximately 20 h [32]. Considering that AP was induced 24 h after the last HBO exposure and the half life of L-NAME is about 20 h, we speculate that L-NAME has little influence on the pancreatitis and associated lung injury. TNF-α is an important pro-inflammatory cytokine involved in the systemic

ACCEPTED MANUSCRIPT inflammation secondary to AP [33] because sterile, cytokine-free ascitic fluid from rats with pancreatitis failed to induce lung injury in IL-1β/TNF-α receptor double knockout mice [34]. MCP-1, also known as CCL-2, is an important chemokine involving in leucocyte trafficking and also closely related to the AP associated ALI

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because CCL-2 and CCR-2 levels rise during pancreatitis, and both pancreatitis and the associated lung injury are blunted in CCL-2 deficient mice [35]. ICAM-1 is an

endothelial- and leukocyte-associated transmembrane protein and play important roles

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in stabilizing cell-cell interactions and facilitating leukocyte endothelial

transmigration. Study also confirms the important of ICAM-1 in AP associated ALI

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because pulmonary expression of ICAM-1 is increased in pancreatitis and that ICAM-1 deficient mice with pancreatitis evince less lung injury than their disease-free counterparts [36].

On the basis of importance of oxidative stress in the pathogenesis of AP

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associated lung injury, investigators attempt to treat ALI secondary to AP by targeting ROS. Yang et al found free radical scavenger edaravone was able to protect the lung against AP associated injury [37]. Lv et al also found thalidomide could reduce the

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expression of adhesion molecules via inhibition of oxidative stress to alleviate

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AP-associated lung injury in a rat model [38]. It is clear that Nrf2 plays a key role in orchestrating cellular antioxidant defenses and in maintaining redox homeostasis. Generally, Nrf2 is regulated by a cytosolic repressor protein Keap1 which facilitates the Nrf2 ubiquitination and proteosomal degradation in basal conditions. Once the reactive cysteine residues are oxidized, Nrf2 is released and then translocates into the nucleu where it binds to the antioxidant response element/electrophiles response element (ARE/EpRE), leading to the transcriptional activation of phase 2 genes, such as HO-1, NADPH-quinone oxidoreductase-1 (NQO1) and γ-glutamyl-cystein

ACCEPTED MANUSCRIPT synthase (γ-GCS), which provide efficient cytoprotection by regulating the intracellular redox state [39]. Some studies have confirmed that HBO-PC can protect organ injury by activating Nrf2 expression [20,40,41]. Our results also confirmed that HBO-PC could increase the Nrf2 expression and the expression of its downstream

effects of HBO-PC on ALI secondary to AP.

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genes (HO-1 and SOD-1), which may be, at least partially, related to the protective

Taken together, HBO-PC is able to enhance the anti-inflammatory and

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anti-oxidative capabilities to exert protective effects on ALI secondary to AP. Our previous study showed NO content of the brain and spinal cord increased immediately

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after HBO preconditioning and thereafter reduced [42]. In the present study, we also detected the NO content and NOS activity of the lung. Results showed the lung NO content and NOS activity increased significantly after HBO-PC, and L-NAME partially inhibits the protective effects of HBO-PC on the ALI associated with AP,

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suggesting that the protective effects of HBO-PC are related to the NOS activation and consequent NO production in the lung. These results were consistent with previous findings that L-NAME also attenuated the protective effects of HBO-PC on

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decompression sickness in a rat model [43,44].

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There were still limitations in this study. First, which isoform of NOS is involved in the protective effects of HBO-PC was not further investigated. Second, there is evidence showing that the protective effects of HBO-PC are time dependent [43]. In this study, AP was induced at 24 h after the last HBO exposure. In future studies, more time points are needed to find the optimal time interval between last HBO exposure and injury.

Acknowledgement This work was supported by the National Natural Science Foundation of China (No. 81400668)

ACCEPTED MANUSCRIPT Conflict of interest There is no conflict of interest in this paper.

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Figure 1 HE staining of the lung and pancreas and serum activities of lipase and amylase. In AP group, there were disruption of pancreatic acini and lobular structure, massive hemorrhagic and necrotic areas, and infiltration of a large amount of neutrophils. The pancreatic pathology was improved by HBO-PC, but it was deteriorated in the presence of L-NAME during HBO-PC. In AP group, there were diffuse interstitial edema of the lung, broadening of lung interstitium, infiltration of a large amount of neutrophils and mononuclear cells into lung interstitium and alveolar space, and disruption and disordered structure of some alveoli. The lung pathology was improved by HBO-PC, but it was deteriorated in in the presence of L-NAME during HBO-PC. AP significantly increased the serum lipase and amylase activities, but HBO-PC attenuated the increases in serum lipase and amylase activities after AP. However, L-NAME administered before HBO-PC abolished this effect. Note: *P<0.05 vs AP group; # P<0.05 vs HBO+AP group; Scale bar: 50 µm

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Figure 2 Lung wet / dry weight ratio, BALF LDH activity and BALF protein content AP significantly increased the lung wet / dry weight ratio, BALF LDH activity and BALF protein content, suggesting the lung edema and increase in vascular permeability. In the presence of HBO-PC, the lung edema was improved, accompanied by reductions in LDH activity and protein content of BALF. However, this effect of HBO-PC was partially abolished by L-NAME administered before HBO-PC, and significant differences were still observed between L-NAME group and AP group. Note: *P<0.05 vs AP group; # P<0.05 vs HBO+AP group

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Figure 3 Inflammatory cytokines, MDA content and SOD activity of the blood and lung AP significantly increased inflammation related cytokines, elevated MDA content and reduced SOD activity of the blood and lung. In the presence of HBO-PC, the inflammatory cytokines reduced markedly, MDA content decreased dramatically and SOD activity increased significantly in the blood and lung. However, L-NAME administered before HBO-PC partially inhibited these effects of HBO-PC, and significant differences were observed between L-NAME group and AP group (P<0.05). Note: *P<0.01 vs AP group; # P<0.05 vs HBO+AP group; Figure 4 Anti-oxidase protein expression in the lung AP significantly reduced Nrf2, HO-1 and SOD-1 protein expression in the lung, which was markedly increased in the presence of HBO-PC. However, L-NAME administered before HBO-PC partially, but significantly abolished this effect of HBO-PC, and significantly difference was observed in the protein expression of Nrf2, HO-1 and SOD-1 between HBO-PC group and L-NAME group (P<0.05).

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Note: *P<0.01 vs AP group; # P<0.05 vs HBO+AP group

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ACCEPTED MANUSCRIPT Highlights 1. Hyperbaric oxygen preconditioning (HBO-PC) is able to protect the lung against acute pancreatitis induced injury.

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2. Protective effects of HBO-PC are related to anti-inflammation and anti-oxidation.

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3. Protective effects of HBO-PC are partially mediated by nitric oxide.