Maresin Conjugates in Tissue Regeneration 1 improves alveolar fluid clearance by up-regulating alveolar ENaC, Na, K-ATPase in lipopolysaccharide-induced acute lung injury.

Abstract Maresin Conjugates in Tissue Regeneration 1 (MCTR1) is a newly identified macrophage‐derived sulfido‐conjugated mediator that stimulates the resolution of inflammation. This study assessed the role of MCTR1 in alveolar fluid clearance (AFC) in a rat model of acute lung injury (ALI) induced by lipopolysaccharide (LPS). Rats were intravenously injected with MCTR1 at a dose of 200 ng/rat, 8 hours after administration of 14 mg/kg LPS. The level of AFC was then determined in live rats. Primary rat ATII (Alveolar Type II) epithelial cells were also treated with MCTR1 (100 nmol/L) in a culture medium containing LPS for 8 hours. MCTR1 treatment improved AFC (18.85 ± 2.07 vs 10.11 ± 1.08, P < .0001) and ameliorated ALI in rats. MCTR1 also significantly promoted AFC by up‐regulating epithelial sodium channel (ENaC) and Na+‐K+‐adenosine triphosphatase (Na, K‐ATPase) expressions in vivo. MCTR1 also activated Na, K‐ATPase and elevated phosphorylated‐Akt (P‐Akt) by up‐regulating the expression of phosphorylated Nedd4‐2 (P‐Nedd4‐2) in vivo and in vitro. However, BOC‐2 (ALX inhibitor), KH7 (cAMP inhibitor) and LY294002 (PI3K inhibitor) abrogated the improved AFC induced by MCTR1. Based on the findings of this study, MCTR1 may be a novel therapeutic approach to improve reabsorption of pulmonary oedema during ALI/acute respiratory distress syndrome (ARDS).


Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS)
is a life-threatening clinical condition. It damages the alveolar epithelial tissues leading to non-cardiogenic pulmonary oedema. 1,2 Recent pathophysiological advancements of ALI/ARDS have improved its clinical outcome. 3 However, effective management and treatment of ALI/ARDS are challenging. As such, it has significantly high morbidity and mortality rates. 4,5 Previous studies report that the majority of ALI/ARDS patients have impaired alveolar fluid clearance (AFC). 6 Early control of increased alveolar fluid is therefore important for proper clinical prognosis of ALI/ARDS. Alveolar fluid clearance is pegged on the active transport of sodium ions (Na + ) across the alveolar epithelium via the apical alveolar epithelial sodium channel (ENaC) and basolateral Na + -K + -adenosine triphosphatase (Na, K-ATPase). 7,8 ENaC is the primary driving force for removal of lung fluid. 6 Na, K-ATPase plays the role of sodium pump that works in concert with apical ENaC in regulating AFC. 7 A 2 B adenosine receptor agonists, angiotensin, tri-Iodo-L-thyronine and oestradiol have exhibited increasing AFC effects via promoting ENaC or Na, K-ATPase activity in animal studies. [9][10][11][12] However, these studies remain unsuccessful in humans. [9][10][11][12] Previously, we reported that administration of a β-agonist salbutamol (iv) decreased extravascular pulmonary fluid in ALI/ARDS patients. However, some side-effects of salbutamol such as lactic acidosis, arrhythmia and tachycardia were significantly up-regulated in a 28-day mortality rate in a multi-centred randomized controlled clinical trial. 13 This necessitated the need to identify a new therapeutic approach.
Maresin Conjugates in Tissue Regeneration 1 (MCTR1) is a specialized family of macrophage-derived sulfido-conjugated mediators. It possesses organ protective function as well as the ability to enhance the resolution of inflammation in vivo. 14,15 MCTR1 (50 ng/mouse, IP) decreased the neutrophil infiltration and duration of resolving inflammation in mice pre-treated with lipopolysaccharide (LPS). It also enhanced the phagocytosis of E Coli by macrophages. 16 Levy BD et al 17 reported that MCTRs improved the recovery of the allergic airway responses in mice. However, the effects of MCTR1 on AFC are yet to be explored.
In this study, the beneficial effects of MCTR1 on AFC in ALI caused by LPS treatment were explored. Moreover, the effects of MCTR1 on levels of phosphorylated-Akt (P-Akt), Na, K-ATPase, ENaC and phosphorylated Nedd4-2 (P-Nedd 4-2) in the rat lungs and alveolar type II (ATII) cells were explored. Furthermore, the signalling pathways of MCTR1 were studied to assess its response to ALX receptor inhibitor (BOC-2), cAMP inhibitor (KH7), PI3K antagonist (LY294002) and PKA blocker (H89) on AFC.

| Experimental animals
All the experiments were performed using adult male Sprague-Dawley (SD) rats weighing between 250 and 300 g. The rats were procured from SLAC Laboratory Animal (

| Pulmonary histopathology evaluation
The left lung tissues of the killed rats from each group were fixed in 4% paraformaldehyde for 24 hours. This was done for light microscopy analysis. Subsequently, the fixed lung tissues were embedded in liquid paraffin and stained with haematoxylin and eosin (H&E).
Lung injury scores were determined by a semi-quantitative scoring system based on several indicators. 18 The indicators were as follows: alveolar congestions, alveolar haemorrhage, infiltration of neutrophils, thickness of the alveolar wall/hyaline membrane, aggregation in the airspace or vessel wall and infiltration of inflammatory cells.
Light microscopy pathologic findings were graded between 0 and 4.

| ELISA
The right lung tissues from each group were homogenized and centrifuged as per the manufacturer's guidelines. The R&D systems ELISA kits were then used to determine the concentrations of MPO, IL-10, TNF-α and IL-1β in the supernatants.

| Measurement of alveolar fluid clearance in live rats
Alveolar fluid clearance is expressed as percentage of the total instilled volume cleared within 60 minutes. Alveolar fluid clearance was determined based on concentration changes in the Evan's bluetagged-albumin as previously described by our laboratory. 18 For alveolar instillation, 50 mg/mL BSA was dissolved in modi-

| Extraction and preparation of primary rat ATII cells
Lung tissues were digested using elastase digestion and differentially adhered to IgG-coated plates as described by Dobbs et al 19 The purity of ATII cells was then determined using the modified Papanicolaou stain by identifying the presence of dark blue inclusions. The trypan blue exclusion assay was used to evaluate ATII cell viability (>95%). ATII cells were seeded on plastic dishes containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum, 0.1 mg/mL streptomycin, 100 U/mL penicillin and 2 mmol/L l-glutamine at a density of 1 × 10 6 per cm 2 .
The cells were cultured in 5% CO 2 and 95% air in six-well plates and grown to 80% confluence. The cells were subsequently deprived of serum for 24 hours after which 1 μg/mL LPS was added in the presence or absence of MCTR1 (100 nmol/L).
Electrochemiluminescence (ECL) was then used to detect the protein bands while band visualization was done using a UVP gel imaging system (Upland, CA). AlphaEaseFC (version 4.0) was used to analyse the band intensities.

| Measurement of Na, K-ATPase activity in rat lung tissues
The ouabain-sensitive ATP hydrolysis was used to assess the hydrolytic activity of Na, K-ATPase under maximal velocity conditions. Lung tissues were harvested and processed by centrifugation and homogenization. The activity of Na, K-ATPase was then measured using the minimal ATP enzyme test kit (Jian Cheng Company) following the manufacturer's instructions.

| Measurement of cAMP concentrations
Isobutyryl methylxanthine (Sigma-Aldrich) was added to the cells to suppress phosphodiesterase. The cells were then homogenized in ice-cold 1 mol/L TCA and centrifuged at 2500 g RCF to precipitate the cells. The cAMP ELISA kit (R&D Systems) was then used to determine the concentration of cAMP in the lysates.

| Blinding method
A completely randomized design was adopted in the blinding of patient. The SAS/STAT software was used to generate a randomization list. The separation function was employed to achieve blinding. One researcher dispensed and administered treatments to the rat models while a different group of researchers was involved in harvesting the lung tissues for Western blot, alveolar fluid clearance measurement, transmission electron microscopy and pathological examination.

| Statistical analysis
All statistical analyses were done by one-way anova using Prism 7.0 software (GraphPad Software). All data are presented as mean ± SD.

| MCTR1 ameliorates LPS-Induced ALI
We assessed pathological changes in lung tissue by HE staining.
While the lung tissues of the control group had a normal histology, tissues from the LPS group were substantially injured ( Figure 1A,B).
These injured tissues were characterized by haemorrhage, interstitial oedema and alveolar wall thickening. Inflammatory cell infiltration into the alveolar space and the interstitium was also observed in the injured tissues. This was revealed by increased lung injury scores ( Figure 1C). Tissue injuries suggested that ALI occurred in this model. The morphological changes were comparatively lower in the LPS+MCTR1 group than the LPS group. However, there was no significant difference between the control and the MCTR1 groups.

| MCTR1 up-regulates the clearance of alveolar fluid in LPS-induced ALI
This study assessed the effect of MCTR1 on the clearance of alveolar fluid in LPS-induced ALI in vivo. Experimental results indicated that the LPS group had a lower alveolar fluid clearance rate than the control group (10.11 ± 1.08 vs 25.64 ± 2.92). Notably, administration of MCTR1 increased alveolar fluid clearance after LPS-induced ALI (18.85 ± 2.07 vs 10.11 ± 1.08). However, there was no significant difference between the control and MCTR1 groups ( Figure 1H). F I G U R E 1 MCTR1 ameliorates lung injury and enhances alveolar fluid clearance in a rat model of ALI induced by LPS. MCTR1 (200 ng/rat) was administered to Sprague-Dawley (SD) rats eight hours post-stimulation with LPS (14 mg/kg). Lung tissue sections were stained with haematoxylin and eosin (Magnification ×200, ×400) to evaluate the effects of MCTRI (A, B). Scores of lung injury (C) ranging from 0 to 16 as described in Section 2. Zero indicates no damage whereas 16 indicates the highest damage. Expression of MPO (D), TNF-α (E), IL-1β (F) and IL-10 (G) in lung tissues determined using ELISA. Following intratracheal instillation with 5% of albumin solution comprising albumin labelled with Evans blue (5 mL/kg) via a left lung tracheostomy, clearance of alveolar fluid was measured for 60 min in pre-ventilated animals. (H) All data are presented as mean ± SD. n = 7, ***P < .001. MCTR1, Maresin Conjugates in Tissue Regeneration 1; MPO, myeloperoxidase F I G U R E 2 Effects of MCTR1 on ultrastructure of lung tissue in LPS-induced lung injury in vivo. MCTR1 (200 ng/rat) was administered to SD rats 8 h after LPS injected (14 mg/kg). Next, 60-min ventilation of the rats was performed. Photomicrographs of lung tissues taken by electron microscope were used to explore the effects of MCTR1. Severe vacuolation of the lamellar bodies was observed in the LPS group in contrast to the mock group; but, MCTR1 administration alleviated the damage to the lamellar body (A). In the mock group, the air-blood barrier was normal but it was damaged in the LPS group as indicated by broken capillary walls and epithelial bridges in the lung tissues. LPS damaged the air-blood barrier and this effect was eliminated by MCTR1 treatment (B). ac, air capillary; bc, blood capillary; cn, cell nucleus; ecm, extracellular matrix of the capillary wall; en, endothelial cell; ep, epithelial bridge; er, erythrocyte; lb, lamellar body

| MCTR1 reversed the LPS-induced injury to the air-blood barrier
Further determination of the impact of MCTR1 on the ultrastructure of the lung tissues revealed that the samples obtained from rats treated with LPS showed high vacuolation of lamellar bodies compared to the control group. However, administration of MCTR1 ameliorated this pathological change (Figure 2A). Notably, although air-blood barrier was intact in the control group, it was damaged in the LPS group as demonstrated by broken capillary walls and epithelial bridges ( Figure 2B). Therefore, MCTR1 substantially reversed the LPS-induced injury to the air-blood barrier.

| MCTR1 up-regulates Na, K-ATPase and sodium channels in LPS-induced ALI in vivo
The LPS+MCTR1 group has a higher protein expression of the α, β, γ sodium channel subunits and the Na, K-ATPase α1, β1 subunits compared with the LPS group ( Figure 3A-E). Besides this, the activity of the Na, K-ATPase was significantly reduced in the LPS group compared with the control group. However, the activity of the Na, K-ATPase was significantly increased by administration of MCTR1 ( Figure 3F).

| MCTR1 facilitates clearance of alveolar fluid by activating the ALX/cAMP/PI3K/P-Nedd4-2 pathway in vivo
This study further assessed the ALX/cAMP/PI3K/P-Nedd4-2 pathway-dependent actions of MCTR1 in vivo by determining the concentrations of cAMP in the lung tissues. Experimental result revealed that the concentration of cAMP in the control group was higher than in the LPS group. cAMP concentrations were increased by MCTR1 treatment in LPS-treated lungs ( Figure 4A). Further to this, the expression levels of the Ser 473 -phosphorylated-Akt protein (P-Akt) in rat lung tissue homogenates were also assessed. The LPS group had significantly lower expression of the P-Akt protein compared with the control group. However, the P-Akt protein expression levels were considerably lower in the LPS group than the LPS+MCTR1 group ( Figure 4B). Nedd 4-2 is critical for the negative control of Na + transport. It is an E3 ubiquitin-protein ligase that primes the degradation of the protein channel. However, phosphorylation of Nedd4-2 by P-Akt nullifies its priming effect. 20 In this study, LPStreated rats showed lower expression of P-Nedd4-2 compared with the control group. However, its expression was significantly higher in the MCTR1 group compared to the LPS group ( Figure 4C).

| MCTR1 up-regulated the expression of the Na and Na, K-ATPase pump in primary rat ATII cells
Rat primary ATII epithelial cells were incubated with MCTR1 for 8 hours at 37°C with and without LPS. Confocal laser-scanning microscopy results revealed that MCTR1 increased the expression of sodium channel α ( Figure 7A) and Na, K-ATPase α1 ( Figure 7B).
The LPS+MCTR1 group had a higher protein expression of sodium channel α, β, γ subunits and Na, K-ATPase α1, β1 subunits than the LPS group ( Figure 7C-G). Consistent with the protein expression in lung tissue homogenates, the expression of P-Akt and P-Nedd4-2 proteins was higher in the LPS+MCTR1 group compared to the LPS group in primary ATII cells. (Figure 7H,I).

| D ISCUSS I ON
This study revealed that MCTR1 ameliorated lung injury and stimulated AFC of LPS-induced ALI in a rat model. This led to reduction of lung oedema. Besides this, the study showed that MCTR1 not only up-regulated the in vivo and in vitro expression of ENaC and Na, K-ATPase proteins, but also increased Na, K-ATPase activity in vivo.  ALI/ARDS exhibits increased alveolar-capillary permeability and impaired alveolar fluid clearance. 21 The currently available therapies are ineffective in modulating this inflammatory response. 3 It is known that the clearance of alveolar oedema is vital to ALI/ARDS patient survival. 22 In this study, we found that MCTR1 significantly protected the lamellar body structure and alveolar-capillary barrier from being damaged by LPS. Importantly, MCTR1 improved the AFC 8 hours after treatment with LPS. Previous studies reported that excessive levels of cytokines might cause alveolar injury and impaired AFC. 23,24 These results were consistent with those of this study.
MCTR1 reduced the lung inflammatory cytokines such as MPO, TNF-α, IL-1β and IL-10. These findings proved that lung inflammatory cytokines are associated with severity of ALI/ARDS. 25 These results suggest that MCTR1 promoted the resolution of inflammation in ALI/ARDS. ENaC is the primary driving force of AFC. It promotes removal of lung fluid from alveolar spaces and thus is beneficial for management of ALI/ARDS. 26 It is expressed in both ATI and ATII cells and is the key player in the Na + transport system. 27 The three homologous subunits of ENaC (Na + channel-α, β and γ) allow the flow of Na + ions through the high resistance epithelia thus enabling monitoring of the salt and water homeostasis in the body.
Mice genetically lacking the Na + channel-α, they are unable to clear the alveolar oedema fluid resulting to death within 40 hours of birth. 28 MCTR1 improved the expression of Na + channel-α, β and γ subunits in pulmonary tissue homogenates in LPS-induced ALI. It also improvised the three homologous subunits in LPSstimulated ATII cells. The ATII cells showed similar results on F I G U R E 3 MCTR1 increases sodium channel and Na, K-ATPase expression and Na, K-ATPase activity in lung injury induced by LPS in vivo. MCTR1 (200 ng/ rat) was administered to SD rats 8 h post-LPS induction (14 mg/kg); after which 60-min ventilation of the rats was carried out. Next, tissues of right lung were harvested and used to determine the protein expression levels of the α, β, and γ sodium channel subunits (A, B, C) and Na, K-ATPase α1 and β1 subunits (D, E) using Western blot assay. The activity of Na, K-ATPase in the homogenates of lung tissue (F). All data are presented as mean ± SD. n = 7, ***P < .001. MCTR1, Maresin Conjugates in Tissue Regeneration 1

F I G U R E 4
The effect of MCTR1 is partly mediated by ALX, cAMP and PI3K pathways in vivo. MCTR1 (200 ng/rat) was administered to SD rats 8 h post-LPS induction (14 mg/kg); after which 60-min ventilation of the rats was carried out. The right lung tissues were harvested and used to perform ELISA for determination of cAMP concentration (A), and Western blot to determine the protein expressions of phosphorylated-Akt (B) and Nedd4-2 (C). All data are presented as mean ± SD. n = 7, ***P < .001. MCTR1, Maresin Conjugates in Tissue Regeneration 1 confocal LASER-scanning microscopy. It has been established that the up-regulation of Na + channel aid pulmonary oedema fluid reabsorption while down-regulation of Na + slow down the process after thiourea-induced lung injury. 29 As such, Na + channel up-regulation by MCTR1 promotes AFC.
Na, K-ATPase works in concert with apical ENaC in regulating clearance of lung oedema. 26 Na + gain entry to the cell through either cationic or amiloride-sensitive Na + channels at the apical surface and expelled at the basolateral surface through Na, K-ATPase. Fluid and solute conveyance in alveoli are severely decreased with the inhibition or loss of Na, K-ATPase. 7 Increased Na + transport through up-regulation of Na, K-ATPase and Na + channels results in the ability to clear pulmonary oedema. 21,30 Even in the preclinical stage, impaired Na, K-ATPase functions serve as a hallmark of pulmonary injury. 21,31 Herein, expression of primary ATII cells and K-ATPase in lung tissues of the rats was enhanced by MCTR1 after being treated with LPS. MCTR1 promoted the clearance of alveolar fluid via the Na, K-ATPase and sodium channel essential mechanisms.
Generally, it is thought that the bioactions of specialized pro-resolving mediators (SPMs) are mediated via specific high-affinity G protein-coupled receptor (GPCR). 32 ALX/FPR2 is a well-known GPCR bound by SPMs such as LXA4, RvD1 and their stable analogues. 33 Herein, the clearance of alveolar fluid was enhanced by MCTR1. However, BOC-2 (ALX antagonist) abrogated its beneficial effects in vivo. This was an indication that response to MCTR1 was partly dependent on ALX.
Up-regulation of intracellular cAMP levels is a sign of GPCRs pathway activation. Previous studies revealed that cAMP-regulated AFC enhancement by activating Na + transport and Na, K-ATPase. It is thus a vital mechanism in lung oedema reabsorption. 29 Our previous studies F I G U R E 5 Inhibition of ALX, cAMP and PI3K pathways decreases the role of MCTR1 on AFC in vivo. BOC-2 (600 ng/ kg, ALX receptor inhibitor), KH7 (1 mg/kg, cAMP inhibitor), H89 (1 mg/kg, PKA inhibitor) or LY294002 (3 mg/kg, PI3K inhibitor) were administered to SD rats 30 min prior to MCTR1 administration (200 ng/rat). Subsequently, we inoculated 5% albumin solution comprising albumin tagged with Evans blue (5 mL/kg) into intratracheal of rats via the left lung tracheostomy to determine AFC (A). Lung tissues were then harvested and used to perform ELISA for determination of cAMP concentration (B), also for Western blot to determine expression levels of P-Akt (C) and P-Nedd4-2 (D). All data are presented as mean ± SD. n = 7, ***P < .001, **P < .01. *P < .05. MCTR1, Maresin Conjugates in Tissue Regeneration 1 F I G U R E 6 MCTR1 regulates the expression of Na, K-ATPase in primary ATII cells in a time and dose-dependent manner. Western blot assay was used to explore how LPS (1 μg/mL) affects the expression level of Na, K-ATPase in primary ATII cells. Cells were treated with various concentrations of MCTR1 for 8 h, including 1 nmol/L, 10 nmol/L, 100 mol/L and 200 nmol/L and then used to perform Western blot (A). ATII cells were treated with LPS (1 μg/ mL) for 1, 2, 4, 8 h at 100 nmol/L MCTR1 and the expression levels of Na, K-ATPase α1 subunit was determined (B). All data are presented as mean ± SD. n = 7, ***P < .001. MCTR1, Maresin Conjugates in Tissue Regeneration 1 F I G U R E 7 MCTR1 enhances the expression level of sodium channel and Na, K-ATPase in primary ATII cells treated with LPS in vitro. Briefly, primary ATII cells from SD rats were exposed to LPS (1 μg/mL) in the presence or absence of MCTR1 (100 nmol/L) for 8 h. Thereafter, cells were sonicated and incubated with specific antibodies. Cells were examined with Confocal laser-scanning microscopy to detect the expression levels of sodium channel α (A) and Na, K-ATPase α1 (B) (original magnification ×400). Western blot assay was also performed to evaluate the expression level of sodium channel α, β, and γ subunit (C-E), Na, K-ATPase α1 and β1 subunit (F, G), P-Akt (H) and P-Nedd4-2 (I) in the cell lysates. All data are presented as mean ± SD. n = 7, ***P < .001. MCTR1, Maresin Conjugates in Tissue Regeneration 1 It is widely accepted that both PKA and PI3K can be activated by cAMP. 35,36 PI3K signals are associated with the regulation of sodium channel trafficking and activity. 37 PI3K also regulates clearance of alveolar fluid by insulin through sodium channel mediation. 38 This study revealed that LY294002 inhibited the elevation of cAMP level through MCTR1 induction. Additionally, LY294002 restricted the increasing AFC in the MCTR1 treatment group. Akt is a critical effector kinase downstream of PI3K signalling pathway that influences many physiological processes. 39 Experimental results revealed that stimulation of LPS led to a reduction in P-Akt (ser 473 ). However, the reduction was attenuated by MCTR1 Akt (ser 473 ). Notably, LY294002, BOC-2 and KH7 suppressed these effects. We therefore concluded that MCTR1 enhances AFC through activation of P-Akt in an ALX/ cAMP/PI3K dependent pathway.
The mechanism that underlies insulin-induced Akt regulation of ENaC activity involves phosphorylation of Nedd4-2. 40 Lee IH et al,40 reported that Akt increased ENaC activity by inducing Nedd4-2 phosphorylation consequently increasing Na + absorption. Similarly, we found that MCTR1 increased the expression level of P-Nedd4-2 in the LPS group. However, the beneficial effects of MCTR1 were inhibited by BOC-2, LY294002 and KH7 inhibitors in vivo.

| CON CLUS ION
Results of this study revealed that administration of MCTR1 increases AFC in rat lungs injured by LPS. These findings also indicate that AFC is dependent on enhanced expression of Na + /K + -ATPase and ENaC via the ALX/cAMP/PI3K/P-Nedd4-2 signalling pathways following administration of MCTR1. Furthermore, the findings of this study reveal a new mechanism for reabsorption of pulmonary oedema. Evidently, MCTR1 may be a novel mediator of improving the "reabsorption targeted" treatments. These types of treatments are more precise in management of ALI/ARDS. F I G U R E 8 MCTR1 ameliorates acute lung injury induced by LPS in vitro and in vivo. In this study, we established the acute lung injury model induced by LPS in rats and primary ATII cells. MCTR1 activates ALX, cAMP, PI3K, P-Akt and P-Nedd4-2. In addition, it increases the expression of ENaC and Na, K-ATPase thereby improve the alveolar fluid clearance. ATII, Alveolar Type II; LPS, lipopolysaccharide; MCTR1, Maresin Conjugates in Tissue Regeneration 1