Protective role of resolvin D1, a pro-resolving lipid mediator, in nonsteroidal anti-inflammatory drug-induced small intestinal damage

Takuya Kuzumoto; Tetsuya Tanigawa; Akira Higashimori; Hiroyuki Kitamura; Yuji Nadatani; Koji Otani; Shusei Fukunaga; Shuhei Hosomi; Fumio Tanaka; Noriko Kamata; Yasuaki Nagami; Koichi Taira; Toshio Watanabe; Yasuhiro Fujiwara

doi:10.1371/journal.pone.0250862

. 2021 May 4;16(5):e0250862. doi: 10.1371/journal.pone.0250862

Protective role of resolvin D1, a pro-resolving lipid mediator, in nonsteroidal anti-inflammatory drug-induced small intestinal damage

Takuya Kuzumoto ¹, Tetsuya Tanigawa ^1,^2,^*, Akira Higashimori ¹, Hiroyuki Kitamura ¹, Yuji Nadatani ¹, Koji Otani ¹, Shusei Fukunaga ¹, Shuhei Hosomi ¹, Fumio Tanaka ¹, Noriko Kamata ¹, Yasuaki Nagami ¹, Koichi Taira ¹, Toshio Watanabe ¹, Yasuhiro Fujiwara ¹

Editor: Hiroyasu Nakano³

PMCID: PMC8096073 PMID: 33945545

Abstract

Resolvin D1, a specialized pro-resolving lipid mediator produced from docosahexaenoic acid by 15- and 5-lipoxygenase, exerts anti-inflammatory effects driving to the resolution of inflammation. The present study aimed to elucidate its role in small intestinal damage induced by nonsteroidal anti-inflammatory drug (NSAID). Indomethacin was administered orally to C57BL/6J male mice, which were sacrificed 24 h later to collect small intestine specimens. Before administration of indomethacin, mice were subjected to intraperitoneal treatment with resolvin D1 or oral administration of baicalein, a 15-lipoxygenase inhibitor. Small intestinal damage induced by indomethacin was attenuated by pretreatment with resolvin D1. Furthermore, resolvin D1 reduced the gene expression levels of interleukin-1β, tumor necrosis factor-α, and CXCL1/keratinocyte chemoattractant. Conversely, the inhibition of 15-lipoxygenase activity by baicalein increased the expression of genes coding for these inflammatory cytokines and chemokine, leading to exacerbated small intestinal damage, and reduced the concentration of resolvin D1 in the small intestinal tissue. Exogenous treatment with resolvin D1 negated the deleterious effect of baicalein. 15-lipoxygenase was mainly expressed in the epithelium and inflammatory cells of the small intestine, and its gene and protein expression was not affected by the administration of indomethacin. Inhibition of the resolvin D1 receptor, lipoxin A4 receptor /formyl peptide receptor 2, by its specific inhibitors Boc-1 and WRW4 aggravated indomethacin-induced small intestinal damage. Collectively, these results indicate that resolvin D1 produced by 15-lipoxygenase contributes to mucoprotection against NSAID-induced small intestinal damage through its anti-inflammatory effect.

Introduction

Inflammation can be resolved through elimination of pathogens and foreign harmful substances and reduction of inflammatory mediators and cytokines via non-specific metabolic process. However, recent accumulating evidence has revealed that specialized pro-resolving lipid mediators (SPMs) play key roles in regulating the resolution of acute inflammation [1, 2]. SPMs include lipoxins, which are derived from the ω-6 polyunsaturated fatty acid (PUFA) arachidonic acid, and resolvins and protectins, which are derived from the ω-3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Resolvins are categorized into D-series resolvins synthesized from DHA and E-series resolvins synthesized from EPA [3, 4]. In mice, DHA is converted to 17(S)-hydroxy-DHA via 12/15-lipoxygenase, an ortholog of the human 15-lipoxygenase, which is further converted to endogenous resolvin D by 5-lipoxygenase [5, 6].

Resolvin D1 (7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-DHA) is one of the most studied resolvins. Resolvin D1 can exert anti-inflammatory effects, such as inhibition of neutrophil recruitment and suppression of production of inflammatory cytokines, via two G protein-coupled receptors, lipoxin A4 receptor (ALX)/formyl peptide receptor 2 (FPR2), and G protein-coupled receptor 32 (GPR32) [7–9]. Moreover, resolvin D1 has been shown to ameliorate various acute inflammatory diseases in animal models, including lung injury, kidney injury, pancreatitis, hepatitis, and peritonitis [10–14].

Nonsteroidal anti-inflammatory drugs (NSAIDs) are one class of frequently prescribed drugs with antipyretic analgesic properties. However, these drugs induce gastrointestinal mucosal damage, mainly in the upper gastrointestinal tract, as an adverse effect. Furthermore, recent technological advances in small intestinal endoscopy have revealed that NSAIDs frequently induce mucosal damage also in the small intestine [15, 16]. In particular, we previously reported mild mucosal damage in 25% and severe mucosal damage in 27.8% of patients with rheumatoid arthritis who took NSAID orally for more than 3 months, as revealed by small intestinal capsule endoscopy [17]. NSAID-induced small intestinal damage occurs as a result of prostaglandin deficiency due to NSAID-dependent inhibition of cyclooxygenase activity and mitochondrial dysfunction [18]. However, the significance of other lipid mediators, including resolvin D1, in NSAID-induced small intestinal damage remains unknown. In this study, we investigated the role of resolvin D1 in NSAID-induced small intestinal damage using an experimental animal model.

Materials and methods

Animals

Male C57BL/6 mice, 8–10 weeks old, were purchased from CLEA Japan Inc. (Tokyo, Japan). All mice were kept in polycarbonate cages, maintained under a 12-h light/12-h dark cycle, and allowed free access to standard rodent diet (CE-2; CLEA Japan Inc.) and water. When performing invasive procedures, including tail vein injection and euthanasia, mice were anesthetized via inhalation of isoflurane. Euthanasia was performed by cervical dislocation under deep anesthesia with isoflurane. All procedures were approved by the Institutional Animal Care and Use Committee of the Osaka City University Graduate School of Medicine (Approval No. 18073).

Induction of NSAID-induced small intestinal damage

To induce small intestinal damage, 10 mg/kg body weight (BW) of indomethacin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) suspended in a 0.5% carboxymethylcellulose (CMC) solution was orally administered to non-fasted mice, which were sacrificed 0, 3, 6, 12, or 24 h after administration. In some experiments, 60 mg/kg BW of diclofenac with vehicle (0.5% CMC) was orally administered instead of indomethacin. The assessment of animal health and well-being was done 24 h before indomethacin administration and 3,6, and 24 h after indomethacin administration. At each time point, the critical problem about general status of the animal health was not observed in all of the animals. For evaluation of macroscopic intestinal damage, 1% Evans blue was injected intravenously 30 min before sacrifice to delineate the lesions. The entire small intestine was collected and cut open along the antimesenteric side. The areas of lesions delineated with Evans blue were measured macroscopically, summed for each small intestine, and expressed in terms of lesion index. The lesions were measured by two investigators (T.K. and T.T.) in a blinded manner. For histological evaluation, each of the small intestinal tissue samples that exhibited typical mucosal damage was fixed with 10% buffered formalin, and 4 μm-thick tissue sections were mounted on glass slides and subjected to hematoxylin and eosin (H&E) staining.

Histological evaluation of small intestinal damage

Tissue sections stained with H&E were viewed under high power using a white-light microscope. For each mouse, at least 10 random villi in the damaged areas were scored independently by two investigators (H.K. and T.T.) in a masked fashion. For evaluation, a modified histological scoring system was used [19]. Histological scores ranged from 0 to 13 and were divided into the following six categories: epithelium (0 = normal, 1 = flattened, 2 = loss of epithelial continuity, 3 = severe denudation), villus shape (0 = normal, 1 = short and rounded, 2 = extremely short and thick), villus tip (0 = normal, 1 = damaged, 2 = severely damaged), stroma (0 = normal, 1 = slightly retracted, 2 = severely retracted), inflammation (0 = no infiltration, 1 = mild infiltration, 2 = severe infiltration), and crypt status (0 = normal, 1 = mild crypt loss, 2 = severe crypt loss).

Reagents for analysis of the roles of resolvin D1 and 12/15-lipoxygenase

For the experiments to evaluate the effect of exogenous resolvin D1 on NSAID-induced small intestinal damage, 10 μg/kg BW of resolvin D1 (Cayman Chemical Company, Ann Arbor, MI, USA) in phosphate-buffered saline (PBS) was intraperitoneally administered 3 and 24 h before administration of indomethacin. To examine the effect of endogenous resolvin D1, 200 mg/kg BW of baicalein (Tokyo Chemical Industry, Tokyo, Japan), the 12/15-lipoxygenase inhibitor, in PBS was administered 3 and 24 h before administration of indomethacin. Boc-1 (Bachem AG, Bubendorf, Switzerland) and WRW4 (R&D Systems, Minneapolis, MN, USA) were used as inhibitors of the resolvin D1 receptor ALX/FPR2. Boc-1 (5 mg/kg BW) or WRW4 (1 mg/kg BW) was intraperitoneally administered 3 and 24 h before administration of indomethacin.

Measurement of resolvin D1 concentration in small intestinal tissue

Small intestinal tissue samples were weighed and placed in tubes containing cold 100% methanol. Samples were then homogenized on ice and centrifuged at 20,000 ×g for 20 min at 4°C. After the supernatant was evaporated with N₂ gas on ice, assay buffer was added to the residue. The concentrations of resolvin D1 were measured with an enzyme immunoassay (Resolvin D1 EIA kit; Cayman Chemical Company) according to the manufacturer’s protocol.

Quantitative real‑time reverse transcription polymerase chain reaction (RT-PCR) analysis

Total RNA was extracted from intestinal tissue using ISOGEN II (Nippon Gene Co., Ltd., Tokyo, Japan). Complementary DNA was synthesized from RNA using the High Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Quantitative real-time RT-PCR analyses were performed using an Applied Biosystems 7500 Fast Real-Time PCR system with TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific Inc.). The thermal cycling conditions consisted of 45 cycles of 95°C for 15 s and 60°C for 1 min. The mRNA levels of interleukin 1β (Il1b), tumor necrosis factor α (Tnfa), the mouse IL-8 homolog CXCL1/keratinocyte chemoattractant (Cxcl1), and 12/15-lipoxygenase (Alox15) in small intestinal tissue were quantified by quantitative real-time RT-PCR and normalized to the gene expression levels of glyceraldehyde-3-phosphate dehydrogenase (Gapdh; Thermo Fisher Scientific Inc.). The mRNA levels were expressed as ratios of the mean values for the control group of mice. The sequences of PCR primers and TaqMan probes for Il1b, Tnfa, Cxcl1, and Alox15 are shown in S1 Table. RT-PCR analysis was performed at the Research Support Platform of Osaka City University Graduate School of Medicine.

Western blotting analysis

Intestinal tissue was homogenized on ice in a solution containing TNE buffer (50 mM Tris-HCl pH 7.8, 1% NP-40, 150 mM NaCl, 10 mM ethylenediaminetetraacetic acid), PhosSTOP (one tablet per 10 mL of TNE buffer; Roche Applied Science, Indianapolis, IN, USA), and cOmplete™ Mini Protease Inhibitor Cocktail (one tablet per 10 mL of TNE buffer; Thermo Fisher Scientific Inc.). The supernatant was collected after centrifugation at 10,000 ×g for 10 min at 4°C. Protein concentrations were measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc.). Proteins were denatured with sample buffer solution (FUJIFILM Wako Pure Chemical Corporation) at 95°C for 5 min, subjected to 15% SDS-polyacrylamide gel electrophoresis, and transferred to an Immune-Blot polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA, USA). DynaMarker Protein MultiColor Stable (BioDynamics Laboratory, Tokyo, Japan) was used as a protein ladder marker. The membranes were blocked with blocking buffer (5% skim milk and Tris-buffered saline with 0.1% Tween 20 [TBS-T]) for 1 h at room temperature and then incubated with rabbit polyclonal antibodies against 12/15-lipoxygenase (#Bs-6505R; Bioss Antibodies, Woburn, MA, USA) and ALX/FPR2 (#NBP1-90180; Novus Biologicals, Littleton, CO, USA) overnight at 4°C. After washing with TBS-T, the membranes were incubated with anti-rabbit IgG-HRP (#NA934-1ML; GE Healthcare, Little Chalfont, UK) in blocking buffer for 1 h at room temperature. The antigen-antibody complexes were visualized by enhanced chemiluminescence with ECL Prime Western Blotting Detection Reagent (GE Healthcare) and detected using an ImageQuant LAS 4000 Mini instrument (GE Healthcare).

Immunohistochemistry

For immunohistochemical staining, tissue samples were fixed with 4% paraformaldehyde in 0.1 μM phosphate buffer (pH 7.4) and embedded in Tissue-Tek® O.C.T. Compound (Sakura Finetek Japan, Tokyo, Japan). Six-micrometer-thick cryostat sections were placed on silanized slides (Dako, Tokyo, Japan). Tissue samples were then incubated overnight at 4°C with primary antibodies, including rabbit polyclonal anti-12/15-lipoxygenase (diluted 1:800, #Bs-6505R, Bioss Antibodies), rabbit polyclonal anti-FPR2 (diluted 1:300, #NBP1-90180, Novus Biologicals), and rat monoclonal anti-F4/80, targeting a marker for mature macrophages (diluted 1:500, #MCA497GA, AbD Serotec, Oxford, UK). For 12/15-lipoxygenase detection, the sections were incubated for 30 min with the secondary antibody EnVision+ System-HRP Labeled Polymer Anti-Rabbit (Dako), treated for 1 min with the DAB substrate of a Liquid DAB+ Substrate Chromogen System (Dako), and counterstained with Mayer’s hematoxylin. For FPR2 and F4/80 detection, the sections were incubated with the corresponding secondary fluorescent dye-conjugated antibodies (Invitrogen, Carlsbad, CA, USA) for 2 h and then 4’,6-diamidino-2-phenylindole (DAPI) Fluoromount-G (Southern Biotech, Birmingham, AL, USA) was added for visualization of the nucleus. The sections were examined using a confocal microscope equipped with argon and argon–krypton laser sources (Keyence, Osaka, Japan).

Statistical analysis

Values are expressed as the mean ± standard error of the mean. One-way analysis of variance was used to test for significant differences among the means of the treatment groups, and results were analyzed using a protected Fisher’s least significant difference test. Differences with p values < 0.05 were considered statistically significant. All statistical analyses were performed using EZR v.1.40 (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a modified graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria) designed to add statistical functions frequently used in biostatistics [20].

Results

Effects of exogenous resolvin D1 on NSAIDs-induced small intestinal damage

Pretreatment with resolvin D1 attenuated small intestinal damage induced by indomethacin to 58% in terms of lesion index and 86% in terms of histological score (Fig 1A and 1B). Indomethacin increased the gene expression levels of Il1b, Tnfa, and Cxcl1, while resolvin D1 reduced the gene expression levels of Il1b, Tnfa, and Cxcl1 to 57%, 72%, and 44%, respectively, in comparison with those of the indomethacin-administered vehicle-treated group (Fig 1C). Similar to indomethacin-dependent damage, diclofenac-induced small intestinal damage was also attenuated by pretreatment with resolvin D1. Indeed, resolvin D1 reduced the lesion index to 52% and the histological score to 84% (Fig 1D and 1E). Moreover, the gene expression levels of Il1b, Tnfa, and Cxcl1 were reduced to 35%, 61%, and 24%, respectively, in comparison with those of the diclofenac-administered vehicle-treated group (Fig 1F).

Fig 1 — (a) Lesion index and histological score of the small intestines 24 h after administration of indomethacin (10 mg/kg BW) in mice pretreated with vehicle or resolvin D1 (RvD1: 10 μg/kg BW). N = 7–8. (b) Representative macroscopic and microscopic images of small intestine 24 h after administration of indomethacin in mice pre-administered with RvD1 (10 μg/kg BW) or vehicle. Bars in histological images: 500 μm. (c) The gene expression levels of interleukin-1β (*Il1b*), CXCL1/keratinocyte chemoattractant (*Cxcl1*), and tumor necrosis factor-α (*Tnfa*) 6 h after administration of indomethacin in mice pretreated with vehicle or RvD1 (10 μg/kg BW). The mRNA levels were determined by quantitative reverse transcription polymerase chain reaction and expressed as ratios relative to the mean value for small intestinal tissue from non-treated control mice. N = 6–8. (d) Lesion index and histological score of small intestines 24 h after administration of diclofenac (60 mg/kg BW) in mice pretreated with vehicle or RvD1 (10 μg/kg BW). (e) Representative macroscopic and microscopic images of small intestine 24 h after administration of diclofenac in mice pre-administered with RvD1 (10 μg/kg BW) or vehicle. (f) The gene expression levels of *Il1b*, *Cxcl1*, and *Tnfa* 6 h after administration of diclofenac with vehicle or RvD1 (10 μg/kg BW). N = 6–8. All data are from a single experiment, representative of at least two independent experiments and expressed as mean ± standard error of mean. * p < 0.05, ** p < 0.01.

Dynamics of 12/15-lipoxygenase expression in small intestinal tissue

Immunoreactivity against 12/15-lipoxygenase was observed in intestinal epithelial cells as well as in inflammatory cells of the lamina propria (Fig 2A). The mRNA levels of Il1b, Tnfa, and Cxcl1 in the small intestine peaked 6 to 12 h after indomethacin administration. In contrast, the mRNA levels of 12/15-lipoxygenase (Alox15) in the small intestine did not change over time (Fig 2B). Similar to the mRNA profile, the protein concentration of 12/15-lipoxygenase in the small intestine was almost constant after the administration of indomethacin (Fig 2C and 2D). Consistently, there was no difference in concentration of resolvin D1 in the small intestine between the vehicle-administered control group and indomethacin-administered group (Fig 2E).

Fig 2 — (a) Representative image of immunohistochemical staining for localization analysis of 12/15-lipoxygenase protein in the small intestinal tissue. Bars in histological images: 200 μm. The figure on the right is a magnified view showing inflammatory cells with arrows. Bars in histological images: 50 μm. (b) Time course of gene expression levels of interleukin-1β (*Il1b*), CXCL1/keratinocyte chemoattractant (*Cxcl1*), and tumor necrosis factor-α (*Tnfa*) and 12/15-lipoxygenase (*Alox15*) after administration of indomethacin. N = 5–8. *p < 0.05, **p < 0.01 vs. untreated control group (0 h group). (c) Representative images of western blotting analysis for time course of 12/15-lipoxygenase protein expression after administration of indomethacin. (d) Relative amount of normalized 12/15-lipoxygenase protein expressed as a percentage of untreated control (0 h group). N = 4. (e) Concentration of resolvin D1 in the small intestinal tissue of untreated control mice (0 h group) and mice administered with indomethacin (24 h group). Small intestinal tissue samples were obtained from mice 24 h after indomethacin administration. N = 6. All data are from a single experiment, representative of at least two independent experiments and expressed as mean ± standard error of mean. * p < 0.05, ** p < 0.01.

Inhibition of 12/15-lipoxygenase by baicalein aggravates indomethacin-induced small intestinal damage

Pre-administration of baicalein, an inhibitor of the activity of 12/15-lipoxygenase, aggravated indomethacin-induced small intestinal damage. In particular, the lesion index increased by 1.6-fold compared with that of vehicle-treated mice (Fig 3A and 3B). Baicalein also promoted the expression of Il1b, Tnfa, and Cxcl1 (Fig 3C). In addition, exogenous administration of resolvin D1 prevented the aggravation of intestinal damage by baicalein (Fig 3D). To corroborate the above experimental results, we confirmed that the administration of baicalein to the control group reduced the concentration of resolvin D1 in the small intestinal tissue in a dose-dependent manner (Fig 3E).

Fig 3 — (a) The effect of baicalein, a 12/15-lipoxygenase inhibitor, on indomethacin-induced small intestinal damage. Lesion index and histological score of the group pre-administered with baicalein and the group pre-administered baicalein (200 mg/kg BW) and resolvin D1 (RvD1: 10 μg/kg BW). N = 7–8. Baicalein and resolvin D1 were administered 3 and 24 h before administration of indomethacin. (b) Representative macroscopic and microscopic images of small intestine 24 h after administration of indomethacin in mice pre-administered with baicalein (200 mg/kg BW) and RvD1 (10 μg/kg BW). Bars in histological images: 500 μm. (c) The gene expression levels of interleukin-1β (*Il1b*), CXCL1/keratinocyte chemoattractant (*Cxcl1*), and tumor necrosis factor-α (*Tnfa*) 6 h after administration of indomethacin in mice pre-administered with baicalein (0, 200 mg/kg BW). The mRNA levels were determined by quantitative reverse transcription polymerase chain reaction and expressed as ratios relative to the mean value for small intestinal tissue from untreated control mice. N = 6–8. (d) The gene expression levels of *Il1b*, *Cxcl1*, and *Tnfa* 6 h after administration of indomethacin (10 mg/kg BW) in mice pre-administered with baicalein (200 mg/kg BW) concomitant with RvD1 (0, 10 μg/kg BW). Baicalein and resolvin D1 were administered 3 and 24 h before administration of indomethacin. N = 5–8. (e) Concentration of resolvin D1 in the small intestinal tissue of the group pre-administered with baicalein (100 and 200 mg/kg BW) and the control group. Baicalein was administered 24 and 48 h before sacrifice. N = 6. All data are from a single experiment, representative of at least two independent experiments and expressed as mean ± standard error of mean. * p < 0.05, ** p < 0.01.

Dynamics of ALX/FPR2 expression in small intestinal tissue

Similar to the expression of inflammatory cytokines, ALX/FPR2 protein expression peaked 6 to 12 h after administration of indomethacin (Fig 4A and 4B). Immunoreactivity against ALX/FPR2 was observed in intestinal epithelial cells and inflammatory cells of the lamina propria (Fig 4C). In particular, double staining for ALX/FPR2 with F4/80 showed that ALX/FPR2 was expressed in macrophages in the lamina propria (Fig 4D).

Pharmacological inhibition of the resolvin D1 receptor ALX/FPR2 aggravates indomethacin-induced small intestinal damage

We further investigated the effects of Boc-1 and WRW4, inhibitors of the resolvin D1 receptor ALX/FPR2, on indomethacin-induced small intestinal damage. Exacerbation of mucosal lesions was observed after pretreatment with each of the inhibitors; specifically, administration of Boc-1 and WRW4 induced a 1.8-fold and 1.5-fold increase, respectively, in the lesion index (Fig 5A and 5B).

Fig 5 — (a) Lesion index and histological score of small intestines 24 h after administration of indomethacin (10 mg/kg BW) in mice pre-administered with vehicle or ALX/FPR2 inhibitor Boc-1 (5 mg/kg BW). N = 8. (b) Lesion index and histological score of small intestines 24 h after administration of indomethacin (10 mg/kg BW) pre-administered with vehicle or ALX/FPR2 inhibitor WRW4 (1 mg/kg BW). N = 6–8. All data are from a single experiment, representative of at least two independent experiments and expressed as mean ± standard error of mean. * p < 0.05, ** p < 0.01.

Discussion

In the present study, we demonstrated that resolvin D1 exerts protective properties against NSAID-induced small intestinal damage. Exogenous supplementation of resolvin D1 protected the small intestine from damage induced by NSAIDs, while inhibition of 12/15-lipoxygenase, the enzyme responsible for the production of resolvin D1, exacerbated NSAID-induced small intestinal damage. Moreover, inhibition of ALX/FPR2, the receptor for resolvin D1, resulted in further deterioration of NSAID-induced small intestinal damage.

Accumulating evidence indicates that resolvin D1 is a key player in the resolution of inflammation [1]. One of the mechanisms through which resolvin D1 promotes resolution of inflammation is linked to its anti-inflammatory properties. In fact, resolvin D1 inhibits lipopolysaccharide (LPS)-induced transcriptional activation of TNF-α by modulating the nuclear factor κB (NF-κB) signaling pathway in human macrophages [21]. In addition, resolvin D1 downregulates the expression of IL-1β via inhibition of NOD-like receptor family proteins such as pyrin domain-1 containing 3 (NLRP3), thereby inhibiting the inflammasome pathway in rats with streptozotocin-induced diabetic retinopathy or hyperhomocysteinemia-induced podocyte injury [22, 23]. With regard to the pathophysiology of NSAID-induced small intestinal damage, we previously revealed that LPS and high-mobility group box 1 (HMGB1) liberated from damaged epithelial cells induce activation of Toll-like receptor (TLR) 4. Subsequently, TLR4 stimulates NF-κB to produce TNF-α through an MyD88-dependent pathway, resulting in NSAID-induced small intestinal damage [24, 25]. In addition, we have previously demonstrated that the NLRP3 inflammasome activates IL-1β via the TLR4 signaling pathway, which is a critical step for induction and promotion of NSAID-induced small intestinal damage [26]. Altogether, inhibition of the expression of TNF-α and IL-1β via inhibition of the TLR4- NF-κB and NLRP3 inflammasome signaling pathways could be proposed as the possible mechanism through which resolvin D1 ameliorates NSAID-induced small intestinal damage.

In the present study, we demonstrated that 12/15-lipoxygenase, a major synthetic enzyme for the production of resolvin D1 in mice, was mainly present in the small intestinal epithelium and inflammatory cells, consistent with a previous report [27]. The 12/15-lipoxygenase gene was constitutively expressed regardless of the time after indomethacin administration. We further showed that administration of baicalein, an inhibitor of the activity of 12/15-lipoxygenase, exacerbated NSAID-induced small intestinal damage and was accompanied by a reduced accumulation of endogenous resolvin D1 in small intestinal tissue; conversely, exogenous supplementation of resolvin D1 negated the deleterious effect of baicalein on NSAID-induced small intestinal damage. These results indicate that 12/15-lipoxygenase is constitutively expressed in the small intestinal mucosa to contribute to the regulation of inflammation via the production of resolvin D1. Consistent with the present study, the significance of 12/15-lipoxygenase in physiological homeostasis has been reported for a variety of pathologies in other organs. For example, 12/15-lipoxygenase contributes to skin homeostasis by regulating the infiltration of proinflammatory macrophages and proinflammatory signaling in dermal adipose tissue and in the dorsal skin [28]. Furthermore, deletion of 12/15-lipoxygenase accelerates the development of aging-associated and instability-induced osteoarthritis [29].

The contribution of 12/15-lipoxygenase-derived SPMs other than resolvin D1 to mucosal defense and repair against intestinal inflammation has also been reported. For instance, resolvin E1 exerts anti-inflammatory effect against experimental dextran sodium sulfate (DSS)-induced colitis and 2,4,6-trinitrobenzene sulfonic acid-induced colitis, and promoted intestinal wound healing; in addition, lipoxin A4 also exerts anti-inflammatory effect in a DSS-induced inflammatory bowel disease model and a ischemic/reperfusion injury model, counteracting LPS-induced acute intestinal inflammation [30–35]. With regard to NSAID-induced small intestinal damage, the present study suggests that the effect of 12/15-lipoxygenase on indomethacin-induced small intestinal damage is, at least in part, dependent on resolvin D1 because its exacerbation upon inhibition of 12/15-lipoxygenase was fully neutralized by exogenous supplementation of resolvin D1.

Recent studies have revealed that resolvin D1 exerts its anti-inflammatory effect via two G protein-coupled receptors, ALX/FPR2 and GPR32 [9]. Although the ortholog of human ALX/FPR2 is present in mice and rats, the murine ortholog of human GPR32 is absent [36]. ALX/FPR2 is expressed on various cells, including macrophages and intestinal epithelial cells, and recognizes resolvin D1, thereby promoting resolution of inflammation [9]. In the present study, we confirmed the expression of ALX/FPR2 in these cell types and found that the expression of this receptor peaked 6 to 12 h after indomethacin administration, similar to the expression of inflammatory cytokines. Such an increase in ALX/FPR2 expression was considered to be mainly because of indomethacin-promoted infiltration of inflammatory cells into the small intestinal tissue. Furthermore, indomethacin-induced small intestinal damage was aggravated by pre-administration of two different ALX/FPR2 inhibitors, Boc-1 and WRW4, indicating that these inhibitors blocked the anti-inflammatory effect of endogenous resolvin D1. Several reports have revealed the contribution of ALX/FPR2 in SPM-mediated resolution of inflammation in vitro and in animal models. For example, Bento and colleagues reported that the ALX/FPR2 inhibitor Boc-1 abolishes the anti-inflammatory effects of aspirin-triggered resolvin D1 on LPS-stimulated macrophages derived from murine bone marrow, thereby exacerbating murine DSS-induced colitis [37]. In addition, Gobbetti and colleagues reported that the blockade of ALX/FPR2 annuls its anti-inflammatory effects in an intestinal ischemia/reperfusion-induced injury mouse model [34]. Moreover, Alx/Fpr2-deficient mice show delayed mucosal restoration after DSS-induced colitis [38]. Consistently, Birkl and colleagues demonstrated that ALX/FPR2 regulates monocyte recruitment to promote intestinal mucosal wound repair [39]. In agreement with these studies, we also demonstrated that inhibition of ALX/FPR2 abolished the inhibitory effect of endogenous resolvin D1 against indomethacin-induced small intestinal damage, leading to the exacerbation of such damage.

In conclusion, the present study demonstrated that 12/15-lipoxygenase-derived resolvin D1 contributes to mucoprotection against NSAID-induced small intestinal damage by exerting anti-inflammatory effects via activation of the ALX/FPR2 receptor.

Supporting information

S1 Table. The PCR primers and TaqMan probes used in the present study.

(DOCX)

Click here for additional data file.^{(16.1KB, docx)}

S1 Fig. The original uncropped and unadjusted images of the western blotting analysis for 12/15 lipoxygenase.

(TIF)

Click here for additional data file.^{(2.5MB, tif)}

S2 Fig. The original uncropped and unadjusted images of the western blotting analysis for ALX FRP2.

(TIF)

Click here for additional data file.^{(1.7MB, tif)}

Acknowledgments

We thank Emi Yoshioka for providing technical assistance.

Data Availability

All relevant data are available in figshare (https://figshare.com/), DOI: 10.6084/m9.figshare.13514146.

Funding Statement

The authors received no specific funding for this work.

References

1.Serhan CN. Pro-resolving lipid mediators are leads for resolution physiology. Nature. 2014;510(7503):92–101. 10.1038/nature13479 [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Basil MC, Levy BD. Specialized pro-resolving mediators: endogenous regulators of infection and inflammation. Nat Rev Immunol. 2016;16(1):51–67. 10.1038/nri.2015.4 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Sansbury BE, Spite M. Resolution of Acute Inflammation and the Role of Resolvins in Immunity, Thrombosis, and Vascular Biology. Circ Res. 2016;119(1):113–130. 10.1161/CIRCRESAHA.116.307308 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Kohli P, Levy BD. Resolvins and protectins: mediating solutions to inflammation. Br J Pharmacol. 2009;158(4):960–971. 10.1111/j.1476-5381.2009.00290.x [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Hong S, Gronert K, Devchand PR, Moussignac RL, Serhan CN. Novel docosatrienes and 17S-resolvins generated from docosahexaenoic acid in murine brain, human blood, and glial cells. Autacoids in anti-inflammation. J Biol Chem. 2003;278(17):14677–14687. 10.1074/jbc.M300218200 [DOI] [PubMed] [Google Scholar]
6.Serhan CN, Hong S, Gronert K, Colgan SP, Devchand PR, Mirick G, et al. Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals. J Exp Med. 2002;196(8):1025–1037. 10.1084/jem.20020760 [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Serhan CN, Chiang N, Van Dyke TE. Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators. Nat Rev Immunol. 2008;8(5):349–361. 10.1038/nri2294 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Schif-Zuck S, Gross N, Assi S, Rostoker R, Serhan CN, Ariel A. Saturated-efferocytosis generates pro-resolving CD11b low macrophages: modulation by resolvins and glucocorticoids. Eur J Immunol. 2011;41(2):366–379. 10.1002/eji.201040801 [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Krishnamoorthy S, Recchiuti A, Chiang N, Yacoubian S, Lee CH, Yang R, et al. Resolvin D1 binds human phagocytes with evidence for proresolving receptors. Proc Natl Acad Sci U S A. 2010;107(4):1660–1665. 10.1073/pnas.0907342107 [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Hu X, Shen H, Wang Y, Zhang L, Zhao M. Aspirin-triggered resolvin D1 alleviates paraquat-induced acute lung injury in mice. Life Sci. 2019;218:38–46. 10.1016/j.lfs.2018.12.028 [DOI] [PubMed] [Google Scholar]
11.Zhao YL, Zhang L, Yang YY, Tang Y, Zhou JJ, Feng YY, et al. Resolvin D1 Protects Lipopolysaccharide-induced Acute Kidney Injury by Down-regulating Nuclear Factor-kappa B Signal and Inhibiting Apoptosis. Chin Med J (Engl). 2016;129(9):1100–1107. 10.4103/0366-6999.180517 [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Liu Y, Zhou D, Long FW, Chen KL, Yang HW, Lv ZY, et al. Resolvin D1 protects against inflammation in experimental acute pancreatitis and associated lung injury. Am J Physiol Gastrointest Liver Physiol. 2016;310(5):G303–G309. 10.1152/ajpgi.00355.2014 [DOI] [PubMed] [Google Scholar]
13.Kuang H, Hua X, Zhou J, Yang R. Resolvin D1 and E1 alleviate the progress of hepatitis toward liver cancer in long-term concanavalin A-induced mice through inhibition of NF-κB activity. Oncol Rep. 2016;35(1):307–317. 10.3892/or.2015.4389 [DOI] [PubMed] [Google Scholar]
14.Sun YP, Oh SF, Uddin J, Yang R, Gotlinger K, Campbell E, et al. Resolvin D1 and its aspirin-triggered 17R epimer. Stereochemical assignments, anti-inflammatory properties, and enzymatic inactivation. J Biol Chem. 2007;282(13):9323–9334. 10.1074/jbc.M609212200 [DOI] [PubMed] [Google Scholar]
15.Maiden L, Thjodleifsson B, Theodors A, Gonzalez J, Bjarnason I. A quantitative analysis of NSAID-induced small bowel pathology by capsule enteroscopy. Gastroenterology. 2005;128(5):1172–1178. 10.1053/j.gastro.2005.03.020 [DOI] [PubMed] [Google Scholar]
16.Fujimori S, Seo T, Gudis K, Ehara A, Kobayashi T, Mitsui K, et al. Prevention of nonsteroidal anti-inflammatory drug-induced small-intestinal injury by prostaglandin: a pilot randomized controlled trial evaluated by capsule endoscopy. Gastrointest Endosc. 2009;69(7):1339–1346. 10.1016/j.gie.2008.08.017 [DOI] [PubMed] [Google Scholar]
17.Watanabe T, Tanigawa T, Nadatani Y, Nagami Y, Sugimori S, Okazaki H, et al. Risk factors for severe nonsteroidal anti-inflammatory drug-induced small intestinal damage. Dig Liver Dis. 2013;45(5):390–395. 10.1016/j.dld.2012.12.005 [DOI] [PubMed] [Google Scholar]
18.Arakawa T, Watanabe T, Tanigawa T, Tominaga K, Otani K, Nadatani Y, et al. Small intestinal injury caused by NSAIDs/aspirin: finding new from old. Curr Med Chem. 2012;19(1):77–81. 10.2174/092986712803414105 [DOI] [PubMed] [Google Scholar]
19.Tanigawa T, Watanabe T, Otani K, Nadatani Y, Ohkawa F, Sogawa M, et al. Rebamipide inhibits indomethacin-induced small intestinal injury: possible involvement of intestinal microbiota modulation by upregulation of α-defensin 5. Eur J Pharmacol. 2013;704(1–3):64–69. 10.1016/j.ejphar.2013.02.010 [DOI] [PubMed] [Google Scholar]
20.Kanda Y. Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics. Bone Marrow Transpl. 2013;48:452–8. 10.1038/bmt.2012.244 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Lee HN, Kundu JK, Cha YN, Surh YJ. Resolvin D1 stimulates efferocytosis through p50/p50-mediated suppression of tumor necrosis factor-α expression. J Cell Sci. 2013;126(Pt 17):4037–4047. 10.1242/jcs.131003 [DOI] [PubMed] [Google Scholar]
22.Yin Y, Chen F, Wang W, Wang H, Zhang X. Resolvin D1 inhibits inflammatory response in STZ-induced diabetic retinopathy rats: Possible involvement of NLRP3 inflammasome and NF-κB signaling pathway. Mol Vis. 2017;23:242–250. [PMC free article] [PubMed] [Google Scholar]
23.Li G, Chen Z, Bhat OM, Zhang Q, Abais-Battad JM, Conley SM, et al. NLRP3 inflammasome as a novel target for docosahexaenoic acid metabolites to abrogate glomerular injury. J Lipid Res. 2017;58(6):1080–1090. 10.1194/jlr.M072587 [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Watanabe T, Kobata A, Tanigawa T, Nadatani Y, Yamagami H, Watanabe K, et al. Activation of the MyD88 signaling pathway inhibits ischemia-reperfusion injury in the small intestine. Am J Physiol Gastrointest Liver Physiol. 2012;303(3):G324–G334. 10.1152/ajpgi.00075.2012 [DOI] [PubMed] [Google Scholar]
25.Nadatani Y, Watanabe T, Tanigawa T, Machida H, Okazaki H, Yamagami H, et al. High mobility group box 1 promotes small intestinal damage induced by nonsteroidal anti-inflammatory drugs through Toll-like receptor 4. Am J Pathol. 2012;181(1):98–110. 10.1016/j.ajpath.2012.03.039 [DOI] [PubMed] [Google Scholar]
26.Higashimori A, Watanabe T, Nadatani Y, Takeda S, Otani K, Tanigawa T, et al. Mechanisms of NLRP3 inflammasome activation and its role in NSAID-induced enteropathy. Mucosal Immunol. 2016;9(3):659–668. 10.1038/mi.2015.89 [DOI] [PubMed] [Google Scholar]
27.Ferrer R, Moreno JJ. Role of eicosanoids on intestinal epithelial homeostasis. Biochem Pharmacol. 2010;80(4):431–438. 10.1016/j.bcp.2010.04.033 [DOI] [PubMed] [Google Scholar]
28.Kim SN, Akindehin S, Kwon HJ, Son YH, Saha A, Jung YS, et al. Anti-inflammatory role of 15-lipoxygenase contributes to the maintenance of skin integrity in mice. Sci Rep. 2018. June 11;8(1):8856. 10.1038/s41598-018-27221-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Habouri L, El Mansouri FE, Ouhaddi Y, Lussier B, Pelletier JP, Martel-Pelletier J, et al. Deletion of 12/15-lipoxygenase accelerates the development of aging-associated and instability-induced osteoarthritis. Osteoarthritis Cartilage. 2017. October;25(10):1719–1728. 10.1016/j.joca.2017.07.001 [DOI] [PubMed] [Google Scholar]
30.Ishida T, Yoshida M, Arita M, Nishitani Y, Nishiumi S, Masuda A, et al. Resolvin E1, an endogenous lipid mediator derived from eicosapentaenoic acid, prevents dextran sulfate sodium-induced colitis. Inflamm Bowel Dis. 2010. January;16(1):87–95. 10.1002/ibd.21029 [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Arita M, Yoshida M, Hong S, Tjonahen E, Glickman JN, Petasis NA, et al. Resolvin E1, an endogenous lipid mediator derived from omega-3 eicosapentaenoic acid, protects against 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Proc Natl Acad Sci U S A. 2005. May 24;102(21):7671–6. 10.1073/pnas.0409271102 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Quiros M, Feier D, Birkl D, Agarwal R, Zhou DW, García AJ, et al. Resolvin E1 is a pro-repair molecule that promotes intestinal epithelial wound healing. Proc Natl Acad Sci U S A. 2020. April 28;117(17):9477–9482. 10.1073/pnas.1921335117 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Gewirtz AT, Collier-Hyams LS, Young AN, Kucharzik T, Guilford WJ, Parkinson JF, et al. Lipoxin a4 analogs attenuate induction of intestinal epithelial proinflammatory gene expression and reduce the severity of dextran sodium sulfate-induced colitis. J Immunol. 2002. May 15;168(10):5260–7. 10.4049/jimmunol.168.10.5260 [DOI] [PubMed] [Google Scholar]
34.Gobbetti T, Ducheix S, le Faouder P, Perez T, Riols F, Boue J, et al. Protective effects of n-6 fatty acids-enriched diet on intestinal ischaemia/reperfusion injury involve lipoxin A4 and its receptor. Br J Pharmacol. 2015. February;172(3):910–23. 10.1111/bph.12957 [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Kure I, Nishiumi S, Nishitani Y, Tanoue T, Ishida T, Mizuno M, et al. Lipoxin A(4) reduces lipopolysaccharide-induced inflammation in macrophages and intestinal epithelial cells through inhibition of nuclear factor-kappaB activation. J Pharmacol Exp Ther. 2010. February;332(2):541–8. 10.1124/jpet.109.159046 [DOI] [PubMed] [Google Scholar]
36.Marchese A, Nguyen T, Malik P, Xu S, Cheng R, Xie Z, et al. Cloning genes encoding receptors related to chemoattractant receptors. Genomics. 1998;50(2):281–286. 10.1006/geno.1998.5297 [DOI] [PubMed] [Google Scholar]
37.Bento AF, Claudino RF, Dutra RC, Marcon R, Calixto JB. Omega-3 fatty acid-derived mediators 17(R)-hydroxy docosahexaenoic acid, aspirin-triggered resolvin D1 and resolvin D2 prevent experimental colitis in mice. J Immunol. 2011;187(4):1957–1969. 10.4049/jimmunol.1101305 [DOI] [PubMed] [Google Scholar]
38.Chen K, Liu M, Liu Y, Yoshimura T, Shen W, Le Y, et al. Formylpeptide receptor-2 contributes to colonic epithelial homeostasis, inflammation, and tumorigenesis. J Clin Invest. 2013;123(4):1694–1704. 10.1172/JCI65569 [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Birkl D, O’Leary MN, Quiros M, Azcutia V, Schaller M, Reed M, et al. Formyl peptide receptor 2 regulates monocyte recruitment to promote intestinal mucosal wound repair. FASEB J. 2019. December;33(12):13632–13643. 10.1096/fj.201901163R [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0250862.r001

Decision Letter 0

Hiroyasu Nakano

26 Jan 2021

PONE-D-21-00075

Protective role of resolvin D1, a pro-resolving lipid mediator, in nonsteroidal anti-inflammatory drug-induced small intestinal damage

PLOS ONE

Dear Dr. Tanigawa

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by March 19, 2021. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Hiroyasu Nakano, M.D., Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2.At this time, we request that you please report additional details in your Methods section regarding animal care, as per our editorial guidelines:

(1) Please include the method of euthanasia

(2) Please describe the operative care received by the animals, including the frequency of monitoring and the criteria used to assess animal health and well-being.

Thank you for your attention to these requests.

3. Please provide the product number and any lot numbers of the antibodies purchased for your study.

4.PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

6.Thank you for submitting the above manuscript to PLOS ONE. During our internal evaluation of the manuscript, we found significant text overlap between your submission and the following previously published works, some of which you are an author.

- https://www.gastrojournal.org/article/S0016-5085(20)32578-6/pdf?referrer=https%3A%2F%2Fapi.ithenticate.com%2F

- https://www.intechopen.com/books/municipal-solid-waste-management/life-cycle-inventory-lci-modeling-of-municipal-solid-waste-msw-management-systems-in-kosodrza-commun

https://www.nature.com/articles/mi201589?code=4eb62728-cfde-4eba-97dd-898b4180c86b&error=cookies_not_supported

We would like to make you aware that copying extracts from previous publications, especially outside the methods section, word-for-word is unacceptable. In addition, the reproduction of text from published reports has implications for the copyright that may apply to the publications.

Please revise the manuscript to rephrase the duplicated text, cite your sources, and provide details as to how the current manuscript advances on previous work. Please note that further consideration is dependent on the submission of a manuscript that addresses these concerns about the overlap in text with published work.

We will carefully review your manuscript upon resubmission, so please ensure that your revision is thorough.

Additional Editor Comments:

Although both reviewers feel the study is potentially interesting, one of the reviewers claims a flaw of the authors’ experimental conditions. Specifically, the reviewer strongly recommends the authors to collaborate with lipid biochemists to correctly measure lipid mediators including resolving D1 in their samples.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Kuzumoto et al showed the resolving D1, an anti-inflammatory lipid mediator derived from DHA ameliorated the NSAIDs-induced intestinal damage by inhibiting the intestinal inflammation. An inhibitor for 12/15-lipoxygenase, a critical enzyme for resolving D1 synthesis, increased the expression of inflammatory cytokines and exacerbated the intestinal damage, which was again ameliorated by exogenous resolving D1. Most of the experiments and their results are nicely organized and discussed, except for the quantification of resolving D1 by Elisa.

Major comments

In figure 3a-d, they showed the marked increase in the lesion index, histological score and the expression of inflammatory cytokines by baicalein, a 12/15 lipoxygenase inhibitor, however, they showed the minimal reduction of resolving D1 by baicalein treatment in Figure 3e. This small change in resolving D1 can not explain the huge difference in the clinical scores and inflammation. This is possibly due to the inadequate measurement of resolving D1. They extracted fatty acid fraction from intestinal tissues by methanol and quantified resolving D1 using an Elisa kit, which does not give the reliable values. Currently, lipid mediators including SPM2 should be measured by mass-spectrometry as proposed by Bo Burla et al. in J. Lipid Res. 2018;59:2001-2017. The reviewer strongly recommends the authors to collaborate with lipid biochemists to correctly measure lipid mediators including resolving D1 in their samples.

Reviewer #2: In the manuscript entitled "Protective role of resolvin D1, a pro-resolving lipid mediator, in nonsteroidal anti-inflammatory drug-induced small intestinal damage" by Kuzumoto et al, the authors demonstrate that administration of resolvin D1 ameliorates NSAIDs-induced damages of the small intestine in mice (Fig. 1). The expression of the resolvin D1-producing enzyme (12/15-lipoxygenase) is not affected after administration of indomethacin (Fig. 2). The indomethacin-induced intestinal damages are aggravated in mice that are pre-treated with the inhibitor of 12/15-lipoxygenase, and the aggravation is abrogated by exogenous resolvin D1 (Fig. 3). The indomethacin-induced intestinal damages are also aggravated by the inhibitors of a resolvin D1 receptor (ALX1/FPR2), which is mainly expressed in macrophages (Fig. 4 and Fig. 5).

I feel that the present study provides important findings regarding the role of the endogenous lipid mediator resolvin D1 in regulation of inflammation, and thus seems to be suitable for publication if the following minor issues are adequately addressed.

Specific comments:

1) The authors should measure the concentration of resolvin D1 in the small intestine before and after administration of indomethacin, while they have shown that the expression of 12/15-lipolyxegease is not affected by the administration. Alternatively, appropriate references should be cited in terms of the alteration of the enzymatic activity during the inflammatory responses.

2) The scale bar should be included in the right panel of Fig. 2a.

3) In Figs. 1b and 1e, the time point at the observation after indomethacin administration should be presented (possibly 24 h).

4) For all experiments in Fig. 3, the timing and duration of pre-treatment of baicalein should be provided.

5) The gene names should be formally written for Il1b and Tnf (both in italic), instead of those in the current manuscript.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 May 4;16(5):e0250862. doi: 10.1371/journal.pone.0250862.r002

Author response to Decision Letter 0

27 Mar 2021

Response to the editors and reviewers

PONE-D-21-00075

Protective role of resolvin D1, a pro-resolving lipid mediator, in nonsteroidal anti-inflammatory drug-induced small intestinal damage

Editor’s comments

1.“Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.”

>>>We ensured that our manuscript meets PLOS ONE’s style requirements, including those for file naming.

2. “At this time, we request that you please report additional details in your Methods section regarding animal care, as per our editorial guidelines:

(1) Please include the method of euthanasia

(2) Please describe the operative care received by the animals, including the frequency of monitoring and the criteria used to assess animal health and well-being.”

>>We really appreciate the editor’s advice. In accordance with the editors’ advice, we reported the details of the method of euthanasia and operative care in the revised manuscript.

3. “Please provide the product number and any lot numbers of the antibodies purchased for your study”.

>>>In the revised manuscript, we provided the product number and the lot numbers of the antibodies purchased for our study.

4. “PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files”.

>>>We provided the original uncropped and unadjusted images underlying all blot results reported in a Supporting Information file.

5. “Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly”.

>>> We included captions for your Supporting Information files at the end of our revised manuscript.

6. “Thank you for submitting the above manuscript to PLOS ONE. During our internal evaluation of the manuscript, we found significant text overlap between your submission and the following previously published works, some of which you are an author.

- https://www.gastrojournal.org/article/S0016-5085(20)32578-6/pdf?referrer=https%3A%2F%2Fapi.ithenticate.com%2F

- https://www.intechopen.com/books/municipal-solid-waste-management/life-cycle-inventory-lci-modeling-of-municipal-solid-waste-msw-management-systems-in-kosodrza-commun

https://www.nature.com/articles/mi201589?code=4eb62728-cfde-4eba-97dd-898b4180c86b&error=cookies_not_supported

>>>We revised the manuscript carefully to rephrase the duplicated text in accordance with the results of the present study.

Additional Editor Comments:

“Although both reviewers feel the study is potentially interesting, one of the reviewers claims a flaw of the authors’ experimental conditions. Specifically, the reviewer strongly recommends the authors to collaborate with lipid biochemists to correctly measure lipid mediators including resolvin D1 in their samples.”

>>>Please see “the Response to Reviewer #1’s Comment”.

Comments to the Author

Response to Reviewer #1:

Major comments

“In figure 3a-d, they showed the marked increase in the lesion index, histological score and the expression of inflammatory cytokines by baicalein, a 12/15 lipoxygenase inhibitor, however, they showed the minimal reduction of resolvin D1 by baicalein treatment in Figure 3e. This small change in resolvin D1 can not explain the huge difference in the clinical scores and inflammation. This is possibly due to the inadequate measurement of resolvin D1. They extracted fatty acid fraction from intestinal tissues by methanol and quantified resolvin D1 using an Elisa kit, which does not give the reliable values. Currently, lipid mediators including SPM2 should be measured by mass-spectrometry as proposed by Bo Burla et al. in J. Lipid Res. 2018;59:2001-2017. The reviewer strongly recommends the authors to collaborate with lipid biochemists to correctly measure lipid mediators including resolvin D1 in their samples”.

>>>We really appreciate the reviewer’s advice. In accordance with the reviewer’s advice, we tried to measure the concentration of resolvin D1 in the small intestinal tissue with help of the expert of mass-spectrometory analysis at the Research Support Platform of Osaka City University Graduate School of Medicine. However, the result was not reasonable. At the moment, for our study, the EIA assay provided by Cayman Chemical is the most reliable method, and this assay kit is used successfully in the several studies including the following papers;

Chen YC, Su MC, Chin CH, Lin IC, Hsu PY, Liou CW, Huang KT, Wang TY, Lin YY, Zheng YX, Hsiao CC, Lin MC. Formyl peptide receptor 1 up-regulation and formyl peptide receptor 2/3 down-regulation of blood immune cells along with defective lipoxin A4/resolvin D1 production in obstructive sleep apnea patients. PLoS One. 2019 May 22;14(5):e0216607. doi: 10.1371/journal.pone.0216607. PMID: 31116781; PMCID: PMC6530856.

Parashar K, Schulte F, Hardt M, Baker OJ. Sex-mediated elevation of the specialized pro-resolving lipid mediator levels in a Sjögren's syndrome mouse model. FASEB J. 2020 Jun;34(6):7733-7744. doi: 10.1096/fj.201902196R. Epub 2020 Apr 11. PMID: 32277856.

Nordgren TM, Friemel TD, Heires AJ, Poole JA, Wyatt TA, Romberger DJ. The omega-3 fatty acid docosahexaenoic acid attenuates organic dust-induced airway inflammation. Nutrients. 2014 Nov 27;6(12):5434-52. doi: 10.3390/nu6125434. PMID: 25436433; PMCID: PMC4276977.

Zhang L, Terrando N, Xu ZZ, Bang S, Jordt SE, Maixner W, Serhan CN, Ji RR. Distinct Analgesic Actions of DHA and DHA-Derived Specialized Pro-Resolving Mediators on Post-operative Pain After Bone Fracture in Mice. Front Pharmacol. 2018 May 1;9:412. doi: 10.3389/fphar.2018.00412. PMID: 29765320; PMCID: PMC5938385.

To confirm the validation of the assay system using this EIA kit in the present study, and to response the comment suggested by Reviewer #2 about concentration of resolvin D1, we performed the additional experiment. In the revised manuscript, we have demonstrated the following results.

1. There was no difference in concentration of resolvin D1 in the small intestine between the vehicle-administered control group and indomethacin-administered group (Fig 2e).

2. The inhibitory effect of baicalein on concentration of resolvin D1 in small intestinal tissue is dose-dependent (Fig 3e).

These results support the validation of the methods of measurement of concentration of resolvin D1 in small intestinal tissue by using the EIA assay. In the revised manuscript, we added the results of the additional experiments.

Response to Reviewer #2:

1) “The authors should measure the concentration of resolvin D1 in the small intestine before and after administration of indomethacin, while they have shown that the expression of 12/15-lipolyxegease is not affected by the administration. Alternatively, appropriate references should be cited in terms of the alteration of the enzymatic activity during the inflammatory responses.”

>>>We really appreciate the reviewer’s suggestion. In accordance with the reviewer’s advice, we measured the concentration of resolvin D1 in the small intestine before and after administration of indomethacin. In consistent with the constant expression of 12/15-lipoxygenase, there was no difference in concentration of resolvin D1 in the small intestine between the vehicle-administered control group and indomethacin-administered group (Fig 2e). We added the data in the revised manuscript.

2) “The scale bar should be included in the right panel of Fig. 2a.”

>>>We added the scale bar in the right panel of Fig.2a.

3) “In Figs. 1b and 1e, the time point at the observation after indomethacin administration should be presented (possibly 24 h).”

>>>We added the information of the time point at the observation after indomethacin administration in the Figure legends of the revised manuscript.

4) “For all experiments in Fig. 3, the timing and duration of pre-treatment of baicalein should be provided.”

>>>We provided the information about the timing and duration of pre-treatment of baicalein in the Figure legends of the revised manuscript.

5) “The gene names should be formally written for Il1b and Tnf (both in italic), instead of those in the current manuscript.”

>>>We corrected the gene names in the revised manuscript.

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(26.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0250862.r003

Decision Letter 1

Hiroyasu Nakano

15 Apr 2021

Protective role of resolvin D1, a pro-resolving lipid mediator, in nonsteroidal anti-inflammatory drug-induced small intestinal damage

PONE-D-21-00075R1

Dear Dr. Tanigawa

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Although one reviewer still has a concern about the quantification of the indicated lipids of mass spectrometry, I have agreed with the authors’ response that the quantification did not work well at least under the authors’ experimental conditions. Thus, I have accepted the manuscript.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Hiroyasu Nakano, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

6. Review Comments to the Author

Reviewer #1: As a lipid biochemist, the reviewer can not accept the results drawn by SPM measurements by ELISA, even though some papers using this ELISA have been published.

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

PLoS One. doi: 10.1371/journal.pone.0250862.r004

Acceptance letter

Hiroyasu Nakano

20 Apr 2021

PONE-D-21-00075R1

Protective role of resolvin D1, a pro-resolving lipid mediator, in nonsteroidal anti-inflammatory drug-induced small intestinal damage

Dear Dr. Tanigawa:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Professor Hiroyasu Nakano

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. The PCR primers and TaqMan probes used in the present study.

(DOCX)

Click here for additional data file.^{(16.1KB, docx)}

S1 Fig. The original uncropped and unadjusted images of the western blotting analysis for 12/15 lipoxygenase.

(TIF)

Click here for additional data file.^{(2.5MB, tif)}

S2 Fig. The original uncropped and unadjusted images of the western blotting analysis for ALX FRP2.

(TIF)

Click here for additional data file.^{(1.7MB, tif)}

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(26.9KB, docx)}

Data Availability Statement

All relevant data are available in figshare (https://figshare.com/), DOI: 10.6084/m9.figshare.13514146.

[pone.0250862.ref001] 1.Serhan CN. Pro-resolving lipid mediators are leads for resolution physiology. Nature. 2014;510(7503):92–101. 10.1038/nature13479 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0250862.ref002] 2.Basil MC, Levy BD. Specialized pro-resolving mediators: endogenous regulators of infection and inflammation. Nat Rev Immunol. 2016;16(1):51–67. 10.1038/nri.2015.4 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0250862.ref003] 3.Sansbury BE, Spite M. Resolution of Acute Inflammation and the Role of Resolvins in Immunity, Thrombosis, and Vascular Biology. Circ Res. 2016;119(1):113–130. 10.1161/CIRCRESAHA.116.307308 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0250862.ref004] 4.Kohli P, Levy BD. Resolvins and protectins: mediating solutions to inflammation. Br J Pharmacol. 2009;158(4):960–971. 10.1111/j.1476-5381.2009.00290.x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0250862.ref005] 5.Hong S, Gronert K, Devchand PR, Moussignac RL, Serhan CN. Novel docosatrienes and 17S-resolvins generated from docosahexaenoic acid in murine brain, human blood, and glial cells. Autacoids in anti-inflammation. J Biol Chem. 2003;278(17):14677–14687. 10.1074/jbc.M300218200 [DOI] [PubMed] [Google Scholar]

[pone.0250862.ref006] 6.Serhan CN, Hong S, Gronert K, Colgan SP, Devchand PR, Mirick G, et al. Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals. J Exp Med. 2002;196(8):1025–1037. 10.1084/jem.20020760 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0250862.ref007] 7.Serhan CN, Chiang N, Van Dyke TE. Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators. Nat Rev Immunol. 2008;8(5):349–361. 10.1038/nri2294 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0250862.ref008] 8.Schif-Zuck S, Gross N, Assi S, Rostoker R, Serhan CN, Ariel A. Saturated-efferocytosis generates pro-resolving CD11b low macrophages: modulation by resolvins and glucocorticoids. Eur J Immunol. 2011;41(2):366–379. 10.1002/eji.201040801 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0250862.ref009] 9.Krishnamoorthy S, Recchiuti A, Chiang N, Yacoubian S, Lee CH, Yang R, et al. Resolvin D1 binds human phagocytes with evidence for proresolving receptors. Proc Natl Acad Sci U S A. 2010;107(4):1660–1665. 10.1073/pnas.0907342107 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0250862.ref010] 10.Hu X, Shen H, Wang Y, Zhang L, Zhao M. Aspirin-triggered resolvin D1 alleviates paraquat-induced acute lung injury in mice. Life Sci. 2019;218:38–46. 10.1016/j.lfs.2018.12.028 [DOI] [PubMed] [Google Scholar]

[pone.0250862.ref011] 11.Zhao YL, Zhang L, Yang YY, Tang Y, Zhou JJ, Feng YY, et al. Resolvin D1 Protects Lipopolysaccharide-induced Acute Kidney Injury by Down-regulating Nuclear Factor-kappa B Signal and Inhibiting Apoptosis. Chin Med J (Engl). 2016;129(9):1100–1107. 10.4103/0366-6999.180517 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0250862.ref012] 12.Liu Y, Zhou D, Long FW, Chen KL, Yang HW, Lv ZY, et al. Resolvin D1 protects against inflammation in experimental acute pancreatitis and associated lung injury. Am J Physiol Gastrointest Liver Physiol. 2016;310(5):G303–G309. 10.1152/ajpgi.00355.2014 [DOI] [PubMed] [Google Scholar]

[pone.0250862.ref013] 13.Kuang H, Hua X, Zhou J, Yang R. Resolvin D1 and E1 alleviate the progress of hepatitis toward liver cancer in long-term concanavalin A-induced mice through inhibition of NF-κB activity. Oncol Rep. 2016;35(1):307–317. 10.3892/or.2015.4389 [DOI] [PubMed] [Google Scholar]

[pone.0250862.ref014] 14.Sun YP, Oh SF, Uddin J, Yang R, Gotlinger K, Campbell E, et al. Resolvin D1 and its aspirin-triggered 17R epimer. Stereochemical assignments, anti-inflammatory properties, and enzymatic inactivation. J Biol Chem. 2007;282(13):9323–9334. 10.1074/jbc.M609212200 [DOI] [PubMed] [Google Scholar]

[pone.0250862.ref015] 15.Maiden L, Thjodleifsson B, Theodors A, Gonzalez J, Bjarnason I. A quantitative analysis of NSAID-induced small bowel pathology by capsule enteroscopy. Gastroenterology. 2005;128(5):1172–1178. 10.1053/j.gastro.2005.03.020 [DOI] [PubMed] [Google Scholar]

[pone.0250862.ref016] 16.Fujimori S, Seo T, Gudis K, Ehara A, Kobayashi T, Mitsui K, et al. Prevention of nonsteroidal anti-inflammatory drug-induced small-intestinal injury by prostaglandin: a pilot randomized controlled trial evaluated by capsule endoscopy. Gastrointest Endosc. 2009;69(7):1339–1346. 10.1016/j.gie.2008.08.017 [DOI] [PubMed] [Google Scholar]

[pone.0250862.ref017] 17.Watanabe T, Tanigawa T, Nadatani Y, Nagami Y, Sugimori S, Okazaki H, et al. Risk factors for severe nonsteroidal anti-inflammatory drug-induced small intestinal damage. Dig Liver Dis. 2013;45(5):390–395. 10.1016/j.dld.2012.12.005 [DOI] [PubMed] [Google Scholar]

[pone.0250862.ref018] 18.Arakawa T, Watanabe T, Tanigawa T, Tominaga K, Otani K, Nadatani Y, et al. Small intestinal injury caused by NSAIDs/aspirin: finding new from old. Curr Med Chem. 2012;19(1):77–81. 10.2174/092986712803414105 [DOI] [PubMed] [Google Scholar]

[pone.0250862.ref019] 19.Tanigawa T, Watanabe T, Otani K, Nadatani Y, Ohkawa F, Sogawa M, et al. Rebamipide inhibits indomethacin-induced small intestinal injury: possible involvement of intestinal microbiota modulation by upregulation of α-defensin 5. Eur J Pharmacol. 2013;704(1–3):64–69. 10.1016/j.ejphar.2013.02.010 [DOI] [PubMed] [Google Scholar]

[pone.0250862.ref020] 20.Kanda Y. Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics. Bone Marrow Transpl. 2013;48:452–8. 10.1038/bmt.2012.244 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0250862.ref021] 21.Lee HN, Kundu JK, Cha YN, Surh YJ. Resolvin D1 stimulates efferocytosis through p50/p50-mediated suppression of tumor necrosis factor-α expression. J Cell Sci. 2013;126(Pt 17):4037–4047. 10.1242/jcs.131003 [DOI] [PubMed] [Google Scholar]

[pone.0250862.ref022] 22.Yin Y, Chen F, Wang W, Wang H, Zhang X. Resolvin D1 inhibits inflammatory response in STZ-induced diabetic retinopathy rats: Possible involvement of NLRP3 inflammasome and NF-κB signaling pathway. Mol Vis. 2017;23:242–250. [PMC free article] [PubMed] [Google Scholar]

[pone.0250862.ref023] 23.Li G, Chen Z, Bhat OM, Zhang Q, Abais-Battad JM, Conley SM, et al. NLRP3 inflammasome as a novel target for docosahexaenoic acid metabolites to abrogate glomerular injury. J Lipid Res. 2017;58(6):1080–1090. 10.1194/jlr.M072587 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0250862.ref024] 24.Watanabe T, Kobata A, Tanigawa T, Nadatani Y, Yamagami H, Watanabe K, et al. Activation of the MyD88 signaling pathway inhibits ischemia-reperfusion injury in the small intestine. Am J Physiol Gastrointest Liver Physiol. 2012;303(3):G324–G334. 10.1152/ajpgi.00075.2012 [DOI] [PubMed] [Google Scholar]

[pone.0250862.ref025] 25.Nadatani Y, Watanabe T, Tanigawa T, Machida H, Okazaki H, Yamagami H, et al. High mobility group box 1 promotes small intestinal damage induced by nonsteroidal anti-inflammatory drugs through Toll-like receptor 4. Am J Pathol. 2012;181(1):98–110. 10.1016/j.ajpath.2012.03.039 [DOI] [PubMed] [Google Scholar]

[pone.0250862.ref026] 26.Higashimori A, Watanabe T, Nadatani Y, Takeda S, Otani K, Tanigawa T, et al. Mechanisms of NLRP3 inflammasome activation and its role in NSAID-induced enteropathy. Mucosal Immunol. 2016;9(3):659–668. 10.1038/mi.2015.89 [DOI] [PubMed] [Google Scholar]

[pone.0250862.ref027] 27.Ferrer R, Moreno JJ. Role of eicosanoids on intestinal epithelial homeostasis. Biochem Pharmacol. 2010;80(4):431–438. 10.1016/j.bcp.2010.04.033 [DOI] [PubMed] [Google Scholar]

[pone.0250862.ref028] 28.Kim SN, Akindehin S, Kwon HJ, Son YH, Saha A, Jung YS, et al. Anti-inflammatory role of 15-lipoxygenase contributes to the maintenance of skin integrity in mice. Sci Rep. 2018. June 11;8(1):8856. 10.1038/s41598-018-27221-7 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0250862.ref029] 29.Habouri L, El Mansouri FE, Ouhaddi Y, Lussier B, Pelletier JP, Martel-Pelletier J, et al. Deletion of 12/15-lipoxygenase accelerates the development of aging-associated and instability-induced osteoarthritis. Osteoarthritis Cartilage. 2017. October;25(10):1719–1728. 10.1016/j.joca.2017.07.001 [DOI] [PubMed] [Google Scholar]

[pone.0250862.ref030] 30.Ishida T, Yoshida M, Arita M, Nishitani Y, Nishiumi S, Masuda A, et al. Resolvin E1, an endogenous lipid mediator derived from eicosapentaenoic acid, prevents dextran sulfate sodium-induced colitis. Inflamm Bowel Dis. 2010. January;16(1):87–95. 10.1002/ibd.21029 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0250862.ref031] 31.Arita M, Yoshida M, Hong S, Tjonahen E, Glickman JN, Petasis NA, et al. Resolvin E1, an endogenous lipid mediator derived from omega-3 eicosapentaenoic acid, protects against 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Proc Natl Acad Sci U S A. 2005. May 24;102(21):7671–6. 10.1073/pnas.0409271102 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0250862.ref032] 32.Quiros M, Feier D, Birkl D, Agarwal R, Zhou DW, García AJ, et al. Resolvin E1 is a pro-repair molecule that promotes intestinal epithelial wound healing. Proc Natl Acad Sci U S A. 2020. April 28;117(17):9477–9482. 10.1073/pnas.1921335117 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0250862.ref033] 33.Gewirtz AT, Collier-Hyams LS, Young AN, Kucharzik T, Guilford WJ, Parkinson JF, et al. Lipoxin a4 analogs attenuate induction of intestinal epithelial proinflammatory gene expression and reduce the severity of dextran sodium sulfate-induced colitis. J Immunol. 2002. May 15;168(10):5260–7. 10.4049/jimmunol.168.10.5260 [DOI] [PubMed] [Google Scholar]

[pone.0250862.ref034] 34.Gobbetti T, Ducheix S, le Faouder P, Perez T, Riols F, Boue J, et al. Protective effects of n-6 fatty acids-enriched diet on intestinal ischaemia/reperfusion injury involve lipoxin A4 and its receptor. Br J Pharmacol. 2015. February;172(3):910–23. 10.1111/bph.12957 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0250862.ref035] 35.Kure I, Nishiumi S, Nishitani Y, Tanoue T, Ishida T, Mizuno M, et al. Lipoxin A(4) reduces lipopolysaccharide-induced inflammation in macrophages and intestinal epithelial cells through inhibition of nuclear factor-kappaB activation. J Pharmacol Exp Ther. 2010. February;332(2):541–8. 10.1124/jpet.109.159046 [DOI] [PubMed] [Google Scholar]

[pone.0250862.ref036] 36.Marchese A, Nguyen T, Malik P, Xu S, Cheng R, Xie Z, et al. Cloning genes encoding receptors related to chemoattractant receptors. Genomics. 1998;50(2):281–286. 10.1006/geno.1998.5297 [DOI] [PubMed] [Google Scholar]

[pone.0250862.ref037] 37.Bento AF, Claudino RF, Dutra RC, Marcon R, Calixto JB. Omega-3 fatty acid-derived mediators 17(R)-hydroxy docosahexaenoic acid, aspirin-triggered resolvin D1 and resolvin D2 prevent experimental colitis in mice. J Immunol. 2011;187(4):1957–1969. 10.4049/jimmunol.1101305 [DOI] [PubMed] [Google Scholar]

[pone.0250862.ref038] 38.Chen K, Liu M, Liu Y, Yoshimura T, Shen W, Le Y, et al. Formylpeptide receptor-2 contributes to colonic epithelial homeostasis, inflammation, and tumorigenesis. J Clin Invest. 2013;123(4):1694–1704. 10.1172/JCI65569 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0250862.ref039] 39.Birkl D, O’Leary MN, Quiros M, Azcutia V, Schaller M, Reed M, et al. Formyl peptide receptor 2 regulates monocyte recruitment to promote intestinal mucosal wound repair. FASEB J. 2019. December;33(12):13632–13643. 10.1096/fj.201901163R [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Protective role of resolvin D1, a pro-resolving lipid mediator, in nonsteroidal anti-inflammatory drug-induced small intestinal damage

Takuya Kuzumoto

Tetsuya Tanigawa

Akira Higashimori

Hiroyuki Kitamura

Yuji Nadatani

Koji Otani

Shusei Fukunaga

Shuhei Hosomi

Fumio Tanaka

Noriko Kamata

Yasuaki Nagami

Koichi Taira

Toshio Watanabe

Yasuhiro Fujiwara

Roles

Abstract

Introduction

Materials and methods

Animals

Induction of NSAID-induced small intestinal damage

Histological evaluation of small intestinal damage

Reagents for analysis of the roles of resolvin D1 and 12/15-lipoxygenase

Measurement of resolvin D1 concentration in small intestinal tissue

Quantitative real‑time reverse transcription polymerase chain reaction (RT-PCR) analysis

Western blotting analysis

Immunohistochemistry

Statistical analysis

Results

Effects of exogenous resolvin D1 on NSAIDs-induced small intestinal damage

Fig 1. Effects of exogenous resolvin D1 on NSAID-induced small intestinal damage.

Dynamics of 12/15-lipoxygenase expression in small intestinal tissue

Fig 2. Dynamics and localization of 12/15-lipoxygenase in small intestinal tissue.

Inhibition of 12/15-lipoxygenase by baicalein aggravates indomethacin-induced small intestinal damage

Fig 3. Effect of inhibition of activity of 12/15-lipoxygenase on indomethacin-induced small intestinal damage.

Dynamics of ALX/FPR2 expression in small intestinal tissue

Fig 4. Expression of resolvin D1 receptor, lipoxin A4 receptor /formyl peptide receptor 2 (ALX/FPR2) in the small intestine and its dynamics on indomethacin-induced small intestinal damage.

Pharmacological inhibition of the resolvin D1 receptor ALX/FPR2 aggravates indomethacin-induced small intestinal damage

Fig 5. Effect of inhibition of lipoxin A4 receptor /formyl peptide receptor 2 (ALX/FPR2) on indomethacin-induced small intestinal damage.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Hiroyasu Nakano

Roles

Author response to Decision Letter 0

Decision Letter 1

Hiroyasu Nakano

Roles

Acceptance letter

Hiroyasu Nakano

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases