Suppression of Fpr2 expression protects against endotoxin-induced acute lung injury by interacting with Nrf2-regulated TAK1 activation

Biomedicine & Pharmacotherapy 125 (2020) 109943

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha

Suppression of Fpr2 expression protects against endotoxin-induced acute lung injury by interacting with Nrf2-regulated TAK1 activation

T

Haiyan Liua,1, Zhanyuan Linb,1, Ying Mac,* a

Deparatment of Pediatrics, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, 710004, China Deparatment of Pediatrics, Yangling Demonstration Area Hospital, Xianyang, 712100, China c Deparatment of Neonatology, The Affiliated Children Hospital of Xi’an Jiaotong University, Xi’an, 710004, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Acute lung injury (ALI) Fpr2 Inflammation and oxidative stress Nrf2 TAK1

Acute lung injury (ALI) is caused by severe infection, and urgently needs effective treatments or validated pharmacological targets. Formyl peptide receptor 2 (Fpr2) plays essential roles in immune responses and inflammatory diseases. In the present study, Fpr2 expression was markedly increased in lung tissues of lipopolysaccharide (LPS)-challenged mice, and these effects were confirmed in LPS-stimulated macrophages. Then, the in vitro analysis suggested that Fpr2 knockdown significantly decreased LPS-induced inflammatory response in macrophages. Notably, the in vivo experiments indicated that Fpr2 deficiency alleviated ALI in LPS-treated mice, as evidenced by the improved histological changes in lung, reduced protein concentrations in bronchoalveolar lavage fluid (BALF) and decreased neutrophil infiltration. In addition, LPS-induced pulmonary inflammation was ameliorated by Fpr2 knockout, which was partly through blocking nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) signaling pathways. Furthermore, oxidative stress stimulated by LPS was also attenuated by Fpr2 knockout, as indicated by the reduced malondialdehyde (MDA) levels and reactive oxygen species (ROS) production, accompanied with the elevated glutathione (GSH), superoxide dismutase (SOD), heme oxygenase-1 (HO-1) and NAD (P) H: quinone oxidoreductase (NQO1) levels. These antioxidative processes were mainly via the activation of Nrf2. Importantly, the in vitro results showed that Fpr2 over-expression markedly accelerated the inflammation and ROS production in LPS-incubated macrophages, which could be reversed by restoring the Nrf2 activation, demonstrating that Nrf2 was partially involved in Fpr2-regulated inflammatory response and oxidative stress during ALI progression. Then, we found that Fpr2 inhibition markedly reduced the activation of transforming growth factor beta-activated kinase 1 (TAK1) induced by LPS. What’s more important, immunoprecipitation results demonstrated that Fpr2 directly interacted with the kinase TAK1. Taken together, findings in the present study illustrated that Fpr2 could directly interact with TAK1 to promote ALI through enhancing inflammation and oxidative stress associated with the activation of Nrf2, providing a novel therapeutic target to develop effective treatment against ALI progression.

1. Introduction Acute lung injury (ALI) and respiratory distress syndrome (ARDS), the more serious form of ALI, are acute and severe inflammatory processes existing in lungs due to multiple insults, and still remain high mortality rates [1]. Moreover, accumulating studies indicate that inflammation and oxidative stress are pivotal lung injury pathways, influencing the development and severity of ALI [2]. Presently, suppressing inflammatory response and oxidative stress is considered as promising therapeutic strategies to prevent ALI progression [3]. LPS is derived from the outer membranes of Gram-negative bacteria, and its

exposure to lung induces macrophages activation and inflammatory cells leakage, particularly the neutrophils [4]. A large number of neutrophils influx into the pulmonary tissues not only leads to the secretion of uncontrolled inflammatory factors, but also results in ROS accumulation [5]. However, the molecular mechanism of ALI is not completely understood, and there is no effective treatment for ALI. Formyl peptide receptor 2 (Fpr2) is a chemoattractant receptor, which belongs to the Fpr family [6]. Fpr2 could be activated by an array of ligands, including structurally unrelated lipids and peptide/proteins agonists, which lead to different intracellular responses [7]. As reported, human Fpr2 and its mouse homologue Fpr2 are significantly

⁎

Corresponding author. E-mail address: [email protected] (Y. Ma). 1 The first two authors contributed equally to this study. https://doi.org/10.1016/j.biopha.2020.109943 Received 10 October 2019; Received in revised form 19 January 2020; Accepted 23 January 2020 0753-3322/ © 2020 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Biomedicine & Pharmacotherapy 125 (2020) 109943

H. Liu, et al.

2.3. Protein concentration in bronchoalveolar lavage fluid (BALF) and cell counting

expressed in macrophages, monocytes and neutrophils, which could interact with peptide and lipid ligands, and transduce either pro- or anti-inflammatory actions [8,9]. Fpr2 has been involved in various diseases, such as asthma, brain injury and tumors [10,11]. Recently, Fpr2 knockout was suggested to alleviate murine sepsis by reducing inflammatory response, alleviating associated myocardial dysfunction [12]. So far, the specific roles of Fpr2 in LPS-exposed macrophages and the detailed molecular mechanisms by which Fpr2 mediates these LPSinduced pathologies of ALI remain unclear. In this study, we explored how Fpr2 modulated the progression of ALI by performing both in vivo and in vitro experiments. Results here for the first time demonstrated that Fpr2 expression was significantly increased in lung samples from mice with ALI. Suppression of Fpr2 evidently alleviated ALI development by reducing inflammation and oxidative stress, which was mainly associated with the improvement of Nrf2 signaling. Importantly, the in vitro analysis suggested that Fpr2 directly interacted with TAK1, subsequently affecting inflammatory response. Therefore, Fpr2 could be served as a therapeutic interventional target to prevent ALI.

After LPS challenge, all mice were euthanized for BALF collection. The BALF samples were centrifuged to pellet the cells, lysed by ACK Lysis Buffer (Beyotime, Nanjing, China) for 5 min, washed with ice-cold phosphate buffer solution (PBS) and collected, and then again centrifuged at 3000 rpm for 10 min at 4 °C. The protein concentrations in BALF were determined using BCA protein assay kit (Beyotime) following the manufacturer’s instructions. The cells were counted (Coulter counter; Beckman Coulter, USA) and were normalized according to the volume of recovered BALF. For differentials, cytospin slides were made and analyzed for the presence of neutrophils, macrophages, lymphocytes, and basophils by 2 hematologists who were blinded to genotype and treatment group assignments. 2.4. Western blot and real-time qPCR Cells or tissues were lysed in RIPA lysis buffer (Thermo Fisher, USA) containing 1 % protease inhibitor (Sigma Aldrich). Nucleoprotein Extraction Kit (Sangon Biotech, Nanjing, China) was used for protein extraction from nuclear of tissues or cells following the manufacturer’s instructions. Then, the protein samples (20−50 μg) were separated by SDS-PAGE gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA), followed by blocking with 5 % non-fat milk in Tris-buffer saline with 0.1 % Tween 20 (TBST). Then, membranes were incubated with primary antibodies (Supplementary table S1) overnight at 4 °C. After washing with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, USA). The protein signals were visualized using enhanced chemiluminescence (ECL) chromogenic substrate (Thermo Fisher). GAPDH or Lamin B was used as an internal control. As for RT-qPCR analysis, total RNA was extracted from cells or tissues using TRIzol reagent (Invitrogen) according to the manufacturer’s protocols, followed by reverse transcription with the Advantage RT-for-PCR Kit (Takara, Dalian, China). Aliquots of doublestranded cDNA were amplified by a SYBR Green PCR Kit (Applied Biosystems, USA) for RT-qPCR. The relative expression levels of the targeting genes were calculated using the 2–ΔΔCt method [14]. All reactions were repeated, and the primer sequences were exhibited in Supplementary Table S2. GAPDH expression was used as an internal control.

2. Materials and methods 2.1. Animals and culture Wild-type (WT) C57BL/6 mice (18−22 g, 8-week old) were purchased from Laboratory Animal Centre of China Medical University (Shenyang, China). Fpr2 knockout (KO) C57BL/6 mice (18−22 g, 8week old) were designed and purchased from Cyagen Biosciences Inc (Suzhou, China). All mice were raised under SPF-condition at controlled temperature (22 ± 2 °C) with relative humidity of 50 ± 10 %, 12 h light/dark cycle and free access to water and food. Animal experiments were approved by Ethics Committee of Laboratory Animal Science, Ankang Central Hospital (Shannxi, China). Endotoxemia mouse model was induced by the treatment of LPS (Escherichia coli 055:B5, Sigma-Aldrich, St. Louis, MO, USA). Mice were randomly divided into 4 groups (n = 8 in each group): WT/Con (saline), WT/LPS (0.5 mg/kg, dissolved in saline), KO/Con (saline) and KO/LPS (0.5 mg/ kg, dissolved in saline). Mice were anesthetized using diethyl ether. LPS was then administered intranasally (i.n.) to induce lung injury [13]. After LPS treatment for 24 h, the mice were euthanized by CO2 asphyxiation. The lung tissues from each group of mice were collected for further analysis. 2.2. Cells and treatments

2.5. ROS production

The mouse macrophage of RAW264.7 cells was purchased from the China Cell Line Bank (Beijing, China), and cultured in DMEM (Invitrogen-Gibco, USA) supplemented with 10 % fetal bovine serum (FBS) (Invitrogen-Gibco), 100 U/mL of penicillin, 100 U/mL of streptomycin and 3 mM glutamine at 37 °C in a humidified atmosphere containing 5 % CO2. The small interfering RNAs (siRNAs) against Fpr2 (siFpr2), siRNAs against TAK1 (siTAK1) and siRNA negative control (siCon) were synthesized by Genepharma (Shanghai, China). For Fpr2 and TAK1 overexpression, the entire open reading frame of Fpr2 and TAK1 cloned in the pCDNA3-vector (oe-NC), pCDNA3-Fpr2 (oeFpr2) and pCDNA3-TAK1 (oeTAK1) were purchased from Origene (Rockville, USA). Lipofectamine 3000 (Invitrogen-Gibco) was used for cell transfection following the manufacturer’s instructions. All chemicals, including tBHQ (Nrf2 activator) and ML385 (Nrf2 inhibitor), were offered by Sigma-Aldrich. Nrf2 selective agonist (3S)-1-[4-[(2,3,5,6-tetramethylphenyl) sulfonylamino]-1-naphthyl]pyrrolidine-3-carboxylic acid (RA-839) was purchased from R&D System (purity ≥ 98 %, USA). Plasmids encoding full-length mouse Flag-Fpr2 were obtained by cloning the cDNA of Fpr2 into the psi-Flag vectors, which were used for further in vitro analysis.

BALF was lysed using ACK Lysis Buffer, and then the sedimented cells were resuspended in PBS for ROS calculation. Briefly, the cells were stained with 50 μM of DCFH-DA (KeyGen Biotech, Nanjing, China) at 37 °C in the dark for 30 min. DCF fluorescence intensities were measured through flow cytometry with a multi-detection reader (BD Bioscences, San Diego, USA). As for intracellular ROS production, after various treatments, RAW264.7 cells were stained with DCFH-DA (KeyGen Biotech) at 37 °C in the dark for 30 min, and then were observed under a fluorescent microscope. 2.6. Immunoprecipitation and glutathione S-transferase (GST) pull-down GST-Fpr2 and pcDNA5-HA-Fpr2 were generated by cloning the human Fpr2 gene into pGEX-4T-1 and pcDNA5-HA-C1, respectively. pcDNA5-Flag-TAK1 were generated by cloning the human TAK1 gene into pcDNA5-Flag-C1. Immunoprecipitation and GST pull-down assays were performed to determine protein-protein interactions [15]. As for immunoprecipitation, cells were rinsed with cold PBS and lysed with lysis buffer (Thermo Fisher) containing Protease Inhibitor (Sigma Aldrich). After being pre-cleared with normal mouse or rabbit 2

Biomedicine & Pharmacotherapy 125 (2020) 109943

H. Liu, et al.

2.9. The wet/dry weight (W/D) ratio

immunoglobulin G and protein A/G-agarose beads (Thermo Fisher), lysates were incubated with the indicated primary antibodies and Protein G-agarose (Thermo Fisher) at 4 °C overnight with gentle shaking. The immunoprecipitated proteins were further washed with lysis buffer, boiled with 2 × SDS loading buffer, separated with SDSPAGE and electrophoretically transferred to PVDF membranes (Millipore). The membranes were then blocked with 5 % BSA in TBST and were immunoreacted with the indicated primary antibodies and secondary antibodies conjugated to HRP. As for the GST pull-down analysis, immunopurified Flag-TAK1 was prepared according the IP assay followed by incubation in elution buffer for 2 h at 4 °C. The Rosetta (DE3) Escherichia coli was transformed with vectors pGEX-4T-1-GST-Fpr2, and then were induced using isopropyl-bD thiogalactopyranoside (IPTG) at an optical density at 600 nm (OD600) of 0.8. E. coli extracts were prepared in PBS containing protease inhibitor cocktail tablets (Sigma Aldrich) and incubated with glutathione-Sepharose 4B beads (GE Healthcare Biosciences AB, USA) for 1 h at 4 °C. Then, the beads loaded with the proteins were washed with PBS and incubated for an additional 4 h at 4 °C with immunopurified Flag-TAK1. The glutathione-Sepharose 4B beads were then rinsed with the IP lysis buffer without cocktail, and the eluted proteins, in buffer containing 20 mM reduced glutathione were resolved through SDS-PAGE and analyzed by western blot using anti-Flag antibodies. A GST tag was served as the negative control under the same conditions.

The W/D ratio was measured to evaluate the moisture content of lung tissue. Briefly, the surface liquid of lung section was carefully drained with clean filter paper and then recorded as the wet weight, which were then stored in a thermostatic drier at constant 80 °C for 48 h. The dry weight was obtained. 2.10. ELISA analysis and calculation of MPO, MDA, GSH and SOD The concentration of pro- and anti-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10, IL-12 and IL-4, was measured using commercial kits purchased from Cloud-Clone Corp. (Wuhan, China) according to the manufacturer’s protocols. Myeloperoxidase (MPO), MDA, GSH and SOD levels or activities in lung tissues were measured using commercial kits obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the respective manufacturer’s instructions. 2.11. Statistical analysis All statistical analyses used in this study were performed using GraphPad Prism (GraphPad Software, CA, USA). The data were presented as the mean ± SEM. The statistical differences between groups were analyzed by Student’s t-test or one-way analysis of variance (ANOVA). P value < 0.05 was considered significant. Animal feeding and treatment, as well as histological analysis, were performed in a single-blinded fashion. All in vitro experiments were performed in triplicate unless specified in the figure legends.

2.7. Immunofluorescence (IF) After various treatments, cells were washed with PBS and fixed in 4 % paraformaldehyde. Then, the cells were permeabilized using 0.1 % Triton-X. Then, the cells were blocked using 3 % BSA (Sigma Aldrich) and probed with primary antibody (Fpr2, 1:100 dilutions, XY13448-1, XYbscience, USA) overnight at 4 °C. Cells were then incubated with secondary antibody (1:200 dilutions, Abcam) for 1.5 h at room temperature. The nuclei were labeled with DAPI (Sigma Aldrich). Images were obtained with a fluorescence microscope.

3. Results 3.1. Fpr2 expression is up-regulated during ALI progression in vivo and in vitro To explore the effects of Fpr2 on ALI progression, its expression change was measured. As shown in Fig. 1A and B, Fpr2 expression from mRNA and protein levels was markedly increased in pulmonary samples of LPS-treated WT mice. Similar results were obtained using IHC staining (Fig. 1C). Then, LPS-enhanced Fpr2 expression levels were verified in RAW264.7 cells (Fig. 1D and E). IF staining further confirmed the up-regulation of Fpr2 in LPS-exposed RAW264.7 cells (Fig. 1F). Given the significant role of inflammatory response in ALI progression and the regulatory effects of Fpr2 on inflammation, Fpr2 expression was knocked down in RAW264.7 cells by transfecting with its specific siRNA. Transfection efficacy was confirmed by RT-qPCR and western blot analysis (Fig. 1G and H). Subsequently, we found that the mRNA levels of pro-inflammatory factors (CD68, CD11b, F4/80, TNF-α, IL-1β, IL-6, IL-18, CXCL1, CXCL2, CCL2 and MCP1) were markedly increased by LPS in RAW264.7 cells. Notably, these effects induced by LPS were clearly reversed by Fpr2 knockdown (Fig. 1I). Therefore, findings above demonstrated that Fpr2 was involved in ALI progression. Herein, global deletion of Fpr2 mice was used to further explore the effects of Fpr2 on ALI. Western blot analysis suggested that Fpr2 expression was undetectable in lung, liver, kidney and brain samples of KO mice (Fig. 1J).

2.8. Histopathological analysis and immunohistochemistry (IHC) The 5-micron lung tissue sections were stained with hematoxylin followed by eosin (H&E) to observe pathological and morphological alterations in lung tissues. A semiquantitative histopathologic scoring was used to calculate the lung structural and cellular changes [16,17]. As for lung barrier permeability, the mice were injected with 2 % Evans Blue Dye (100 mg/kg, Sigma-Aldrich) via the tail vein 30 min before euthanization. After euthanization, the pulmonary tissues were removed en bloc and homogenized. The homogenate was then incubated at 37 °C for 24 h. Next, the blue dye was conducted at 620 nm using a microplate reader. IHC was conducted as per standard protocol reported earlier [18]. The sections were blocked with 3 % BSA (Sigma Aldrich) to avoid non-specific binding. Then, the lung sections were incubated with Fpr2 (1:150 dilutions, XY13448-1, XYbscience), F4/80 (1:150 dilutions, ab6640, Abcam, USA), CD68 (1:150 dilutions, ab125212, Abcam), p-IκBα (1:150 dilutions, ab133462, Abcam) and pNF-κB (1:150 dilutions, ab86299, Abcam) primary antibodies overnight at 4 °C. Then, tissue sections were incubated with the HRP-conjugated secondary antibodies (Abcam). The immune reactions were finally visualized through adding the 3,3′-diaminobenzidine tetrachloride (DAB, Sigma Aldrich) and all sections were counterstained using hematoxylin. The number of Fpr2+, F4/80+, CD68+, p-IκBα+ and p-NF-κB+ cells in the lung sections was counted in 15 equivalent high-power fields and was expressed as the average number of cells per field. Quantitative analysis of immunohistochemical staining was measured by ImagePro Plus 6.0 and presented as a fold value of integrated optical density compared to the normal control group [19].

3.2. Fpr2 deficiency alleviates ALI by reducing inflammatory response and oxidative stress H&E staining indicated that LPS caused apparently pathologic alterations through elevating accumulation of inflammatory cells and alveolar hemorrhage compared to the WT/Con group, which were, however, obviously weakened by Fpr2 knockout measured by the greater lung injury score (Fig. 2A). LPS induced lung vascular leakage and Fpr2 deletion significantly inhibited LPS-induced elevation of 3

Biomedicine & Pharmacotherapy 125 (2020) 109943

H. Liu, et al.

Fig. 1. Fpr2 expression is up-regulated during ALI progression in vivo and in vitro. (A) The mRNA and (B) protein levels of Fpr2 in lung tissue from WT mice challenged with LPS using RT-qPCR and western blot analysis, respectively. ***P < 0.001. (C) IHC staining of Fpr2 in lung sections from LPS-treated WT mice. Scale bar, 50 μm. (D) The mRNA and (E) protein levels of Fpr2 in RAW264.7 cells treated with 100 ng/ml of LPS for the indicated time. (F) IF staining of Fpr2 in RAW264.7 cells stimulated with LPS (100 ng/ml) for 12 or 24 h. Scale bar, 50 μm. (G) The mRNA and (H) protein levels of Fpr2 in RAW264.7 cells transfected with siCon or siFpr2 for 24 h. ***P < 0.001. (I) The mRNA levels of pro-inflammatory factors in RAW264.7 cells were measured using RT-qPCR analysis. Here, RAW264.7 cells were transfected with siFpr2 for 24 h, and then were exposed to LPS (100 ng/ml) for another 24 h. **P < 0.01 and ***P < 0.001 vs Con/siCon group; +P < 0.05, + + P < 0.01 and +++P < 0.001 vs LPS/siCon group. (J) Western blot analysis of Fpr2 expression in different organs (lung, liver, kidney and brain) from WT or KO mice. Data represented as mean ± SEM.

neutrophils, macrophages, lymphocytes and basophils compared to the WT/Con group. Of note, Fpr2 deficiency significantly reduced the number of these cells in BALF from LPS-stimulated mice (Fig. 2F). ELISA analysis demonstrated that LPS-stimulated pro-inflammatory cytokines TNF-α, IL-1β and IL-6 in BALF were markedly alleviated by Fpr2 knockout. However, Fpr2 deletion significantly improved the contents of anti-inflammatory cytokines IL-10, IL-12 and IL-4 in BALF from LPS-challenged mice compared with WT/LPS group (Fig. 2G). Furthermore, Fpr2 knockout effectively reduced the mRNA expression levels of pro-inflammatory factors (CD68, CD11b, F4/80, TNF-α, IL-1β,

permeability as evidenced by the obvious Evans blue extravasation into the lung tissue (Fig. 2B). Lung wet/dry (W/D) weight ratio was remarkably increased following LPS stimulation, whereas being decreased in mice with Fpr2 ablation (Fig. 2C). MPO activation represents polymorphonuclear leukocyte accumulation in pulmonary samples [20]. As shown in Fig. 2D, LPS-stimulated MPO activities in lung tissues were remarkably alleviated by Fpr2 deficiency. Additionally, LPS-increased protein concentrations in BALF were higher than that of the WT/Con group, which were markedly reduced in Fpr2-knockout mice (Fig. 2E). BALF from WT mice exposed to LPS showed more total cells, 4

Biomedicine & Pharmacotherapy 125 (2020) 109943

H. Liu, et al.

Fig. 2. Fpr2 deficiency alleviates ALI in LPS-treated mice by reducing inflammatory response and oxidative stress. (A) H&E staining of pulmonary sections. Scale bar, 200 μm. (B) Evan’s blue dye for lung tissues. Measurements of (C) W/D ratio of lung, and (D) MPO activity in lung. (E) Protein concentrations in BALF were tested. (F) Determination of the number of total cells, neutrophils, macrophages, lymphocytes and basophils in BALF. (G) Calculation of pro- and anti-inflammatory cytokines in BALF by ELISA analysis. (H) The mRNA levels of pro-inflammatory factors in lung tissues were examined by RT-qPCR. Calculation of (I) GSH, SOD, and (J) MDA in lung. (K) DCF-DA analysis for ROS production in BALF using flow cytometry. (L) HO1, NQO1, Nrf2 and iNOS protein levels in lung samples were determined by western blot. (M) Western blot analysis was performed to calculate the nuclear Nrf2 expression levels from lung tissues of each group. Data represented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 vs Con/WT group; +P < 0.05 and ++P < 0.01 vs LPS/WT group.

CD68, known as essential markers of macrophages controlling inflammatory response, was markedly induced by LPS in WT mice, which were, however, clearly reduced by Fpr2 knockout (Fig. 3A). A considerable number of studies have underscored the crucial role of the NFκB and MAPKs signaling pathways in regulating ALI progression [4,22]. Our results also showed that LPS activated NF-κB signaling both in the lung of WT mice and siCon RAW264.7 cells, as indicated by the increased expression of p-IKKα, p-I-κBα and p-NF-κB (p65). Surprisingly, Fpr2 knockout or knockdown significantly inhibited the phosphorylation of IKKα, I-κBα and NF-κB (Fig. 3B and C). Consistently, western blotting analysis demonstrated that NF-κB translocation to nuclear was markedly induced by LPS treatment, which was, however, alleviated by the suppression of Fpr2 both in lung tissues and in the RAW264.7 cells (Fig. 3D). Moreover, we found that LPS-challenged WT mice showed higher expression of phosphorylated p38, ERK1/2 and JNK in lung. Notably, these effects were clearly reversed by Fpr2 deletion. The role of Fpr2 inhibition in blocking MAPKs was verified in LPS-stimulated RAW264.7 cells (Fig. 3E). Results above indicated that suppressing Fpr2 expression could alleviate LPS-induced inflammation through restraining the activation of NF-κB and MAPKs signaling pathways to alleviate acute lung injury.

IL-6, IL-18, CXCL1, CXCL2, CCL2 and MCP1) and promoted the expression of anti-inflammatory cytokines (IL-10, IL-12 and IL-4) in lung tissues from LPS-treated mice (Fig. 2H). Subsequently, GSH and SOD levels in lung of LPS-challenged mice were markedly improved by Fpr2 deletion (Fig. 2I). However, LPS-stimulated MDA levels in pulmonary samples were clearly reduced by Fpr2 knockout (Fig. 2J). Fpr2-KO mice showed a significantly reduced ROS production in BALF after LPS challenge compared to the LPS/WT mice (Fig. 2K). Finally, LPS-induced down-regulation of HO1, NQO1 and Nrf2 was markedly rescued by Fpr2 knockout, while an opposite result was detected in the change of iNOS expression (Fig. 2L). Upon exposure to stressors and inducers, the release of Nrf2 into the nucleus could result in the expression of various cytoprotective genes [21]. Herein, the expression levels of Nrf2 in nuclear were measured by western blotting assays. As shown in Fig. 2M, we found that nuclear Nrf2 expression levels were markedly reduced in lung tissues of mice challenged with LPS; however, Fpr2 deletion significantly reversed this process, as evidenced by the obvious decreases in nuclear Nrf2. Therefore, results above demonstrated that Fpr2 knockout could alleviate LPS-induced acute lung injury through suppressing oxidative stress.

3.3. Fpr2 knockout reduces the activation of NF-κB and MAPKs signaling pathways At first, IHC staining indicated that the expression of F4/80 and 5

Biomedicine & Pharmacotherapy 125 (2020) 109943

H. Liu, et al.

Fig. 3. Fpr2 knockout reduces the activation of NF-κB and MAPKs signaling pathways. IHC staining of (A) F4/80, CD68, (B) p-I-κBα and p-NF-κB in lung tissues, as well as the quantification of these signals. Scale bar, 100 μm. (C) Western blotting results for p-IKKα, p-I-κBα and p-NF-κB in lung tissues isolated from the indicated groups of mice, and in 24 h of LPS (100 ng/ml)-treated RAW264.7 cells following transfection with siFpr2 for 24 h. (D) Nuclear NF-κB expression in lung tissues and RAW264.7 cells was measured by western blotting analysis. Here, RAW264.7 cells were transfected with siFpr2 for 24 h, and then were subjected to LPS (100 ng/ml) exposure for another 24 h. (E) The protein expression levels of p-p38, p-ERK1/2 and p-JNK in lung tissues or RAW264.7 cells were measured using western blot. RAW264.7 cells were subjected to siFpr2 transfection for 24 h, followed by LPS (100 ng/ml) stimulation for another 24 h. Data represented as mean ± SEM. **P < 0.01 and ***P < 0.001 vs Con/WT or Con/siCon group; +P < 0.05 and ++P < 0.01 vs LPS/WT or LPS/siCon group.

6

Biomedicine & Pharmacotherapy 125 (2020) 109943

H. Liu, et al.

(caption on next page)

inhibitor ML385 was then pre-treated to RAW264.7 cells. As shown in Fig. 4B, Fpr2 knockdown-reduced ROS production in LPS-stimulated cells was significantly restored by ML385. Oppositely, Fpr2 silenceimproved Nrf2 expression was clearly abolished by ML385 (Fig. 4C). Then, we demonstrated that Nrf2 suppression using ML385 remarkably diminished the inhibitory role of Fpr2 knockdown in regulating inflammation, as indicated by the restored mRNA levels of pro-

3.4. Fpr2 directly interacts with TAK1 to control LPS-induced inflammation and ROS production regulated by Nrf2 In this regard, we found that Fpr2 knockdown markedly reduced LPS-induced ROS accumulation in RAW264.7 cells (Fig. 4A). Nrf2 plays a critical role in controlling ROS production [23]. Thus, to further evaluate the effects of ROS regulated by Nrf2 on ALI through Fpr2, Nrf2 7

Biomedicine & Pharmacotherapy 125 (2020) 109943

H. Liu, et al.

Fig. 4. Fpr2 directly interacts with TAK1 to control LPS-induced inflammation and ROS production regulated by Nrf2. (A) RAW264.7 cells were transfected with siFpr2 for 24 h, and then were exposed to LPS (100 ng/ml) for another 24 h, followed by DCF-DA fluorescence to calculate ROS production. Scale bar, 100 μm. (B–D) RAW264.7 cells were ptr-treated with MLB385 (10 μM) for 4 h, and then were transfected with siFpr2 for 24 h, which were subjected to LPS (100 ng/ml) for another 24 h. Then, (B) DCF-DA fluorescence was used to calculate ROS production. (C) The mRNA levels of Nrf2 and (D) pro-inflammatory factors in cells were examined by RT-qPCR. (E) The protein levels of Fpr2 in RAW264.7 cells transfected with pCDNA3-vector (oeNC) or pCDNA3-Fpr2 (oeFpr2) to over-express Fpr2 expression. (F–G) RAW264.7 cells were pre-treated with Nrf2 selective agonist RA-839 (6 μM) for 4 h, and then were transfected with pCDNA3-Fpr2 for 24 h, which were exposed to LPS (100 ng/ml) for final 24 h. Then, (F) DCF-DA fluorescence was used to calculate ROS production. The mRNA levels of (G) Nrf2 and (H) pro-inflammatory cytokines in cells were measured using RT-qPCR. (I) p-TAK1 protein levels in lung tissues or LPS-incubated RAW264.7 cells with or without Fpr2 knockdown were measured by western blot. HEK293 T cells were transfected with pcDNA5-FLAG-TAK1 and/or pcDNA5-HA-Fpr2. 48 h later, cells were collected, and cellular lysates were subjected to IP using antibodies against (J) HA or (K) Flag. (L) GST-Fpr2 or GST was retained on glutathione-sepharose beads, followed by incubation with immunopurified Flag-TAK1 from HEK293 T cells, which were then immunoblotted using the antibody against Flag. Data represented as mean ± SEM. ***P < 0.001 vs Con/WT or Con/siCon group; ++P < 0.01 vs LPS/WT or LPS/siCon group; #P < 0.05 and ##P < 0.01.

knockdown in RAW264.7 cells cultured in LPS (Fig. 5E). At the same time, oeFpr2-enhanced activation of NF-κB (p65) and MAPKs was markedly abrogated in LPS-treated cells with TAK1 knockdown (Fig. 5F and G). Taken together, these in vitro experiments illustrated that Fpr2regulated ALI was largely associated with the activation of TAK1 signaling.

inflammatory factors (Fig. 4D). To further calculate the role of Fpr2 in mediating ALI, we over-expressed Fpr2 in RAW264.7 cells (Fig. 4E). In Addition, Nrf2 selective activator RA-839 was pre-treated to cells with or without Fpr2 over-expression to further investigate the role of Nrf2 during ALI progression regulated by Fpr2. As shown in Fig. 4F, LPSinduced ROS accumulation was further promoted by the over-expression of Fpr2. However, this effect was markedly abrogated by RA-839 pre-treatment. Also, Fpr2 over-expression significantly decreased Nrf2 mRNA levels compared to oeNC/LPS group, which was restored by RA839 (Fig. 4G). Then, RT-qPCR analysis suggested that LPS-stimulated expression of TNF-α, IL-1β and IL-6 was further accelerated by Fpr2 over-expression, and of note, RA-839 pre-treatment significantly abolished these effects potentiated by oeFpr2 (Fig. 4H). Another Nrf2 activator tBHQ was used to confirm the potential relationship between Fpr2 and Nrf2, and obvious nuclear Nrf2 translocation was observed in RAW264.7 cells treated with tBHQ (Supplementary Fig. 1A). Consistently, tBHQ showed similar effects as RA-839 performed in RAW264.7 cells with or without Fpr2 over-expression following LPS stimulation (Supplementary Fig. 1B–D). These results demonstrated that Fpr2-induced ROS production could be partially reversed by Nrf2 activation. TAK1 activation plays a pivotal role in regulating NF-κB and MAPKs signaling, and it has been reported to be meditated by Fpr2 during obesity development [24–26]. Thus, we supposed that TAK1 activation might be also involved in Fpr2-modulated ALI. As illustrated in Fig. 4I, LPS-induced up-regulation of p-TAK1 was markedly reduced by Fpr2 knockout or knockdown following the in vivo or in vitro experiments. Then, we further sought to identify the intermediate mechanism linking Fpr2 and TAK1 activation. Immunoprecipitation analysis indicated that the ectopically expressed Fpr2 could interact with TAK1, but the TAK1 phosphorylation levels were indistinguishable between these two groups (Fig. 4J and K). Next, a GST pull-down analysis with GST-tagged Fpr2 to explore if the binding of Fpr2 to TAK1 occurred through a direct interaction. In line with the immunoprecipitation findings, TAK1 was eluted along with Fpr2 (Fig. 4L). Collectively, blocking ROS could reverse Fpr2-regulated exacerbation of ALI by Nrf2, and Fpr2 could directly interact with TAK1.

4. Discussion Here, we for the first time revealed the previously unrecognized biological function of Fpr2 in ALI and provided evidence supporting the role of Fpr2 in ALI as a critical factor that functioned to promote inflammation and ROS accumulation. During ALI induced by LPS, a progressive increase in Fpr2 expression was detected. Fpr2 deletion significantly alleviated LPS-induced ALI, as evidenced by the improved histological changes, the reduced W/D ratio and protein level in BALF, which account for an elevated pulmonary permeability and lung edema in LPS-instillation lungs [27]. Murine macrophage RAW264.7 cells are widely used to mimic inflammation to evaluate the potential protective effect of drugs in vitro for conditions such as wound healing in diabetic rats [28], acute lung injury [29], and mice ear oedema [30]. Previous studies have indicated that macrophages release many pro-inflammatory cytokines, such as IL-1β, TNF-α and IL-6, in the early stages of inflammation induced by various pathogenic stimulants, such as LPS; excessive production of pro-inflammatory cytokines increases the extent of immune responses, which in turn results in inflammatory cascades and tissue injury [31,32]. Thus, RAW264.7 cells were used as the in vitro model to mimic ALI. Based on the in vivo and in vitro analysis, we clearly indicated that Fpr2 expression from mRNA and protein levels were markedly up-regulated during ALI progression induced by LPS. Suppressing Fpr2 expression could alleviate LPS-triggered ALI, which might be associated with the blockage of NF-κB and MAPKs signaling pathways and the activation of Nrf2 signaling. Importantly, Fpr2regulated ROS production was largely associated with the activation of Nrf2, which could subsequently modulate the pro-inflammatory response. Finally, by the use of Co-IP and GST pull down assays, we found that Fpr2 could directly interact with TAK1, which might be involved in ALI progression, subsequently meditating NF-κB and MAPKs signaling and inflammation (Fig. 5H). Thus, reducing Fpr2 expression represented a promising therapeutic strategy for ALI treatment. Fpr2 is a critical regulator in macrophage M1 polarization. Fpr2 knockout ameliorates high fat diet-induced inflammation in white adipose tissues [26]. Additionally, the deletion of Fpr2 attenuates sepsis in mice challenged by LPS, contributing to the improvement of cardiac dysfunction, which is associated with the suppression of inflammatory response [12]. Thus, Fpr2 plays an important role in regulating inflammatory response, and might be involved in LPS-induced ALI. It has been suggested that LPS-induced an ALI model is similar with pathological features to ALI in humans through inducing excessive releases of inflammatory regulators and chemokines [3,4,33]. The inflammatory cells, including dendritic cells, macrophages, neutrophils, lymphocytes and basophils, impair the endothelial barrier [34]. In our study, we also found that Fpr2 deletion markedly reduced LPS-induced inflammation

3.5. Fpr2-mediated ALI requires activation of TAK1 signaling To further confirm the effects of Fpr2/TAK1 signaling pathway on ALI progression, we then transfected Flag-Fpr2 in RAW264.7 cells. After LPS stimulation, we found that p-TAK1 expression levels were dose-dependently up-regulated by Fpr2 (Fig. 5A). Subsequently, we performed gain and loss-of-function studies in RAW264.7 cells. RAW264.7 cells were transfected with either siTAK1 to knockdown TAK1 or oeTAK1 to overexpress TAK1 (Fig. 5B). RT-qPCR analysis demonstrated that Fpr2 knockdown-reduced mRNA levels of TNF-α, IL1β and IL-6 were markedly rescued by oeTAK1 in LPS-stimulated RAW264.7 cells, which were accompanied with significantly restored protein expression levels of p-NF-κB (p65), p-p38, p-ERK1/2 and p-JNK (Fig. 5C and D). However, we also found that oeFpr2-promoted expression of TNF-α, IL-1β and IL-6 was highly alleviated by TAK1 8

Biomedicine & Pharmacotherapy 125 (2020) 109943

H. Liu, et al.

Fig. 5. Fpr2-mediated ALI requires activation of TAK1 signaling. (A) RAW264.7 cells were transfected with varying amounts of Flag-tagged Fpr2 for 24 h, and then were incubated in the absence or presence of LPS (100 ng/ml) for another 24 h. Then, all cells were harvested for western blot analysis of p-TAK1 and Flag. (B) RAW264.7 cells were transfected with siTAK1 or oeTAK1 for 24 h, followed by transfection efficacy analysis using western blotting. (C,D) RAW264.7 cells were transfected with siFpr2 combined with or without oeTAK1 for 24 h, and then were subjected to LPS (100 ng/ml) for another 24 h. (C) RT-qPCR analysis was used to calculate TNF-α, IL-1β and IL-6 mRNA expression levels in cells. (D) Western blot analysis was performed to determine p-NF-κB, p-p38, p-ERK1/2 and p-JNK protein expression levels in cells. (E–G) RAW264.7 cells were transfected with oeFpr2 combined with or without siTAK1 for 24 h, followed by LPS (100 ng/ml) incubation for another 24 h. (E) TNF-α, IL-1β and IL-6 mRNA levels were measured by RT-qPCR analysis. (F,G) The protein expression levels of p-NF-κB, p-p38, p-ERK1/2 and pJNK in cells were assessed using western blot analysis. (H) A model demonstrating how Fpr2 signaling regulated LPS-induced ALI. Data represented as mean ± SEM. # P < 0.05 and ##P < 0.01.

9

Biomedicine & Pharmacotherapy 125 (2020) 109943

H. Liu, et al.

inflammatory response eventually [49–51]. Notably, here the in vitro analysis showed that TAK1 over-expression could markedly abrogate the role of Fpr2 knockdown in attenuating inflammation, NF-κB and MAPKs activation in RAW264.7 cells treated with LPS. However, reducing TAK1 significantly alleviated Fpr2-promoted inflammatory injury in LPS-incubated RAW264.7 cells. Thus, consistent with previous analysis [49–51], TAK1 could meditate NF-κB and MAPKs signaling pathways to control the inflammatory response in RAW264.7 cells with LPS stimulation. Collectively, Fpr2-regulated inflammation during ALI progression induced by LPS was largely dependent on TAK1 activation. Suppression of TAK1 pathway could counteract the inflammatory endothelial cell phenotype induced by oxidative stress [52]. Thus, we supposed that Fpr2-regulated ALI was largely through its interaction with TAK1. In addition, there are many or different signaling pathways that could contribute to the progression of ALI, such as endoplasmic reticulum (ER) stress, apoptosis and autophagy [53–55]. Meanwhile, Fpr2 was also implicated in the development of cancer by regulating the metastasis process [56,57]. Considering all of these factors, further studies are still warranted either in ALI or in other inflammatory diseases in future to fully reveal the molecular mechanism through which Fpr2 regulates the progression of diseases. In conclusion, we further provided the evidence that Fpr2 was a pivotal molecular for the progression of pathological ALI. Fpr2 suppression could reduce oxidtaive stress and inflammation, which were largely associated with the activation of Nrf2 signaling. Importantly, Fpr2 could directly interact with TAK1 to control the inflammatory response mainly through regulating NF-κB and MAPKs signaling pathways (Fig. 5H). Therefore, reducing Fpr2 may represent a promising therapeutic strategy for ALI treatment.

in lung tissues, as evidenced by the decreased expression levels of proinflammatory factors, including CD68, CD11b, F4/80, TNF-α, IL-1β, IL6, IL-18, CXCL1, CXCL2, CCL2 and MCP1 [35]. NF-κB and MAPKs signaling pathways play crucial roles in regulating inflammation, which are involved in ALI development [4,36,37]. IKK-α is activated by LPS, which then leads to the phosphorylation of IκB, resulting in NF-κB (p65) nuclear translocation and eventually inducing inflammatory cytokine gene expression [4]. The MAPK pathway includes p38, JNK and ERK1/ 2, which have important functions in acute lung injury. Accordingly, p38 activation leads to a large amount of inflammatory events [38]. The ERK1/2 also has significant roles in modulating various cytokines [39]. Here in our present study, we also found that Fpr2 deletion-alleviated inflammatory response was also attributed to its blockage of NF-κB and MAPKs signaling pathways, as proved by the reduced phosphorylation of IKKα, I-κBα, NF-κB, p38, ERK1/2 and JNK, which were verified in LPS-incubated RAW264.7 cells transfected with siFpr2. Oxidative stress could exaggerate pro-inflammatory gene expression, and also inflammatory cells could similarly induce overproduction of ROS, leading to a vicious cycle to provoke the occurrence and progression of various diseases, including ALI [5,40]. The stimulation of LPS results in the accumulation of free radicals that causes lipid peroxidation and the over-generation of oxidation markers such as MDA. At the same time, the activities of anti-oxidant enzymes, such as SOD and GSH, are repressed, and the balance is impaired [41]. The Nrf2 signaling is one of the most essential pathways that counteract oxidative stress. The oxidative-stress responsive enzymes HO1 and NQO1 are down-streaming targets of Nrf2 [6]. Mice lacking Nrf2 are extremely susceptible to oxidative damage [42]. Fpr2 could bind to a wide variety of amyloid-like ligands to induce NADPH oxidase-dependent oxidative stress, which is critically involved in Alzheimer’s disease progression and irreversible neurodegeneration [43]. Here in the study, we notably found that Fpr2 deletion-alleviated ALI was also attributed to the suppression of oxidative stress, as shown by the reduced MDA levels and ROS production, while the improved SOD, GSH, HO1 and NQO1 expression levels. These effects were largely through improving the Nrf2 signaling. Nrf2 plays a critical role in regulating ROS production, which in turn could meditate inflammation in various types of diseases, including ALI [44]. Here, our in vitro studies suggested that Fpr2 knockdown-alleviated ROS generation and inflammation was rescued by Nrf2 suppression using ML385; however, promoting Fpr2 expression in LPSincubated RAW264.7 cells significantly further enhanced ROS accumulation and inflammatory response. As expected, these effects could be reversed by Nrf2 promotion using RA-839 or tBHQ. Therefore, Fpr2 deletion-ameliorated ALI was largely associated with Nrf2 activation to subsequently inhibit oxidative stress and inflammation. However, whether Fpr2 regulated Nrf2 in a direct or indirect manner, more studies are still required. TAK1 is an indispensable modulator of a wide variety of physiological events, especially in progression and the immune response [45]. TAK1-regulated effects range from metabolic disturbances to inflammation and cell death, depending on the specific stimulus and context [46]. Previous study has identified TAK1 as a central signal transducer that regulates the development of ALI [47]. Recently, Fpr2 was suggested to modulate TAK1 activation to control the progression of inflammation in adipose white tissues, which was involved in high fat diet-induced obesity [26]. Here, we found that Fpr2 deletion could repress TAK1 phosphorylation in pulmonary tissues and RAW264.7 stimulated by LPS, which were in line with previous study [12,26]. Intriguingly, our immunoprecipitation and GST pull-down analysis indicated that Fpr2 could directly interact with TAK1. TAK1 has been implicated in inflammation and oxidative stress. Accumulating studies have reported that TAK1 activation plays a critical role in meditating oxidative stress, which is also associated with Nrf2 nuclear translocation under different stresses [48]. Moreover, TAK1 activation was reported to regulate the phosphorylation of NF-κB and MAPKs signaling pathways under different stresses, contributing to the progression of

Declaration of Competing Interest The authors declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2020.109943. References [1] T. Zhu, et al., Andrographolide protects against LPS-induced acute lung injury by inactivation of NF-κB, PLoS One 8 (2013) e56407. [2] W. Jiang, et al., The protective effect of Trillin LPS-induced acute lung injury by the regulations of inflammation and oxidative state, Chem. Biol. Interact. 243 (2016) 127–134. [3] L. Wang, et al., Effects of resolvin D1 on inflammatory responses and oxidative stress of lipopolysaccharide-induced acute lung injury in mice, Chin. Med. J. 127 (2014) 803–809. [4] X. Chen, et al., Kaempferol regulates MAPKs and NF-κB signaling pathways to attenuate LPS-induced acute lung injury in mice, Int. Immunopharmacol. 14 (2012) 209–216. [5] J. Grommes, et al., Simvastatin reduces endotoxin-induced acute lung injury by decreasing neutrophil recruitment and radical formation, PLoS One 7 (6) (2012) e38917. [6] S.N. Cooray, et al., Ligand-specific conformational change of the G-protein-coupled receptor ALX/FPR2 determines proresolving functional responses, Proc. Natl. Acad. Sci. 110 (2013) 18232–18237. [7] S. Bena, et al., Annexin A1 interaction with the FPR2/ALX receptor identification of distinct domains and downstream associated signaling, J. Biol. Chem. 287 (2012) 24690–24697. [8] D.Y. Richard, et al., International Union of Basic and Clinical Pharmacology. LXXIII. Nomenclature for the formyl peptide receptor (FPR) family, Pharmacol. Rev. 61 (2009) 119–161. [9] K. Chen, et al., Regulation of inflammation by members of the formyl-peptide receptor family, J. Autoimmun. 85 (2017) 64–77. [10] H.Q. He, et al., The formyl peptide receptors: diversity of ligands and mechanism for recognition, Molecules 22 (2017) 455. [11] S.N. Cooray, et al., Ligand-specific conformational change of the G-protein–coupled receptor ALX/FPR2 determines proresolving functional responses, Proc. Natl. Acad.

10

Biomedicine & Pharmacotherapy 125 (2020) 109943

H. Liu, et al.

L333–L341. [34] J.W. Card, et al., Gender differences in murine airway responsiveness and lipopolysaccharide-induced inflammation, J. Immunol. 177 (2006) 621–630. [35] A. Willeford, et al., CaMKIIδ-mediated inflammatory gene expression and inflammasome activation in cardiomyocytes initiate inflammation and induce fibrosis, JCI Insight 3 (2018). [36] Y. Yan, et al., MicroRNA-223 attenuates LPS-induced inflammation in an acute lung injury model via the NLRP3 inflammasome and TLR4/NF-κB signaling pathway via RHOB, Int. J. Mol. Med. 43 (2019) 1467–1477. [37] K.C. Wu, et al., Ugonin M, a Helminthostachys zeylanica constituent, prevents LPSinduced acute lung injury through TLR4-mediated MAPK and NF-κB signaling pathways, Molecules 22 (2017) 573. [38] J.F. Schindler, et al., p38 pathway kinases as anti-inflammatory drug targets, J. Dent. Res. 86 (2007) 800–811. [39] X. Shi, et al., Curcumin inhibits Aβ-induced microglial inflammatory responses in vitro: involvement of ERK1/2 and p38 signaling pathways, Neurosci. Lett. 594 (2015) 105–110. [40] Y. Imai, et al., Identification of oxidative stress and Toll-like receptor 4 signaling as a key pathway of acute lung injury, Cell 133 (2008) 235–249. [41] H.S. Park, et al., Impact of oxidative stress on lung diseases, Respirology 14 (2009) 27–38. [42] J. Yan, et al., Nrf2 protects against acute lung injury and inflammation by modulating TLR4 and Akt signaling, Free Radic. Biol. Med. 121 (2018) 78–85. [43] P. Iribarren, et al., Role of formyl peptide receptor-like 1 (FPRL1/FPR2) in mononuclear phagocyte responses in Alzheimer disease, Immunol. Res. 31 (2005) 165–176. [44] N.M. Reddy, et al., Conditional deletion of Nrf2 in airway epithelium exacerbates acute lung injury and impairs the resolution of inflammation, Am. J. Respir. Cell Mol. Biol. 45 (2011) 1161–1168. [45] S. Sato, et al., Essential function for the kinase TAK1 in innate and adaptive immune responses, Nat. Immunol. 6 (2005) 1087. [46] A.A. Ajibade, et al., Cell type-specific function of TAK1 in innate immune signaling, Trends Immunol. 34 (2013) 307–316. [47] S. Yang, et al., Therapeutic effect of methyl salicylate 2-O-β-d-lactoside on LPSinduced acute lung injury by inhibiting TAK1/NF-kappaB phosphorylation and NLRP3 expression, Int. Immunopharmacol. 40 (2016) 219–228. [48] K. Hashimoto, et al., TAK1 regulates the Nrf2 antioxidant system through modulating p62/SQSTM1, Antioxid. Redox Signal. 25 (2016) 953–964. [49] W. Zou, et al., A novel biological role of α-mangostin via TAK1–NF-κB pathway against inflammatory, Inflammation 42 (2019) 103–112. [50] S. Chen, et al., Wogonin inhibits LPS-induced inflammatory responses in rat dorsal root ganglion neurons via inhibiting TLR4–MyD88–TAK1-mediated NF-κB and MAPK signaling pathway, Cell. Mol. Neurobiol. 35 (2015) 523–531. [51] Z.B. Zhang, et al., Curcumin’s metabolites, tetrahydrocurcumin and octahydrocurcumin, possess superior anti-inflammatory effects in vivo through suppression of TAK1-NF-κB pathway, Front. Pharmacol. 9 (2018) 1181. [52] E.S. Lee, et al., Suppression of TAK1 pathway by shear stress counteracts the inflammatory endothelial cell phenotype induced by oxidative stress and TGF-β1, Sci. Rep. 7 (2017) 42487. [53] M. Zeng, et al., 4-PBA inhibits LPS-induced inflammation through regulating ER stress and autophagy in acute lung injury models, Toxicol. Lett. 271 (2017) 26–37. [54] M. Xu, et al., Tanshinone IIA therapeutically reduces LPS-induced acute lung injury by inhibiting inflammation and apoptosis in mice, Acta Pharmacol. Sin. 36 (2015) 179. [55] X. Yang, et al., Hydroxytyrosol attenuates LPS-induced acute lung injury in mice by regulating autophagy and sirtuin expression, Curr. Mol. Med. 17 (2017) 149–159. [56] T.S. Gastardelo, et al., Inflammation and cancer: role of annexin A1 and FPR2/ALX in proliferation and metastasis in human laryngeal squamous cell carcinoma, PLoS One 9 (2014) e111317. [57] X.L. Hou, et al., FPR2 promotes invasion and metastasis of gastric cancer cells and predicts the prognosis of patients, Sci. Rep. 7 (2017) 3153.

Sci. 110 (2013) 18232–18237. [12] T. Gobbetti, et al., Nonredundant protective properties of FPR2/ALX in polymicrobial murine sepsis, Proc. Natl. Acad. Sci. 111 (2014) 18685–18690. [13] X. Chen, et al., Kaempferol regulates MAPKs and NF-κB signaling pathways to attenuate LPS-induced acute lung injury in mice, Int. Immunopharmacol. 14 (2012) 209–216. [14] M. Ritz, et al., Determination of rpoA as the most suitable internal control to study stress response in C. jejuni by RT-qPCR and application to oxidative stress, J. Microbiol. Methods 76 (2009) 196–200. [15] J. Lu, et al., Interferon regulatory factor 3 is a negative regulator of pathological cardiac hypertrophy, Basic Res. Cardiol. 108 (2013) 326. [16] A. Kotanidou, et al., Inhaled activated protein C attenuates lung injury induced by aerosolized endotoxin in mice, Vascul. Pharmacol. 45 (2006) 134–140. [17] J.W. Card, et al., Gender differences in murine airway responsiveness and lipopolysaccharide-induced inflammation, J. Immunol. 177 (2006) 621–630. [18] C. Godugu, et al., Inhalation delivery of Telmisartan enhances intratumoral distribution of nanoparticles in lung cancer models, J. Control. Release 172 (2013) 86–95. [19] M. Wei, et al., Expression of phosphatidylinositol-3 kinase and effects of inhibitor Wortmannin on expression of tumor necrosis factor-α in severe acute pancreatitis associated with acute lung injury, World J. Emerg. Med. 6 (2015) 299. [20] C. Nussbaum, et al., Myeloperoxidase: a leukocyte-derived protagonist of inflammation and cardiovascular disease, Antioxid. Redox Signal. 18 (2013) 692–713. [21] X. Zhang, et al., Berberine activates Nrf2 nuclear translocation and inhibits apoptosis induced by high glucose in renal tubular epithelial cells through a phosphatidylinositol 3-kinase/Akt-dependent mechanism, Apoptosis 21 (2016) 721–736. [22] C.J. Liou, et al., Water extract of Helminthostachys zeylanica attenuates LPS-induced acute lung injury in mice by modulating NF-κB and MAPK pathways, J. Ethnopharmacol. 199 (2017) 30–38. [23] R.L. Cho, et al., Haem oxygenase-1 up-regulation by rosiglitazone via ROS-dependent Nrf2-antioxidant response elements axis or PPARγ attenuates LPS-mediated lung inflammation, Br. J. Pharmacol. 175 (2018) 3928–3946. [24] F.T. Huang, et al., miR-143 targeting TAK1 attenuates pancreatic ductal adenocarcinoma progression via MAPK and NF-κB pathway in vitro, Dig. Dis. Sci. 62 (2017) 944–957. [25] R. Mao, et al., TAK1 lysine 158 is required for TGF-β-induced TRAF6-mediated Smad-independent IKK/NF-κB and JNK/AP-1 activation, Cell. Signal. 23 (2011) 222–227. [26] X. Chen, et al., Fpr2 deficiency alleviates diet-induced insulin resistance through reducing body weight gain and inhibiting inflammation mediated by macrophage chemotaxis and M1 polarization, Diabetes (2019) db180469. [27] C.L. Tsai, et al., Baicalein, an active component of Scutellaria baicalensis, protects against lipopolysaccharide-induced acute lung injury in rats, J. Ethnopharmacol. 153 (2014) 197–206. [28] Q. Zhang, et al., Effects of dimethyloxalylglycine-embedded poly (ε-caprolactone) fiber meshes on wound healing in diabetic rats, ACS Appl. Mater. Interfaces 9 (2017) 7950–7963. [29] S. Yang, et al., The natural product bergenin ameliorates lipopolysaccharide-induced acute lung injury by inhibiting NF-kappaB activition, J. Ethnopharmacol. 200 (2017) 147–155. [30] Y.S. Jung, et al., Anti-inflammatory effect of tricin 4’-O-(threo-β-guaiacylglyceryl) ether, a novel flavonolignan compound isolated from Njavara on in RAW264. 7 cells and in ear mice edema, Toxicol. Appl. Pharmacol. 277 (2014) 67–76. [31] G. Zhao, et al., The anti-inflammatory effects of interferon tau by suppressing NFκB/MMP9 in macrophages stimulated with Staphylococcus aureus, J. Interferon Cytokine Res. 36 (2016) 516–524. [32] T. Wang, et al., Preventative effect of OMZ-SPT on lipopolysaccharide-induced acute lung injury and inflammation via nuclear factor-kappa B signaling in mice, Biochem. Biophys. Res. Commun. 485 (2017) 284–289. [33] M. Rojas, et al., Endotoxin-induced lung injury in mice: structural, functional, and biochemical responses, Am. J. Physiol. Lung Cell. Mol. Physiol. 288 (2005)

11