Selenium-Containing Compound Ameliorates Lipopolysaccharide-Induced Acute Lung Injury via Regulating the MAPK/AP-1 Pathway

Abstract—Acute lung injury (ALI) is characterized by a series of inflammatory reactions and serves as the main cause of mortality in intensive care unit patients. Although great progress has been made in understanding the pathophysiology of ALI, there are no effective treatments in clinic. Recently, we have synthesized a selenium-containing compound, which possesses obvious anti-inflammatory activity. The aim of the present study is to evaluate the protective effects of the selenium-containing compound 34# in LPS-induced ALI in mice as well as its underlying mechanism. Compound 34# was found to inhibit LPS-induced macrophage inflammatory cytokine release. These effects were observed to be produced via suppression of the MAPK/AP-1 pathway. Compound 34# was also noted to attenuate the LPS-induced lung inflammation in mice with ALI. The corresponding results suggested that compound 34# possesses remarkable protective effects on LPS-induced ALI. Furthermore, the MAPK/AP-1 pathway may prove to be the underlying mechanism. Accordingly, compound 34# may serve as a potential candidate for the prevention of ALI.


INTRODUCTION
Acute lung injury (ALI) is characterized by acute diffuse lung injury due to various internal and external lung factors. ALI always follows a series of inflammatory reactions, such as increased pulmonary capillary permeability, release of inflammatory mediators, and pulmonary interstitial and alveolar edema. Diffuse alveolar injury increases pulmonary microvascular permeability, leading to pulmonary edema and hyaline membrane formation, which may also be accompanied by pulmonary interstitial fibrosis. ALI physiologically manifests as decreased lung volume, decreased lung compliance, and severe ventilation/blood flow imbalance, which clinically manifests as acute and Wenjing Jia and Wenting Ding authors contribute equally to this work. 1 To whom correspondence should be addressed at Medicine and Health Care Center, First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325035, Zhejiang, China. Email: zhuzaisheng@ wmu.edu.cn progressive hypoxic respiratory insufficiency. When ALI progresses further and shows signs of obvious respiratory distress, refractory hypoxemia, or even respiratory failure, it is termed acute respiratory distress syndrome (ARDS) [1]. A previous study has demonstrated that among ICUs in 50 countries, the period prevalence of ARDS was 10.4% of ICU admissions, having a high mortality rate [2]. Current treatment methods and effects of ALI/ARDS are very limited. In addition to treating the primary disease, the main treatments are respiratory support treatment, fluid management, and other supportive therapies [3]. Therefore, determining effective drugs for the treatment of ALI is of clinical significance.
Excessive inflammation is known to play an important role in ALI. Excessive inflammation includes the infiltration of inflammatory cells and release of cytokines, such as inflammatory factors, adhesion molecules, and chemokines [4]. Lipopolysaccharide (LPS) is the main cell wall component of Gram-negative bacteria. LPS is commonly used to establish the ALI model, which explores the mechanism and potential therapies of ALI. There is growing evidence that LPS can activate MAPK pathways, such as c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 pathways, which stimulate the production of various inflammatory factors, leading to lung tissue injury [5].
Selenium is an important trace element found in the human body. Selenium decrease was found to be correlated with many severe diseases. Manzanares et al. have reported that selenium decreased early in serum/plasma in critically ill patients suffering from sepsis/septic shock [6]. Selenium administration can diminish many diseases, such as apoptosis [7], cancers [8], diabetes [9], liver injury [10], and lung injury [11,12]. Meanwhile, compounds containing selenium in their structure have demonstrated significant antifungal [13], anticancer [14,15], and antiinflammatory [16] activities. Recently, we have synthesized a selenium-containing compound 3-(phenylselanyl)-1H-pyrrolo [2,3-b]pyridine 34#, (Fig. 1a), whose detailed information is exhibited in the Supplementary information. In this study, we illustrate its anti-inflammatory activities and its underlying mechanism in conjunction with its protective effects on LPS-induced ALI.

Reagents
Lipopolysaccharide (LPS) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for P38, p-P38, JNK, p-JNK, ERK, p-ERK, p-c-jun, GAPDH, and IκB were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-F4/80 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-c-jun and anti-LY6G were obtained from Abcam (Cambridge, UK). Antibodies for CD11B, CD45, and LY6C were obtained from BD Pharmingen (New York, USA). The mouse TNF-α and IL-6 ELISA kits were purchased from eBioscience (San Diego, CA, USA). Compound 34# was synthesized in our lab and structurally identified using MS and 1 H-NMR analyses. For in vivo studies, 34# was dissolved in 0.5% carboxymethylcellulose sodium (CMCNa) in water solution and 0.5% CMCNa alone was used as vehicle control. For cell culture studies, 34# was dissolved in dimethyl sulfoxide (DMSO), and the same volume of DMSO alone was used as the vehicle control.

Cell Culture
Mouse primary peritoneal macrophages (MPMs) were prepared from male C57BL/6 mice. C57BL/6 mice were intraperitoneal (i.p.) injected of 2.5 mL starch broth which was constituted with 1 g tryptone, 0.5 g NaCl, 6 g soluble starch, and 0.3 g beef extract, and boiled in 100 mL double-distilled H 2 O. After 2 days, peritoneal macrophages were collected by washing the peritoneal cavity with 10 mL RPMI-1640 medium (Gibco, Eggenstein, Germany) per mouse. The cells were centrifuged and resuspended in RPMI-1640 medium with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA). The macrophages were cultured at 37 °C in 5% CO 2 -humidified air. Four hours later, the non-adherent cells were removed by washing with PBS. Firmly adhered macrophages were used for experiments.

Animal Care and ALI Mouse Model
Male C57BL/6 mice (18-20 g) were obtained from the Animal Centre of Wenzhou Medical University (Wenzhou, China). Animals were housed at a constant room temperature with a 12:12 h light-dark cycle and fed with a standard rodent diet for at least 7 days before being used at the Animal Centre of Wenzhou Medical University. All animal care and experimental procedures were approved by the Wenzhou Medical University Animal Policy and Welfare Committee (Approval Document No. wydw2019-0438). All animals received humane care according to the National Institutes of Health (USA) guidelines.

MTT Assay
MPMs were seeded into 96-well plates at a density of 5 × 10 4 cells per well in RPMI-1640 medium with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin. Cells were incubated with different concentrations of 34# (2.5, 5, 10, 20, and 40 μM) for 24 h. Then, 20 μL MTT (5 mg/mL) was added to all wells, and the plate was incubated in 5% CO 2 at 37 °C for another 4 h. Cells were dissolved with 150 μL DMSO and were then analyzed in a multi-well-plate reader at 490 nm.

Determination of Cytokines
After treatment of cells with 34# and LPS, TNF-α and IL-6 content in culture medium and serum was determined by ELISA according to the manufacturer's instructions (Bioscience, San Diego, CA). The total amount of TNF-α or IL-6 in medium was normalized to the protein concentration of lysates.

RNA Extraction and Real-time Quantitative PCR
MPMs and lung tissues (10-20 mg) were homogenized in TRIzol (Invitrogen, Carlsbad, CA, USA) for total RNA extraction. The purity of the sample was estimated by calculating the OD ratio (A260/A280, ranging from 1.8 to 2.2). Both reverse transcription and quantitative PCR (qPCR) was carried out using IQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). QuantStudio® 3 Real Time PCR Systems (ABI, CA, USA) was used for qPCR analysis. The primers of the target genes are listed in Table 1 and were obtained from Invitrogen (Shanghai, China).

Western Blot Assay
MPMs were pretreated with DMSO (vehicle) or 10 μM 34# for 30 min, which was followed by 0.5 μg/mL LPS for 15 min. Collected cells or homogenized lung tissues were then lysed. Concentration of total proteins was determined using the Bradford assay (Bio-Rad, Hercules, CA, USA).
Lysates were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and were electrotransferred to a nitrocellulose membrane. The membranes were then blocked for 1.5 h at room temperature in Trisbuffered saline (pH 7.6) containing 0.05% Tween 20 and 5% non-fat dry milk. Primary antibody incubations were carried out at 4 °C overnight. Secondary antibodies were then introduced for 1 h at room temperature. Immunoreactivity was visualized using enhanced chemiluminescence reagents (BI, Beit Haemek, Israel) and was quantified using ImageJ analysis software version 1.38e (NIH, Bethesda, MD, USA). Values were normalized to their respective protein controls.

BALF Analysis
A tracheal cannula was inserted into the primary bronchus, and BALF was performed through the cannula using Ca 2+ /Mg 2+ -free PBS. Approximately 0.8 mL BALF was acquired. The collected BALF was centrifuged at 3000 rpm for 10 min at 4 °C. The supernatant was then immediately stored at −80 °C for protein concentration and cytokine determination. The sediment was resuspended in 50 μL physiological saline in order to determine the number of total cells using a cell counting instrument (Countstar, Shanghai, China).

Flow Cytometric Analysis
The cells were resuspended in the collected BALF in PBS without Ca 2+ /Mg 2+ . The cells were mixed in the BALF of each group of mice together in pairs, after which 100 μL cell suspension was taken from each tube. Next, CD11B, CD45, and LY6C antibodies were used to stain the cells for 30 min. The cells were then washed with 1 mL PBS, centrifuged at 5000 rpm for 5 min, and resuspended with 500 μL PBS. Accuri C6 Plus (488 nm) flow cytometer and FlowJo software were then used to analyze the cell subpopulations.

Histopathology and Immunohistochemistry
Lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 μm thickness. After dehydration, sections were stained with hematoxylin and eosin (H&E) and were evaluated for general histopathological damage using light microscopy (Nikon, Japan).
Paraffin sections were also used to perform immunohistochemistry for CD68 and LY6G using routine techniques. Sections were then deparaffinized, rehydrated, treated with 3% H 2 O 2 for 30 min to block endogenous peroxidase activity, and blocked with 1% BSA for 30 min. Slides were incubated overnight at 4 °C with primary antibodies, and immunoreactivity was detected by diaminobenzidine (DAB). Slides were counterstained with hematoxylin for 5 min, dehydrated, and mounted for viewing by bright-field microscopy (Nikon, Japan). The percentage expression was measured using ImageJ software (NIH, Bethesda, MD, USA).

Myeloperoxidase
The myeloperoxidase (MPO) test kit was used to measure MPO activity in lung tissue. The lung tissue was homogenized according to the kit's instructions, and was centrifuged at 12,000 rpm at 4 °C for 10 min. The supernatant was then taken for MPO activity detection and indicated in the form of units per gram of protein. BCA assay was used to determine the total protein content in the sample.

Statistical Analysis
All experiments were randomized and blinded. In vitro experiments were repeated at least three times. All data are expressed as mean ± SEM. All statistical analyses were performed using GraphPad Pro Prism 8.0 (Graph-Pad, San Diego, CA). We used one-way ANOVA, followed by Dunn's post hoc test when comparing multiple independent groups. Differences between group means were considered statistically significant at p < 0.05.

Compound 34# Inhibits LPS-Induced Expression of Inflammatory Cytokines in MPMs
In order to test the cytotoxicity of compound 34#, the MTT assay was used. Figure 1b illustrates that compound 34# at 2.5, 5, 10, 20, and 40 μM did not show obvious cytotoxicity. The effects of compound 34# on LPS-induced tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) secretion were evaluated through an ELISA assay. MPMs were pretreated with different concentrations (2.5, 5, 10, 20, and 40 μM) of 34# for 30 min and were stimulated with LPS for 24 h. The corresponding results demonstrated that, compared to the blank control group, LPS significantly increased the production of TNF-α (Fig. 1c) and IL-6 ( Fig. 1d). Moreover, the 34# pretreatment dose-dependently inhibited LPS-induced inflammatory cytokine release.
Meanwhile, the gene expression of inflammatory mediators was detected by RT-qPCR. MPMs were pretreated with different concentrations (5, 10, and 20 μM) of 34# for 30 min and were stimulated by LPS for 6 h. The results (Fig. 1e and f) showed that LPS significantly increased the gene expression of inflammatory cytokines (TNF-α, IL-6, IL-1β), adhesion molecules (ICAM-1, VCAM-1), and chemokine (MCP-1). However, pretreatment with 34# dose-dependently inhibited the LPSinduced increase.  TNF-α  Mouse  CAG GGG CCA CCA CGC TCT TC  TTT GTG AGT GTG AGG GTC TGG   IL-6  Mouse  GAG GAT ACC ACT CCC AAC AGACC  AAG TGC ATC ATC GTT GTT CAT ACA   IL-1β  Mouse  ACT CCT TAG TCC TCG  Macrophages were pretreated with various concentrations of 34# for 24 h, and cell viability was analyzed via MTT assay. c, d Macrophages were plated at a density of 5 × 10 5 /plate and were cultured overnight in medium containing 10% serum, which was pretreated with 34# at 2.5, 5, 10, 20, or 40 μM for 30 min followed by stimulation with 0.5 μg/mL LPS for 24 h. DMSO was used as the vehicle control. Supernatants were collected and analyzed for TNF-α c and IL-6 d release using ELISA. e, f MPMs were pretreated with the vehicle control (DMSO) and 34# (5, 10, or 20 μM) for 30 min, after which they were incubated with LPS at 0.5 μg/mL for 6 h. The mRNA levels of TNF-α, IL-6, IL-1β, VCAM-1, ICAM-1, and MCP-1 were quantified by RT-qPCR. Data were normalized to β-actin and were expressed as percentage of LPS group. Data were normalized to total protein concentration from the same plate and were expressed as fold change relative to LPS group. *P < 0.05, **P < 0.01, ***P < 0.001 vs LPS group.

Compound 34# Inhibited LPS-Induced MAPK Phosphorylation in MPMs
MAPK/AP-1 and NF-κB signaling pathways are two typical downstream pathways of LPS/TLR4. The effects of 34# on LPS-stimulated MAPK phosphorylation and NF-κB activation were further analyzed, which demonstrated that LPS significantly increased the phosphorylation of JNK, ERK, and P38, which were obviously inhibited by 34# treatment (Fig. 2a-c). The phosphorylation of c-jun, an AP-1 subunit, was found to be increased after LPS stimulation, whereas it was observed to be decreased by 34# pretreatment (Fig. 2d). Unfortunately, compound 34# had no effect on LPSinduced degradation of IκB-α and phosphorylation of P65 (Fig. 2e and f). The results indicated that the MAPK/AP-1 signaling pathway may mediate the antiinflammatory activity of 34#, whereas NF-κB does not.

34# Diminishes the Severity of LPS-Induced ALI in Mice
Since 34# has a significant effect on LPS-induced inflammation in vitro, its ability to protect against LPSinduced lung inflammation in vivo was examined, thus establishing an ALI model. First, the severity of lung injury was measured via H&E staining (Fig. 3a). Histological analyses demonstrated marked inflammatory cell infiltration and alveolar wall thickening. In addition, LPS exposed mice were found to have vascular congestion in the lungs, which were dramatically reduced after pretreatment with 34#. The lung injury scores (Fig. 3b) represent the degree of damage to lung tissues. The corresponding results suggested the protective effect of 34# in LPS-induced lung injury. Moreover, the lung hyperpermeability marker, total protein concentration in BALF, exhibited the same results as that of H&E (Fig. 3c).

34# Reduced LPS-Induced Inflammatory Cell Infiltration in Lung Tissue
The previous results suggested that LPS can induce lung hyperpermeability and infiltration of inflammatory cells. Here, the effect of 34# was evaluated and showed that 34# pretreatment can reduce total cell number in BALF, which was found to be induced by LPS (Fig. 4a). The number of neutrophils (Fig. 4b) in BALF was verified in regard to the effect of 34# in relieving inflammatory cell infiltration. MPO is a marker of neutrophil activity.
The corresponding results showed that the activity of MPO was reduced after pretreatment with 34# (Fig. 4d). Furthermore, flow cytometry was used to detect the content of monocytes in BALF (Fig. 4c). The number of monocytes was found to be remarkably increased 6 h after LPS exposure, which was significantly reduced following 34# administration. Moreover, immunohistochemical staining was performed in order to evaluate the recruitment of macrophages and neutrophils in lung tissues. Moreover, the macrophage marker CD68 (Fig. 4e and f) and neutrophil marker LY6G (Fig. 4g and h) were found to be increased in the LPS group and were reduced by 34# pretreatment.

34# Reduced the Level of Inflammatory Cytokine in the Lung Tissue of LPS-Induced ALI
The effect of 34# on inflammatory cytokine expression was evaluated using an ELISA assay. The amount of TNF-α ( Fig. 5a and c) and IL-6 ( Fig. 5b and d) in serum and BALF was noted to be significantly increased but was markedly reduced following 34# pretreatment. Furthermore, RT-qPCR was used to evaluate the gene expression of TNF-α, IL-6, IL-1β, ICAM-1, VCAM-1, and MCP-1 (Fig. 5e, f, g, h, i and j). As expected, the LPS-induced gene expressions of inflammatory cytokines and adhesion molecules were found to be ameliorated by 34# pretreatment. In order to determine how 34# worked in LPS-induced ALI, the protein levels of P-JNK, P-ERK, and P-p38 in lung tissue were ascertained by Western blot analysis (Fig. 5k). The results were found to be the same as those in the in vitro experiment, where 34# was found to reduce the phosphorylation of JNK, ERK, and P38 in LPS-stimulated lung tissues.

DISCUSSION
MAPKs are a family of serine/threonine protein kinases found in organisms, which include the JNK, P38, and ERK pathways. They are able to activate the production of a series of stress-related inflammatory mediators [17]. Additionally, the MAPK/AP-1 signaling pathway regulates the expression of multiple genes, which play a vital role in various pathological processes such as inflammation [18,19], photoaging [20], and tumors [21]. LPS activates the MAPK/AP-1 pathway; induces the phosphorylation of JNK, ERK, and P38; and further activates AP-1 and c-jun, triggering a series of inflammatory reactions. Finkin-Groner et al. showed that indoline-3-propionate can reduce the production of LPS-induced pro-inflammatory factors by inhibiting the P38 MAPK/AP-1 pathway [22]. In addition, studies have shown that MAPK/AP-1 signaling pathways are crucial in O 3 -induced TNF-R-mediated pulmonary toxicity [18], which was also demonstrated by the present experiments. The MAPK/AP-1 pathway plays an important role in LPSinduced lung injury, and 34# can inhibit the activation of the MAPK/AP-1 pathway while serving a protective role. However, our compound was found to have no effect on the NF-κB pathway, which should be further explored. Inflammatory cell infiltration of lung tissue is an important pathological process of ALI. In ALI, inflammatory cells, such as macrophages, monocytes, and neutrophils, are recruited to the lungs [23]. Intrapulmonary inflammatory responses [24]. The accumulation and infiltration of neutrophils in lung tissue are also a known indication of ALI [25]. The increased expression of vascular endothelial cell adhesion molecules at the inflammatory reaction site results in the easy recruitment of inflammatory cells in the lung to the alveoli and lung interstitium,  . e, f, g, h, i and j The mRNA levels of inflammatory cytokines and adhesion molecule in lung tissues were determined by RT-qPCR after LPS treatment. Cytokine expression was normalized to β-actin. k The protein levels of p-JNK, p-ERK, and p-P38 in lung tissue was ascertained by western blot analysis. Each bar represents mean ± SEM of independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001 compared with LPS group). which accumulate in the lungs following their activation [26,27]. The recruited and activated neutrophils are able to release a large number of metabolites, such as elastase, ROS, and arachidonic acid, resulting in the destruction of alveolar walls and capillaries, reduction of the production of alveolar surfactants, and lung cell inflammation and infiltration, which leads to lung tissue damage [28]. Therefore, immunohistochemistry was utilized in order to detect CD68, a marker of monocytes and macrophages, and LY6G, a marker of neutrophils. The corresponding results demonstrated that 34# can inhibit inflammatory cell infiltration of ALI. In addition, the total cell, monocyte, and neutrophil counts in BALF exhibited the inhibitory effect of 34# on inflammatory cell recruitment.
ALI is also characterized by increased permeability of the alveolar-capillary barrier as well as the presence of a large number of cytokines and pro-inflammatory mediators, which further amplify the inflammatory response of ALI [4]. In the early stages of ALI, alveolar macrophages secrete inflammatory factors and chemokines, such as TNF-α and IL-6 [17]. In the middle stage of ALI, inflammatory factors and chemokines produced by macrophages, platelets, and vascular endothelial cells can chemoattract and recruit granulocytes [22]. In this study, 34# was found to significantly inhibit the secretion of inflammatory factors in serum and BALF. Meanwhile, the LPS-induced gene expression of adhesion molecules ICAM-1, VCAM-1, and chemokines MCP-1 in the lung was also observed to be inhibited by 34# administration. Elastase secreted by neutrophils is a serine protease, which can act on the extracellular matrix of endothelial tissue, damage the integrity of the vascular-endothelial barrier, and increase the permeability of the vascular endothelium [23,29]. The present results showed that 34# administration significantly reduced the protein concentration and total cell counts in BALF after exposure to LPS, which indicate that 34# may serve as a potential treatment for ALI.

CONCLUSION
In conclusion, the in vitro experiment demonstrated that 34# can obviously inhibit LPS-induced inflammatory cytokine expression, which may occur through the inhibition of the MAPK/AP-1 pathway. Meanwhile, 34# treatment was found to attenuate LPS-induced ALI in mice by reducing inflammatory cell infiltration as well as the inflammatory response, indicating that 34# may serve as a candidate in preventing acute lung inflammation and injury.