Selenium-containing compound ameliorates lipopolysaccharide-induced acute lung injury via regulating MAPKs/AP-1 pathway

Acute lung injury (ALI) characterized by a series of inammatory reactions and served as the main cause of mortality in intensive care unit patients. Although great progress have been made in understanding the pathophysiology of ALI, there are no effective treatment in clinic. Recently, we have synthesized a selenium-containing compounds, which possess obvious anti-inammatory 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 inammatory cytokine release. These effects were observed to be produced via suppression of the MAPKs/AP-1 pathway. Compound 34# was also noted to attenuate the LPS-induced lung inammation in mice with ALI. The corresponding results suggested that compound 34# possesses remarkable protective effects on LPS-induced ALI. Furthermore, the MAPKs/AP-1 pathway may prove to be its 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 in ammatory reactions, such as increased pulmonary capillary permeability, release of in ammatory 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 brosis. ALI physiologically manifests as decreased lung volume, decreased lung compliance and severe ventilation/blood ow imbalance, which clinically manifests as acute and progressive hypoxic respiratory insu ciency. 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, uid management and other supportive therapies [3]. Therefore, determining effective drugs for the treatment of ALI is of clinical signi cance.
Excessive in ammation is known to play an important role in ALI. Excessive in ammation includes the in ltration of in ammatory cells and release of cytokines, such as in ammatory factors, adhesion molecules and chemokines [4]. Lipopolysaccharide (LPS) is the main cell wall component of Gramnegative 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 stimulates the production of various in ammatory 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. has 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 signi cant antifungal [13], anticancer [14,15] and anti-in ammatory [16] activities. Recently, we have synthesized a selenium-containing compound 3-(phenylselanyl)-1H-pyrrolo [2,3-b]pyridine (34#, Fig. 1a). In this study, we illustrate its anti-in ammatory activities and its underlying mechanism in conjunction with its protective effects on LPS-induced ALI.

Materials And Methods
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, 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-LY6G was obtained from Abcam (Cambridge, UK). Antibodies for CD11B, CD45 and LY6Cwere 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 identi ed 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 macrophage (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 -humidi ed 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 used in 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 x 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 were determined by ELISA according to 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 the 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 sulfate-polyacrylamide gel electrophoresis and were electrotransferred to a nitrocellulose membrane. The membranes were then blocked for 1.5 h at room temperature in Tris-buffered 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 quanti ed using Image J 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 (Count star, 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) ow cytometer and Flow Jo software were then used to analyze the cell subpopulations.

Histopathology and immunohistochemistry
Lung tissues were xed in 4% paraformaldehyde, embedded in para n 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).
Para n sections were also used to perform immunohistochemistry for CD68 and LY6G using routine techniques. Sections were then depara nized, 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 eld microscopy (Nikon, Japan). The percentage expression was measured using Image J software (NIH, Bethesda, MD, USA).

MPO
MPO test kit was used to measure myeloperoxidase (MPO) activity in lung tissue. The lung tissue was homogenized according to the kit's instructions, and was centrifuged at 12000 rpm at 4°C for 10 min. The supernatant was then taken for MPO activity detection and indicated in the form of U/g 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 (GraphPad, 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 signi cant at p < 0.05.

Compound 34# inhibited LPS-induced MAPKs phosphorylation in MPMs
MAPKs/AP-1 and NF-κB signaling pathways are two typical down-stream pathways of LPS/TLR4. The effects of 34# on LPS-stimulated MAPKs phosphorylation and NF-κB activation were further analyzed, which demonstrated that LPS signi cantly 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 LPS-induced degradation of IκB-α and phosphorylation of P65 ( Fig. 2e and f). The results indicated that the MAPKs/AP-1 signaling pathway may mediate the anti-in ammatory activity of 34#, whereas NF-κB does not.

34# diminishes the severity of LPS-induced ALI in mice
Since 34# has a signi cant effect on LPS-induced in ammation in vitro, its ability to protect against LPSinduced lung in ammation 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 in ammatory cell in ltration 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 in ammatory cell in ltration in lung tissue
The previous results suggested that LPS can induce lung hyperpermeability and in ltration of in ammatory 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 veri ed in regard to the effect of 34# in relieving in ammatory cell in ltration. MPO is a marker of neutrophil activity. The corresponding results showed that the activity of MPO was reduced after treatment with 34# (Fig. 4d). Furthermore, ow 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 signi cantly 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 4f) and neutrophil marker LY6G (Fig. 4g and 4h) were found to be increased in the LPS group and were reduced by 34# treatment.
34# reduced the level of in ammatory cytokine in the lung tissue of LPS-induced ALI.
The effect of 34# on in ammatory 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 were noted to be signi cantly 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-j). As expected, LPS-induced gene expression of in ammatory cytokines and adhesion molecules were found to be ameliorated by 34# treatment. In order to determine how 34# worked in LPS-induced ALI, the protein level of P-JNK, P-ERK and P-p38 in lung tissue was 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 is a family of serine/threonine protein kinases found in organisms, which includes the JNK, P38, and ERK pathways. They are able to activate the production of a series of stress-related in ammatory mediators [17]. Additionally, the MAPKs/AP-1 signaling pathway regulates the expression of multiple genes, which play a vital role in various pathological processes such as in ammation [18,19], photoaging [20] and tumors [21]. LPS activates the MAPKs/AP-1 pathway, induces the phosphorylation of JNK, ERK, P38 and further activates AP-1 and c-jun, triggering a series of in ammatory reactions. E Finkin-Groner et al. showed that Indoline-3-propionate can reduce the production of LPS-induced pro-in ammatory factors by inhibiting the P38 MAPKs/AP-1 pathway [22]. In addition, studies have shown that MAPKs/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 MAPKs/AP-1 pathway plays an important role in LPS-induced lung injury, and 34# can inhibit the activation of the MAPKs/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.
In ammatory cell in ltration of lung tissue is an important pathological process of ALI. In ALI, in ammatory cells, such as macrophages, monocytes, and neutrophils, are recruited to the lungs [23].
Intrapulmonary macrophages mainly refer to alveolar macrophages, which are the rst line of defense in lung tissue, have functions of phagocytosis and secretion, can synthesize in ammatory factors like tumor necrosis factor, and trigger a cascade of in ammatory responses [24]. The accumulation and in ltration of neutrophils in lung tissue is also a known indication of ALI [25]. The increased expression of vascular endothelial cell adhesion molecules at in ammatory reaction site results in the easy recruitment of in ammatory cells in the lung to the alveoli and lung interstitium, 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 in ammation and in ltration, 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 in ammatory cell in ltration of ALI. In addition, the total cell, monocyte and neutrophil counts in BALF exhibited the inhibitory effect of 34# on in ammatory 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-in ammatory mediators, which further amplify the in ammatory response of ALI [4]. In the early stages of ALI, alveolar macrophages secrete in ammatory factors and chemokines, such as TNF-α and IL-6 [17]. In the middle stage of ALI, in ammatory factors and chemokines produced by macrophages, platelets, and vascular endothelial cells can chemoattract and recruit granulocytes [22]. In this study, 34# was found to signi cantly inhibit the secretion of in ammatory 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 signi cantly reduced 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 in ammatory cytokines 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 in ammatory cell in ltration as well as the in ammatory response, indicating that 34# may serve as a candidate in treating acute lung in ammation and injury.   Figure 2 Effect of 34# on LPS-induced MAPK phosphorylation and NF-κB activation in macrophages. MPMs were pretreated with 34# at 10 μM for 30 min followed by incubation with LPS (0.5 μg/mL) for 15 min. The protein levels of p-JNK (a), p-ERK (b), p-P38 (c) and IκB-α (e) were measured by western blot. Total protein of JNK, ERK, P38 and GAPDH were used as loading control. Protein levels of p-c-jun (d) and p-P65 (f) were examined by Western blot. MPMs were pretreated with 34# at 10 μM for 30 min followed by stimulation with LPS (0.5 μg/mL) for 30 min. Each bar represents mean ± SEM of independent experiments. (**P < 0.01, ***P < 0.001 compared with LPS group).