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Original Research Article

DTT 2023; 2(1): 30-40

Published online March 31, 2023 https://doi.org/10.58502/DTT.23.0001

Copyright © The Pharmaceutical Society of Korea.

Anti-Inflammatory Effect of Phosphodiesterase 2 Inhibitor in Lipopolysaccharide (LPS)-Stimulated RAW 264.7 Macrophages

Jin Yong Song* , Wang Tae Lee*, Oh Seong Kwon, Yeon Jin Lee, Su Hyun Lee, Yubin Lee, Ji-Yun Lee

Laboratory of Pathophysiology, College of Pharmacy, Chung-Ang University, Seoul, Korea

Correspondence to:Ji-Yun Lee, jylee98@cau.ac.kr
*These authors contributed equally to this work.

Received: January 25, 2023; Revised: March 3, 2023; Accepted: March 4, 2023

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ((http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Although the inflammatory response is an important process in the host defense system, it has been found to cause serious complications in disease in response to various stresses. Cyclic nucleotide monophosphates (cAMP and cGMP) are related to the inflammatory response, however, their mechanism is unclear. Although the antioxidative and neuroprotective mechanisms of the phosphodiesterase 2 (PDE2) inhibitor have been extensively studied in relation to heart failure and neuropsychiatric diseases, the PDE2 inhibitor’s role in inflammatory immune responses caused by bacterial endotoxin has not been elucidated. In the present study, the anti-inflammatory effects of the PDE2 selective inhibitor BAY 60-7550 were investigated in the murine macrophage cell line RAW 264.7 after being induced by lipopolysaccharide (LPS). BAY 60-7550 showed no cytotoxicity in RAW 264.7 cells and suppressed the LPS-induced overexpression of oxidative stress factors such as an inducible nitric oxide synthase (iNOS), nitric oxide (NO), and reactive oxygen species (ROS). The oxidative stress-induced activation of the mitogen-activated protein kinase (MAPK) pathway, especially the phosphorylation of the extracellular signal-regulated kinases (ERK)1/2 and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways, was also inhibited by the application of BAY 60-7550. Moreover, the expression of COX-2, PGE₂, and pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β was significantly reduced by the application of BAY 60-7550. In conclusion, BAY 60-7550 has an anti-inflammatory effect through the regulation of the ERK1/2-associated MAPK and NF-κB pathways. Thus, this finding shows the potential of the PDE2 selective inhibitor as a new therapeutic candidate for infectious inflammatory diseases.

Keywordsphosphodiesterase 2, lipopolysaccharide, BAY 60-7550, inflammation, ROS

Inflammation is a primary immune response to physical or chemical stimuli such as pathogens, damaged cells, and irritants (Dong et al. 2018). Inflammation can cause many diseases, including arthritis and asthma (Chen et al. 2017). Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in the clinical setting to combat inflammatory diseases (Al-Shidhani et al. 2015), but they have several side effects making them unsuitable for the treatment of all inflammatory diseases (Yao et al. 2019). Therefore, it is necessary to develop new safe drugs that can be treated for inflammatory diseases.

Along with neutrophils and dendritic cells, macrophages play a major role in the immune system as they are the first cell mediators of the innate immune response (Carralot et al. 2009). When the body got pathological damage, activated macrophages release numerous pro-inflammatory cytokines and inflammatory mediators such as interleukin-1-beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), nitric oxide (NO), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) (Du et al. 2018). The pro-inflammatory mediators, NO and prostaglandin E2 (PGE2), are produced by iNOS and COX-2 respectively (Kwon et al. 2017). NO is regulated by NO synthase (NOS) and iNOS which is significantly upregulated in inflammatory diseases (Muniandy et al. 2018). Another enzyme that plays an important role in mediating inflammation is COX-2, which catalyzes the rate-determining step of the synthesis of PGE2 from the arachidonic acid metabolism of cell membrane phospholipids (Guerrero et al. 2015).

Phosphodiesterase (PDE) is an enzyme that restricts the intracellular signaling of second messenger molecules by hydrolyzing cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) (Knott et al. 2017). cAMP and cGMP play important roles in regulating many cellular functions in physiology and pathology (Kilanowska and Ziółkowska 2020), including airway smooth muscle tone, cell proliferation, differentiation, apoptosis, migration, inflammatory mediator secretion, extracellular deposits, matrix, and endothelial and epithelial barriers (Zuo et al. 2019). Among the PDE isotypes, PDE4 is the dominant family of PDEs expressed in inflammatory cells, which include eosinophils, T lymphocytes, macrophages, neutrophils, and dendritic cells (Giembycz 2002). As a result, Roflumilast, a selective and long-acting PDE4 inhibitor, was developed as a treatment of inflammatory lung diseases such as asthma and COPD for preventing their exacerbation (Ilango et al. 2013). Our previous study also showed that PDE7 inhibitor, BRL-50481, alleviates ovalbumin-induced asthmatic lung inflammation (Kim et al. 2022).

In contrast, BAY 60-7550, the PDE2 selective inhibitor, is known to be distributed in the nervous system (Wang et al. 2017) and has been extensively studied in relation to neuropsychiatric disorders such as depression, anxiety, and drug addiction as an antioxidant and neuroprotective mechanism (Huang et al. 2018). Recently, the effects of NO, guanylyl cyclase, and cGMP signaling in heart failure have also been studied (Baliga et al. 2018). In addition, research has shown that PDE2 is also involved in the differentiation of monocytes into macrophages (Hertz and Beavo 2011), and it has also been found to be a key PDE in activated human and rat primary macrophages (Rentsendorj et al. 2014). Nevertheless, no studies have been conducted to elucidate the crucial role of PDE2 selective inhibition in the inflammatory response.

This study demonstrates the anti-inflammatory properties of BAY 60-7550 against Lipopolysaccharide (LPS)-induced inflammatory damage in the RAW 264.7 macrophage cell line. In addition, this study investigated the ability of BAY 60-7550 to inhibit the activation of Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) and elucidated the mechanism of the anti-inflammatory effects. Through this study, the anti-inflammatory effect of PDE2 selective inhibitors and their pathways are revealed.

Materials

BAY 60-7550, LPS, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM), antibiotic-antimycotic solution (10,000 units/mL penicillin, 10,000 µg/mL streptomycin, and 25 µg/mL amphotericin B), 2’,7’-dichlorodihydrofluorescein diacetate (H2DCF-DA), and a RIPA buffer were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) and Dulbecco’s phosphate buffered saline (DPBS) were purchased from WELGENE, Inc. (Daegu, Republic of Korea). iNOS (cat. no. 2982S), phosphorylated (p)-ERK1/2 (cat. no. 4370S), total (t)-ERK1/2 (cat. no. 4695S), p-NF-κB (cat. no. 3033S), t-NF-κB (cat. no. 8242S), and COX-2 (cat. no. 12282S) primary antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). β-actin (cat. no. sc-47778) was purchased from Santacruz Biotechnology, Inc. (Dallas, TX, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies, goat anti-mouse IgG (cat .no. 7076S), and goat anti-rabbit IgG (cat. no. 7074S) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Cell culture

The RAW 264.7 macrophage cell line was obtained from the Korea Cell Line Bank (KCLB, Seoul, Republic of Korea). The cells were cultured at 37℃ in DMEM supplemented with 2 mM of glutamine, 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 10% fetal bovine serum (FBS) in 5% CO2 humidified environment.

MTT assay

Cell viability was performed using the MTT assay. Cells were cultured for 24 h in a 24-well plate (8 × 104 cells/well), and treated with BAY 60-7550 (1, 3, 10, and 30 μM). To evaluate cytotoxicity of BAY 60-7550, Cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM), and then treated with LPS that had been dissolved in DPBS. After being washed with DPBS, MTT (0.5 mg/mL) was applied to cells, and they were incubated for 2 h at 37℃. The supernatant was removed, and DMSO was added to each well to dissolve the formazan. The absorbance was measured at 570 nm using a microplate absorbance reader (Synergy HTX Multi-Mode Microplate Reader, BioTek®, Winooski, VT, USA).

H2DCF-DA assay

Cells were cultured for 24 h on poly-L-lysine-coated cover glass (5 × 105 cells/glass) and 96-well dark plate (3.2 × 104 cells/well) and pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, after that they were treated with LPS (0.1 μg/mL) for 15 h. 40 μM of H2DCF-DA was added to cells for 30 min. Cells on poly-L-lysine-coated cover glass were stained with DAPI for 5 min. After the cover glass is placed on the slide glass, images were taken with a Leica DM 480 camera (Leica Microsystems, Wetzlar, Germany). ROS was detected by a fluorescence microplate reader (Synergy HTX Multi-Mode Microplate Reader, BioTek®, Winooski, VT, USA), and the excitation and emission wavelengths were 485 and 535 nm, respectively.

Western blot

Cells were cultured for 24 h in a six-well plate (3.2 × 105 cells/well) and pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h and then treated with LPS (0.1 ng/mL). After being washed with DPBS, cells were lysed using a RIPA buffer containing 100 mM of vanadate and 100 mM of PMSF, which were on ice for 15 min. The lysate was centrifuged at 16,200 g for 15 min at 4℃, after which the supernatant was collected. The protein content was measured by BCA protein assay reagent (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA). 20 μg of protein samples were loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoreses (SDS-PAGE) and separated at 60 V for 30 min and 90 V for 2 h. When the electrophoresis was completed, the protein samples were transferred to polyvinylidene fluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany) for 90 min. The membranes were blocked with 5% bovine serum albumin (BSA) in TBS-T (tris-buffered saline with 0.1% Tween 20) at room temperature for 1 h. Then, the membranes were incubated with primary antibodies overnight at 4℃. Primary antibodies were diluted at 1:1000. After being washed with TBS-T, the primary antibodies were incubated with HRP-conjugated secondary antibodies at room temperature for 2 h. Secondary antibodies were diluted at 1:3000. Finally, detection was performed using an ECL detection kit (Western Lightning ECL Pro; PerkinElmer Inc, Waltham, MA, USA), and the band signal was detected by Fusion Solo X (Vilber, Paris, France). The band intensity was analyzed with ImageJ software (version 1.52a; National Institute of Health).

NO production assay

Cells were cultured for 24 h in a six-well plate (3.2 × 105 cells/well) and pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, and 0.1 ng/mL of LPS was added before they were left overnight. The following day, the culture supernatant from each well was collected. 100 μL each of the supernatant and the modified Griess reagent (Sigma-Aldrich, MO, USA) were reacted in a 96-well plate at room temperature (RT) for 10 min. The absorbance of each well was measured with a microplate reader (Synergy HTX Multi-Mode Microplate Reader, BioTek®, Winooski, VT, USA) at 540 nm. NO concentration was calculated using a serial dilution of the sodium NO standard curve.

Enzyme-linked immunosorbent assay (ELISA)

Cells were cultured for 24 h in a six-well plate (3.2 × 105 cells/well) and pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, after which 0.1 ng/mL of LPS was added. Next, the culture supernatant from each well was collected, centrifuged at 15,000 g for 15 min, and used to measure the IL-6, IL-1β, TNF-α, and PGE2 concentrations. IL-6, IL-1β, TNF-α, and PGE2 Parameter Assay Kits (R&D Systems, Minneapolis, MN, USA) were used according to the manufacturer’s protocol.

Statistical analysis

All data were presented as mean ± standard deviation (SD). Statistical analyses were performed using the GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA). Quantitative measured values were analyzed to determine the significance of each group using one-way ANOVA with Dunnett’s post hoc test. The differences among groups were determined as statistically significant at p < 0.05, p <0.01, and p < 0.001.

Cytotoxicity of BAY 60-7550 and LPS in RAW 264.7 cells

The MTT assay was performed to evaluate the cytotoxicity of BAY 60-7550 and LPS in RAW 264.7 cells. To evaluate the cytotoxicity of treatment with BAY 60-7550, RAW 264.7 cells were treated with BAY 60-7550 (1, 3, 10, and 30 μM) for 15 h (Fig. 1A). To evaluate the cytotoxicity of co-treatment with BAY 60-7550 and LPS, RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h and treated with LPS (0.1 μg/mL) for 4, 15, and 24 h, respectively. BAY 60-7550 was not cytotoxic at either 4 h or 15 h treatment conditions. However, a statistically significant cytotoxic effect was observed with 24 h treatment. Therefore, the experiment was conducted using 15 h treatment (Fig. 1B).

Figure 1.Cytotoxicity of BAY 60-7550 and LPS in RAW 264.7 cells. The cells were treated with BAY 60-7550 (1, 3, 10, and 30 μM) for 15 h (A). The cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h and treated with LPS (0.1 μg/mL) for 4, 15, or 24 h to observe the cytotoxicity of BAY 60-7550 and LPS (B). The number of viable cells was assessed by MTT assay. Results were expressed as a percentage (%) in comparison with the control. Each value represents the mean ± S.D from four independent experiments.

The inhibitory effect of BAY 60-7550 on the LPS-induced intracellular oxidative stress in RAW 264.7 cells

The cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM), followed by treatment with LPS (0.1 μg/mL). The protein expression of iNOS was significantly increased by 13.18-fold (p < 0.001) after stimulation with LPS (0.1 μg/mL) for 15 h. Pretreatment with BAY 60-7550 significantly downregulated the protein expression level of iNOS in the LPS-induced RAW 264.7 cells. In the experimental groups treated with 10 and 30 μM of BAY 60-7550, iNOS was significantly reduced by 52% and 77%, respectively (Fig. 2A).

Figure 2.The inhibitory effect of BAY 60-7550 on the LPS-induced intracellular oxidative stress in RAW 264.7 cells. The cells were pretreated with various concentrations of BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h and treated with LPS (0.1 μg/mL) for 15 h. The expression levels of iNOS were determined by western blot analysis (A). The culture supernatant was analyzed for NO. The NO content was determined by Griess reagent (B). The cells were pretreated with various concentrations of BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h and treated with LPS (0.1 μg /mL) for 15 h. ROS levels were assessed with the H2DCF-DA assay. ROS levels were expressed as a percentage (%) relative to the level in the vehicle control (C). Each value represents the mean ± S.D from experiments. ### indicates a significant difference from the vehicle control, which was treated with DMSO alone (###p < 0.001). * and *** indicate significant differences from the cells treated with 0.1 μg/mL of LPS alone (*p < 0.05 & ***p < 0.001).

The release of NO from RAW 264.7 cells was significantly increased by 8.47-fold (p < 0.001) after stimulation with LPS (0.1 μg/mL) for 15 h. Pretreatment with BAY 60-7550 downregulated the LPS-induced NO production. In the experimental group treated with 3 μM of BAY 60-7550, NO was significantly reduced by 14% (21.1 μM to 18.05 μM) (Fig. 2B).

Furthermore, the production of reactive oxygen species (ROS) in RAW 264.7 cells was significantly increased by 1.80-fold (p < 0.001) after stimulation with LPS (0.1 μg/mL) for 15 h. Pretreatment with BAY 60-7550 downregulated the ROS production in the LPS-induced RAW 264.7 cells. In the experimental groups treated with 3, 10, and 30 μM of BAY 60-7550, ROS production was significantly reduced by 18%, 30%, and 43%, respectively. These results can be confirmed in the fluorescence staining image of the cells (Fig. 2C).

The inhibitory effect of BAY 60-7550 on LPS-induced overexpression of COX-2 and PGE2 in RAW 264.7 cells

To determine the inhibitory mechanism of BAY 60-7550 on LPS-induced RAW 264.7 cells, the expression level of the COX-2 enzyme, which is involved in arachidonic acid (AA) metabolism, was examined by western blot analysis. In response to LPS, COX-2 protein was upregulated 5.17-fold compared to that in the vehicle control group. Furthermore, pretreatment with BAY 60-7550 (3 μM, 10 μM, and 30 μM) showed significant inhibitory effects on COX-2 expression of 36%, 49%, and 44%, respectively (Fig. 3A).

Figure 3.The inhibitory effect of BAY 60-7550 on LPS-induced overexpression of COX-2 and PGE2 in RAW 264.7 cells.
The protein expression levels of COX-2 were confirmed by western blot assay (A). RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 1h. The band density of COX-2 was quantified relative to the β-actin. PGE2 production was evaluated by ELISA assay in LPS-treated RAW 264.7 cells (B). RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 1 h. The absorbance at 450 nm was measured by ELISA protocol, and the concentration was measured according to the PGE2 absorbance standard curve. Each value represents the mean ± S.D from experiments. # & ### indicate significant differences from the vehicle control, which was treated with DMSO alone (#p < 0.05 and ###p < 0.001). *, **, and *** indicate significant differences from the cells treated with 0.1 μg/mL of LPS alone (*p < 0.05, **p < 0.01, and ***p < 0.001).

The PGE2 concentration of LPS-induced RAW 264.7 cells was analyzed by ELISA assay. These results indicate that the treatment of BAY 60-7550 inhibited the expression of COX-2 in LPS-induced RAW 264.7 cells. The group treated with 0.1 μg/mL LPS alone for 1 h showed upregulated PGE2 levels by 1.40-fold compared to the control group (857.97 pg/mL to 1205.19 pg/mL). Furthermore, pretreatment with BAY 60-7550 (3 μM, 10 μM, and 30 μM) showed significant inhibitory effects on PGE2 release with 20%, 17%, and 25%, respectively (Fig. 3B).

The inhibitory effect of BAY 60-7550 on LPS-induced overproduction of pro-inflammatory cytokines in RAW 264.7 cells

The TNF-α, IL-6, and IL-1β concentrations of LPS-induced RAW 264.7 cells were analyzed by ELISA. The group treated with 0.1 μg/mL of LPS alone for 4 h showed a significantly upregulated TNF-α level by 16.68-fold compared to the control group (1315.625 pg/mL to 21940.625 pg/mL). However, the group treated with BAY 60-7550 (1 μM) showed a decrease in TNF-α release (21940.625 pg/mL to 19412.5 pg/mL) (Fig. 4A). The group treated with 0.1 μg/mL of LPS alone for 4 h showed a significantly upregulated IL-6 level of 51.51-fold compared to the control group (6.36 pg/mL to 327.66 pg/mL). However, the group treated with BAY 60-7550 (1 μM) showed a decrease in IL-6 release (327.66 pg/mL to 266.76 pg/mL) (Fig. 4B). The group treated with 0.1 μg/mL of LPS alone for 24 h showed an upregulated IL-1β level by 1.50-fold compared to the control group (5.38 pg/mL to 8.08 pg/mL). However, the group treated with BAY 60-7550 showed decrease in IL-1β release (Fig. 4C). These results indicate that treatment with BAY 60-7550 inhibited overproduction of TNF-α, IL-6, and IL-1β in RAW 264.7 cells treated with LPS.

Figure 4.The inhibitory effect of BAY 60-7550 on LPS-induced overproduction of pro-inflammatory cytokines in RAW 264.7 cells. The levels of TNF-α, IL-6, and IL-1β were confirmed by ELISA. RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 4 h. The absorbance at 450 nm was measured by ELISA protocol, and the concentration was measured according to the TNF-α absorbance standard curve (A). RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 4 h. The absorbance at 450 nm was measured by ELISA protocol, and the concentration was measured according to the IL-6 absorbance standard curve (B). RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 15 h. The absorbance at 450 nm was measured by ELISA protocol, and the concentration was measured according to the IL-1β absorbance standard curve (C). Each value represents the mean ± S.D from three separate experiments. ### indicates a significant difference from the vehicle control, which was treated with DMSO alone (###p < 0.001). * and *** indicate significant differences from the cells treated with 0.1 μg/mL of LPS alone (*p < 0.05 and ***p < 0.001).

The inhibitory effect of BAY 60-7550 on LPS-induced activation of MAPK-ERK1/2 and NF-κB signaling pathways in RAW 264.7 cells

To determine the inhibitory mechanism of BAY 60-7550 on RAW 264.7 cells, the expression levels of ERK1/2 and NF-κB were examined by western blot analysis. In response to LPS, the expression level of phosphorylated ERK1/2 protein was significantly upregulated by 5.20-fold compared to that of the vehicle control group. Furthermore, pretreatment with BAY 60-7550 (10 μM and 30 μM) showed 35% and 63% inhibitory effects on ERK1/2 phosphorylation, respecively (Fig. 5A).

Figure 5.The inhibitory effect of BAY 60-7550 on LPS-induced activation of MAPK-ERK1/2 and NF-κB signaling pathways in RAW 264.7 cells. The levels of phosphorylated-ERK1/2, total-ERK1/2 (A), phosphorylated-NF-κB, and total-NF-κB (B) were confirmed by western blotting assay. RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 4 h. The band density of phosphorylated-ERK1/2 and the phosphorylated-NF-κB protein level was quantified relative to the total-ERK1/2 and total-NF-κB, respectively. NF-κB levels were expressed as a percentage (%) relative to the level in the vehicle control, and each value represents the mean ± S.D from experiments. ### indicates a significant difference from the vehicle control, which was treated with DMSO alone (###p < 0.001). *, **, and *** indicate significant differences from the cells treated with 0.1 μg/mL of LPS alone (*p < 0.05, **p < 0.01, and ***p < 0.001).

In response to LPS, the expression level of phosphorylated NF-κB protein was significantly upregulated by 4.68-fold compared to that of the vehicle control group. Furthermore, pretreatment with BAY 60-7550 (3 μM, 10 μM, and 30 μM) showed 34%, 31%,and 63% inhibitory effects on NF-κB phosphorylation, respecively (Fig. 5B).

LPS is an important component in the Gram-negative bacteria’s outer membrane, as it reacts with CD14 on the surface of macrophages to produce inflammatory cytokines and activates the MAPK and NF-κB pathways (Meng and Lowell 1997). To observe anti-inflammatory effects and mechanisms of drugs, we applied the in vitro model, LPS-stimulated RAW 264.7 macrophages

Concentrations of intracellular cyclic nucleotides are primarily determined by PDEs, which hydrolyze cAMP and cGMP and prevent them from diffusing to other compartments (Zuo et al. 2019). PDE2 hydrolyzes both cAMP and cGMP (Meng and Lowell 1997) and is expressed in the peripheral and central nervous system (CNS) in a variety of tissues and cell types (Zhang et al. 2015). In the context of cognition, activation of the cAMP, protein kinase A (PKA), element-sensitive cAMP, and cGMP has been associated with long-term potentiation and the formation of memory (Xu et al. 2011). However, there have been no studies on the anti-inflammatory effects of PDE2 inhibitors. Therefore, this study focused on the anti-inflammatory activity of BAY 60-7550 which we determine to occur through the activation of MAPK (ERK1/2) and NF-κB pathways.

In this study, examination of cell viability using the MTT assay was performed first to investigate the cytotoxicity of LPS and BAY 60-7550 on RAW 264.7 cells. BAY 60-7550 did not show significant toxicity. Furthermore, when BAY 60-7550 and LPS were co-treated under the same conditions in our experiment, the cell viability was more than 80% in all experimental groups. This suggests that BAY 60-7550 and LPS have no cytotoxic effects in our experimental conditions.

Oxidative stress occurs when the balance between ROS and antioxidant compounds is disturbed (Adwas et al. 2019). Macrophages stimulated by LPS are known to initially promote the production of ROS, such as superoxides and H₂O₂, through the activation of NADPH oxidase (Mittal et al. 2014). While high levels of ROS promote cytotoxicity, non-toxic levels of ROS activate many signaling mediators, including phosphatidyl inositol 3-kinase (PI3K), phosphatase and tensin homolog (PTEN), NF-kB-inducing kinase (NIK), and MAPK, leading to a wide range of responses, such as inflammation and survival. ROS may also be involved in the activation of the NF-κB signaling pathway (Li and Engelhardt 2006). In particular, ERK1/2, also known as p44/42 in the MAPK family, is primarily activated by inflammation and growth factors (Mebratu and Tesfaigzi 2009). Moreover, NO is one of the most important inflammatory mediators involved in inflammatory processes (Waltz et al. 2015). Exposure of cells to bacterial products such as LPS, lipoteichoic acid (LTA), peptidoglycans, and bacterial DNA or whole bacteria will induce a high expression of iNOS and thereby increase the production of NO (Ginsburg 2002). In these situations, NO forms peroxynitrite, which acts as a cytotoxic molecule, resists invading microorganisms, and acts as a killer (Yao et al. 2019). This study revealed that BAY 60-7550 has anti-inflammatory effects, such as reducing iNOS, NO, and ROS which is increased by LPS.

This study confirmed that phosphorylation of ERK1/2 was effectively reduced by treatment with BAY 60-7550 through western blotting assay. This result suggests that BAY 60-7550 has anti-inflammatory effects by inhibiting the MAPK pathway. In addition, NF-κB acts as an important regulator in inflammation and as a transcription factor for iNOS, COX-2, and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) (Seok et al. 2021). When NF-κB is activated by LPS, it enters the nucleus, binds to the DNA, and expresses the target gene (Liu et al. 2017). Therefore, this study investigated the inhibitory effect of BAY 60-7550 on LPS-induced NF-κB. Our study showed that BAY 60-7550 reduces the activity of NF-κB, which downregulates the activity of inflammatory mediators and pro-inflammatory cytokines.

The upregulated level of NF-κB expression increases the expression of the COX-2 protein (Liu et al. 2017). COX-2 is an enzyme that catalyzes the first step in the conversion of AAs to PGE2 (Ha et al. 2022). Under inflammatory conditions, COX-2 is expressed and increases the conversion of AA to PGE2 (Liu et al. 2015). Because research has shown that LPS upregulates PGE2 production and COX-2 expression, ELISA and western blot tests were performed to measure the expression levels of PGE2 and COX-2. In the present study, the expressions of COX-2 and PGE2 were increased by treatment with LPS, and the levels of expression decreased after treatment with BAY 60-7550.

LPS-stimulated RAW 264.7 macrophages trigger an acute inflammatory response, which upregulates the MAPK and NF-κB pathways, thereby promoting the production of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β (Xu et al. 2014). This is beneficial in attracting circulating immune effector cells, such as neutrophils, to fight infection, but excessive inflammatory responses can damage tissues and organs (Chen et al. 2017). It is therefore important that the expression of these pro-inflammatory cytokines is properly regulated. This study has found that LPS increases TNF-α, IL-6, and IL-1β in macrophages and that BAY 60-7550 effectively reduces them.

Taken together, it was found that BAY 60-7550 significantly regulates the inflammatory response of RAW 264.7 cells induced by LPS. LPS triggered the MAPK and NF-κB pathways in RAW 264.7 cells, and BAY 60-7550 significantly inhibited their cellular signaling. In addition, various inflammatory mediators and pro-inflammatory cytokines, which are sub-regulators of the MAPK and NF-κB pathways, were also regulated by BAY 60-7550 treatment. This study revealed that the PDE2 selective inhibitor, BAY 60-7550, could have the potential to be an anti-inflammatory therapeutic.

The authors declare that they have no conflict of interest.

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (Grant Number NRF-2022R1F1A1076528).

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Article

Original Research Article

DTT 2023; 2(1): 30-40

Published online March 31, 2023 https://doi.org/10.58502/DTT.23.0001

Copyright © The Pharmaceutical Society of Korea.

Anti-Inflammatory Effect of Phosphodiesterase 2 Inhibitor in Lipopolysaccharide (LPS)-Stimulated RAW 264.7 Macrophages

Jin Yong Song* , Wang Tae Lee*, Oh Seong Kwon, Yeon Jin Lee, Su Hyun Lee, Yubin Lee, Ji-Yun Lee

Laboratory of Pathophysiology, College of Pharmacy, Chung-Ang University, Seoul, Korea

Correspondence to:Ji-Yun Lee, jylee98@cau.ac.kr
*These authors contributed equally to this work.

Received: January 25, 2023; Revised: March 3, 2023; Accepted: March 4, 2023

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ((http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Although the inflammatory response is an important process in the host defense system, it has been found to cause serious complications in disease in response to various stresses. Cyclic nucleotide monophosphates (cAMP and cGMP) are related to the inflammatory response, however, their mechanism is unclear. Although the antioxidative and neuroprotective mechanisms of the phosphodiesterase 2 (PDE2) inhibitor have been extensively studied in relation to heart failure and neuropsychiatric diseases, the PDE2 inhibitor’s role in inflammatory immune responses caused by bacterial endotoxin has not been elucidated. In the present study, the anti-inflammatory effects of the PDE2 selective inhibitor BAY 60-7550 were investigated in the murine macrophage cell line RAW 264.7 after being induced by lipopolysaccharide (LPS). BAY 60-7550 showed no cytotoxicity in RAW 264.7 cells and suppressed the LPS-induced overexpression of oxidative stress factors such as an inducible nitric oxide synthase (iNOS), nitric oxide (NO), and reactive oxygen species (ROS). The oxidative stress-induced activation of the mitogen-activated protein kinase (MAPK) pathway, especially the phosphorylation of the extracellular signal-regulated kinases (ERK)1/2 and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways, was also inhibited by the application of BAY 60-7550. Moreover, the expression of COX-2, PGE₂, and pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β was significantly reduced by the application of BAY 60-7550. In conclusion, BAY 60-7550 has an anti-inflammatory effect through the regulation of the ERK1/2-associated MAPK and NF-κB pathways. Thus, this finding shows the potential of the PDE2 selective inhibitor as a new therapeutic candidate for infectious inflammatory diseases.

Keywords: phosphodiesterase 2, lipopolysaccharide, BAY 60-7550, inflammation, ROS

Introduction

Inflammation is a primary immune response to physical or chemical stimuli such as pathogens, damaged cells, and irritants (Dong et al. 2018). Inflammation can cause many diseases, including arthritis and asthma (Chen et al. 2017). Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in the clinical setting to combat inflammatory diseases (Al-Shidhani et al. 2015), but they have several side effects making them unsuitable for the treatment of all inflammatory diseases (Yao et al. 2019). Therefore, it is necessary to develop new safe drugs that can be treated for inflammatory diseases.

Along with neutrophils and dendritic cells, macrophages play a major role in the immune system as they are the first cell mediators of the innate immune response (Carralot et al. 2009). When the body got pathological damage, activated macrophages release numerous pro-inflammatory cytokines and inflammatory mediators such as interleukin-1-beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), nitric oxide (NO), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) (Du et al. 2018). The pro-inflammatory mediators, NO and prostaglandin E2 (PGE2), are produced by iNOS and COX-2 respectively (Kwon et al. 2017). NO is regulated by NO synthase (NOS) and iNOS which is significantly upregulated in inflammatory diseases (Muniandy et al. 2018). Another enzyme that plays an important role in mediating inflammation is COX-2, which catalyzes the rate-determining step of the synthesis of PGE2 from the arachidonic acid metabolism of cell membrane phospholipids (Guerrero et al. 2015).

Phosphodiesterase (PDE) is an enzyme that restricts the intracellular signaling of second messenger molecules by hydrolyzing cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) (Knott et al. 2017). cAMP and cGMP play important roles in regulating many cellular functions in physiology and pathology (Kilanowska and Ziółkowska 2020), including airway smooth muscle tone, cell proliferation, differentiation, apoptosis, migration, inflammatory mediator secretion, extracellular deposits, matrix, and endothelial and epithelial barriers (Zuo et al. 2019). Among the PDE isotypes, PDE4 is the dominant family of PDEs expressed in inflammatory cells, which include eosinophils, T lymphocytes, macrophages, neutrophils, and dendritic cells (Giembycz 2002). As a result, Roflumilast, a selective and long-acting PDE4 inhibitor, was developed as a treatment of inflammatory lung diseases such as asthma and COPD for preventing their exacerbation (Ilango et al. 2013). Our previous study also showed that PDE7 inhibitor, BRL-50481, alleviates ovalbumin-induced asthmatic lung inflammation (Kim et al. 2022).

In contrast, BAY 60-7550, the PDE2 selective inhibitor, is known to be distributed in the nervous system (Wang et al. 2017) and has been extensively studied in relation to neuropsychiatric disorders such as depression, anxiety, and drug addiction as an antioxidant and neuroprotective mechanism (Huang et al. 2018). Recently, the effects of NO, guanylyl cyclase, and cGMP signaling in heart failure have also been studied (Baliga et al. 2018). In addition, research has shown that PDE2 is also involved in the differentiation of monocytes into macrophages (Hertz and Beavo 2011), and it has also been found to be a key PDE in activated human and rat primary macrophages (Rentsendorj et al. 2014). Nevertheless, no studies have been conducted to elucidate the crucial role of PDE2 selective inhibition in the inflammatory response.

This study demonstrates the anti-inflammatory properties of BAY 60-7550 against Lipopolysaccharide (LPS)-induced inflammatory damage in the RAW 264.7 macrophage cell line. In addition, this study investigated the ability of BAY 60-7550 to inhibit the activation of Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) and elucidated the mechanism of the anti-inflammatory effects. Through this study, the anti-inflammatory effect of PDE2 selective inhibitors and their pathways are revealed.

Materials|Methods

Materials

BAY 60-7550, LPS, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM), antibiotic-antimycotic solution (10,000 units/mL penicillin, 10,000 µg/mL streptomycin, and 25 µg/mL amphotericin B), 2’,7’-dichlorodihydrofluorescein diacetate (H2DCF-DA), and a RIPA buffer were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) and Dulbecco’s phosphate buffered saline (DPBS) were purchased from WELGENE, Inc. (Daegu, Republic of Korea). iNOS (cat. no. 2982S), phosphorylated (p)-ERK1/2 (cat. no. 4370S), total (t)-ERK1/2 (cat. no. 4695S), p-NF-κB (cat. no. 3033S), t-NF-κB (cat. no. 8242S), and COX-2 (cat. no. 12282S) primary antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). β-actin (cat. no. sc-47778) was purchased from Santacruz Biotechnology, Inc. (Dallas, TX, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies, goat anti-mouse IgG (cat .no. 7076S), and goat anti-rabbit IgG (cat. no. 7074S) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Cell culture

The RAW 264.7 macrophage cell line was obtained from the Korea Cell Line Bank (KCLB, Seoul, Republic of Korea). The cells were cultured at 37℃ in DMEM supplemented with 2 mM of glutamine, 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 10% fetal bovine serum (FBS) in 5% CO2 humidified environment.

MTT assay

Cell viability was performed using the MTT assay. Cells were cultured for 24 h in a 24-well plate (8 × 104 cells/well), and treated with BAY 60-7550 (1, 3, 10, and 30 μM). To evaluate cytotoxicity of BAY 60-7550, Cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM), and then treated with LPS that had been dissolved in DPBS. After being washed with DPBS, MTT (0.5 mg/mL) was applied to cells, and they were incubated for 2 h at 37℃. The supernatant was removed, and DMSO was added to each well to dissolve the formazan. The absorbance was measured at 570 nm using a microplate absorbance reader (Synergy HTX Multi-Mode Microplate Reader, BioTek®, Winooski, VT, USA).

H2DCF-DA assay

Cells were cultured for 24 h on poly-L-lysine-coated cover glass (5 × 105 cells/glass) and 96-well dark plate (3.2 × 104 cells/well) and pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, after that they were treated with LPS (0.1 μg/mL) for 15 h. 40 μM of H2DCF-DA was added to cells for 30 min. Cells on poly-L-lysine-coated cover glass were stained with DAPI for 5 min. After the cover glass is placed on the slide glass, images were taken with a Leica DM 480 camera (Leica Microsystems, Wetzlar, Germany). ROS was detected by a fluorescence microplate reader (Synergy HTX Multi-Mode Microplate Reader, BioTek®, Winooski, VT, USA), and the excitation and emission wavelengths were 485 and 535 nm, respectively.

Western blot

Cells were cultured for 24 h in a six-well plate (3.2 × 105 cells/well) and pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h and then treated with LPS (0.1 ng/mL). After being washed with DPBS, cells were lysed using a RIPA buffer containing 100 mM of vanadate and 100 mM of PMSF, which were on ice for 15 min. The lysate was centrifuged at 16,200 g for 15 min at 4℃, after which the supernatant was collected. The protein content was measured by BCA protein assay reagent (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA). 20 μg of protein samples were loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoreses (SDS-PAGE) and separated at 60 V for 30 min and 90 V for 2 h. When the electrophoresis was completed, the protein samples were transferred to polyvinylidene fluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany) for 90 min. The membranes were blocked with 5% bovine serum albumin (BSA) in TBS-T (tris-buffered saline with 0.1% Tween 20) at room temperature for 1 h. Then, the membranes were incubated with primary antibodies overnight at 4℃. Primary antibodies were diluted at 1:1000. After being washed with TBS-T, the primary antibodies were incubated with HRP-conjugated secondary antibodies at room temperature for 2 h. Secondary antibodies were diluted at 1:3000. Finally, detection was performed using an ECL detection kit (Western Lightning ECL Pro; PerkinElmer Inc, Waltham, MA, USA), and the band signal was detected by Fusion Solo X (Vilber, Paris, France). The band intensity was analyzed with ImageJ software (version 1.52a; National Institute of Health).

NO production assay

Cells were cultured for 24 h in a six-well plate (3.2 × 105 cells/well) and pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, and 0.1 ng/mL of LPS was added before they were left overnight. The following day, the culture supernatant from each well was collected. 100 μL each of the supernatant and the modified Griess reagent (Sigma-Aldrich, MO, USA) were reacted in a 96-well plate at room temperature (RT) for 10 min. The absorbance of each well was measured with a microplate reader (Synergy HTX Multi-Mode Microplate Reader, BioTek®, Winooski, VT, USA) at 540 nm. NO concentration was calculated using a serial dilution of the sodium NO standard curve.

Enzyme-linked immunosorbent assay (ELISA)

Cells were cultured for 24 h in a six-well plate (3.2 × 105 cells/well) and pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, after which 0.1 ng/mL of LPS was added. Next, the culture supernatant from each well was collected, centrifuged at 15,000 g for 15 min, and used to measure the IL-6, IL-1β, TNF-α, and PGE2 concentrations. IL-6, IL-1β, TNF-α, and PGE2 Parameter Assay Kits (R&D Systems, Minneapolis, MN, USA) were used according to the manufacturer’s protocol.

Statistical analysis

All data were presented as mean ± standard deviation (SD). Statistical analyses were performed using the GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA). Quantitative measured values were analyzed to determine the significance of each group using one-way ANOVA with Dunnett’s post hoc test. The differences among groups were determined as statistically significant at p < 0.05, p <0.01, and p < 0.001.

Results

Cytotoxicity of BAY 60-7550 and LPS in RAW 264.7 cells

The MTT assay was performed to evaluate the cytotoxicity of BAY 60-7550 and LPS in RAW 264.7 cells. To evaluate the cytotoxicity of treatment with BAY 60-7550, RAW 264.7 cells were treated with BAY 60-7550 (1, 3, 10, and 30 μM) for 15 h (Fig. 1A). To evaluate the cytotoxicity of co-treatment with BAY 60-7550 and LPS, RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h and treated with LPS (0.1 μg/mL) for 4, 15, and 24 h, respectively. BAY 60-7550 was not cytotoxic at either 4 h or 15 h treatment conditions. However, a statistically significant cytotoxic effect was observed with 24 h treatment. Therefore, the experiment was conducted using 15 h treatment (Fig. 1B).

Figure 1. Cytotoxicity of BAY 60-7550 and LPS in RAW 264.7 cells. The cells were treated with BAY 60-7550 (1, 3, 10, and 30 μM) for 15 h (A). The cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h and treated with LPS (0.1 μg/mL) for 4, 15, or 24 h to observe the cytotoxicity of BAY 60-7550 and LPS (B). The number of viable cells was assessed by MTT assay. Results were expressed as a percentage (%) in comparison with the control. Each value represents the mean ± S.D from four independent experiments.

The inhibitory effect of BAY 60-7550 on the LPS-induced intracellular oxidative stress in RAW 264.7 cells

The cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM), followed by treatment with LPS (0.1 μg/mL). The protein expression of iNOS was significantly increased by 13.18-fold (p < 0.001) after stimulation with LPS (0.1 μg/mL) for 15 h. Pretreatment with BAY 60-7550 significantly downregulated the protein expression level of iNOS in the LPS-induced RAW 264.7 cells. In the experimental groups treated with 10 and 30 μM of BAY 60-7550, iNOS was significantly reduced by 52% and 77%, respectively (Fig. 2A).

Figure 2. The inhibitory effect of BAY 60-7550 on the LPS-induced intracellular oxidative stress in RAW 264.7 cells. The cells were pretreated with various concentrations of BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h and treated with LPS (0.1 μg/mL) for 15 h. The expression levels of iNOS were determined by western blot analysis (A). The culture supernatant was analyzed for NO. The NO content was determined by Griess reagent (B). The cells were pretreated with various concentrations of BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h and treated with LPS (0.1 μg /mL) for 15 h. ROS levels were assessed with the H2DCF-DA assay. ROS levels were expressed as a percentage (%) relative to the level in the vehicle control (C). Each value represents the mean ± S.D from experiments. ### indicates a significant difference from the vehicle control, which was treated with DMSO alone (###p < 0.001). * and *** indicate significant differences from the cells treated with 0.1 μg/mL of LPS alone (*p < 0.05 & ***p < 0.001).

The release of NO from RAW 264.7 cells was significantly increased by 8.47-fold (p < 0.001) after stimulation with LPS (0.1 μg/mL) for 15 h. Pretreatment with BAY 60-7550 downregulated the LPS-induced NO production. In the experimental group treated with 3 μM of BAY 60-7550, NO was significantly reduced by 14% (21.1 μM to 18.05 μM) (Fig. 2B).

Furthermore, the production of reactive oxygen species (ROS) in RAW 264.7 cells was significantly increased by 1.80-fold (p < 0.001) after stimulation with LPS (0.1 μg/mL) for 15 h. Pretreatment with BAY 60-7550 downregulated the ROS production in the LPS-induced RAW 264.7 cells. In the experimental groups treated with 3, 10, and 30 μM of BAY 60-7550, ROS production was significantly reduced by 18%, 30%, and 43%, respectively. These results can be confirmed in the fluorescence staining image of the cells (Fig. 2C).

The inhibitory effect of BAY 60-7550 on LPS-induced overexpression of COX-2 and PGE2 in RAW 264.7 cells

To determine the inhibitory mechanism of BAY 60-7550 on LPS-induced RAW 264.7 cells, the expression level of the COX-2 enzyme, which is involved in arachidonic acid (AA) metabolism, was examined by western blot analysis. In response to LPS, COX-2 protein was upregulated 5.17-fold compared to that in the vehicle control group. Furthermore, pretreatment with BAY 60-7550 (3 μM, 10 μM, and 30 μM) showed significant inhibitory effects on COX-2 expression of 36%, 49%, and 44%, respectively (Fig. 3A).

Figure 3. The inhibitory effect of BAY 60-7550 on LPS-induced overexpression of COX-2 and PGE2 in RAW 264.7 cells.
The protein expression levels of COX-2 were confirmed by western blot assay (A). RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 1h. The band density of COX-2 was quantified relative to the β-actin. PGE2 production was evaluated by ELISA assay in LPS-treated RAW 264.7 cells (B). RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 1 h. The absorbance at 450 nm was measured by ELISA protocol, and the concentration was measured according to the PGE2 absorbance standard curve. Each value represents the mean ± S.D from experiments. # & ### indicate significant differences from the vehicle control, which was treated with DMSO alone (#p < 0.05 and ###p < 0.001). *, **, and *** indicate significant differences from the cells treated with 0.1 μg/mL of LPS alone (*p < 0.05, **p < 0.01, and ***p < 0.001).

The PGE2 concentration of LPS-induced RAW 264.7 cells was analyzed by ELISA assay. These results indicate that the treatment of BAY 60-7550 inhibited the expression of COX-2 in LPS-induced RAW 264.7 cells. The group treated with 0.1 μg/mL LPS alone for 1 h showed upregulated PGE2 levels by 1.40-fold compared to the control group (857.97 pg/mL to 1205.19 pg/mL). Furthermore, pretreatment with BAY 60-7550 (3 μM, 10 μM, and 30 μM) showed significant inhibitory effects on PGE2 release with 20%, 17%, and 25%, respectively (Fig. 3B).

The inhibitory effect of BAY 60-7550 on LPS-induced overproduction of pro-inflammatory cytokines in RAW 264.7 cells

The TNF-α, IL-6, and IL-1β concentrations of LPS-induced RAW 264.7 cells were analyzed by ELISA. The group treated with 0.1 μg/mL of LPS alone for 4 h showed a significantly upregulated TNF-α level by 16.68-fold compared to the control group (1315.625 pg/mL to 21940.625 pg/mL). However, the group treated with BAY 60-7550 (1 μM) showed a decrease in TNF-α release (21940.625 pg/mL to 19412.5 pg/mL) (Fig. 4A). The group treated with 0.1 μg/mL of LPS alone for 4 h showed a significantly upregulated IL-6 level of 51.51-fold compared to the control group (6.36 pg/mL to 327.66 pg/mL). However, the group treated with BAY 60-7550 (1 μM) showed a decrease in IL-6 release (327.66 pg/mL to 266.76 pg/mL) (Fig. 4B). The group treated with 0.1 μg/mL of LPS alone for 24 h showed an upregulated IL-1β level by 1.50-fold compared to the control group (5.38 pg/mL to 8.08 pg/mL). However, the group treated with BAY 60-7550 showed decrease in IL-1β release (Fig. 4C). These results indicate that treatment with BAY 60-7550 inhibited overproduction of TNF-α, IL-6, and IL-1β in RAW 264.7 cells treated with LPS.

Figure 4. The inhibitory effect of BAY 60-7550 on LPS-induced overproduction of pro-inflammatory cytokines in RAW 264.7 cells. The levels of TNF-α, IL-6, and IL-1β were confirmed by ELISA. RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 4 h. The absorbance at 450 nm was measured by ELISA protocol, and the concentration was measured according to the TNF-α absorbance standard curve (A). RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 4 h. The absorbance at 450 nm was measured by ELISA protocol, and the concentration was measured according to the IL-6 absorbance standard curve (B). RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 15 h. The absorbance at 450 nm was measured by ELISA protocol, and the concentration was measured according to the IL-1β absorbance standard curve (C). Each value represents the mean ± S.D from three separate experiments. ### indicates a significant difference from the vehicle control, which was treated with DMSO alone (###p < 0.001). * and *** indicate significant differences from the cells treated with 0.1 μg/mL of LPS alone (*p < 0.05 and ***p < 0.001).

The inhibitory effect of BAY 60-7550 on LPS-induced activation of MAPK-ERK1/2 and NF-κB signaling pathways in RAW 264.7 cells

To determine the inhibitory mechanism of BAY 60-7550 on RAW 264.7 cells, the expression levels of ERK1/2 and NF-κB were examined by western blot analysis. In response to LPS, the expression level of phosphorylated ERK1/2 protein was significantly upregulated by 5.20-fold compared to that of the vehicle control group. Furthermore, pretreatment with BAY 60-7550 (10 μM and 30 μM) showed 35% and 63% inhibitory effects on ERK1/2 phosphorylation, respecively (Fig. 5A).

Figure 5. The inhibitory effect of BAY 60-7550 on LPS-induced activation of MAPK-ERK1/2 and NF-κB signaling pathways in RAW 264.7 cells. The levels of phosphorylated-ERK1/2, total-ERK1/2 (A), phosphorylated-NF-κB, and total-NF-κB (B) were confirmed by western blotting assay. RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 4 h. The band density of phosphorylated-ERK1/2 and the phosphorylated-NF-κB protein level was quantified relative to the total-ERK1/2 and total-NF-κB, respectively. NF-κB levels were expressed as a percentage (%) relative to the level in the vehicle control, and each value represents the mean ± S.D from experiments. ### indicates a significant difference from the vehicle control, which was treated with DMSO alone (###p < 0.001). *, **, and *** indicate significant differences from the cells treated with 0.1 μg/mL of LPS alone (*p < 0.05, **p < 0.01, and ***p < 0.001).

In response to LPS, the expression level of phosphorylated NF-κB protein was significantly upregulated by 4.68-fold compared to that of the vehicle control group. Furthermore, pretreatment with BAY 60-7550 (3 μM, 10 μM, and 30 μM) showed 34%, 31%,and 63% inhibitory effects on NF-κB phosphorylation, respecively (Fig. 5B).

Discussion

LPS is an important component in the Gram-negative bacteria’s outer membrane, as it reacts with CD14 on the surface of macrophages to produce inflammatory cytokines and activates the MAPK and NF-κB pathways (Meng and Lowell 1997). To observe anti-inflammatory effects and mechanisms of drugs, we applied the in vitro model, LPS-stimulated RAW 264.7 macrophages

Concentrations of intracellular cyclic nucleotides are primarily determined by PDEs, which hydrolyze cAMP and cGMP and prevent them from diffusing to other compartments (Zuo et al. 2019). PDE2 hydrolyzes both cAMP and cGMP (Meng and Lowell 1997) and is expressed in the peripheral and central nervous system (CNS) in a variety of tissues and cell types (Zhang et al. 2015). In the context of cognition, activation of the cAMP, protein kinase A (PKA), element-sensitive cAMP, and cGMP has been associated with long-term potentiation and the formation of memory (Xu et al. 2011). However, there have been no studies on the anti-inflammatory effects of PDE2 inhibitors. Therefore, this study focused on the anti-inflammatory activity of BAY 60-7550 which we determine to occur through the activation of MAPK (ERK1/2) and NF-κB pathways.

In this study, examination of cell viability using the MTT assay was performed first to investigate the cytotoxicity of LPS and BAY 60-7550 on RAW 264.7 cells. BAY 60-7550 did not show significant toxicity. Furthermore, when BAY 60-7550 and LPS were co-treated under the same conditions in our experiment, the cell viability was more than 80% in all experimental groups. This suggests that BAY 60-7550 and LPS have no cytotoxic effects in our experimental conditions.

Oxidative stress occurs when the balance between ROS and antioxidant compounds is disturbed (Adwas et al. 2019). Macrophages stimulated by LPS are known to initially promote the production of ROS, such as superoxides and H₂O₂, through the activation of NADPH oxidase (Mittal et al. 2014). While high levels of ROS promote cytotoxicity, non-toxic levels of ROS activate many signaling mediators, including phosphatidyl inositol 3-kinase (PI3K), phosphatase and tensin homolog (PTEN), NF-kB-inducing kinase (NIK), and MAPK, leading to a wide range of responses, such as inflammation and survival. ROS may also be involved in the activation of the NF-κB signaling pathway (Li and Engelhardt 2006). In particular, ERK1/2, also known as p44/42 in the MAPK family, is primarily activated by inflammation and growth factors (Mebratu and Tesfaigzi 2009). Moreover, NO is one of the most important inflammatory mediators involved in inflammatory processes (Waltz et al. 2015). Exposure of cells to bacterial products such as LPS, lipoteichoic acid (LTA), peptidoglycans, and bacterial DNA or whole bacteria will induce a high expression of iNOS and thereby increase the production of NO (Ginsburg 2002). In these situations, NO forms peroxynitrite, which acts as a cytotoxic molecule, resists invading microorganisms, and acts as a killer (Yao et al. 2019). This study revealed that BAY 60-7550 has anti-inflammatory effects, such as reducing iNOS, NO, and ROS which is increased by LPS.

This study confirmed that phosphorylation of ERK1/2 was effectively reduced by treatment with BAY 60-7550 through western blotting assay. This result suggests that BAY 60-7550 has anti-inflammatory effects by inhibiting the MAPK pathway. In addition, NF-κB acts as an important regulator in inflammation and as a transcription factor for iNOS, COX-2, and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) (Seok et al. 2021). When NF-κB is activated by LPS, it enters the nucleus, binds to the DNA, and expresses the target gene (Liu et al. 2017). Therefore, this study investigated the inhibitory effect of BAY 60-7550 on LPS-induced NF-κB. Our study showed that BAY 60-7550 reduces the activity of NF-κB, which downregulates the activity of inflammatory mediators and pro-inflammatory cytokines.

The upregulated level of NF-κB expression increases the expression of the COX-2 protein (Liu et al. 2017). COX-2 is an enzyme that catalyzes the first step in the conversion of AAs to PGE2 (Ha et al. 2022). Under inflammatory conditions, COX-2 is expressed and increases the conversion of AA to PGE2 (Liu et al. 2015). Because research has shown that LPS upregulates PGE2 production and COX-2 expression, ELISA and western blot tests were performed to measure the expression levels of PGE2 and COX-2. In the present study, the expressions of COX-2 and PGE2 were increased by treatment with LPS, and the levels of expression decreased after treatment with BAY 60-7550.

LPS-stimulated RAW 264.7 macrophages trigger an acute inflammatory response, which upregulates the MAPK and NF-κB pathways, thereby promoting the production of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β (Xu et al. 2014). This is beneficial in attracting circulating immune effector cells, such as neutrophils, to fight infection, but excessive inflammatory responses can damage tissues and organs (Chen et al. 2017). It is therefore important that the expression of these pro-inflammatory cytokines is properly regulated. This study has found that LPS increases TNF-α, IL-6, and IL-1β in macrophages and that BAY 60-7550 effectively reduces them.

Taken together, it was found that BAY 60-7550 significantly regulates the inflammatory response of RAW 264.7 cells induced by LPS. LPS triggered the MAPK and NF-κB pathways in RAW 264.7 cells, and BAY 60-7550 significantly inhibited their cellular signaling. In addition, various inflammatory mediators and pro-inflammatory cytokines, which are sub-regulators of the MAPK and NF-κB pathways, were also regulated by BAY 60-7550 treatment. This study revealed that the PDE2 selective inhibitor, BAY 60-7550, could have the potential to be an anti-inflammatory therapeutic.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (Grant Number NRF-2022R1F1A1076528).

Fig 1.

Figure 1.Cytotoxicity of BAY 60-7550 and LPS in RAW 264.7 cells. The cells were treated with BAY 60-7550 (1, 3, 10, and 30 μM) for 15 h (A). The cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h and treated with LPS (0.1 μg/mL) for 4, 15, or 24 h to observe the cytotoxicity of BAY 60-7550 and LPS (B). The number of viable cells was assessed by MTT assay. Results were expressed as a percentage (%) in comparison with the control. Each value represents the mean ± S.D from four independent experiments.
Drug Targets and Therapeutics 2023; 2: 30-40https://doi.org/10.58502/DTT.23.0001

Fig 2.

Figure 2.The inhibitory effect of BAY 60-7550 on the LPS-induced intracellular oxidative stress in RAW 264.7 cells. The cells were pretreated with various concentrations of BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h and treated with LPS (0.1 μg/mL) for 15 h. The expression levels of iNOS were determined by western blot analysis (A). The culture supernatant was analyzed for NO. The NO content was determined by Griess reagent (B). The cells were pretreated with various concentrations of BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h and treated with LPS (0.1 μg /mL) for 15 h. ROS levels were assessed with the H2DCF-DA assay. ROS levels were expressed as a percentage (%) relative to the level in the vehicle control (C). Each value represents the mean ± S.D from experiments. ### indicates a significant difference from the vehicle control, which was treated with DMSO alone (###p < 0.001). * and *** indicate significant differences from the cells treated with 0.1 μg/mL of LPS alone (*p < 0.05 & ***p < 0.001).
Drug Targets and Therapeutics 2023; 2: 30-40https://doi.org/10.58502/DTT.23.0001

Fig 3.

Figure 3.The inhibitory effect of BAY 60-7550 on LPS-induced overexpression of COX-2 and PGE2 in RAW 264.7 cells.
The protein expression levels of COX-2 were confirmed by western blot assay (A). RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 1h. The band density of COX-2 was quantified relative to the β-actin. PGE2 production was evaluated by ELISA assay in LPS-treated RAW 264.7 cells (B). RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 1 h. The absorbance at 450 nm was measured by ELISA protocol, and the concentration was measured according to the PGE2 absorbance standard curve. Each value represents the mean ± S.D from experiments. # & ### indicate significant differences from the vehicle control, which was treated with DMSO alone (#p < 0.05 and ###p < 0.001). *, **, and *** indicate significant differences from the cells treated with 0.1 μg/mL of LPS alone (*p < 0.05, **p < 0.01, and ***p < 0.001).
Drug Targets and Therapeutics 2023; 2: 30-40https://doi.org/10.58502/DTT.23.0001

Fig 4.

Figure 4.The inhibitory effect of BAY 60-7550 on LPS-induced overproduction of pro-inflammatory cytokines in RAW 264.7 cells. The levels of TNF-α, IL-6, and IL-1β were confirmed by ELISA. RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 4 h. The absorbance at 450 nm was measured by ELISA protocol, and the concentration was measured according to the TNF-α absorbance standard curve (A). RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 4 h. The absorbance at 450 nm was measured by ELISA protocol, and the concentration was measured according to the IL-6 absorbance standard curve (B). RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 15 h. The absorbance at 450 nm was measured by ELISA protocol, and the concentration was measured according to the IL-1β absorbance standard curve (C). Each value represents the mean ± S.D from three separate experiments. ### indicates a significant difference from the vehicle control, which was treated with DMSO alone (###p < 0.001). * and *** indicate significant differences from the cells treated with 0.1 μg/mL of LPS alone (*p < 0.05 and ***p < 0.001).
Drug Targets and Therapeutics 2023; 2: 30-40https://doi.org/10.58502/DTT.23.0001

Fig 5.

Figure 5.The inhibitory effect of BAY 60-7550 on LPS-induced activation of MAPK-ERK1/2 and NF-κB signaling pathways in RAW 264.7 cells. The levels of phosphorylated-ERK1/2, total-ERK1/2 (A), phosphorylated-NF-κB, and total-NF-κB (B) were confirmed by western blotting assay. RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 4 h. The band density of phosphorylated-ERK1/2 and the phosphorylated-NF-κB protein level was quantified relative to the total-ERK1/2 and total-NF-κB, respectively. NF-κB levels were expressed as a percentage (%) relative to the level in the vehicle control, and each value represents the mean ± S.D from experiments. ### indicates a significant difference from the vehicle control, which was treated with DMSO alone (###p < 0.001). *, **, and *** indicate significant differences from the cells treated with 0.1 μg/mL of LPS alone (*p < 0.05, **p < 0.01, and ***p < 0.001).
Drug Targets and Therapeutics 2023; 2: 30-40https://doi.org/10.58502/DTT.23.0001

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