GEN-27 exhibits anti-inflammatory effects by suppressing the activation of NLRP3 inflammasome and NF-κB pathway

Cell culture and treatment

RAW264.7 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and grown in Dulbecco's modified Eagle's medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 mg/mL streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified atmosphere with 5% CO₂. To stimulate the cells, the medium was exchanged for fresh DMEM containing 500 ng/mL of LPS.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

RAW264.7 cells were seeded into 96-well plates at a density of 2 × 10⁴ cells per well and then treated with different concentrations of GEN-27 (1, 5, 10, 20 μM) (purity >99.5%; Organic Chemistry Laboratory of China Pharmaceutical University, Nanjing, China) for 1 h, followed by stimulation with or without LPS for another 12 or 24 h. Untreated RAW264.7 cells were used as a control. Subsequently, MTT solution (0.5 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) was added to each well and incubated for 4 h at 37°C. Then, the culture medium was removed and 200 μL of dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added to dissolve the formazan dye. The absorbance at 490 nm was recorded by using a Microplate Reader (Infinite® F200; Tecan Group Ltd., Maennedorf, Switzerland).

Determination of nitrite and NO

RAW264.7 cells were plated in 24-well plates at a density of 5 × 10⁵ cells per well overnight and pretreated with GEN-27 (1, 5, 10 μM) for 1 h prior to stimulation with LPS (1 μg/mL) for 24 h. In brief, 100 μL of Griess reagent was mixed with an equal amount of culture supernatant and then the mixture was incubated for 15 min. The absorbance at 540 nm were determined using a Microplate Reader. The nitrite levels in culture supernatant were calculated as an indicator of NO production according to a sodium nitrite standard curve.

Treated RAW264.7 cells were collected and loaded with 1 μM fluorescent NO indicator 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM diacetate) (Molecular probes, Eugene, OR, USA) for 1 h in the dark. DAF fluorescence (excitation 492 nm, emission 515 nm) was determined by a fluorescence microscope (FV1000; Olympus, Tokyo, Japan).

Transient transfections and luciferase assays

RAW264.7 cells were seeded in 24-well plates and transiently transfected with 1 μg/mL pGL3 empty plasmids, or the indicated genes were harbored including iNOS promoter (iNOS-Luc; Bioworld, Nanjing, China) or NF-κB promoter (NF-κB-Luc; Biomyx Technology, San Diego, CA, USA), along with 0.25 μg/mL pRL-TK plasmid (as an internal control) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. After 24 h of transfection, the cells were treated with GEN-27 (1, 5, 10 μM) for 1 h, followed by stimulation with 1 μg/mL LPS for a further 24 h. Cell lysates were collected and luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer's instruction.

Inflammatory cytokine detection

The RAW264.7 cells were treated as above and the concentrations of pro-inflammatory cytokines including IL-6, TNF-α, IL-1β, and IL-18 in the cell supernatant were determined using corresponding commercial enzyme-linked immunosorbent assay (ELISA) kits (BioLegend, Cambridge, UK).

Measurement of intracellular reactive oxygen species (ROS)

RAW264.7 cells were cultured in a six-well plate at a density of 2 × 10⁶ per well, and treated as above. Then, the cells were harvested, washed twice with PBS, and incubated with 20 μM 2,7-dichlorofluorescein diacetate (DCFH-DA; BestBio, Shanghai, China) at 37°C for 30 h in the dark. Then cells were washed twice with cold PBS to rinse the unconjugated dye and DCF fluorescence distribution was monitored by a fluorescence microscope at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

Western blot analysis

RAW264.7 cells were treated with GEN-27 (1, 5, 10 μM) for 1 h, followed by stimulation with 1 μg/mL LPS or 5 mM adenosine triphosphate (ATP) for 24 h. Then, cells were collected and lysed with radioimmunoprecipitation lysis buffer (Beyotime, Shanghai, China) supplemented with protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany). An equal amount of protein samples (20 µg/lane) was separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA, USA). Non-specific sites on the membrane were blocked with 5% non-fat milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST) buffer for 2 h at room temperature and the membranes were immunoblotted with primary antibodies including anti-iNOS (1:1,000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-COX-2 (1:1,000 dilution; Cell Signaling Technology), anti-NLRP3 (1:2,000 dilution; Cell Signaling Technology), anti-apoptosis-associated speck-like protein-containing a CRAD (ASC) (1:2,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-caspase 1 (1:1,000 dilution; Cell Signaling Technology), ani-IL-1β (1:1,000 dilution; Cell Signaling Technology), anti-phosphorylated p65 (p-p65, 1:1,000 dilution; Cell Signaling Technology), anti-p65 (1:1,000 dilution; Cell Signaling Technology), or anti-β-actin (1:2,000 dilution; Abcam, Cambridge, MA, USA) at 4°C overnight, followed by incubation with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2,000 dilution; Abcam) at room temperature for 1 h. The protein bands were then visualized using enhanced chemiluminescence reagent (GE Healthcare Life Sciences, Little Chalfont, UK).

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was isolated from the treated RAW264.7 cells using the TRIzol reagent (Invitrogen). Complementary DNA was synthesized from 1 μg of total RNA using the PrimeScript RT Reagent kit (Takara Bio Inc., Otsu, Japan). Expressions of iNOS and COX-2 were determined using Fast SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) on an ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, California, USA), with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control. The primers used were as follows: iNOS, forward, 5′-TTGTGCATCGACCTAGGCTGGAA-3′ and reverse, 5′-GACCTTTCGCATTAGCATGGAAGC-3′; COX-2, forward, 5′-GAAGTCTTTGGTCTGGTGCCTG-3′ and reverse, 5′-GTCTGCTGGTTTGGAATAGTTGC-3′; GAPDH, forward, 5′-GGATGCAGGGATGATGTTC-3′ and reverse, 5′-TGCACCACCAACTGCTTAG-3′. The relative gene expression was calculated using method.

Caspase 1 activity assay

RAW264.7 cells treated as above were harvested and homogenized with PBS in microcentrifuge tubes. The caspase 1 activity in cell supernatant was determined using a commercial assay kit (R&D Systems, Minneapolis, MN, USA).

Statistical analysis

Data were expressed as means ± standard deviation from three repeated experiments. Significant differences were performed by Student's t test or one-way analysis of variance. All statistical analyses were carried out using SPSS version 19.0 (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered to be statistically significant.

GEN-27 exhibited anti-inflammatory effects in LPS-stimulated RAW264.7 cells

As shown in Figure 1A, no cytotoxicity was observed with GEN-27 treatment at 1, 5, or 10 μM in RAW264.7 cells with or without LPS stimulation, as demonstrated by MTT assay. However, 20 μM GEN-27 was found to be toxic for RAW264.7 cells. Therefore, non-toxic concentrations were used for further analyses. To explore the anti-inflammatory effects of GEN-27 on LPS-induced inflammation, we detected the levels of inflammatory mediators including nitrite and NO in RAW264.7 cells treated with GEN-27 (1, 5, or 10 μM) for 1 h, followed by 1 μg/mL LPS stimulation. The results demonstrated that GEN-27 dramatically reduced LPS-induced nitrite level in RAW264.7 cells in a dose-dependent manner (Figure 1B). Similarly, NO level was also dose-dependently inhibited following GEN-27 administration in LPS-stimulated RAW264.7 cells (Figure 1C). These results demonstrated that GEN-27 exhibited anti-inflammatory effects in LPS-stimulated RAW264.7 cells.

GEN-27 suppressed the release of LPS-induced pro-inflammatory cytokines in RAW264.7 cells

Next, we further analyzed the effects of GEN-27 on LPS-induced pro-inflammatory cytokines such as IL-6, TNF-α, IL-1β, and IL-18. Consistently with the above results, ELISA results manifested that LPS stimulation distinctly enhanced the secreted levels of IL-6 (Figure 2A), TNF-α (Figure 2B), IL-1β (Figure 2C), and IL-18 (Figure 2D) in the cell supernatant of RAW264.7 cells. However, GEN-27 treatment remarkably reversed LPS-induced increase of pro-inflammatory cytokine levels in RAW264.7 cells in a dose-dependent manner.

GEN-27 suppressed LPS-induced iNOS and COX-2 expressions in RAW264.7 cells

The iNOS and COX-2 are well-known to play major roles in the biosynthesis of NO (Lee et al., 2015). Therefore, we explored the effect of GEN-27 on the expressions of iNOS and COX-2. Western blot and qRT-PCR analysis demonstrated that LPS significantly increased the protein and messenger RNA levels of iNOS and COX-2 in RAW264.7 cells, while the efficacy was attenuated by addition of GEN-27 (Figures 3A and 3C). To clarify the effect of GEN-27 on the transcriptional activation of iNOS gene, luciferase reporter assay was performed in RAW264.7 cells. Results showed that GEN-27 notably blocked the promoter activity of iNOS induced by LPS in a concentration-dependent manner (Figure 3D). Collectively, these results revealed that GEN-27 suppressed the expressions of iNOS and COX-2 in LPS-induced RAW264.7 cells.

GEN-27 inhibited LPS-induced activation of NLRP3 inflammasome in RAW264.7 cells

NLRP3 inflammasome, as one of the most well-characterized inflammasomes, is a multiprotein complex that orchestrates an innate immune response to infection and mediates activation of inflammatory caspases (Elliott and Sutterwala, 2015). It is known that the formation of NLRP3 inflammasome requires the oligomerization of NLRP3, ASC, and caspase 1, which was subsequent production of cytokine IL-1β. Thus, we investigated the effects of GEN-27 on the NLRP3 inflammasome in LPS-stimulated RAW264.7 cells. It has been proposed that ROS is essential for NLRP3 inflammasome activation and subsequent production of cytokine IL-1β (Biasi et al., 2013). We found that LPS stimulation obviously increased ROS production in RAW264.7 cells compared with the control group, which was concentration-dependently retarded following GEN-27 treatment (Figure 4A). Moreover, GEN-27 drastically repressed LPS-induced activation of caspase 1 activity in RAW264.7 cells in a dose-dependent manner (Figure 4B). Western blot analysis further implicated that the protein levels of NLRP3, ASC, and caspase 1 were strikingly augmented in LPS-treated RAW264.7 cells, while these changes were substantially suppressed after GEN-24 administration (Figures 4C and 4D). To further validate this effect, RAW264.7 cells were exposed to GEN-27 (1, 5, or 10 μM) for 1 h before treatment with 5 mM ATP for 24 h, which has been recognized as an NLRP3 inflammasome activator (Ozaki et al., 2015). The results demonstrated that the protein levels of NLRP3 and the subsequent release of IL-1β were conspicuously improved by ATP treatment in RAW264.7 cells, which was prominently suppressed by cotreatment with GEN-27 and ATP in a dose-dependent manner (Figures 4E and 4F). These results suggested that GEN-27 inhibited LPS-induced activation of NLRP3 inflammasome in RAW264.7 cells.

GEN-27 inhibited LPS-induced NF-κB pathway activation in RAW264.7 cells

It is well-known that activation of NF-κB pathway and nuclear translocation of NF-κB p65 play a crucial role in controlling inflammatory mediator expression and mediating the inflammatory response (Yenari and Han, 2006). To further analyze the anti-inflammatory mechanism of GEN-27, western blot was performed to measure the expressions of characteristic proteins in NF-κB pathway including p65 and p-p65. The results showed that LPS stimulation significantly enhanced the ratio of p-p65/p65 in RAW264.7 cells, while GEN-27 treatment strikingly attenuated LPS-induced increase of p-p65/p65 ratio (Figures 5A and 5B). Moreover, GEN-27 also concentration-dependently suppressed the increase of transcriptional activity of NF-κB induced by LPS in RAW264.7 cells (Figure 5C). Collectively, we concluded that GEN-27 remarkably impeded LPS-induced activation of NF-κB pathway in RAW264.7 cells.