Anti-inflammatory effect of nestorone in a lipopolysaccharide-induced acute lung injury model through regulation of the TLR-4/Myd88/NF-κB signaling pathway

Abstract

Progesterone plays a crucial and indispensable role in regulating immunity and attenuating inflammation. Nestorone^® (NES, segesterone acetate) is a steroidal progestin and a 19-norprogesterone derivative with no -CH₃ group radical at the 6-position. Here, we showed that NES enhanced the viability of lipopolysaccharide (LPS)-stimulated THP-1 cell-derived macrophages, potently inhibiting both arms of the Toll-like receptor 4 (TLR-4) signaling cascade triggered by LPS, especially the TLR-4/MyD88/NF-κB pathway. In addition, NES exerted an anti-inflammatory effect by significantly decreasing the secretion of inflammatory cytokines and chemokines in type II alveolar epithelial A549 cells and THP-1 cell-derived macrophages stimulated by LPS. Furthermore, we evaluated the potential of NES pre-treatment, administered 2 h prior to LPS exposure, to mitigate acute lung injury induced by LPS, using an LPS-induced acute lung injury (ALI) mouse model. In this study, NES alleviated lung inflammation and damage by reducing leukocyte infiltration and inflammatory cytokines in the bronchoalveolar lavage fluid (BALF) and lung tissues of mice. Interestingly, our findings indicate that NES at a dosage of 1 mg/kg (91.67%) was more effective than at dosages of 0.1 mg/kg (70.83%) or 10 mg/kg (87.50%), as well as more effective than dexamethasone (DEX, 5 mg/kg, 83.34%), in extending survival in mice subjected to lethal LPS-induced injury. Additionally, this dosage was more successful in reducing acute lung inflammation and alleviating diffuse alveolar damage in the lungs of C57 mice. Our study indicates that concentration is a critical determinant of the anti-inflammatory efficacy of NES. Consequently, NES emerges as a potentially promising therapeutic agent for the treatment of pulmonary inflammatory conditions through the modulation of TLR-4 signaling pathways.

Keywords: A549; Acute lung injury; Anti-inflammatory; Nestorone; Reporter cell; Toll-like receptor.

Fig. 1

LPS and progestogen as modulators of TLR-4/NF-κB/AP-1 signaling. A–D The viability of THP1-Blue™ cells stimulated by LPS at different concentrations (0.001–1000 ng/mL) for 6, 12, 24, or 48 h. E–H NF-κB/AP-1 activation in response to different doses of LPS. Effects of a concentration gradient of DEX, P4 and NES on I the viability of THP1-Blue™ cells, J the viability of THP1-Blue™ cells after LPS (10 ng/mL) stimulation, and K the NF-kB/AP-1 activation after 24 h. The results are presented as the mean ± SD from three separate experiments with triplet repeat represented by each data point. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. vehicle, and *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS

Fig. 2

NES as a modulator of TLR-4/IRF signaling. A–D The viability of THP-1 cell-derived macrophages after stimulation with 0.001–1000 ng/mL LPS for 6, 12, 24, or 48 h. E–H The effect of different doses of LPS on IRF activation at 6, 12, 24, and 48 h. I–K The effect of DEX, P4, and NES on THP1-Dual™ cells with or without LPS (10 ng/mL) stimulation for 24 h: I viability of THP-1 cell-derived macrophages was assessed at 24 h after treatment with different concentrations (0.0001–1000 µmol/L) of DEX, P4, or NES. J Viability of THP-1 cell-derived macrophages treated with the indicated concentrations (0.01–100 µmol/L) of DEX, P4, or NES for 24 h after 2 h of LPS (10 ng/mL) stimulation. K Effects of DEX, P4, and NES (0.01–100 µmol/L) on LPS (10 ng/mL)-induced IRF activation. Values shown are from triplicate wells per data point obtained in three separate cultures. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. vehicle, and *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS

Fig. 3

The anti-inflammatory effect of NES in type II alveolar epithelial A549 cells and THP1-Dual™ cell-derived macrophages. The levels of pro-inflammatory cytokines IL-6, KC, TNF-α, and MCP-1 in A549 cells (A–D) or THP-1 cell-derived macrophages (E–H) stimulated with LPS (10 ng/mL) for 24 h in the presence and absence of DEX, P4 or NES treatment. These results were typical of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS

Fig. 4

The inhibitory activity of NES in an ALI mouse model induced by LPS. A Schematic diagram of the animal experimental protocol. Mice were pretreated with DEX (5 mg/kg) or NES (0.1, 1, and 10 mg/kg) 2 h before LPS challenge, then sacrificed 24 h after stimulation with LPS (10 mg/kg), and biological measurements including differential BALF cell counting and epithelial permeability were performed for further analysis. C–F BALF in each group was collected for evaluation of C the total number of cells, as well as numbers of D neutrophils, E macrophages, F lymphocytes, G total protein content, and H lung W/D ratio. n = 8/group, *p < 0.05, **p < 0.01, ***p < 0.001 relative to the LPS group

Fig. 5

The protective effect of NES on lung damage in LPS-induced ALI mice. A–F Representative histological images of lung sections from mice, stained with H&E for histological evaluation, are presented at magnifications of ×10 and ×40: A sham control group (administered PBS only), B LPS + PBS group, C LPS + DEX group (5 mg/kg), D LPS + NES group (0.1 mg/kg), E LPS + NES group (1 mg/kg), and F LPS + NES group (10 mg/kg). Arrows (red) denote representative regions, highlighting features such as alveolar septal thickening, the presence of neutrophils within the alveolar and interstitial spaces, hyaline membranes, and proteinaceous debris. The scale bar in the left panel = 200 μm while that in the right panel = 50 μm. Lung damage was assessed by examination of five pathophysiological features to obtain the total injury score (G–K). G NES markedly decreased the neutrophil count within the alveolar space. H Higher doses of NES (1 and 10 mg/kg) markedly decreased the neutrophil count within the interstitial spaces, I diminished the formation of hyaline membranes and J proteinaceous debris, and K attenuated alveolar septal thickening. n = 8 per group, ns not significant

Fig. 6

The effect of NES on cytokine production in the BALF of mice with LPS-induced ALI. A Heat map showing cytokine profiles of the BALF under LPS challenge (10 mg/kg, 24 h) with/without NES pre-treatment (0.1, 1, and 10 mg/kg, 2 h before LPS stimulation) using a multiplexed cytokine assay. Different color codes represent the mean fold changes normalized to the PBS group, n = 6 (six were randomly selected from the BALF of eight mice in each group). B–G The levels of selected cytokines in the BALF: B IL-6, C G-CSF, D IFN-γ, E KC, F MCP-1, and G TNF-α. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS group. ns not significant

Fig. 7

The effect of NES on cytokine production in the serum of mice with LPS-induced ALI. Multiplexed cytokine array profile of serum (A) from mice stimulated with LPS with or without NES (0.1, 1, and 10 mg/kg) pre-treatment, different color codes represent the mean fold changes normalized to the PBS group. B–G The levels of selected cytokines in the serum: B IL-6, C G-CSF, D IFN-γ, E KC, F MCP-1, and G TNF-α; n = 6. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS group. ns not significant

Fig. 8

Effect of NES or DEX on the short-term and long-term survival of LPS-treated ALI mice (A–D): A A scheme of the prophylactic treatments of NES (0.1, 1, and 10 mg/kg) or DEX (5 mg/kg) in an LPS-induced ALI model. The mice received an intratracheal pretreatment with NES, DEX or PBS 2 h prior to the initial LPS challenge (20 mg/kg) on Day 0. Subsequently, the surviving mice were administered a second LPS dose (25 mg/kg) on Day 10. The remainder of the time (days 2–8 and days 12–20) treatments were administered at a fixed time (9:00 am) every other day; B the mouse body weights before and during the experiments; C the effects of NES and DEX on the short-term survival of mice with severe ALI; D the long-term survival of mice with severe ALI after secondary LPS (25 mg/kg) challenge with prophylactic treatments with NES or DEX. n = 8 per group, repeated three times. E and F The augmented tolerance to LPS, elicited by DEX and NES in reporter THP-1 cell-derived macrophages, is characterized by the suppression of E NF-κB activation and F IRF activation following a second LPS exposure 24 h subsequent to the initial LPS stimulation. n = 3, repeated three times. Values shown were obtained in three separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. double LPS stimulus

Fig. 9

Schematic diagram showing that LPS, as a ligand of TLR-4, can activate the TLR-4/MyD88/NF-κB and TLR-4/IRF-3 pathways and then upregulate the level of IL-6, TNF-α, MCP-1, IFN-γ, IL-1β, and KC, promoting lung damage. NES reverses this effect of LPS and protects against LPS-induced acute lung injury (ALI) due to its anti-inflammatory and immunomodulatory actions, which are possibly mediated by modulation of the TLR-4/NF-κB signaling pathways