Agmatine suppresses the imidazoline I 2 receptor/ribosomal S6 kinase 2/NF-κB signaling pathway regulating alveolar macrophage polarization and ameliorating sepsis-associated acute lung injury

Abstract

Acute respiratory distress syndrome (ARDS) is an acute diffuse inflammatory lung injury characterized by damage to alveolar epithelial cells and pulmonary capillary endothelial cells. Compared with ARDS caused by other causes, the subtypes of ARDS caused by sepsis are more serious and lead to poor prognosis and higher mortality. Agmatine (AGM) is a biological metabolite of L-arginine decarboxylation, proven to ameliorate sepsis-induced acute lung injury (SALI), but the mechanism remains unclear. Therefore, this study aims to explore the role of AGM in SALI, clarify the relationship between the I₂R/RSK2/NF-κB signaling pathway regulated by AGM and macrophage polarization, and provide a theoretical basis for the clinical treatment of SALI. Cellular and animal models of lung injury in sepsis were established with lipopolysaccharide (LPS). We conducted a series of experiments to examine the oxygenation index (OI), wet/dry ratio (W/D) of the lung, pathological changes, levels of inflammation, Apoptosis and related protein expression in different groups of mice. Finally, we found that AGM can ameliorate sepsis-induced acute lung injury by suppressing the I₂R/RSK2/NF-κB signaling pathway and modulating polarization of alveolar macrophage.

Introduction

Sepsis, a syndrome of organ dysfunction characterized by inflammation disorders associated with infections, has been identified by WHO as a key global health problem, with a reported mortality rate of 30%–45% in hospitalized patients.^1,2 Acute lung injury (ALI) is the most common severe manifestation of sepsis and the leading cause of short-term death and long-term decline in quality of life. ³ The pathogenesis of sepsis-induced lung injury (SLI) is the damage of alveolar epithelium and pulmonary vascular endothelium, which results in increased alveolar-capillary permeability and acute respiratory distress syndrome (ARDS). ⁴ At present, the treatment of SLI mainly consists of prevention, symptomatic and supportive treatment, which lacks specificity. It is urgent to find new targets for the prevention and treatment of SLI. ⁵ Agmatine (AGM) is an endogenous polyamine synthesized by decarboxylation of L-arginine. ⁶ AGM has a variety of biological effects such as neuroprotection, antidepressant, and anti-cancer cell proliferation. ⁷ Studies have shown that endogenous AGM is associated with the progression of sepsis, can ameliorate lipopolysaccharide (LPS)-induced sepsis^8,9 and inhibit the apoptosis of spleen cells and dendritic cells. ¹⁰ In addition, our previous study also confirmed that AGM could decrease hyperoxia-induced lung injury by regulating the expression of mitochondrial fusion protein 1. ¹¹ As an endogenous ligand of α2-adrenergic receptor, ¹² imidazoline receptor, ¹³ and N-methyl-D-aspartate (NMDA) glutamate receptor. ¹⁴ A large number of studies have confirmed that AGM can exert neuroprotective effects by regulating various signaling pathways through the binding of Imidazoline I2 receptors.^15–17 Furthermore, Li et al. ¹⁰ found that AGM could improve sepsis by regulating the Imidazoline I₂ receptor (I₂R)/Ribosomal S6 kinase 2 (RSK2)/nuclear factor-κB (NF-κB) pathway in 2020, but its mechanism in the direction of lung injury protection has not been further studied. On the other hand, macrophage polarization is crucial in the progression of SLI, among which NF-κB can mediate macrophage polarization to M1 type, aggravating lung injury. ¹⁸ Therefore, this study aims to explore the role of AGM in SLI, clarify the relationship between the I₂R/RSK2/NF-κB signaling pathway regulated by AGM and macrophage polarization, and provide a theoretical basis for the clinical treatment of SLI.

Materials and methods

Animal and animal model

The SPF healthy male C57BL/6 mice (6–8 weeks old, 18–22 g) were provided by the Laboratory Animal Center of Zunyi Medical University (Animal License No. SYXK Qian 2021-0004). All animals were housed in the SPF facility of the Biological Research Center under controlled conditions: temperature 18–22°C, humidity 50%–60%, and a 12 h light/dark cycle, with free access to food and water. A total of 30 mice were randomly assigned into 6 groups (n = 5) ¹⁹ : Control group, LPS group, LPS + AGM group, LPS + AGM + 2-BFI group, LPS + AGM + RSK2 group, LPS + AGM + Empty vector group.

To establish the sepsis-induced lung injury model, mice received an intraperitoneal injection of LPS (10 mg/kg). After 12 h, AGM (400 mg/kg)^10,11 was administered via tail vein injection. At 24 h AGM treatment, all mice were anesthetized with sodium pentobarbital (150 mg/kg), followed by bronchoalveolar lavage and lung tissue collection.

The experimental procedures were approved by the Experimental Animal Ethics Committee of Zunyi Medical University (Approval No. KLL-2021-095).

Cell culture and treatment

The mouse alveolar macrophage cell line MH-S (ATCC, CRL-2019) was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum under standard conditions.

To establish the in vitro model of sepsis-induced lung injury, MH-S cells were stimulated with LPS (1 μg/ml) for 12 h. For AGM pretreatment, cells were incubated with AGM (100 μM) for 1 h prior to LPS exposure. For the combined treatment group, cells were pretreated with both AGM (100 μM) and 2-BFI (10 μM) for 1 h before LPS stimulation.

After 12 h of LPS exposure, the cell culture supernatant was collected, and cytoplasmic and nuclear proteins were extracted for further analysis.

In vitro cytotoxicity assay

To assess potential cytotoxicity of AGM and 2-BFI, MH-S alveolar macrophages (ATCC CRL-2019) were seeded in 96-well plates (5 × 10³ cells/well) and treated with AGM (0, 25, 50, 75, 100, 125, 150, 175, 200 μM) or 2-BFI (0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20 μM) for 1h. Cell viability was evaluated using the CCK-8 assay, and absorbance was measured at 450 nm. Results were calculated as percentage viability relative to vehicle controls.

siRNA transfection

The complex of siRNA used for RSK2 silencing was obtained from Hanheng Biotechnology and was prepared according to the manufacturer’s instructions. Untransfected MH-S cells silenced the RSK2 gene after being treated with a siRNA complex for 24 h. The expression of the RSK2 gene in lung tissue was then treated with a siRNA complex by airway administration for 48 h. The expression level of RSK2 was detected using RT-PCR.

Reagents

Cell incubators were purchased from Thermo Fisher Scientific, USA; Flow cytometer was purchased from Beckman Coulter, USA; Fluorescence microscope was purchased from Leica, USA; Bio-Rad ChemiDoc MP gel imaging System was purchased from Bio-Rad, USA; Electrophoresis apparatus was purchased from Bio-Rad, USA; The microplate reader was purchased from Bio-Rad Company, USA. PVDF membrane was purchased from Sigma Company in the United States; Enhanced chemiluminescence reagent (ECL) cartridges were purchased from Affinity Biosciences, USA; LPS was purchased from Sigma Company, USA; Imidazoline I2 receptor agonist (2-BFI) were purchased from MCE Corporation, USA; TUNEL kit was purchased from Thermo Fisher Scientific, USA; ELISA kits for TNF-α, IL-1β, IL-6, IL-10 and IL-10 were purchased from Wuhan Ilerite Biotechnology. CD11b, F4/80, CD206, iNOS antibody, β-actin, ribosomal S6 kinase 2 (RSK2), phospho-RSK2 (p-RSK2), inhibitor of nuclear factor-κBα (IκBα), phospho-nuclear factor-κBα (p-IκBα), and phospho-P65 (p-p65) and secondary antibodies were purchased from CST, USA.

Determination of the oxygenation index (OI)

After the anesthesia of mice, the carotid artery was taken for blood gas analysis to detect OI, corrected OI = PaO₂/[FiO₂ × (P/760)]. PaO₂ is the partial pressure of arterial oxygen; FiO₂ is the inhaled oxygen concentration; P in Zunyi is 680 mmHg.

Wet/dry ratio (W/D) of lung

After the mice were anesthetized, the upper lobe of the left lung was removed, and the excess water was wiped off by absorbent paper and weighed as a wet mass. Then, the left lung was dried at 60℃ for 48 h and weighed as dry mass, and W/D was calculated.

Hematoxylin and eosin staining and pathological scoring of lung tissue

After anesthesia, the lower lobe of the left lung was removed, the excess water was wiped off by absorbent paper, cut into small pieces (1 mm), fixed in 4% paraformaldehyde, embedded and sectioned, stained with hematoxylin and eosin (HE), and finally observed under a light microscope for pathological scoring. ²⁰

Detection of inflammatory mediators in bronchoalveolar lavage fluid

Whole lung lavage was performed by injecting 1 ml of normal saline into the trachea using a syringe. After 30 s, the bronchoalveolar lavage fluid was withdrawn and repeated three times. The levels of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interleukin-6(IL-6) and IL-10 were detected at 450 nm by a microplate reader.

Flow cytometric analysis of macrophages in bronchoalveolar lavage fluid

Whole lung lavage was performed by injecting 1 ml of normal saline into the trachea. After 30, the bronchoalveolar lavage fluid was collected and centrifuged to obtain cell precipitation. The cells were resuspended and fixed, ruptured, and incubated with CD11b and F4/80 mixed antibodies for 30 min. After 30 min of incubation with FITC-labeled iNOS antibody and PE-labeled CD206 antibody, the percentages of iNOS⁺ and CD206⁺ cells in bronchoalveolar lavage fluid (BALF) were detected.

Detection of apoptotic cells with TUNEL

After the mice were anesthetized, the right upper lobe of the lung was removed, and the operation steps were according to the instructions of the TUNEL kit. Finally, the slices were sealed with a DAPI-containing anti-fluorescence quenching agent and photographed under a fluorescence microscope.

Western blotting

Total protein was extracted from the MH-S cell line after adding lysate. Bicinchoninic Acid (BCA) was used for protein quantification. The gel was prepared, and the sample(35 μg) was loaded for electrophoresis separation. RSK2, p-RSK2, IκBα, p-IκBα, p-p65 and β-actin (1:1000), incubated overnight at 4℃ and incubated for 2 h at room temperature for 2 h. The ECL working solution was developed and then exposed to the gel imaging system. Image J software was used to process the experimental images. The β-actin was used as an internal control.

Statistical analysis

Normality of data distribution was assessed using the Shapiro–Wilk test, and homogeneity of variance was evaluated using Levene’s test. If both assumptions were met, data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparisons test. For data that did not satisfy normality or homoscedasticity, the Kruskal–Wallis test was applied. Statistical analyses were performed using GraphPad Prism (version 10.0), and p value < 0.05 was considered statistically significant.

Results

AGM alleviates sepsis-induced lung injury

As illustrated in Figure 1, AGM treatment conferred significant protection against LPS-induced lung injury across multiple functional and structural metrics. AGM markedly improved arterial oxygenation (OI) compared to the LPS group, an effect abolished by 2-BFI (Figure 1(a)). It also reduced pulmonary edema, evidenced by a lower lung wet/dry weight ratio, which was similarly reversed upon 2-BFI co-administration (Figure 1(b)). At the inflammatory level, AGM downregulated pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and elevated anti-inflammatory IL-10 in BALF, effects that were also blocked by 2-BFI (Figure 1(c)–(f)). Histopathological and apoptotic analyses further demonstrated that AGM alleviated alveolar damage, inflammatory infiltration, and apoptosis—as indicated by TUNEL staining and apoptotic index—relative to the LPS-injured group. These improvements were attenuated by 2-BFI (Figure 1(g)–(j)). Collectively, these results indicate that AGM mitigates sepsis-induced lung injury by enhancing gas exchange, limiting vascular leakage, suppressing structural damage and apoptosis, and modulating inflammation, primarily through a 2-BFI-sensitive mechanism.

Figure 1.

AGM alleviates sepsis-induced lung injury. (a) Oxygenation index (OI) of each group. (b) Wet/dry weight ratio of lung tissue in each group. (c–f) Levels of inflammatory cytokines in bronchoalveolar lavage fluid were determined in each group. (g–j) Histopathological changes and apoptosis were evaluated via H&E staining and TUNEL staining in each group.

AGM can induce the polarization of macrophages

The phenotype of alveolar macrophages in bronchoalveolar lavage fluid was detected by flow cytometry, and the results are shown in Figure 2. Compared with the Control group, the proportion of M1 and M2 alveolar macrophages in the LPS group was increased. Compared with the LPS group, the proportion of M1 type in the LPS+AGM group was decreased, and the proportion of M2 type was increased. Compared with the LPS+AGM group, the proportion of M1 type in the LPS+AGM+2-BFI group was increased, and the proportion of M2 was decreased. These results indicate that AGM can induce the transformation of alveolar macrophages into M2, and 2-BFI can block the above process.

Figure 2.

The phenotype of alveolar macrophages in bronchoalveolar lavage fluid was detected by flow cytometry.

Effect of AGM on the expression of I₂R/RSK2/NF-κB signaling pathway-related proteins in alveolar macrophages

Western blot analysis of I₂R/RSK2/NF-κB signaling pathway-related proteins is shown in Figure 3. Compared with the Control group, the LPS group, the expression of p-RSK2, p-p65, p-IκBα, and nuclear p-p65 were up-regulated, and IκBα was down-regulated. Compared with the LPS group and LPS+AGM group, the expression of p-RSK2, p-IκBα, and nuclear p-p65 were down-regulated, and IκBα was up-regulated. Compared with the LPS+AGM group and LPS+AGM+2-BFI group, the expression of p-RSK2, p-p65, p-IκBα, and nuclear p-p65 were up-regulated, and IκBα was down-regulated. These results indicate that AGM can inhibit the I₂R, and 2-BFI can block the I₂R inhibition effect of AGM.

Figure 3.

Expression of I₂R/RSK2/NF-κB signaling pathway-related proteins in alveolar macrophages.

In vitro cytotoxicity of AGM and 2-BFI

A CCK-8 assay performed in MH-S cells showed that AGM (100 μM) and 2-BFI (10 μM) did not reduce cell viability within the concentrations used in this study, and cell viability remained >95%, indicating no overt cytotoxicity (Supplemental Figure S1).

Silencing RSK2 effectively blocks the inhibitory impact of AGM on the I2R/RSK2/NF-κB signaling pathway

As is shown in Figure 4, the expression level of RSK2 was detected using RT-PCR. Compared with the control group, the expression level of RSK2 in the si-RSK2 group was significantly reduced (1.037 ± 0.074 vs 0.191 ± 0.044). Compared with the LPS+AGM group and LPS+AGM+si-RSK2 group, the expression of RSK, p-RSK2, IκBα were down-regulated, and p-IκBα, nuclear p-p65 were up-regulated. The silencing of RSK2 abrogated the inhibitory effect of AGM on the phosphorylation and degradation of IκB, as well as the nuclear translocation of p65. These results indicate that RSK2 mediates the regulation of AGM on the I₂R/RSK2/NF-κB signaling pathway. AGM can inhibit the I₂R/RSK2/NF-κB pathway, thereby inhibiting the nuclear translocation of p65.

Figure 4.

After Silencing RSK2 the expression of I₂R/RSK2/NF-κB signaling pathway related proteins and the silencing efficiency of RSK2 in alveolar macrophages.

Silencing RSK2 inhibits the amelioration of lung injury and the polarization of macrophages induced by AGM

As is shown in Figure 5, compared with the LPS+AGM group, the pathological score of lung tissue in the LPS+AGM+si-RSK2 group was increased. Compared with the LPS+AGM group, the proportion of M1 type in the LPS+AGM+si-RSK2 group was increased, and the proportion of M2 was decreased. These data indicate that silencing RSK2 abrogates the protective function of AGM in the lung and inhibits macrophage polarization toward the M2 phenotype.

Figure 5.

After Silencing RSK2 the lung tissue injury and the phenotype of alveolar macrophages in bronchoalveolar lavage fluid. (a, c) Histopathological changes were evaluated via H&E staining in each group. (b, d, e) The phenotype of alveolar macrophages in bronchoalveolar lavage fluid was detected by flow cytometry. CD11b⁺F4/80⁺ cells indicate macrophages. iNOS⁺ cells indicate M1 macrophages. CD206⁺ cells indicate M2 macrophages.

Discussion

ARDS is an acute diffuse inflammatory lung injury characterized by damage to alveolar epithelial cells and pulmonary capillary endothelial cells, which is mainly manifested as non-cardiogenic pulmonary edema caused by extrapulmonary and intrapulmonary risk factors. ²¹ ARDS is a common heterogeneous disease in critical care medicine, and clinical treatment failure is a common challenge. Its subtype targeted therapy is helpful for the treatment of ARDS. Compared with ARDS caused by other causes, the subtypes of ARDS caused by sepsis are more serious and lead to poor prognosis and higher mortality.^22,23 Sepsis is a systemic inflammatory response to severe infection, an imbalance between pro-inflammatory cytokines (such as TNF-α and IL-1β) and anti-inflammatory cytokines (IL-10), and is increasingly recognized as an important component of sepsis pathogenesis. ²⁴ SLI is an injury of the alveolar- capillary endothelium and alveolar epithelium on the basis of sepsis, resulting in increased alveolar-capillary permeability and decreased alveolar surfactant. ²⁵ Relevant epidemiological studies have shown that the incidence of ALI in sepsis patients is 68.2%, and the 90-day mortality rate of patients with ALI is as high as 35.5%. ²⁶ Therefore, early detection or intervention of inflammatory response disorders may be a promising approach to effectively alleviate sepsis. In the present study, LPS could induce the production of pro-inflammatory cytokines and apoptosis in the lung tissue of mice, increase alveolar capillary leakage, promote pulmonary edema, and seriously affect oxygenation capacity. AGM reduced LPS-induced production of pro-inflammatory cytokines and increased anti-inflammatory cytokines in vivo while reducing pulmonary edema, inhibiting apoptosis, and improving oxygenation.

AGM is a biological metabolite of L-arginine decarboxylation, which can induce a variety of biological effects in vivo, such as neuroprotective effect,^27–29 and protective effect of hepatic ischemia-reperfusion injury. ³⁰ It is well known that receptors that AGM can bind include α2-adrenergic receptors, ¹² imidazoline receptors, ¹³ and N-methyl-D-aspartate (NMDA) glutamate receptors. ¹⁴ In SLI, activated α2-adrenergic receptors can inhibit lung inflammation and protect against LPS-induced lung injury by inhibiting inflammasome 3 activity and promoting M2-type macrophage polarization. ³¹ In pulmonary fibrosis, activation of the NMDA receptor can increase ferroptosis levels and promote alveolar type Ⅱ epithelial cell injury and pulmonary fibrosis. Inhibition of NMDA receptors can reduce bleomycin-induced lung injury and fibrosis in mice. ³² I₂R is involved in analgesia, glial tumors, inflammation, and a large number of brain disorders such as Alzheimer’s disease, Parkinson’s disease, and different psychiatric disorders. ³³ One study showed that AGM can decrease lung injury, ³⁴ and the effect could be concerned with α2-adrenergic receptor and imidazoline receptor.^35,36 At present, there is no research report on the correlation between I₂R and acute lung injury. Li et al. ¹⁰ used antagonists and agonists of α2-adrenergic, imidazoline, and NMDA receptors in a sepsis model and found that the protective effect of AGM on systemic inflammatory response induced by LPS was exerted in an I₂R-dependent manner. In the present study, AGM can improve lung oxygenation function and injury in SLI mice, and the pharmacological effects of AGM can be blocked by using the I₂R agonist 2-BFI, suggesting that AGM may be a potential antagonist in SLI. Consistent with previous findings, agmatine alone does not cause detectable pulmonary toxicity. In the study by Liu et al., ¹¹ agmatine administration in the absence of hyperoxic exposure did not induce lung inflammation, edema, or pathological injury, supporting that AGM exerts no harmful effect under baseline physiological conditions. For this reason, an AGM-only group was not incorporated into the present study.

NF-κB plays a key role in the pathogenesis of sepsis. In the NF-κB signaling pathway, LPS-induced inflammation upregulates the level of IκBα and NF-κB subunit p65 complex (IκBα/p65) through Toll-like receptor 4 (TLR4), IκBα is degraded after phosphorylation, p65 nuclear translocation and DNA binding. It promotes the transcription of inflammatory factors. ³⁷ Studies have shown that NF-κB can inhibit the release of a variety of pro-inflammatory factors by regulating the polarization of macrophages to M2 type, thus reducing nerve cell damage. ³⁸ In SLI, neutrophil exosome-derived miR-30d-5p promotes the polarization of M1 macrophages and aggravates lung injury by activating the NF-κB signaling pathway. Inhibition of the NF-κB signaling pathway promotes M2 macrophages to reduce the inflammatory response and contributes to the alleviation of SLI.^18,39 Our results showed that AGM could inhibit the release of inflammatory cytokines and regulate the polarization of macrophages to the M2 type. Moreover, the anti-inflammatory effect of AGM on macrophage polarization was achieved by inhibiting the nuclear translocation of p65 and the binding activity of NF-κB but not by directly inhibiting the expression and phosphorylation of NF-κB subunits. In addition, although AGM did not down-regulate the expression level of IκBα, AGM down-regulated the phosphorylation level of IκBα, blocked its degradation, and inhibited the nuclear translocation of p65, thereby inhibiting LPS-induced M1-type macrophage polarization and inflammatory factor production.

RSK2 is a serine/threonine kinase that can phosphorylate the ser32 site of IκBα, leading to the degradation of IκBα. ⁴⁰ We found that AGM inhibited the phosphorylation of RSK2, and the use of 2-BFI blocked the phosphorylation effects of AGM on RSK2, including degradation of IκBα phosphorylation and promotion of p65 nuclear translocation, thereby activating the NF-κB signaling pathway. Our findings indicate that AGM inhibits the phosphorylation of RSK2. Furthermore, silencing RSK2 abrogates the impact of AGM on the phosphorylation and degradation of IκB. Additionally, silencing RSK2 prevents the ameliorative effects of AGM on SLI and inhibits the M2-type polarization of macrophages. These results suggest that AGM may protect LPS-induced SLI by inhibiting the I₂R/RSK2/NF-κB signaling pathway.

In summary, we demonstrated the important role of AGM in SLI using a mouse animal model. High levels of M1 macrophages and inflammatory mediators, increased alveolar-capillary leakage, increased apoptotic cells, and poor oxygenation were detected in the lungs of SLI mice. AGM could block the above effects and protect the mice from SLI. Mechanistically, SLI reduction by AGM may be mediated through the I₂R/RSK2/NF-κB signaling pathway. However, this study has certain limitations. First, we did not regulate the expression level of I₂R to observe the phenotypic changes of SLI, and we are currently constructing an in vitro knockout model of I₂R. Then, we did not study the mRNA level of IκBα. Although the phosphorylation level of IκBα was down-regulated by the application of AGM, the expression of IκBα was increased, and whether there was an increase in IκBα mRNA expression was not known. In view of the above shortcomings, we only observed that AGM in SLI could regulate the I₂R/RSK2/NF-κB signaling pathway in macrophages to improve lung injury, and we need to further improve relevant content in the future.

Conclusion

Agmatine can ameliorate sepsis-induced lung injury by suppressing the I₂R/RSK2/NF-κB signaling pathway and modulating polarization of alveolar macrophage.

Supplemental Material

sj-docx-1-iji-10.1177_03946320261425360 – Supplemental material for Agmatine suppresses the imidazoline I2 receptor/ribosomal S6 kinase 2/NF-κB signaling pathway regulating alveolar macrophage polarization and ameliorating sepsis-associated acute lung injury

Supplemental material, sj-docx-1-iji-10.1177_03946320261425360 for Agmatine suppresses the imidazoline I2 receptor/ribosomal S6 kinase 2/NF-κB signaling pathway regulating alveolar macrophage polarization and ameliorating sepsis-associated acute lung injury by Linguo Bai, Kun Yu, Qiuyu Dai, Jie Zheng, Kangjie Qin, Junjie Li, Feiyan Li, Song Qin, Hong Mei, Xinxin Liu, Tao Chen and Liting Cheng in International Journal of Immunopathology and Pharmacology