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Punicalagin (PUN) is a polyphenol derived from the pomegranate peel. It has been reported to have many beneficial effects, including anti-inflammatory, anti-oxidant, and anti-proliferation. However, the role of PUN in macrophage phagocytosis is currently unknown. In this study, we found that pre-treatment with PUN significantly enhanced phagocytosis by macrophages in a time- and dose-dependent manner in vitro. Moreover, KEGG enrichment analysis by RNA-sequencing showed that differentially expressed genes following PUN treatment were significantly enriched in phagocyte-related receptors, such as the C-type lectin receptor signaling pathway. Among the C-type lectin receptor family, Mincle (Clec4e) significantly increased at the mRNA and protein level after PUN treatment, as shown by qRT-PCR and western blotting. Small interfering RNA (siRNA) mediated knockdown of Mincle in macrophages resulted in down regulation of phagocytosis. Furthermore, western blotting showed that PUN treatment enhanced the phosphorylation of nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) in macrophages at the early stage. Mincle-mediated phagocytosis by PUN was inhibited by PDTC (a NF-κB inhibitor) and SB203580 (a p38 MAPK inhibitor). In addition, PUN pre-treatment enhanced phagocytosis by peritoneal and alveolar macrophages in vivo. After intraperitoneal injection of Escherichia coli (E.coli), the bacterial load of peritoneal lavage fluid and peripheral blood in PUN pre-treated mice decreased significantly. Similarly, the number of bacteria in the lung tissue significantly reduced after intranasal administration of Pseudomonas aeruginosa (PAO1). Taken together, our results reveal that PUN enhances bacterial clearance in mice by activating the NF-κB and MAPK pathways and upregulating C-type lectin receptor expression to enhance phagocytosis by macrophages., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The aryl hydrocarbon receptor (AhR) is an important immune regulator with a role in inflammatory response. However, the role of AhR in IL-10 production by inflammatory macrophages is currently unknown. In this study, we investigated LPS-induced IL-10 expression in macrophages from AhR-KO mice and AhR-overexpressing RAW264.7 cells. AhR was highly expressed after LPS stimulation through NF-κB pathway. Loss of AhR resulted in reduced IL-10 expression in LPS-induced macrophages. Moreover, the IL-10 expression was elevated in LPS-induced AhR-overexpressing RAW264.7 cells. Maximal IL-10 expression was dependent on an AhR non-genomic pathway closely related to Src and STAT3. Furthermore, AhR-associated Src activity was responsible for tyrosine phosphorylation of STAT3 and IL-10 expression by inflammatory macrophages. Adoptive transfer of AhR-expressing macrophages protected mice against LPS-induced peritonitis associated with high IL-10 production. In conclusion, we identified the AhR-Src-STAT3-IL-10 signaling pathway as a critical pathway in the immune regulation of inflammatory macrophages, It suggests that AhR may be a potential therapeutic target in immune response.

Objective: To study the anti-inflammatory effects of idazoxan (IDA) on endotoxin lipopolysaccharide (LPS) challenged mice in vivo and activated macrophages in vitro, and explore its potential molecular mechanisms., Methods: To do the experiments in vivo,30 adult male C57BL/6 mice were randomly divided into control group, model group, and low,medium and high doses IDA groups (IDA-L,IDA-M, and IDA-H groups),n =6 in each group. The inflammatory model was reproduced by intraperitoneal injection of LPS 10 mg/kg, and the control group was injected with the same amount of normal saline. The IDA groups received LPS (10 mg/kg) and IDA 0.3,1.0 and 3.0 mg/kg, respectively. The blood samples of mice in each group were collected at 6 hours after the reproduction of the model .For the in vitro experiments, primary peritoneal macrophages were collected from 20 adult male C57BL/6 mouse cells and they were divided into control group, LPS group (10 mg/L) and LPS+IDA-L,IDA-M,IDA-H groups (10 mg/L LPS + 5,25,100 μmol/L IDA, respectively).Cell culture supernatants were collected at 24 hours after the reproduction of the model. Detection methods: enzyme linked immunosorbent assay (ELISA) was used to determine the levels of serum tumor necrosis factor-α (TNF-α),interleukin-6 (IL-6),monocyte chemotactic protein-1 (MCP-1) and nitric oxide (NO).Western Blot was used to determine the effect of IDA on the expression levels of nuclear factor-κB (NF-κB) in macrophages., Results: ① For the in vivo experiment, the serum levels of TNF-α and IL-6 were significantly elevated in the model group as compared with those in the control group [TNF-o (ng/L):403.96 ± 40.98 vs.17.50 ± 8.68;IL-6 (ng/L):61 400.31 ± 7 826.61 vs.2 436.30 ± 448.89;both P ＜ 0.01].IDA treatment could inhibit the elevation of inflammatory cytokines in a dose-dependent manner, with the most significant decrease in LPS+IDA-H group [TNF-α (ng/L):170.09 ± 28.53 vs.403.96 ± 40.98,IL-6 (ng/L):16 570.81 ± 1 083.65 vs.61 400.31± 7 826.61;both P ＜ 0.01].② For the in vitro experiment, the levels of TNF-α,IL-6,MCP-1,and NO secreted by LPS-stimulated macrophages were distinctly higher in the LPS group than those in the control group [TNF-α (ng/L):7 259.14 ± 320.70 vs.28.50±27.08,IL-6 (ng/L):14809.60±5852.73 vs.1 113.47±465.53,MCP-1 (ng/L):20847.37± 1 788.33 vs.447.37± 395.69,NO (μmol/L):1 900.00 ± 144.31 vs.603.03 ± 102.18;all P ＜ 0.01]. However, IDA intervention could lower the secretion of TNF-α,IL-6,MCP-1 and NO in a dose-dependent manner, with the most notable decrease in the LPS+IDA-H group [TNF-α (ng/L):784.40±281.90 vs.7259.14±320.70,IL-6 (ng/L):1 802.96± 1 534.18 vs.14 809.60± 5 852.73,MCP-1 (ng/L):2005.26± 1 534.28 vs.20847.37 ± 1 788.33,NO (μ mol/L):654.54± 150.21 vs.1 900.00 ± 144.31;all P ＜ 0.05].In addition, IDA at the concentration of 100 μmol/L could promote the translocation of NF-κBp65 in macrophages into the nucleus 15 minutes early and lead to increased NF-κBp65 expression (gray value:18.70 ± 2.29 vs.1.09 ± 0.36,P ＜ 0.05),hut significantly reduce the expression levels of NF-κBp50 in the nucleus at 45 minutes after treatment (gray value:1.99 ± 0.14 vs.2.94 ± 0.54,P ＜ 0.05)., Conclusions: IDA could significantly reduce inflammation of mice challenged with LPS and inhibit inflammatory cytokines and mediators secreted by macrophage in a dose-dependent manner. High concentration of IDA (100 μmol/L) exhibited the greatest anti-inflammatory effects. The anti-inflammatory effect of IDA may be worked through NF-κB signaling pathway.

Objective To explore the regulative effect and mechanism of cytochrome P450 1A1 (CYP1A1) on regulating lipopolysaccharide (LPS)-induced arginase-1 (Arg-1) expression in macrophages. Methods After mouse macrophage-like cell line RAW264.7 (RAW) was treated with 10 mg/L LPS for 0, 12 and 24 h, respectively (n=3 for each time point), the expression of CYP1A1 and Arg-1 at mRNA and protein levels were measured by RT-PCR and Western blotting. Negative control RAW(NC/RAW) cells, CYP1A1-overexpression RAW (CYP1A1/RAW) cells, CYP1A1-knockout RAW (CYP1A1 KO/RAW) cells and CYP1A1 hydroxylase-activity mutant RAW (CYP1A1m/RAW) cells were constructed, respectively, and then the cells were treated with LPS and further detected with RT-PCR and Western blotting for the expression and activation levels of Arg-1 and signal transducer and activators of transcription-6 (STAT6). Then STAT 6 inhibitor AS1517499 was added to treat the CYP1A1 KO/RAW cells and NC/RAW cells, and the mRNA and protein expression of Arg-1 was measured again after LPS or PBS treatment. CYP1A1 epoxidase inhibitor, nordihydroguaiaretic acid (NDGA) and 12 (S)-HETE were introduced to treat normal RAW cells, the mRNA and protein levels of Arg-1 and activity of STAT6 were detected after LPS or PBS treatment. Results LPS-induced CYP1A1 and Arg-1 were highly expressed in staggering peaks in RAW cells (P < 0.05). Overexpression of CYP1A1 inhibited LPS-induced Arg-1 expression and STAT6 activation in RAW cells, while CYP1A1 knockout enhanced above effects (P < 0.05). STAT6 inhibitor AS1517499 abolished the enhancement of CYP1A1 knockout (P < 0.05). CYP1A1 hydroxylase-activity mutation or 12(S)-HETE simulation had no effect on the expression of Arg-1 (P>0.05), while CYP1A1 epoxidase-activity inhibitor NDGA enhanced Arg-1 expression and STAT6 activation (P < 0.05). Conclusion CYP1A1 can inhibit Arg-1 expression by reducing STAT6 activation in LPS-induced macrophages, and CYP1A1 hydroxylase-activity or its proinflammatory metabolism 12(S)-HETE has no effect on this regulation, while CYP1A1 epoxidase-activity may be involved in this effect.

Objectives: The knowledge that agmatine is found in the human body has existed for several years; however, its role in sepsis has not yet been studied. In the present study, we investigate the role of agmatine in the progression and treatment of sepsis.Design: Clinical/laboratory investigations.Setting: Medical centers/University-based research laboratory.Subjects: Elective ICU patients with severe sepsis and healthy volunteers; C57BL/6 mice weighing 18-22 g.Interventions: Serum agmatine level and its associations with inflammatory markers were assessed in patients with sepsis. Agmatine was administered intraperitoneally to mice before a lipopolysaccharide challenge. Human peripheral blood mononuclear cells and murine macrophages were pretreated with agmatine followed by lipopolysaccharide stimulation.Measurements and Main Results: Serum agmatine levels were significantly decreased in patients with sepsis and lipopolysaccharide-induced mice, and correlated with Acute Physiology and Chronic Health Evaluation II score, procalcitonin, tumor necrosis factor-α, and interleukin-6 levels. In a therapeutic experiment, exogenous agmatine attenuated the cytokine production of peripheral blood mononuclear cells from patients with sepsis and healthy controls. Agmatine also exerted a significant beneficial effect in the inflammatory response and organ damage and reduced the death rate in lipopolysaccharide-induced mice. Imidazoline I2 receptor agonist 2-benzofuran-2-yl blocked the pharmacological action of agmatine; whereas, other imidazoline receptor ligands did not. Furthermore, agmatine significantly impaired the inflammatory response by inactivating nuclear factor-κB, but not protein 38 mitogen-activated protein kinase, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and inducible nitric oxide synthase signaling in macrophages. Activation of imidazoline I2 receptor or knockdown of ribosomal S6 kinase 2 counteracted the effects of agmatine on phosphorylation and degradation of inhibitor of nuclear factor-κBα.Conclusions: Endogenous agmatine metabolism correlated with the progression of sepsis. Supplemental exogenous agmatine could ameliorate the lipopolysaccharide-induced systemic inflammatory responses and multiple organ injuries through the imidazoline I2 receptor-ribosomal S6 kinase 2-nuclear factor-κB pathway. Agmatine could be used as both a clinical biomarker and a promising pharmaconutrient in patients with severe sepsis. [ABSTRACT FROM AUTHOR]

Macrophages are key responders of inflammation and are closely related with oxidative stress. Activated macrophages can enhance oxygen depletion, which causes an overproduction of reactive oxygen species (ROS) and leads to further excessive inflammatory response and tissue damage. Agmatine, an endogenous metabolite of L-arginine, has recently been shown to have neuroprotective effects based on its antioxidant properties. However, the antioxidant effects of agmatine in peripheral tissues and cells, especially macrophages, remain unclear. In this study we explored the role of agmatine in mediating antioxidant effects in RAW 264.7 cells and studied its antioxidant mechanism. Our data demonstrate that agmatine is an activator of Nrf2 signaling that markedly enhances Nrf2 nuclear translocation, increases nuclear Nrf2 protein level, up-regulates the expression of the Nrf2 downstream effector HO-1, and attenuates ROS generation induced by Lipopolysaccharide (LPS). We further demonstrated that the agmatine-induced activation of Nrf2 is likely through the PI3K/Akt pathway. LY294002, a specific PI3K/Akt inhibitor, abolished agmatine-induced HO-1 up-regulation and ROS suppression significantly. Inhibiting HO-1 pathway significantly attenuated the antioxidant effect of agmatine which the products of HO-1 enzymatic activity contributed to. Furthermore, the common membrane receptors of agmatine were evaluated, revealing that α2-adrenoceptor, I1-imidazoline receptor or I2-imidazoline receptor are not required by the antioxidant properties of agmatine. Taken together, our findings revealed that agmatine has antioxidant activity against LPS-induced ROS accumulation in RAW 264.7 cells involving HO-1 expression induced by Nrf2 via PI3K/Akt pathway activation. [ABSTRACT FROM AUTHOR]

Jiao Mei Gu (JMG), Cayratia albifolia C.L.Li, is a type of Dong plant widely growing in Dong autonomous counties, Hunan province, China. As a type of traditional herbal medicine, the root of JMG plant has been used to treat inflammatory-related diseases such as arthritis because of its prominent anti-inflammatory effects in Dong medicine. This work investigated the anti-inflammatory effects and mechanisms of the water extract from the root of JMG on lipopolysaccharide (LPS)-induced inflammatory models. Endotoxemia was induced in C57BL/6 mice by intraperitoneal injection of LPS (20 mg/kg), meanwhile intraperitoneal administration of safe doses of JMG. The survival curve of mice was determined. Serum inflammatory cytokines were detected by the Bio-Plex Mouse Cytokine 23-Plex Panel Kit and enzyme-linked immunosorbent assay (ELISA) at 6 h after drug treatment. Hematoxylin-eosin (HE) staining of important organs was completed at 24 h after treatment. The mechanism of inflammatory action was investigated in vitro on LPS-stimulated macrophages. Macrophage inflammation was then induced using 10 μg/mL LPS. The anti-inflammatory effect of JMG was investigated by the quantitative polymerase chain reaction (qPCR) and ELISA. The anti-inflammatory mechanism was determined using western blotting, the electrophoretic mobility shift assay, and immunocytochemistry. Finally, the antimicrobial activity of JMG was verified by survival experiments in vivo and by bacterial culture experiments in vitro. A 200 mg/kg water extract of JMG was safe for mice and had a significant protective effect on LPS-induced sepsis. Organ damage of heart, liver, lung and kidney was also significantly reduced at 24 h in the JMG group, when compared with the LPS group. The serum MIP-1α (CCL-3), MIP-1β (CCL-4), IL-1β, and TNFα cytokines were significantly decreased at 6 h in the JMG group, when compared with the LPS group. In a similar manner, 0.2μg/ml JMG significantly reduced mRNA and protein levels of MIP-1α (CCL-3), MIP-1β (CCL-4), IL-1β, and TNFα in LPS-stimulated macrophage. JMG treatment inhibited the phosphorylation of NF-κB p65 and reduced nuclear transduction, thus reducing transcriptional activity. At the same time, we showed that JMG had no protective effect on Escherichia coli -induced sepsis, as well as no antimicrobial activity. Our results showed that a water-soluble extract of JMG inhibited LPS-induced inflammation via attenuating the NF-κB signaling pathway, which provides an important rationale for the treatment of inflammation-related diseases. Image 1 [ABSTRACT FROM AUTHOR]