Primary Peritoneal Macrophages Research Articles

Synthesis and anti-inflammatory activities of two new N-acetyl glucosamine derivatives

N-acetyl glucosamine (NAG) is a natural amino sugar found in various human tissues with previously described anti-inflammatory effects. Various chemical modifications of NAG have been made to promote its biomedical applications. In this study, we synthesized two bi-deoxygenated NAG, BNAG1 and BNAG2 and investigated their anti-inflammatory properties, using an in vivo and in vitro inflammation mouse model induced by lipopolysaccharide (LPS). Among the parent molecule NAG, BNAG1 and BNAG2, BNAG1 showed the highest inhibition against serum levels of IL-6 and TNF α and the leukocyte migration to lungs and peritoneal cavity in LPS challenged mice, as well as IL-6 and TNF α production in LPS-stimulated primary peritoneal macrophages. BNAG2 displayed an anti-inflammatory effect which was comparable to NAG. These findings implied potential application of these novel NAG derivatives, especially BNAG1, in treatment of certain inflammation-related diseases.

Bicyclol mitigates lipopolysaccharide-induced acute lung injury through myeloid differentiation factor 88 inhibition

Acute lung injury (ALI) remains a significant clinical challenge due to the absence of effective treatment alternatives. This study presents a new method that employs a screening platform focusing on MyD88 affinity, anti-inflammatory properties, and toxicity. This platform was used to evaluate a 300-compound library known for its anti-inflammatory potential. Among the screened compounds, Bicyclol emerged as a standout, exhibiting MyD88 binding and a significant reduction in LPS-stimulated pro-inflammatory factors production in mouse primary peritoneal macrophages. By targeting MyD88, Bicyclol disrupts the MyD88/TLR4 complex and MyD88 polymer formation, thereby mitigating the MAPKs and NF-κB signaling pathways. In vivo experiments further confirmed Bicyclol's efficacy, demonstrating alleviated ALI symptoms, decreased inflammatory cytokines level, and reduced inflammatory cells presence in lung tissues. These findings were associated with a decrease in mortality in LPS-challenged mice. Overall, Bicyclol represents a promising treatment option for ALI by specifically targeting MyD88 and limiting inflammatory responses.

N-Pyrazolyl- and N-Triazolylamines and -ureas as Antileishmanial and Antitrypanosomal Drugs.

Three types of modifications of antileishmanial pyrazole lead compounds 7 and 8 were conducted to expand the relationships between structural features and antileishmanial/antitrypanosomal activity: (1) the pyrazole core was retained or replaced by a 1,2,4-triazole ring; (2) various aryl moieties including 2-fluorophenyl, pyridin-3-yl and pyrazin-2-yl rings were attached at 3-position of the core azole; (3) either arylmethylamino or ureido substituents were introduced at 5-position of the azole core. The synthesis followed established routes starting with esters 9 or 15 and anhydride 21. The synthesized 3-arylpyrazoles and 3-aryl-1,2,4-triazoles showed only very low antileishmanial activity. The 2-fluorophenyl-substituted pyrazole 18c revealed the highest antileishmanial activity of this series of compounds, but its IC50 value (20 µM) still indicates low activity. However, low micromolar antitrypanosomal activity was detected for the pyridin-3-yl-substituted pyrazoles 12b (IC50 = 4.7 µM) and 14a (IC50 = 2.1 µM). Their IC50 values are comparable with the IC50 values of the reference compounds benznidazole and nifurtimox. Whereas only low unspecific cytotoxicity at the primary peritoneal mouse macrophages (PMM) was detected, considerable cytotoxicity at MRC-5 human fibroblast cells was found for both pyrazoles 12b an 14a. The activity of pyrazole 12b against T. cruzi is 4-fold higher than its unspecific MRC-5 cytotoxicity.

A Novel IRAK4 Inhibitor DW18134 Ameliorates Peritonitis and Inflammatory Bowel Disease.

IRAK4 is a critical mediator in NF-κB-regulated inflammatory signaling and has emerged as a promising therapeutic target for the treatment of autoimmune diseases; however, none of its inhibitors have received FDA approval. In this study, we identified a novel small-molecule IRAK4 kinase inhibitor, DW18134, with an IC50 value of 11.2 nM. DW18134 dose-dependently inhibited the phosphorylation of IRAK4 and IKK in primary peritoneal macrophages and RAW264.7 cells, inhibiting the secretion of TNF-α and IL-6 in both cell lines. The in vivo study demonstrated the efficacy of DW18134, significantly attenuating behavioral scores in an LPS-induced peritonitis model. Mechanistically, DW18134 reduced serum TNF-α and IL-6 levels and attenuated inflammatory tissue injury. By directly blocking IRAK4 activation, DW18134 diminished liver macrophage infiltration and the expression of related inflammatory cytokines in peritonitis mice. Additionally, in the DSS-induced colitis model, DW18134 significantly reduced the disease activity index (DAI) and normalized food and water intake and body weight. Furthermore, DW18134 restored intestinal damage and reduced inflammatory cytokine expression in mice by blocking the IRAK4 signaling pathway. Notably, DW18134 protected DSS-threatened intestinal barrier function by upregulating tight junction gene expression. In conclusion, our findings reported a novel IRAK4 inhibitor, DW18134, as a promising candidate for treating inflammatory diseases, including peritonitis and IBD.

The mRNA-Binding Protein KSRP Limits the Inflammatory Response of Macrophages.

KH-type splicing regulatory protein (KSRP) is a single-stranded nucleic acid-binding protein with multiple functions. It is known to bind AU-rich motifs within the 3'-untranslated region of mRNA species, which in many cases encode dynamically regulated proteins like cytokines. In the present study, we investigated the role of KSRP for the immunophenotype of macrophages using bone marrow-derived macrophages (BMDM) from wild-type (WT) and KSRP-/- mice. RNA sequencing revealed that KSRP-/- BMDM displayed significantly higher mRNA expression levels of genes involved in inflammatory and immune responses, particularly type I interferon responses, following LPS stimulation. In line, time kinetics studies revealed increased levels of interferon-γ (IFN-γ), interleukin (IL)-1β and IL-6 mRNA in KSRP-/- macrophages after 6 h subsequent to LPS stimulation as compared to WT cultures. At the protein level, KSRP-/- BMDM displayed higher levels of these cytokines after overnight stimulation. Matching results were observed for primary peritoneal macrophages of KSRP-/- mice. These showed higher IL-6, tumor necrosis factor-α (TNF-α), C-X-C motif chemokine 1 (CXCL1) and CC-chemokine ligand 5 (CCL5) protein levels in response to LPS stimulation than the WT controls. As macrophages play a key role in sepsis, the in vivo relevance of KSRP deficiency for cytokine/chemokine production was analyzed in an acute inflammation model. In agreement with our in vitro findings, KSRP-deficient animals showed higher cytokine production upon LPS administration in comparison to WT mice. Taken together, these findings demonstrate that KSRP constitutes an important negative regulator of cytokine expression in macrophages.

METTL3-deficiency m6A-dependently degrades MALAT1 to suppress NLRP3-mediated pyroptotic cell death and inflammation in Mycobacterium tuberculosis (H37Ra strain)-infected mouse macrophages

Mycobacterium tuberculosis (Mtb)-infected macrophages aggravated the development of pulmonary tuberculosis, but its detailed molecular mechanisms are still largely unknown. Here, the mouse primary peritoneal macrophages were infected with the attenuated strain of Mtb H37Ra, and we firstly verified that targeting a novel METTL3/N6-Methyladenosine (m6A)/LncRNA MALAT1/miR-125b/TLR4 axis was effective to suppress pyroptotic cell death in the Mtb-infected macrophages. Specifically, through performing Real-Time qPCR and Western Blot analysis, we validated that METTL3, LncRNA MALAT1 and TLR4 were elevated, whereas miR-125b and the anti-oxidant agents (Nrf2 and HO-1) were downregulated in Mtb-infected mouse macrophages. In addition, functional experiments confirmed that both ROS scavenger NAC and METTL3-ablation downregulated NLRP3, GSDMD-C, cleaved Caspase-1 and ASC to restrain pyroptotic cell death and decreased the expression levels of IL-1β, IL-18, IL-6 and TNF-α to restrain inflammatory cytokines expression in Mtb-infected macrophages. Next, METTL3-ablation induced m6A-demethylation and instability in LncRNA MALAT1, and low-expressed LncRNA MALAT1 caused TLR4 downregulation through sponging miR-125b, resulting in the inactivation of NLRP3 inflammasome. Finally, silencing of METTL3-induced protective effects in Mtb-infected macrophages were all abrogated by overexpressing LncRNA MALAT1 and downregulating miR-125b. Thus, we concluded that targeting METTL3-mediated m6A modifications suppressed Mtb-induced pyroptotic cell death in mouse macrophages, and the downstream LncRNA MALAT1/miR-125b/TLR4 axis played critical role in this process.

Macrophage SHP2 Deficiency Alleviates Diabetic Nephropathy via Suppression of MAPK/NF-κB- Dependent Inflammation.

Increasing evidence implicates chronic inflammation as the main pathological cause of diabetic nephropathy (DN). Exploration of key targets in the inflammatory pathway may provide new treatment options for DN. Here, we aim to investigate the role of Src Homology 2 Containing Protein Tyrosine Phosphatase 2 (SHP2) in macrophages and its association with DN. The upregulated phosphorylation of SHP2 was detected in macrophages of both diabetic patients and mouse model. Using the macrophage-specific SHP2 knockout mice (SHP2-MKO) and SHP2fl/fl mice injected with streptozotocin (STZ), we showed that SHP2-MKO significantly attenuated renal dysfunction, collagen deposition, fibrosis, and inflammatory response in STZ-induced diabetic mice. RNA-sequencing analysis using primary mouse peritoneal macrophages (MPMs) showed that SHP2 deletion mainly affects mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-ĸB) signaling pathway and MAPKs/NF-ĸB-dependent inflammatory cytokine release in MPMs. Further study indicated that SHP2-deficient macrophages failed to release cytokines that induce phenotype transition and fibrosis in renal cells. Administration with a pharmacological SHP2 inhibitor, SHP099, remarkably protected kidneys in both type 1 and type 2 diabetic mice. In conclusion, these results identify macrophage SHP2 as a new accelerator of DN and suggest that SHP2 inhibition may be a therapeutic option for DN patients.

Astaxanthin Inhibits STING Carbonylation and Enhances Antiviral Responses.

STING-mediated DNA sensing pathway plays a crucial role in the innate antiviral immune responses. Clarifying its regulatory mechanism and searching STING agonists has potential clinical implications. Although multiple STING agonists have been developed to target cancer, there are few for the treatment of infectious diseases. Astaxanthin, a natural and powerful antioxidant, serves many biological functions and as a potential candidate drug for many diseases. However, how astaxanthin combats viruses and whether astaxanthin regulates the cyclic GMP-AMP synthase-STING pathway remains unclear. In this study, we showed that astaxanthin markedly inhibited HSV-1-induced lipid peroxidation and inflammatory responses and enhanced the induction of type I IFN in C57BL/6J mice and mouse primary peritoneal macrophages. Mechanistically, astaxanthin inhibited HSV-1 infection and oxidative stress-induced STING carbonylation and consequently promoted STING translocation to the Golgi apparatus and oligomerization, which activated STING-dependent host defenses. Thus, our study reveals that astaxanthin displays a strong antiviral activity by targeting STING, suggesting that astaxanthin might be a promising STING agonist and a therapeutic target for viral infectious diseases.

Deacetylase Sirtuin 1 mitigates type I IFN- and type II IFN-induced signaling and antiviral immunity.

Type I and type II IFNs are important immune modulators in both innate and adaptive immunity. They transmit signaling by activating JAK-STAT pathways. Sirtuin 1 (SIRT1), a class III NAD+-dependent deacetylase, has multiple functions in a variety of physiological processes. Here, we characterized the novel functions of SIRT1 in the regulation of type I and type II IFN-induced signaling. Overexpression of SIRT1 inhibited type I and type II IFN-induced interferon-stimulated response element activation. In contrast, knockout of SIRT1 promoted type I and type II IFN-induced expression of ISGs and inhibited viral replication. Treatment with SIRT1 inhibitor EX527 had similar positive effects. SIRT1 physically associated with STAT1 or STAT3, and this interaction was enhanced by IFN stimulation or viral infection. By deacetylating STAT1 at K673 and STAT3 at K679/K685/K707/K709, SIRT1 downregulated the phosphorylation of STAT1 (Y701) and STAT3 (Y705). Sirt1+/- primary peritoneal macrophages and Sirt1+/- mice exhibited enhanced IFN-induced signaling and antiviral activity. Thus, SIRT1 is a novel negative regulator of type I and type II IFN-induced signaling through its deacetylase activity.IMPORTANCESIRT1 has been reported in the precise regulation of antiviral (RNA and DNA) immunity. However, its functions in type I and type II IFN-induced signaling are still unclear. In this study, we deciphered the important functions of SIRT1 in both type I and type II IFN-induced JAK-STAT signaling and explored the potential acting mechanisms. It is helpful for understanding the regulatory roles of SIRT1 at different levels of IFN signaling. It also consolidates the notion that SIRT1 is an important target for intervention in viral infection, inflammatory diseases, or even interferon-related therapies.

Pyrazole derivative Z10 ameliorates acute pancreatitis by inhibiting the ERK/Ddt pathway

Acute pancreatitis (AP) can lead to death; however, there is no specific treatment for AP. Screening of drugs for AP treatment is rarely performed. Compounds were screened in a primary pancreatic acinar cell and peritoneal macrophage coculture system. Compounds were used in vitro and in vivo. Compound targets were predicted and validated. Among the 18 nitrogen-containing heterocycles, Z10 was shown to decrease the cerulein plus lipopolysaccharide (CL)–induced secretion of both acinar digestive enzymes and macrophage cytokines. Z10 was also shown to ameliorate CL-induced or sodium taurocholate–induced AP in mice. Proteomics analysis and enzyme linked immunosorbent assay (ELISA) revealed that Z10 decreased the levels of D-dopachrome tautomerase (Ddt) within macrophages and those in the extracellular milieu under CL treatment. Z10 also decreased Ddt expression in AP mice. Moreover, exogenous Ddt induced cytokine and digestive enzyme secretion, which could be inhibited by Z10. Ddt knockdown inhibited CL-induced cytokine secretion. Medium from CL-treated macrophages induced the release of amylase by acinar cells, and Ddt knockdown medium decreased amylase secretion. The target of Z10 was predicted to be ERK2. Z10 increased the thermostability of ERK1/2 but not ERK1 K72A/ERK2 K52A. The docking poses of ERK1 and ERK2 with Z10 were similar. Z10 inhibited ERK1/2 phosphorylation, and Ddt levels and cytokines were regulated by ERK1/2 during AP. Additionally, Z10 could not further inhibit cytokines under ERK1/2 knockdown with CL. Thus, this study revealed that Z10-mediated ERK1/2 inhibition decreased Ddt expression and secretion by macrophages. Ddt inhibition decreased cytokine release and digestive enzyme secretion.

Macrophage-derived GSDMD promotes abdominal aortic aneurysm and aortic smooth muscle cells pyroptosis

Macrophage is a vital factor in determining the fate of abdominal aortic aneurysm (AAA). The crosstalk between macrophage and other cells plays a crucial role in the development of aneurysm. Gasdermin D (GSDMD) is a vital executive protein of pyroptosis, which is a novel programmed cell death associated with inflammation. In this study, we identified aortic macrophage as the main expressing cell of GSDMD in AAA. Using Gsdmd-/-ApoE-/- mouse and AAV-F4/80-shGSDMD, we demonstrated the potential role of macrophage-derived GSDMD in AAA and aortic pyroptosis induced by Ang II in vivo. In vitro experiments showed that GSDMD promotes the pyroptosis of mouse primary peritoneal macrophages (MPMs), murine aortic vascular smooth muscle cells (MOVAS) and primary smooth muscle cells. Mechanistically, a mouse cytokine antibody array showed that Gsdmd-/- inhibited LPS + nigericin (LN)- induced secretion of multiple cytokines from MPMs. Furthermore, GSDMD is involved in the crosstalk between MPMs and MOVAS via cytokine secretion. This study provides a novel fundamental insight into macrophage-derived GSDMD in AAA and showed that GSDMD could be a promising therapeutic target for AAA.

Pterostilbene alleviates abdominal aortic aneurysm via inhibiting macrophage pyroptosis by activating the miR-146a-5p/TRAF6 axis.

Pterostilbene (PTE), a natural stilbene found in blueberries and several varieties of grapes, has several pharmacological activities, including anti-inflammatory and antioxidative activities. However, its role in abdominal aortic aneurysm (AAA), which is a severe inflammatory vascular disease, remains incompletely understood. In this study, we investigated the protective effects of natural stilbene PTE on AAA formation and the underlying mechanism. Two AAA mouse models (Ang II-induced model and PPE-induced model) were used to examine the effect of PTE on AAA formation. We showed that PTE administration attenuated AAA formation in mice. Furthermore, we found that PTE significantly inhibited inflammatory responses in mouse aortas, as PTE suppressed macrophage pyroptosis and prevented macrophage infiltration in aortas, resulting in reduced expression of pro-inflammatory cytokines in aortas. We also observed similar results in LPS + ATP-treated Raw 264.7 cells (a macrophage cell line) and primary peritoneal macrophages in vitro. We showed that pretreatment with PTE restrained inflammatory responses in macrophages by inhibiting macrophage pyroptosis. Mechanistically, miR-146a-5p and TRAF6 interventions in vivo and in vitro were used to investigate the role of the miR-146a-5p/TRAF6 axis in the beneficial effect of PTE on macrophage pyroptosis and AAA. We found that PTE inhibited macrophage pyroptosis by miR-146a-5p-mediated suppression of downstream TRAF6 expression. Moreover, miR-146a-5p knockout or TRAF6 overexpression abrogated the protective effect of PTE on macrophage pyroptosis and AAA formation. These findings suggest that miR-146a-5p/TRAF6 axis activation by PTE protects against macrophage pyroptosis and AAA formation. PTE might be a promising agent for preventing inflammatory vascular diseases, including AAA.

Correlation between secondary metabolites of Iris confusa Sealy and Iris pseudacorus L. and their newly explored antiprotozoal potentials

BackgroundIn the last few decades, the use of plant extracts and their phytochemicals as candidates for the management of parasitic diseases has increased tremendously. Irises are aromatic and medicinal plants that have long been employed in the treatment of different infectious diseases by traditional healers in many cultures. This study aims to explore the potential of three common Iris species (I. confusa Sealy, I. pseudacorus L. and I. germanica L.) against infectious diseases. Their in vitro antiprotozoal potency against Plasmodium falciparum, Trypanosoma brucei brucei, T. b. rhodesiense, T. cruzi and Leishmania infantum beside their cytotoxicity on MRC-5 fibroblasts and primary peritoneal murine macrophages were examined.MethodsThe secondary metabolites of the tested extracts were characterized by UPLC-HRMS/MS and Pearsons correlation was used to correlate them with the antiprotozoal activity.ResultsOverall, the non-polar fractions (NPF) showed a significant antiprotozoal activity (score: sc 2 to 5) in contrast to the polar fractions (PF). I. confusa NPF was the most active extract against P. falciparum [IC50 of 1.08 μg/mL, selectivity index (S.I. 26.11) and sc 5] and L. infantum (IC50 of 12.7 μg/mL, S.I. 2.22 and sc 2). I. pseudacorus NPF was the most potent fraction against T. b. rhodesiense (IC50 of 8.17 μg/mL, S.I. 3.67 and sc 3). Monogalactosyldiacylglycerol glycolipid (18:3/18:3), triaceylglycerol (18:2/18:2/18:3), oleic acid, and triterpenoid irridals (spirioiridoconfal C and iso-iridobelamal A) were the top positively correlated metabolites with antiplasmodium and antileishmanial activities of I. confusa NPF. Tumulosic acid, ceramide sphingolipids, corosolic, maslinic, moreollic acids, pheophytin a, triaceylglycerols, mono- and digalactosyldiacylglycerols, phosphatidylglycerol (22:6/18:3), phosphatidylcholines (18:1/18:2), and triterpenoid irridal iso-iridobelamal A, were highly correlated to I. pseudacorus NPF anti- T. b. rhodesiense activity. The ADME study revealed proper drug likeness properties for certain highly corelated secondary metabolites.ConclusionThis study is the sole map correlating I. confusa and I. pseudacorus secondary metabolites to their newly explored antiprotozoal activity.

The UAF1-USP1 Deubiquitinase Complex Stabilizes cGAS and Facilitates Antiviral Responses.

Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) detects cytoplasmic microbial DNA and self-DNA from genomic instability, initiates innate immunity, and plays fundamental roles in defense against viruses and the development of various diseases. The cellular cGAS level determines the magnitude of the response to DNA. However, the underlying mechanisms of the control of cGAS stability, especially its feedback regulation during viral infection, remain largely unknown. In this study, we show that viral infection induces the expression of the UAF1-USP1 deubiquitinase complex in primary peritoneal macrophages (PMs) of C57BL/6J mice. UAF1-USP interacts with cGAS, selectively cleaves its K48-linked polyubiquitination, and thus stabilizes its protein expression in PMs and HEK293T cells. Concordantly, the UAF1-USP1 deubiquitinase complex enhances cGAS-dependent type I IFN responses in PMs. Uaf1 deficiency and ML323 (a specific inhibitor of UAF1-USP1 deubiquitinase complex) attenuates cGAS-triggered antiviral responses and facilitates viral replication both invitro and invivo. Thus, our study uncovers a positive feedback mechanism of cGAS-dependent antiviral responses and suggests the UAF1-USP1 complex as a potential target for the treatment of diseases caused by aberrant cGAS activation.

Immunotoxicity Evaluation of Trihalophenolic Disinfection By-Products in Mouse and Human Mononuclear Macrophage Systems: The Role of RNA Epitranscriptomic Modification in Mammalian Immunity.

2,4,6-Trichlorophenol (TCP), 2,4,6-tribromophenol (TBP) and 2,4,6-triiodophenol (TIP) are three widely detected trihalophenolic disinfection by-products (DBPs). Previous studies have mainly focused on the carcinogenic risk and developmental toxicity of 2,4,6-trihalophenols. Very little is known about their immunotoxicity in mammals. We investigated the effects of 2,4,6-trihalophenols on mammalian immunity using a mouse macrophage model infected with bacteria or intracellular parasites and aimed to elucidate the underlying mechanisms from an epitranscriptomic perspective. The identified mechanisms were further validated in human peripheral blood mononuclear cells (PBMCs). The mouse macrophage cell line RAW264.7 and primary mouse peritoneal macrophages were exposed to different concentrations of TCP, TBP, and TIP. The pro-inflammatory marker Ly6C, the survival of the bacterium Escherichia coli (E. coli), and the parasite burden of Toxoplasma gondii (T. gondii) were assessed. Furthermore, the global gene expression profiling of macrophages following exposure to 2,4,6-trihalophenols was obtained through RNA-sequencing (RNA-seq). The effects of 2,4,6-trihalophenols on RNA () methyltransferases and total RNA levels were evaluated using Western blotting and dot blot, respectively. Transcriptome-wide methylome was analyzed by . In addition, expression of regulators and total RNA levels in human PBMCs exposed to 2,4,6-trihalophenols were detected using quantitative reverse transcriptase polymerase chain reaction and dot blot, respectively. Mouse macrophages exposed to TCP, TBP, or TIP had lower expression of the pro-inflammatory marker Ly6C, with a greater difference from control observed for TIP-exposed cells. Consistently, macrophages exposed to such DBPs, especially TIP, were susceptible to infection with the bacterium E. coli and the intracellular parasite T. gondii, indicating a compromised ability of macrophages to defend against pathogens. Intriguingly, macrophages exposed to TIP had significantly greater levels, which correlated with the greater expression levels of methyltransferases. Macrophages exposed to each of the three 2,4,6-trihalophenols exhibited transcriptome-wide redistribution of . In particular, the peaks in genes associated with immune-related pathways were altered after exposure. In addition, differences in were also observed in human PBMCs after exposure to 2,4,6-trihalophenols. These findings suggest that 2,4,6-trihalophenol exposure impaired the ability of macrophages to defend against pathogens. This response might be associated with notable differences in after exposure. To the best of our knowledge, this study presents the first landscape across the transcriptome of immune cells exposed to pollutants. However, significant challenges remain in elucidating the mechanisms by which mediates immune dysregulation in infected macrophages after 2,4,6-trihalophenol exposure. https://doi.org/10.1289/EHP11329.

Hypobaric hypoxia induces iron mobilization from liver and spleen and increases serum iron via activation of ghrelin/GHSR1a/MAPK signalling pathway in mice

Hypobaric hypoxia (HH) exposure affects appetite and serum iron levels in both humans and animals. Thus, whether appetite-regulating ghrelin is involved in iron regulation under HH needs to be elucidated. In vivo, C57BL/6J mice were placed in a hypobaric chamber to establish a 6000-m-high altitude exposure animal model. In vitro, mouse primary hepatocytes and peritoneal macrophages were exposed to hypoxia (1% O2) to examine the effects of ghrelin on iron-regulating proteins. HH obviously reduced the body weight of mice and significantly increased the levels of erythrocytes, and also significantly enhanced the levels of serum iron and plasma ghrelin. However, iron content in the liver and spleen was decreased, while ferroportin (Fpn) expression was increased. Moreover, ghrelin significantly induced Fpn and pERK expression in both hepatocytes and macrophages under hypoxia, which were reversed by pretreatment with growth hormone secretagogue receptor 1a (GHSR1a) antagonist or pERK inhibitor. Our findings indicated that HH leads to decreased appetite and insufficient dietary intake, which may negatively regulate the levels of ghrelin. Furthermore, GHSR1a/ERK signalling pathway is further activated to upregulate the expression of Fpn, and then promoting iron mobilization both in the liver/hepatocytes and spleen/macrophages in mice. Thus, these results revealed that ghrelin may be a potential iron regulatory hormone, and raised the possibility of ghrelin as a promising therapeutic target against iron disorders under HH.

Biological characteristics and pathogenicity of a Staphylococcus aureus strain with an incomplete hemolytic phenotype isolated from bovine milk.

Staphylococcus aureus is a common pathogen capable of infecting both humans and animals and causing various severe diseases. Here, we aimed to determine the biological features and pathogenicity of S. aureus strain Sa9, of the incomplete hemolysis phenotype, isolated from bovine milk. Sa9 was classified as ST97 by multilocus sequence typing, and it showed increased β-hemolysin expression and lower Hla and Hld expression levels compared with that in the S. aureus USA300 strain LAC. RT-PCR and ELISA results showed that the expression levels of inflammatory cytokines were higher in Sa9-induced mouse primary peritoneal macrophages compared with those induced by the LAC strain. However, the Sa9 strain also mediated anti-inflammatory effects by upregulating IL-10 and IFN-β in macrophages, which were not apparently induced by S. aureus culture supernatants. Phagocytosis and whole-blood survival assays were also performed to assess the in vitro survival of bacteria, and the virulence was evaluated in mice. Although the Sa9 strain showed lower ability of intracellular survival in macrophages than LAC, similar multiplication in human whole blood and pathogenicity toward mice were observed. Taken together, we report that the distinctive immune response induced by the S. aureus strain with an incomplete hemolysis phenotype occurs in cattle, and its potential pathogenicity and risk of transmission to humans require attention.

Effect fraction of Bletilla striata (Thunb.) Reichb.f. alleviates LPS-induced acute lung injury by inhibiting p47phox/NOX2 and promoting the Nrf2/HO-1 signaling pathway

Background & AimsThe effect fraction of Bletilla striata (Thunb.) Reichb.f. (EFBS), a phenolic-rich extract, has significant protective effects on lipopolysaccharide (LPS)-induced acute lung injury (ALI), but its composition and molecular mechanisms are unclear. This study elucidated its chemical composition and possible protective mechanisms against LPS-induced ALI from an antioxidant perspective. MethodsEFBS was prepared by ethanol extraction, enriched by polyamide column chromatography, and characterized using ultra-performance liquid chromatography/time-of-flight mass spectrometry. The LPS-induced ALI model and the RAW264.7 model were used to evaluate the regulatory effects of EFBS on oxidative stress, and transcriptome analysis was performed to explore its possible molecular mechanism. Then, the pathway by which EFBS regulates oxidative stress was validated through inhibitor intervention, flow cytometry, quantitative PCR, western blotting, and immunofluorescence techniques. ResultsA total of 22 compounds in EFBS were identified. The transcriptome analyses of RAW264.7 cells indicated that EFBS might reduce reactive oxygen species (ROS) production by inhibiting the p47phox/NADPH oxidase 2 (NOX2) pathway and upregulating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. Both in vitro and in vivo data confirmed that EFBS significantly inhibited the expression and phosphorylation of p47phox protein, thereby weakening the p47phox/NOX2 pathway and reducing ROS production. EFBS significantly increased the expression of Nrf2 in primary peritoneal macrophages and lung tissue and promoted its nuclear translocation, dose-dependent increase in HO-1 levels, and enhancement of antioxidant activity. In vitro, both Nrf2 and HO-1 inhibitors significantly reduced the scavenging effects of EFBS on ROS, further confirming that EFBS exerts antioxidant effects at least partially by upregulating the Nrf2/HO-1 pathway. ConclusionsEFBS contains abundant phenanthrenes and dibenzyl polyphenols, which can reduce ROS production by inhibiting the p47phox/NOX2 pathway and enhance ROS clearance activity by upregulating the Nrf2/HO-1 pathway, thereby exerting regulatory effects on oxidative stress and improving LPS-induced ALI.

Cyasterone ameliorates sepsis-related acute lung injury via AKT (Ser473)/GSK3β (Ser9)/Nrf2 pathway

BackgroundAcute lung injury (ALI) is a severe disease that can lead to acute respiratory distress syndrome (ARDS), characterized by intractable hypoxemia, poor lung compliance, and respiratory failure, severely affecting patients' quality of life. The pathogenesis of ALI has not been fully elucidated yet, and sepsis is an important cause of ALI. Among the organ injuries caused by sepsis, the lungs are the earliest damaged ones. Radix cyathulae is reported to have analgesic, anti-inflammatory, and anti-aging effects. Cyasterone is extracted from Radix cyathulae. However, it is not known whether cyasterone has protective effects for ALI. This study aims to investigate the effect of cyasterone on sepsis-related ALI and its mechanism.MethodsWe used the cecal ligation peferation (CLP) method to establish a mouse sepsis model, and cyasterone was given intraperitoneally on days 1–3 to observe its preventive effect on sepsis-related acute lung injury. Primary murine peritoneal macrophages were used to investigate the molecular mechanism of cyasterone in vitro.ResultsCyasterone pretreatment inhibits pro-inflammatory cytokine production, NLRP3 inflammasome activation, and oxidative stress in vivo and in vitro. In addition, cyasterone attenuates sepsis-induced ALI by activating nuclear factor erythroid2-related factor (Nrf2), which may be associated with AKT(Ser473)/GSK3β(Ser9) pathway activation.ConclusionsCyasterone defends against sepsis-induced ALI by inhibiting inflammatory responses and oxidative stress, which depends heavily on the upregulation of the Nrf2 pathway through phosphorylation of AKT(Ser473)/GSK3β(Ser9). These results suggest cyasterone may be a valuable drug candidate for preventing sepsis-related ALI.

Luteolin ameliorates DSS-induced colitis in mice via suppressing macrophage activation and chemotaxis

ObjectivesLuteolin, known for its multifaceted therapeutic properties against inflammatory diseases, holds potential for addressing the unmet need for effective treatments in ulcerative colitis (UC), a prevalent subtype of inflammatory bowel disease (IBD). This study aimed to comprehensively assess luteolin's therapeutic efficacy in a dextran sulfate sodium (DSS)-induced colitis mouse model, shedding light on its anti-UC mechanisms. MethodsOur investigation encompassed in vivo assessments of luteolin's therapeutic potential against DSS-induced colitis through rigorous histopathological examination and biochemical analyses. Furthermore, we scrutinized luteolin's anti-inflammatory prowess in vitro using lipopolysaccharide (LPS)-stimulated RAW264.7 cells and primary peritoneal macrophages. Additionally, we quantitatively evaluated the impact of luteolin on C–C motif chemokine ligand 2 (CCL2)-induced macrophage migration employing Transwell and Zigmond chambers. Furthermore, cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) assay, and molecular docking were employed to identify potential therapeutic targets of luteolin and investigate their binding sites and interaction patterns. ResultsLuteolin demonstrated therapeutic potential against DSS-induced colitis by ameliorating colitis symptoms, restoring intestinal barrier integrity, and inhibiting proinflammatory cytokine production in the colonic tissues. Moreover, luteolin demonstrated robust anti-inflammatory activity in vitro, in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and primary peritoneal macrophages. Notably, luteolin suppressed the phosphorylation of IKKα/β, IκBα, and p65, along with preventing IκBα degradation in LPS-treated RAW264.7 cells and peritoneal macrophages. Furthermore, luteolin impaired the migratory behavior of RAW264.7 cells and peritoneal macrophages, as evidenced by reduced migration distance and velocity of luteolin-treated macrophages. Mechanistically, luteolin was found to antagonize IKKα/β, subsequently inhibiting IKKα/β phosphorylation and the activation of NF-κB signaling. ConclusionLuteolin emerges as a promising lead compound for the clinical therapy of colitis by virtue of its ability to ameliorate DSS-induced colitis, antagonize IKKα/β, suppress NF-κB signaling, and impede macrophage activation and migration.