RESUMEN
In bone tissue engineering, biological scaffolds are designed with structural and functional properties that closely resemble the extracellular environment, aiming to establish a microenvironment conducive to osteogenesis. Macrophages hold significant potential for promoting osteogenesis and modulating the biological behavior of tumor cells. Multiple coculture experiments of macrophages and osteoblasts have demonstrated that macrophage polarization significantly impacts osteogenesis. Therefore, exploring bone biomaterials that can modulate macrophage polarization holds great clinical significance. In this study, heparin was modified with maleimide and was used as a raw material to form a hydrogel with 4-am-PEG-SH. The compound was used to polarize macrophages and promote osteogenesis after combining with interleukin 4 (IL-4) by taking advantage of the electronegativity of heparin. The results revealed overexpressed M2 macrophage-related phenotypic genes and cocultivation with MC3T3-E1 cells demonstrated the osteogenesis-promoting effect of the loaded IL-4 heparin hydrogel. Previous research reported that hydrogel loaded with IL-4 can be used as a biomaterial for osteogenesis promotion. Heparin materials used in this paper are derived from clinically anticoagulant drugs and feature a simple operation. The synthesized hydrogel effectively binds cytokines, regulates macrophages to induce osteogenesis and has many potential clinical applications.
Asunto(s)
Diferenciación Celular , Heparina , Hidrogeles , Interleucina-4 , Macrófagos , Osteogénesis , Heparina/farmacología , Heparina/química , Animales , Interleucina-4/farmacología , Interleucina-4/metabolismo , Ratones , Osteogénesis/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Diferenciación Celular/efectos de los fármacos , Hidrogeles/química , Hidrogeles/farmacología , Células RAW 264.7RESUMEN
Tissue engineering technology offers a promising solution for ear reconstruction; however, it faces the challenge of foreign body reaction and neocartilage malformation. This study investigates the impact of interleukin-4 (IL-4), an anti-inflammatory factor, on cartilage regeneration of hydrogel encapsulating autologous auricular chondrocytes in a rabbit subcutaneous environment. Initially, we assessed the influence of IL-4 on chondrocyte proliferation and determined the appropriate concentration using the CCK-8 test in vitro. Subsequently, we loaded IL-4 into gelatin methacryloyl (GelMA) hydrogel containing chondrocytes and measured its release profile through ELISA. The constructs were then implanted autologously into rabbits' subcutis, and after 3, 7, 14, and 28 days, cartilage matrix formation was evaluated by histological examinations, and gene expression levels were detected by qRT-PCR. Results demonstrated that IL-4 promotes chondrocyte proliferation in vitro, and maximum release from constructs occurred during the first week. In the rabbit subcutaneous implantation model, IL-4-loaded constructs (20 ng/mL) maintained a superior chondrocytic phenotype compared to controls with increased expression of anti-inflammatory factors. These findings highlight IL-4 as a potential strategy for promoting chondrogenesis in a subcutaneous environment and improving ear reconstruction.
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Condrocitos , Condrogénesis , Cartílago Auricular , Gelatina , Hidrogeles , Interleucina-4 , Ingeniería de Tejidos , Animales , Conejos , Gelatina/química , Gelatina/farmacología , Condrogénesis/efectos de los fármacos , Interleucina-4/metabolismo , Interleucina-4/farmacología , Hidrogeles/química , Hidrogeles/farmacología , Condrocitos/metabolismo , Condrocitos/citología , Metacrilatos/química , Metacrilatos/farmacología , Proliferación Celular/efectos de los fármacosRESUMEN
OBJECTIVE: To determine the biological effects of arecoline on oral submucous fibrosis (OSF). DESIGN: The differential genes between OSF tissue and normal oral tissue were collected form GSE64216 dataset, analyzed by Gene Expression Omnibus (GEO) database. Real-time PCR and immunohistochemistry were used to analyze the expression of IL-4 gene and protein in oral tissue. Enzyme-linked immunosorbent assay (ELISA) was used to analyze the expression of exocrine IL-4 protein in human oral fibroblasts (HOF) pre-treated by arecoline. Cell Counting Kit-8 (CCK-8) and transwell assays were used to analyze the proliferation and migration of HOF cells, respectively. After IL-4 was knocked down by short hairpin (sh) plasmid, the proliferation and migration of HOF cells were detected. Flow cytometry was used to analyze the proportion of M2-macrophages. Real-time PCR and immunohistochemistry were used to verify the expression of biomarker proteins of macrophages in OSF tissues. RESULTS: The expression of IL-4 gene and protein were both up-regulated in OSF tissue. Arecoline could enhance the expression of IL-4 gene and exocrine protein in HOF cells, and promote the proliferation and migration of HOF cells. While knockdown of IL-4 could inhibit arecoline-induced proliferation and migration in HOF cells. The results of flow cytometry showed that recombinant human IL-4 (rhIL-4) protein could increase the proportion of M2-macrophages. Similarly, the results of real-time PCR and immunohistochemistry showed the expression of ARG1 (Biomarker proteins of M2-macrophage) was up-regulated in OSF tissues. CONCLUSION: Arecoline promotes activation of fibroblasts and polarization of M2-macrophages by up-regulating the expression of IL-4.
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Arecolina , Movimiento Celular , Proliferación Celular , Fibroblastos , Interleucina-4 , Macrófagos , Reacción en Cadena en Tiempo Real de la Polimerasa , Regulación hacia Arriba , Interleucina-4/farmacología , Humanos , Arecolina/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Proliferación Celular/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Movimiento Celular/efectos de los fármacos , Fibrosis de la Submucosa Bucal/metabolismo , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Citometría de Flujo , Células CultivadasRESUMEN
A culture of cells expressing markers of mesenchymal stem cells (MSC) (CD73, CD90, CD44, CD29, and CD49b), but not hematopoietic cell markers, and capable of multilineage differentiation was isolated from the deciduous tooth pulp. Co-culturing with immature dendritic cells in the presence of LPS did not reveal an ability of the MSC to suppress the maturation of dendritic cells. On the contrary, co-culturing of MSC with monocytes in the presence of granulocyte-macrophage CSF and IL-4 led to complete suppression of monocyte differentiation into dendritic cells. However, long-term culturing of MSC from dental pulp showed that by the passage 11, they almost completely lose their suppressor ability. These results indicate that the immunological properties of MSC can change during culturing without changing their phenotypic markers. This should be taken into account when creating biomedical cell products.
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Diferenciación Celular , Técnicas de Cocultivo , Células Dendríticas , Pulpa Dental , Células Madre Mesenquimatosas , Diente Primario , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Pulpa Dental/citología , Células Dendríticas/citología , Humanos , Diente Primario/citología , Células Cultivadas , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Monocitos/citología , Monocitos/inmunología , Interleucina-4/metabolismo , Interleucina-4/farmacología , Lipopolisacáridos/farmacologíaRESUMEN
Modelling atopic dermatitis (AD) in vitro is paramount to understand the disease pathophysiology and identify novel treatments. Previous studies have shown that the Th2 cytokines IL-4 and IL-13 induce AD-like features in keratinocytes in vitro. However, it has not been systematically researched whether the addition of Th2 cells, their supernatants or a 3D structure is superior to model AD compared to simple 2D cell culture with cytokines. For the first time, we investigated what in vitro option most closely resembles the disease in vivo based on single-cell RNA sequencing data (scRNA-seq) obtained from skin biopsies in a clinical study and published datasets of healthy and AD donors. In vitro models were generated with primary fibroblasts and keratinocytes, subjected to cytokine treatment or Th2 cell cocultures in 2D/3D. Gene expression changes were assessed using qPCR and Multiplex Immunoassays. Of all cytokines tested, incubation of keratinocytes and fibroblasts with IL-4 and IL-13 induced the closest in vivo-like AD phenotype which was observed in the scRNA-seq data. Addition of Th2 cells to fibroblasts failed to model AD due to the downregulation of ECM-associated genes such as POSTN. While keratinocytes cultured in 3D showed better stratification than in 2D, changes induced with AD triggers did not better resemble AD keratinocyte subtypes observed in vivo. Taken together, our comprehensive study shows that the simple model using IL-4 or IL-13 in 2D most accurately models AD in fibroblasts and keratinocytes in vitro, which may aid the discovery of novel treatment options.
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Dermatitis Atópica , Fibroblastos , Interleucina-13 , Interleucina-4 , Queratinocitos , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Células Th2 , Humanos , Fibroblastos/metabolismo , Interleucina-4/farmacología , Interleucina-4/metabolismo , Interleucina-13/metabolismo , Interleucina-13/farmacología , Citocinas/metabolismo , Técnicas de Cocultivo , RNA-Seq , Células Cultivadas , Piel/patologíaRESUMEN
Allergic inflammation, which is the pathogenesis of allergic rhinitis and asthma, is associated with disruption of the airway epithelial barrier due to the effects of type 2 inflammatory cytokines, i.e. interleukin-4 and interleukin-13 (IL-4/13). The anti-allergic inflammatory effect of ß-eudesmol (BE) on the tight junction (TJ) of the airway epithelium has not previously been reported. Herein, the barrier protective effect of BE was determined by measurement of transepithelial electrical resistance and by paracellular permeability assay in an IL-4/13-treated 16HBE14o- monolayer. Pre-treatment of BE concentration- and time- dependently inhibited IL-4/13-induced TJ barrier disruption, with the most significant effect observed at 20 µM. Cytotoxicity analyses showed that BE, either alone or in combination with IL-4/13, had no effect on cell viability. Western blot and immunofluorescence analyses showed that BE inhibited IL-4/13-induced mislocalization of TJ components, including occludin and zonula occludens-1 (ZO-1), without affecting the expression of these two proteins. In addition, the mechanism of the TJ-protective effect of BE was mediated by inhibition of IL-4/13-induced STAT6 phosphorylation, in which BE might serve as an antagonist of cytokine receptors. In silico molecular docking analysis demonstrated that BE potentially interacted with the site I pocket of the type 2 IL-4 receptor, likely at Asn-126 and Tyr-127 amino acid residues. It can therefore be concluded that BE is able to prevent IL-4/13-induced TJ disassembly by interfering with cytokine-receptor interaction, leading to suppression of STAT6-induced mislocalization of occludin and ZO-1. BE is a promising candidate for a therapeutic intervention for inflammatory airway epithelial disorders driven by IL-4/13.
Asunto(s)
Células Epiteliales , Interleucina-13 , Interleucina-4 , Factor de Transcripción STAT6 , Uniones Estrechas , Proteína de la Zonula Occludens-1 , Uniones Estrechas/metabolismo , Uniones Estrechas/efectos de los fármacos , Humanos , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Interleucina-4/metabolismo , Interleucina-4/farmacología , Interleucina-13/metabolismo , Factor de Transcripción STAT6/metabolismo , Proteína de la Zonula Occludens-1/metabolismo , Ocludina/metabolismo , Línea Celular , Simulación del Acoplamiento Molecular , Citocinas/metabolismo , Supervivencia Celular/efectos de los fármacosRESUMEN
BACKGROUND: The preservation of retinal ganglion cells (RGCs) and the facilitation of axon regeneration are crucial considerations in the management of various vision-threatening disorders. Therefore, we investigate the efficacy of interleukin-4 (IL-4), a potential therapeutic agent, in promoting neuroprotection and axon regeneration of retinal ganglion cells (RGCs) as identified through whole transcriptome sequencing in an in vitro axon growth model. METHODS: A low concentration of staurosporine (STS) was employed to induce in vitro axon growth. Whole transcriptome sequencing was utilized to identify key target factors involved in the molecular mechanism underlying axon growth. The efficacy of recombinant IL-4 protein on promoting RGC axon growth was validated through in vitro experiments. The protective effect of recombinant IL-4 protein on somas of RGCs was assessed using RBPMS-specific immunofluorescent staining in mouse models with optic nerve crush (ONC) and N-methyl-D-aspartic acid (NMDA) injury. The protective effect on RGC axons was evaluated by anterograde labeling of cholera toxin subunit B (CTB), while the promotion of RGC axon regeneration was assessed through both anterograde labeling of CTB and immunofluorescent staining for growth associated protein-43 (GAP43). RESULTS: Whole-transcriptome sequencing of staurosporine-treated 661 W cells revealed a significant upregulation in intracellular IL-4 transcription levels during the process of axon regeneration. In vitro experiments demonstrated that recombinant IL-4 protein effectively stimulated axon outgrowth. Subsequent immunostaining with RBPMS revealed a significantly higher survival rate of RGCs in the rIL-4 group compared to the vehicle group in both NMDA and ONC injury models. Axonal tracing with CTB confirmed that recombinant IL-4 protein preserved long-distance projection of RGC axons, and there was a notably higher number of surviving axons in the rIL-4 group compared to the vehicle group following NMDA-induced injury. Moreover, intravitreal delivery of recombinant IL-4 protein substantially facilitated RGC axon regeneration after ONC injury. CONCLUSION: The recombinant IL-4 protein exhibits the potential to enhance the survival rate of RGCs, protect RGC axons against NMDA-induced injury, and facilitate axon regeneration following ONC. This study provides an experimental foundation for further investigation and development of therapeutic agents aimed at protecting the optic nerve and promoting axon regeneration.
Asunto(s)
Axones , Interleucina-4 , Regeneración Nerviosa , Células Ganglionares de la Retina , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/metabolismo , Animales , Interleucina-4/farmacología , Axones/efectos de los fármacos , Axones/metabolismo , Regeneración Nerviosa/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Traumatismos del Nervio Óptico/patología , Traumatismos del Nervio Óptico/tratamiento farmacológico , N-Metilaspartato/farmacología , Estaurosporina/farmacología , Fármacos Neuroprotectores/farmacología , Proteínas Recombinantes/farmacologíaRESUMEN
Spleen tyrosine kinase (Syk) plays a crucial role as a target for allergy treatment due to its involvement in immunoreceptor signaling. The purpose of this study was to identify natural inhibitors of Syk and assess their effects on the IgE-mediated allergic response in mast cells and ICR mice. A list of eight compounds was selected based on pharmacophore and molecular docking, showing potential inhibitory effects through virtual screening. Among these compounds, sophoraflavanone G (SFG) was found to inhibit Syk activity in an enzymatic assay, with an IC50 value of 2.2 µM. To investigate the conformational dynamics of the SYK-SFG system, we performed molecular dynamics simulations. The stability of the binding between SFG and Syk was evaluated using root mean square deviation (RMSD) and root mean square fluctuation (RMSF). In RBL-2H3 cells, SFG demonstrated a dose-dependent suppression of IgE/BSA-induced mast cell degranulation, with no significant cytotoxicity observed at concentrations below 10.0 µM within 24 h. Furthermore, SFG reduced the production of TNF-α and IL-4 in RBL-2H3 cells. Mechanistic investigations revealed that SFG inhibited downstream signaling proteins, including phospholipase Cγ1 (PLCγ1), as well as mitogen-activated protein kinases (AKT, Erk1/2, p38, and JNK), in mast cells in a dose-dependent manner. Passive cutaneous anaphylaxis (PCA) experiments demonstrated that SFG could reduce ear swelling, mast cell degranulation, and the expression of COX-2 and IL-4. Overall, our findings identify naturally occurring SFG as a direct inhibitor of Syk that effectively suppresses mast cell degranulation both in vitro and in vivo.
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Interleucina-4 , Mastocitos , Ratones , Animales , Interleucina-4/metabolismo , Interleucina-4/farmacología , Mastocitos/metabolismo , Anafilaxis Cutánea Pasiva , Simulación del Acoplamiento Molecular , Inmunoglobulina E/metabolismo , Inmunoglobulina E/farmacología , Ratones Endogámicos ICR , Ratones Endogámicos BALB CRESUMEN
OBJECTIVE: To investigate the effect of LAG-3 deficiency (LAG3-/-) on natural killer (NK) cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis. METHODS: C57BL/6 mice, each weighing (20 ± 2) g, were divided into the LAG3-/- and wild type (WT) groups, and each mouse in both groups was inoculated with 3 000 E. multilocularis protoscoleces via the hepatic portal vein. Mouse liver and spleen specimens were collected 12 weeks post-infection, sectioned and stained with sirius red, and the hepatic lesions and fibrosis were observed. Mouse hepatic and splenic lymphocytes were isolated, and flow cytometry was performed to detect the proportions of hepatic and splenic NK cells, the expression of CD44, CD25 and CD69 molecules on NK cell surface, and the secretion of interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin (IL)-4, IL-10 and IL-17A. RESULTS: Sirius red staining showed widening of inflammatory cell bands and hyperplasia of fibrotic connective tissues around mouse hepatic lesions, as well as increased deposition of collagen fibers in the LAG3-/-group relative to the WT group. Flow cytometry revealed lower proportions of mouse hepatic (6.29% ± 1.06% vs. 11.91% ± 1.85%, P < 0.000 1) and splenic NK cells (4.44% ± 1.22% vs. 5.85% ± 1.10%, P > 0.05) in the LAG3-/- group than in the WT group, and the mean fluorescence intensity of CD44 was higher on the surface of mouse hepatic NK cells in the LAG3-/- group than in the WT group (t = -3.234, P < 0.01), while no significant differences were found in the mean fluorescence intensity of CD25 or CD69 on the surface of mouse hepaticNK cells between the LAG3-/- and WT groups (both P values > 0.05). There were significant differences between the LAG3-/- and WT groups in terms of the percentages of IFN-γ (t = -0.723, P > 0.05), TNF-α (t = -0.659, P > 0.05), IL-4 (t = -0.263, P > 0.05), IL-10 (t = -0.455, P > 0.05) or IL-17A secreted by mouse hepatic NK cells (t = 0.091, P > 0.05), and the percentage of IFN-γ secreted by mouse splenic NK cells was higher in the LAG3-/- group than in the WT group (58.40% ± 1.64% vs. 50.40% ± 4.13%; t = -4.042, P < 0.01); however, there were no significant differences between the two groups in terms of the proportions of TNF-α (t = -1.902, P > 0.05), IL-4 (t = -1.333, P > 0.05), IL-10 (t = -1.356, P > 0.05) or IL-17A secreted by mouse splenic NK cells (t = 0.529, P > 0.05). CONCLUSIONS: During the course of E. multilocularis infections, LAG3-/- promotes high-level secretion of IFN-γ by splenic NK cells, which may participate in the reversal the immune function of NK cells, resulting in aggravation of hepatic fibrosis.
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Echinococcus multilocularis , Interleucina-10 , Animales , Ratones , Interleucina-10/metabolismo , Interleucina-17/metabolismo , Interleucina-17/farmacología , Interleucina-4/metabolismo , Interleucina-4/farmacología , Echinococcus multilocularis/genética , Factor de Necrosis Tumoral alfa/metabolismo , Ratones Endogámicos C57BL , Interferón gamma/genética , Interferón gamma/metabolismo , Células Asesinas Naturales/metabolismo , Cirrosis Hepática/genéticaRESUMEN
Neutrophilic pulmonary inflammation in asthmatics substantially exacerbates the severity of the disease leading to resistance to conventional corticosteroid therapy. Many studies established the involvement of Th1- and Th17-cells and cytokines produced by them (IFNg, IL-17A, IL-17F etc.) in neutrophilic pulmonary inflammation. Recent studies revealed that IL-4 - a Th2-cytokine regulates neutrophil effector functions and migration. It was showed that IL-4 substantially reduces neutrophilic inflammation of the skin in a mouse model of cutaneous bacterial infection and blood neutrophilia in a mouse model systemic bacterial infection. However, there are no data available regarding the influence of IL-4 on non-infectious pulmonary inflammation. In the current study we investigated the effects of IL-4 in a previously developed mouse model of neutrophilic bronchial asthma. We showed that systemic administration of IL-4 significantly restricts neutrophilic inflammation of the respiratory tract probably through the suppression of Th1-/Th17-immune responses and downregulation of CXCR2. Additionally, pulmonary neutrophilic inflammation could be alleviated by IL-4-dependant polarization of N2 neutrophils and M2 macrophages, expressing anti-inflammatory TGFß. Considering these, IL-4 might be used for reduction of exaggerated pulmonary neutrophilic inflammation and overcoming corticosteroid insensitivity of asthma patients.
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Asma , Infecciones Bacterianas , Neumonía , Humanos , Animales , Ratones , Interleucina-4/farmacología , Neutrófilos , Citocinas , Inflamación , Susceptibilidad a Enfermedades , Corticoesteroides/farmacologíaRESUMEN
BACKGROUND: Xiao-Qing-Long-Tang (XQLT), a classical Chinese herbal medicine formula, has been extensively used for allergic asthma treatment. However, there is limited research on its anti-inflammatory effects and mechanisms specifically in neutrophilic asthma (NA). PURPOSE: This study aims to investigate the potential therapeutic effects of XQLT against NA using a combination of network pharmacology and experimental validation. STUDY DESIGN: By utilizing traditional Chinese medicine and disease databases, we constructed an XQLT-asthma network to identify potential targets of XQLT for NA. In the experimental phase, we utilized an ovalbumin (OVA)/lipopolysaccharide (LPS)-induced model for neutrophilic asthma and examined the therapeutic effects of XQLT. RESULTS: Our research identified 174 bioactive components within XQLT and obtained 140 target genes of XQLT against asthma. Functional enrichment analysis revealed that these target genes were primarily associated with inflammation and cytokines. In the experimental validation, mice induced with OVA-LPS showcased eosinophilic and neutrophilic cell infiltration in peri-bronchial areas, elevated levels of IL-4 and IL-17 in both serum and lung, increased percentages of Th2 and Th17 cells in the spleen, as well as elevated levels of CD11b+ and CD103+ dendritic cells (DCs) within the lung. Treatment with XQLT effectively reduced IL-4 and IL-17 levels, decreased the percentages of Th2, Th17, CD11b+, and CD103+ DCs, and improved inflammatory cell infiltrations in lung tissues. These findings serve as a foundation for the potential clinical application of XQLT in neutrophilic asthma.
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Asma , Medicamentos Herbarios Chinos , Interleucina-17 , Ratones , Animales , Interleucina-17/farmacología , Interleucina-17/uso terapéutico , Interleucina-4/farmacología , Interleucina-4/uso terapéutico , Lipopolisacáridos/farmacología , Lipopolisacáridos/uso terapéutico , Farmacología en Red , Asma/tratamiento farmacológico , Pulmón , Citocinas , Ovalbúmina , Ratones Endogámicos BALB C , Modelos Animales de Enfermedad , Líquido del Lavado BronquioalveolarRESUMEN
Microglia/macrophages are major contributors to neuroinflammation in the central nervous system (CNS) injury and exhibit either pro- or anti-inflammatory phenotypes in response to specific microenvironmental signals. Our latest in vivo and in vitro studies demonstrated that curcumin-treated olfactory ensheathing cells (aOECs) can effectively enhance neural survival and axonal outgrowth, and transplantation of aOECs improves the neurological outcome after spinal cord injury (SCI). The therapeutic effect is largely attributed to aOEC anti-inflammatory activity through the modulation of microglial polarization from the M1 to M2 phenotype. However, very little is known about what viable molecules from aOECs are actively responsible for the switch of M1 to M2 microglial phenotypes and the underlying mechanisms of microglial polarization. Herein, we show that Interleukin-4 (IL-4) plays a leading role in triggering the M1 to M2 microglial phenotype, appreciably decreasing the levels of M1 markers IL1ß, IL6, tumour necrosis factor-alpha (TNF-α) and inducible nitric oxide synthase (iNOS) and elevating the levels of M2 markers Arg-1, TGF-ß, IL-10, and CD206. Strikingly, blockade of IL-4 signaling by siRNA and a neutralizing antibody in aOEC medium reverses the transition of M1 to M2, and the activated microglia stimulated with the aOEC medium lacking IL-4 significantly decreases neuronal survival and neurite outgrowth. In addition, transplantation of aOECs improved the neurological function deficits after SCI in rats. More importantly, the crosstalk between JAK1/STAT1/3/6-targeted downstream signals and NF-κB/SOCS1/3 signaling predominantly orchestrates IL-4-modulated microglial polarization event. These results provide new insights into the molecular mechanisms of aOECs driving the M1-to-M2 shift of microglia and shed light on new therapies for SCI through the modulation of microglial polarization.
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Curcumina , Traumatismos de la Médula Espinal , Animales , Ratas , Microglía , Interleucina-4/farmacología , Curcumina/farmacología , Macrófagos , Traumatismos de la Médula Espinal/terapia , AntiinflamatoriosRESUMEN
BACKGROUND: Neuroinflammation induced by systemic inflammation is a risk factor for developing chronic neurologic disorders. Oleuropein (OLE) has antioxidant and anti-inflammatory properties; however, its effect on systemic inflammation-related neuroinflammation is unknown. OBJECTIVES: This study aimed to determine whether OLE protects against systemic lipopolysaccharide (LPS)-induced neuroinflammation in rats. METHODS: Six-wk-old Wistar rats were randomly assigned to 1 of the following 5 groups: 1) control, 2) OLE-only, 3) LPS + vehicle, 4) OLE+LPS (O-LPS), and 5) a single-dose OLE + LPS (SO-LPS group). OLE 200 mg/kg or saline as a vehicle was administered via gavage for 7 d. On the seventh day, 2.5 mg/kg LPS was intraperitoneally administered. The rats were decapitated after 24 h of LPS treatment, and serum collection and tissue dissection were performed. The study assessed astrocyte and microglial activation using glial fibrillary acidic protein (GFAP) and CD11b immunohistochemistry, nod-like receptor protein-3, interleukin (IL)-1ß, IL-17A, and IL-4 concentrations in prefrontal and hippocampal tissues via enzyme-linked immunosorbent assay, and total antioxidant/oxidant status (TAS/TOS) in serum and tissues via spectrophotometry. RESULTS: In both the O-LPS and SO-LPS groups, LPS-related activation of microglia and astrocytes was suppressed in the cortex and hippocampus (P < 0.001), excluding cortical astrocyte activation, which was suppressed only in the SO-LPS group (P < 0.001). Hippocampal GFAP immunoreactivity and IL-17A concentrations in the dentate gyrus were higher in the OLE group than those in the control group, but LPS-related increases in these concentrations were suppressed in the O-LPS group. The O-LPS group had higher cortical TAS and IL-4 concentrations. CONCLUSIONS: OLE suppressed LPS-related astrocyte and microglial activation in the hippocampus and cortex. The OLE-induced increase in cortical IL-4 concentrations indicates the induction of an anti-inflammatory phenotype of microglia. OLE may also modulate astrocyte and IL-17A functions, which could explain its opposing effects on hippocampal GFAP immunoreactivity and IL-17A concentrations when administered with or without LPS.
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Interleucina-17 , Glucósidos Iridoides , Lipopolisacáridos , Ratas , Animales , Masculino , Lipopolisacáridos/toxicidad , Ratas Wistar , Interleucina-17/metabolismo , Interleucina-17/farmacología , Interleucina-17/uso terapéutico , Enfermedades Neuroinflamatorias , Antioxidantes/metabolismo , Interleucina-4/metabolismo , Interleucina-4/farmacología , Interleucina-4/uso terapéutico , Hipocampo/metabolismo , Inflamación/metabolismo , Antiinflamatorios/farmacología , Interleucina-1beta/metabolismo , Microglía/metabolismoRESUMEN
Combined allergic rhinitis and asthma syndrome (CARAS) is an airway-type 2 immune response with a profuse inflammatory process widely affecting the world population. Due to the compromise of quality of life and the lack of specific pharmacotherapy, the search for new molecules becomes relevant. This study aimed to evaluate the effectiveness of the Morita-Bailys-Hillman adduct (CISACN) treatment in the CARAS experimental model. Female BALB/c mice were ovalbumin (OVA) -sensitized and -challenged and treated with CISACN. The treatment decreased the eosinophil migration to the nasal and lung cavities and tissues and the goblet cell hyperplasia/hypertrophy, attenuated airway hyperactivity by reducing the hyperplasia/hypertrophy of the smooth muscle and the extracellular matrix's thickness. Also, the treatment reduced the clinical signs of rhinitis as nasal rubbing and sneezing in a histamine-induced nasal hyperreactivity assay. The immunomodulatory effect of CISACN was by reducing OVA-specific IgE serum level, and IL-33, IL-4, IL-13, and TGF-ß production, dependent on IFN-γ increase. Furthermore, the effect of CISACN on lung granulocytes was by decreasing the p-p38MAPK/p65NF-κB signaling pathway. Indeed, CISACN reduced the p38MAPK and p65NF-κB activation. These data demonstrated the anti-inflammatory and immunomodulatory effects of the CISACN with scientific support to become a pharmacological tool to treat airway inflammatory diseases.
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Acrilonitrilo , Asma , Rinitis Alérgica , Animales , Femenino , Ratones , Acrilonitrilo/administración & dosificación , Asma/tratamiento farmacológico , Asma/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Hiperplasia , Hipertrofia , Inmunidad , Inflamación/tratamiento farmacológico , Interleucina-4/farmacología , Pulmón , Ratones Endogámicos BALB C , Ovalbúmina , Calidad de Vida , Rinitis Alérgica/tratamiento farmacológico , Células Th2RESUMEN
OBJECTIVE: To investigate the effects of Echinococcus multilocularis on the phenotypic transformations of glucose metabolism, polarization types and inflammatory responses in macrophages, so as to provide insights into elucidation of echinococcosis pathogenesis. METHODS: Bone marrow cells were isolated from C57BL/6J mice at ages of 6 to 8 weeks, and induced into bone marrow-derived macrophages (BMDMs) with mouse macrophage colony-stimulating factor (M-CSF), which served as controls (BMDMs-M0). BMDMs-M0 induced M2 macrophages by interleukin-4 for 24 hours served as the IL-4 induction group, and BMDMs-M0 co-cultured with 2.4 ng/mL E. multilocularis cystic fluid (CF) served as the BMDM-CF co-culture group, while BMDMs-M0 co-cultured with E. multilocularis protoscolex (PSC) at a ratio of 500:1 served as the BMDM-PSC co-culture group. The types of polarization of BMDMs co-cultured with E. multilocularis CF and PSC were analyzed using flow cytometry, and the expression of macrophage markers, inflammatory factors, and glucose metabolism-related enzymes was quantified using fluorescent quantitative real-time PCR (qPCR) and Western blotting assays. RESULTS: There were significant differences among the four groups in terms of Arginase-1 (Arg1) (F = 1 457.00, P < 0.000 1), macrophages-derived C-C motif chemokine 22 (Ccl22) (F = 22 203.00, P < 0.000 1), resistin-like α (Retnla) (F = 151.90, P < 0.000 1), inducible nitric oxide synthase (iNOS) (F = 107.80, P < 0.001), hexokinase (HK) (F = 9 389.00, P < 0.000 1), pyruvate kinase (PK) (F = 641.40, P < 0.001), phosphofructokinase 1 (PFK1) (F = 43.97, P < 0.01), glucokinase (GK) (F = 432.50, P < 0.000 1), pyruvate dehydrogenase kinases1 (PDK1) (F = 737.30, P < 0.000 1), lactic dehydrogenase (LDH) (F = 3 632.00, P < 0.000 1), glucose transporter 1 (GLUT1) (F = 532.40, P < 0.000 1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (F = 460.00, P < 0.000 1), citrate synthase (CS) (F = 5 642.00, P < 0.01), glycogen synthase1 (GYS1) (F = 273.30, P < 0.000 1), IL-6 (F = 1 823.00, P < 0.000 1), IL-10 (F = 291.70, P < 0.000 1), IL-1ß (F = 986.60, P < 0.000 1), and tumor necrosis factor (TNF)-α (F = 334.80, P < 0.000 1) and transforming growth factor (TGF)-ß mRNA expression (F = 163.30, P < 0.001). The proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-PSC co-culture group [(22.87% ±1.48%) vs. (1.70% ±0.17%); t = 24.61, P < 0.001], and the proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-CF co-culture group [(20.07% ±0.64%) vs. (1.93% ±0.25%); t = 45.73, P < 0.001]. The mRNA expression of M2 macrophages markers Arg1, Ccl22 and Retnla was significantly higher in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all P values < 0.01), and no significant difference was seen in the mRNA expression of the M1 macrophage marker iNOS among the three groups (P > 0.05), while qPCR assay quantified higher mRNA expression of key glycolytic enzymes HK, PK and PFK, as well as inflammatory factors IL-10, IL-1ß, TNF-α and TGF-ß in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all P values < 0.01). Western blotting assay determined higher HK, PK and PFK protein expression in the BMDM-PSC co-culture group than in the control group (all P values < 0.05), and qPCR quantified higher GLUT1, GAPDH and IL-6 mRNA expression in the BMDM-CF co-culture group than in the control group (all P values < 0.05), while higher HK, PK and PFK protein and mRNA expression (all P values < 0.01), as well as lower IL-6 and TNF-α and higher TGF-ß mRNA expression (both P values < 0.05) was detected in the IL-4 induction group than in the control group. Glycolytic stress test showed no significant difference in the extracellular acidification rate (ECAR) of mouse BMDM among the control group, IL-4 induction group and BMDM-PSC co-culture group (F = 124.4, P < 0.05), and a higher ECAR was seen in the BMDM-PSC co-culture group and a lower ECAR was found in the IL-4 induction group than in the control group (both P values < 0.05). CONCLUSIONS: Treatment of E. multilocularis CF or PSC mainly causes polarization of BMDM into M2 macrophages, and phenotypic transformation of glucose metabolism into high-energy and high-glycolytic metabolism, and affects inflammatory responses in BMDM.
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Echinococcus , Interleucina-10 , Animales , Ratones , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Interleucina-4/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Interleucina-6/metabolismo , Interleucina-6/farmacología , Ratones Endogámicos C57BL , Macrófagos , Factor de Crecimiento Transformador beta/metabolismo , Oxidorreductasas/metabolismo , Glucosa/metabolismo , Glucosa/farmacología , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: The epidermic microbiota plays crucial roles in the pathogenesis of atopic dermatitis (AD), a common inflammatory skin disease. Melatonin (MLT) has been shown to ameliorate skin damage in AD patients, yet the underlying mechanism is unclear. METHODS: Using 2,4-dinitrofluorobenzene (DNFB) to induce an AD model, MLT intervention was applied for 14 days to observe its pharmaceutical effect. Skin lesions were observed using HE staining, toluidine blue staining and electron microscopy. Dermal proinflammatory factor (IL-4 and IL-13) and intestinal barrier indices (ZO1 and Occludin) were assessed by immunohistochemistry and RT-qPCR, respectively. The dysbiotic microbiota was analyzed using 16S rRNA sequencing. RESULTS: MLT significantly improved skin lesion size; inflammatory status (mast cells, IgE, IL-4, and IL-13); and the imbalance of the epidermal microbiota in AD mice. Notably, Staphylococcus aureus is the key bacterium associated with dysbiosis of the epidermal microbiota and may be involved in the fine modulation of mast cells, IL-4, IL-13 and IgE. Correlation analysis between AD and the gut revealed that intestinal dysbiosis occurred earlier than that of the pathological structure in the gut. CONCLUSION: Melatonin reverses DNFB-induced skin damage and epidermal dysbiosis, especially in S. aureus.
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Dermatitis Atópica , Melatonina , Microbiota , Enfermedades de la Piel , Humanos , Ratones , Animales , Dermatitis Atópica/inducido químicamente , Dermatitis Atópica/tratamiento farmacológico , Dinitrofluorobenceno/toxicidad , Melatonina/farmacología , Interleucina-13 , Staphylococcus aureus , Interleucina-4/farmacología , ARN Ribosómico 16S/genética , Disbiosis/patología , Piel , Enfermedades de la Piel/patología , Inmunoglobulina ERESUMEN
Riboflavin (RF) as a photosensitizer has been used in corneal surgery and the inactivation of blood products. However, the effect of RF on immune cells after ultraviolet (UV) light stimulation has not been investigated. This study pioneered a novel application method of RF. Firstly, UV-stimulated RF was co-cultured with human peripheral blood mononuclear cells in vitro, and the apoptosis rate of lymphocyte subsets, cell proliferation inhibition rate and concentrations of IL-1ß, IL-6, IL-10, TNF-α were assessed. UV-stimulated RF was then administered intravenously to mice via the tail vein for a consecutive period of 5 days. The levels of immunoglobulin (IgG, IgM, IgA), complement (C3, C4) and cytokines (IFN-γ, IL-4, IL17, TGF-ß) were detected by ELISA. Flow cytometry was employed to analyze the populations of CD3+T, CD4+T, CD8+T and CD4+T/CD8+T cells in spleen lymphocytes of mice. The data showed that UV-stimulated RF can effectively induce apoptosis in lymphocytes, and different lymphocyte subtypes exhibited varying degrees of treatment tolerance. Additionally, the proliferative capacity of lymphocytes was suppressed, while their cytokine secretion capability was augmented. The animal experiments demonstrated that UV-stimulated RF led to a significant reduction observed in serum immunoglobulin and complement levels, accompanied by an elevation in IFN-γ, IL-17 and TGF-ß levels, as well as a decline in IL-4 level. In summary, the results of both in vitro and in vivo experiments have demonstrated that UV-stimulated RF, exhibits the ability to partially inhibit immune function. This novel approach utilizing RF may offer innovative perspectives for diseases requiring immunosuppressive treatment.
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Interleucina-4 , Leucocitos Mononucleares , Humanos , Ratones , Animales , Interleucina-4/farmacología , Ratones Endogámicos BALB C , Citocinas/farmacología , Riboflavina/farmacología , Factor de Crecimiento Transformador beta/farmacología , Inmunoglobulinas/farmacología , Linfocitos T CD4-PositivosRESUMEN
BACKGROUND: Endometritis is a condition usually resulted from the bacterial infection of uterus, causing pelvic disease, sepsis, shock, uterine necrosis and even death if it is inappropriately treated. The aim of this study is to explore the pathogenesis of endometritis, and investigate whether the combination of doxycycline and metronidazole offers stronger protection against lipopolysaccharide (LPS)-induced endometritis, and decipher more about the mechanisms underlying endometritis-related pyroptosis. METHODS: Sprague-Dawley (SD) rats were divided into five groups (n = 8 per group): control, model, metronidazole, doxycycline, and combination groups. In control group, the rats were injected with saline, while in other groups, lipopolysaccharide was injected into uterus of the rats to establish endometritis. Hematoxylin-eosin (H&E) staining was performed as part of the histopathological examination of endometrium. The integrity of chromatin and pyroptosis were evaluated by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay. Western blot and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) were performed to ascertain the activation of toll-like receptors (TLR4)/nuclear factor-kappa B (NF-κB) pathway by detecting protein levels of phosphorylated p50 (p-p50)/p50, phosphorylated nuclear factor-kappa B (p-NF-κB)/NF-κB, phosphorylated IkappaB (p-IκB), and TLR4 protein and mRNA. Development of pyroptosis was also detected by determining the levels of caspase-1 and caspase-5 through Western blot and qRT-PCR. Enzyme-linked immunosorbent assay (ELISA) was used to detect levels of interleukin (IL)-1ß, IL-18, IL-2, IL-4, IL-6 and tumor necrosis factor alpha (TNF-α), and flow cytometry was adopted to determine T-helper (Th)1 and Th2 cell percentage to assess the extent of pyroptosis and Th1/Th2 imbalance. RESULTS: The uterine of the model group exhibited pathological alterations and higher degree of cell apoptosis. Compared with the control rats, model group showed lower protein levels of p-p50/p50 (p < 0.001), p-NF-κB/NF-κB (p < 0.001), p-IκB (p < 0.001), and TLR4 protein (p < 0.001) and mRNA (p < 0.001). Elevated levels of caspase-1 (p < 0.001), caspase-5 (p < 0.001), IL-1ß (p < 0.001), IL-18 (p < 0.001), IL-2 (p < 0.01), TNF-α (p < 0.05) and Th1/Th2 (p < 0.001) as well as reduced levels of IL-4 (p < 0.05) and IL-6 (p < 0.01) were observed in the model group, which could however be reversed by metronidazole (p < 0.01) or doxycycline (p < 0.01), with a more significant effect detected if a combination of the two drugs was administered (p < 0.01). CONCLUSIONS: The combination of doxycycline and metronidazole protects against rat endometritis by inhibiting TLR4/NF-κB pathway-mediated inflammation and suppressing pyroptosis.
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Endometritis , FN-kappa B , Humanos , Femenino , Ratas , Animales , FN-kappa B/metabolismo , FN-kappa B/farmacología , Endometritis/tratamiento farmacológico , Interleucina-18/farmacología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Metronidazol/uso terapéutico , Metronidazol/farmacología , Doxiciclina/farmacología , Transducción de Señal , Factor de Necrosis Tumoral alfa/farmacología , Lipopolisacáridos/farmacología , Interleucina-6/metabolismo , Piroptosis , Interleucina-2/farmacología , Interleucina-4/farmacología , Ratas Sprague-Dawley , Caspasas/metabolismo , Caspasas/farmacología , ARN Mensajero/genéticaRESUMEN
Diffuse large B-cell lymphoma (DLBCL) relapses in approximately 40% of patients following frontline therapy. We reported that STAT6D419 mutations are enriched in relapsed/refractory DLBCL (rrDLBCL) samples, suggesting that JAK/STAT signaling plays a role in therapeutic resistance. We hypothesized that STAT6D419 mutations can improve DLBCL cell survival by reprogramming the microenvironment to sustain STAT6 activation. Thus, we investigated the role of STAT6D419 mutations on DLBCL cell growth and its microenvironment. We found that phospho-STAT6D419N was retained in the nucleus longer than phospho-STAT6WT following IL-4 stimulation, and STAT6D419N recognized a more restricted DNA-consensus sequence than STAT6WT. Upon IL-4 induction, STAT6D419N expression led to a higher magnitude of gene expression changes, but in a more selective list of gene targets compared with STATWT. The most significantly expressed genes induced by STAT6D419N were those implicated in survival, proliferation, migration, and chemotaxis, in particular CCL17. This chemokine, also known as TARC, attracts helper T-cells to the tumor microenvironment, especially in Hodgkin's lymphoma. To this end, in DLBCL, phospho-STAT6+ rrDLBCL cells had a greater proportion of infiltrating CD4+ T-cells than phospho-STAT6- tumors. Our findings suggest that STAT6D419 mutations in DLBCL lead to cell autonomous changes, enhanced signaling, and altered composition of the tumor microenvironment.
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Linfoma de Células B Grandes Difuso , Microambiente Tumoral , Humanos , Microambiente Tumoral/genética , Interleucina-4/genética , Interleucina-4/metabolismo , Interleucina-4/farmacología , Recurrencia Local de Neoplasia , Linfoma de Células B Grandes Difuso/patología , Mutación , Factor de Transcripción STAT6/genética , Factor de Transcripción STAT6/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Lung macrophages (LMs) are critically involved in respiratory diseases. The primary objective of the present study was to determine whether or not an adenosine analog (NECA) and prostaglandin E2 (PGE2) affected the interleukin (IL)-4- and IL-13-induced release of M2a chemokines (CCL13, CCL17, CCL18, and CCL22) by human LMs. EXPERIMENTAL APPROACH: Primary macrophages isolated from resected human lungs were incubated with NECA, PGE2, roflumilast, or vehicle and stimulated with IL-4 or IL-13 for 24 h. The levels of chemokines and PGE2 in the culture supernatants were measured using ELISAs and enzyme immunoassays. KEY RESULTS: Exposure to IL-4 (10 ng/mL) and IL-13 (50 ng/mL) was associated with greater M2a chemokine production but not PGE2 production. PGE2 (10 ng/mL) and NECA (10-6 M) induced the production of M2a chemokines to a lesser extent but significantly enhanced the IL-4/IL-13-induced production of these chemokines. At either a clinically relevant concentration (10-9 M) or at a concentration (10-7 M) that fully inhibited phosphodiesterase 4 (PDE4) activity, roflumilast did not increase the production of M2a chemokines and did not modulate their IL-13-induced production, regardless of the presence or absence of PGE2. CONCLUSIONS: NECA and PGE2 enhanced the IL-4/IL-13-induced production of M2a chemokines. The inhibition of PDE4 by roflumilast did not alter the production of these chemokines. These results contrast totally with the previously reported inhibitory effects of NECA, PGE2, and PDE4 inhibitors on the lipopolysaccharide-induced release of tumor necrosis factor alpha and M1 chemokines in human LMs.