Exploration of Pharmacological Mechanism of Cinnamomum tamala Essential Oil in Treating Inflammation based on Network Pharmacology, Molecular Modelling, and Experimental Validation.

BACKGROUND: Cinnamomum tamala (Buch.-Ham.) T.Nees & Eberm., also known as Indian Bay leaf, holds a distinctive position in complementary and alternative medicinal systems due to its anti-inflammatory properties. However, the active constituents and key molecular targets by which C. tamala essential oil (CTEO) exerts its anti-inflammatory action remain unclear. OBJECTIVE: The present study used network pharmacology and experimental validation to investigate the mechanism of CTEO in the treatment of inflammation. METHODS: GC-MS analysis was used to identify the constituents of CTEO. The key constituents and core targets of CTEO against inflammation were obtained by network pharmacology. The binding mechanism between the active compounds and inflammatory genes was ascertained by molecular docking and molecular dynamics simulation analysis. The pharmacological mechanism predicted by network pharmacology was verified in lipopolysaccharide-stimulated murine macrophage (RAW 264.7) cell lines. RESULTS: Forty-nine constituents were identified by GC-MS analysis, with 44 constituents being drug-like candidates. A total of 549 compounds and 213 inflammation-related genes were obtained, revealing 68 overlapping genes between them. Compound target network analysis revealed cinnamaldehyde as the core bioactive compound with the highest degree score. PPI network analysis demonstrated Il-1ß, TNF-α, IL8, IL6 and TLR4 as key hub anti-inflammatory targets. KEGG enrichment analysis revealed a Toll-like receptor signalling pathway as the principally regulated pathway associated with inflammation. A molecular docking study showed that cinnamaldehyde strongly interacted with the Il-1ß, TNF-α and TLR-4 proteins. Molecular dynamics simulations and MMPBSA analysis revealed that these complexes are stable without much deviation and have better free energy values. In cellular experiments, CTEO showed no cytotoxic effects on RAW 264.7 murine macrophages. The cells treated with LPS exhibited significant reductions in NO, PGE2, IL-6, TNF-α, and IL-1ß levels following treatment with CTEO. Additionally, CTEO treatment reduced the ROS levels and increased the antioxidant enzymes such as SOD, GSH, GPx and CAT. Immunofluorescence analysis revealed that CTEO inhibited LPS-stimulated NF-κB nuclear translocation. The mRNA expression of TLR4, MyD88 and TRAF6 in the CTEO group decreased significantly compared to the LPS-treated group. CONCLUSION: The current findings suggest that CTEO attenuates inflammation by regulating TLR4/MyD88/NF- κB signalling pathway.

Functional Characterisation of Surfactant Protein A as a Novel Prophylactic Means against Oncogenic HPV Infections.

Human papillomavirus (HPV) infection poses a significant health challenge, particularly in low- and middle-income countries (LMIC), where limited healthcare access and awareness hinder vaccine accessibility. To identify alternative HPV targeting interventions, we previously reported on surfactant protein A (SP-A) as a novel molecule capable of recognising HPV16 pseudovirions (HPV16-PsVs) and reducing infection in a murine cervicovaginal HPV challenge model. Building on these findings, our current study aimed to assess SP-A's suitability as a broad-spectrum HPV-targeting molecule and its impact on innate immune responses. We demonstrate SP-A's ability to agglutinate and opsonise multiple oncogenic HPV-PsVs types, enhancing their uptake and clearance by RAW264.7 murine macrophages and THP-1 human-derived immune cells. The SP-A opsonisation of HPV not only led to increased lysosomal accumulation in macrophages and HaCaT keratinocytes but also resulted in a decreased infection of HaCaT cells, which was further decreased when co-cultured with innate immune cells. An analysis of human innate immune cell cytokine profiles revealed a significant inflammatory response upon SP-A exposure, potentially contributing to the overall inhibition of HPV infection. These results highlight the multi-layered impact of SP-A on HPV, innate immune cells and keratinocytes and lay the basis for the development of alternative prophylactic interventions against diverse HPV types.

Molecular characteristics of Echinococcus multilocularis FABP1 and its regulatory functions on murine macrophages.

Fatty acid binding proteins (FABPs) have emerged as attractive vaccination candidates for several platyhelminth species. To explore the physiological functions of Echinococcus multilocularis (E. multilocularis) FABP, the molecular characteristics of EmFABP1 were analyzed by online software, and the regulatory roles of rEmFABP1 protein in murine macrophages were further investigated. The emfabp1 gene encodes 133 amino acids with the characteristic ß-barrel shape of the cytoplasmic FABP family. Natural EmFABP1 protein is predominantly expressed in protoscoleces tegument and germinal layer cells and is also detected in cyst fluid and exosomes of E. multilocularis. rEmFABP1 protein demonstrated a notable suppression of phagocytic activity and nitric oxide production in murine macrophages. Additionally, the protein was observed to promote apoptosis and regulate cytokine expression in macrophages. These findings suggested that E. multilocularis FABP1 is critical in modifying macrophage physiological processes and that this protein may have immunomodulatory roles during infection.

Dichapetalin-type triterpenoids from Dichapetalum longipetalum and their anti-inflammatory activity.

A phytochemical research on the twigs of Dichapetalum longipetalum (Turcz.) Engl. Resulted in five undescribed dichapetalin-type triterpenoids 1-5. Their chemical structures were determined by spectroscopic analysis of HR-ESIMS and NMR spectra and the absolute configuration of compound 1 was completely elucidated by single crystal X-ray crystallography. Through preliminary anti-inflammatory activity assessment, compound 1 exhibited inhibitory effect on LPS-induced NO production in RAW264.7 murine macrophages with an IC50 value of 2.09 µM.

Microemulsions Enhance the In Vitro Antioxidant Activity of Oleanolic Acid in RAW 264.7 Cells.

Oleanolic acid (OA) is the main triterpenic acid of olive leaves known for numerous pharmacological properties, including antioxidant activity. However, it is poorly soluble in water and consequently with low bioavailability, which limits its pharmacological application. Microemulsions (MEs) are dispersed systems consisting of two immiscible phases that promote rapid solubilization and absorption in the gastrointestinal tract. To improve both solubility and intestinal permeability of this molecule, OA has been formulated in two different microemulsions (ME-1 and ME-2). A solubility screening was carried out to select the ME components, and pseudoternary phase diagrams were constructed to evaluate the region of existence and select the appropriate amount of the constituents. ME-1 was prepared using Capmul PG-8/NF as the oily phase, and Transcutol and Tween 20 (7:3) as surfactants, while ME-2 contained Nigella oil and Isopropil myristate as the oily phase, and Transcutol HP and Cremophor EL (2:1) as surfactants. The OA solubility was increased by 1000-fold and 3000-fold in ME-1-OA and ME-2-OA, respectively. The MEs' droplet size and the PdI were evaluated, and the stability was assessed for 8 weeks by monitoring chemical and physical parameters. The parallel artificial membrane permeability assay (PAMPA) also demonstrated an enhanced intestinal permeability of both OA formulations compared with free OA. The potential ability of both MEs to enhance the bioactivity of OA against LPS-induced oxidative stress in RAW 264.7 murine macrophages was also investigated. Overall, this study suggests that both MEs promote a bio-enhancement of the protective action of OA against the LPS-induced pro-oxidant stress in macrophages. Overall, this study suggests that MEs could be an interesting formulation to improve OA oral bioavailability with potential clinical applications.

Cytotoxic Effects Caused by Functionalized Carbon Nanotube in Murine Macrophages.

BACKGROUND/AIMS: The development of new nanomaterials has been growing in recent decades to bring benefits in several areas, especially carbon-based nanoparticles, which have unique physical-chemical properties and allow to take on several applications. Consequently, the use of new nanomaterials without previous toxicological studies raises concern about possible harmful health effects. The aim of this study was to investigate the cytotoxic profile of a new multi-walled carbon nanotube (MWCNT) functionalized with tetraethylenepentamine called OCNT-TEPA using in vitro assays in murine macrophage cells linage J774 A.1. METHODS: OCNT-TEPA was characterized by transmission electron microscopy (TEM) and high resolution TEM (HR-TEM), scanning electron microscopy (SEM), zeta potential and dynamic light scattering (DLS), and its cytotoxic effects were evaluated at 24 and 48 hours by cell viability assays (MTT and NR), morphology and cell recovery (optic microscopy and clonogenic assay), formation of reactive oxygen (ROS) and nitric oxide (NO) species, inflammatory profile (IL-6 and TNF cytokines), mitochondrial membrane potential analysis (MMP), activation of the caspase 3 pathway and cell death (flow cytometry). RESULTS: The data showed a significant decrease in cell viability, increased production of ROS and NO, alteration of mitochondrial membrane potential, increased levels of inflammatory cytokines, alteration of cell morphology, activation of the Caspase 3 pathway and consequently cell death, in the highest concentrations of OCNT-TEPA tested in the periods of 24 and 48 hours. CONCLUSION: The analyses showed that OCNT-TEPA has a dose-dependent cytotoxic profile, which may be harmful to murine macrophages (J774 A.1) and may represent a health risk.

Anti-inflammatory effects of Torin2 on lipopolysaccharide-treated RAW264.7 murine macrophages and potential mechanisms.

Context: Torin2 has various pharmacological properties. However, its anti-inflammatory activity has not been reported. Objective: This study focused on the potential anti-inflammatory properties of Torin2 in lipopolysaccharide (LPS)-evoked RAW264.7 murine macrophages. The study aimed to shed light on the molecular mechanisms that ameliorate these effects. Methods: Torin2 was applied to 100 ng/mL lipopolysaccharide-induced RAW 264.7 macrophages in vitro. Nitric oxide (NO) levels were detected using the Griess reagent kit. Prostaglandin E2 (PGE2), pro-inflammatory cytokines interleukin (IL)-1ß, interleukin (IL)-6, and tumor necrosis factor in the supernatant fraction were determined using enzyme-linked immunosorbent assay (ELISA). Gene expression of pro-inflammatory cytokines, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) were tested using real-time quantitative polymerase chain reaction (qPCR). Cyclooxygenase-2 and inducible nitric oxide synthase proteins, phosphorylation of mitogen-activated protein kinase (MAPK) subgroups, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38, I-kappa-B-alpha (IκBα), and nuclear factor-kappa-B (NF-κB), and activation in extracts were detected via western blotting. Nuclear factor-kappa-B/p65 nuclear translocation was tested using an immunofluorescence assay. Results: The results demonstrated that pre-treatment with Torin2 profoundly attenuated the lipopolysaccharide-stimulated levels of nitric oxide and prostaglandin E2, pro-inflammatory cytokines, messenger ribonucleic acid (mRNA), and protein expression of cyclooxygenase-2 and inducible nitric oxide synthase. Collectively, Torin2 pre-treatment notably weakened lipopolysaccharide-induced damage by reducing the phosphorylation of nuclear factor-kappa-B, p38, c-Jun N-terminal kinase, extracellular signal-regulated kinase proteins, and nuclear factor-kappa-B/p65 nuclear translocation. Conclusion: Numerous pieces of evidence indicated that Torin2 reversed inflammatory activation by regulating nuclear factor-kappa-B and mitogen-activated protein kinase signaling pathways and provided a tentative potential candidate for preventing and treating inflammatory diseases.

MicroRNA-23a-3p targeting of HMGB1 inhibits LPS-induced inflammation in murine macrophages in vitro.

Inflammatory cytokines, including high mobility group box 1 (HMGB1), play a key role in sepsis via various mechanisms, some of which remain unknown. Sepsis is a common cause of death in patients admitted to the intensive care unit. MicroRNAs (miRs) serve an important role in the inflammatory response. The present study aimed to investigate the role of miR-23a-3p in macrophage inflammation and the targeted regulation of HMGB1 expression. The murine macrophage cell line RAW264.7 was subjected to lipopolysaccharide (LPS) treatment to mimic the inflammation involved in sepsis in vitro. Reverse transcription-quantitative PCR was performed to measure miR-23a-3p expression and mRNA expression. Protein levels were determined using ELISA and western blotting. The target binding relationship between miR-23a-3p and the HMGB1 3'untranslated region was predicted and validated with a dual luciferase reporter assay. HMGB1 expression was increased and miR-23a-3p expression significantly reduced in patients with sepsis and in LPS-treated RAW264.7 cells in comparison with controls. Overexpression of miR-23a-3p reduced interleukin (IL)-6 and tumor necrosis factor (TNF)-α expression in RAW264.7 cells under LPS stimulation, while silencing of miR-23a-3p elevated the expression of IL-6 and TNF-α in comparison with controls. The inhibitory effect of miR-23a-3p on LPS-induced inflammation could be abolished by HMGB1 upregulation in RAW264.7 cells. HMGB1 was targeted by miR-23a-3p. miR-23a-3p is expressed at reduced levels during inflammation in sepsis, and overexpression of miR-23a-3p inhibits LPS-induced inflammation in murine macrophages in vitro by directly downregulating HMGB1. The results of the present study provided a novel insight into the molecular mechanism underlying HMGB1 expression at the post-transcriptional level in sepsis.

Molecular characterization of a novel GSTO2 of Fasciola hepatica and its roles in modulating murine macrophages.

Fascioliasis is an important zoonotic helminthic disease caused by Fasciola hepatica and poses a serious threat to global public health. To evade the immune response of its host (humans or animals), F. hepatica secretes various antioxidant enzymes such as glutathione transferase (GST) to facilitate its invasion, migration and parasitism in vivo. To investigate the biological functions of a novel omega-class GST (GSTO), the molecular features of GSTO2 of F. hepatica were analyzed by online software, and the biochemical properties in vitro of recombinant GSTO2 (rGSTO2) were dissected. Then, the regulatory roles of rGSTO2 protein in murine macrophages in vitro were further explored. The results revealed that the GSTO2 gene encodes 254 amino acids, which harbor the characteristic N-terminal domain (ßαßαßßα) and C-terminal domain (α-helical) of the cytoplasmic GST superfamily. GSTO2 was mainly expressed in F. hepatica vitelline follicles, intestinal tract, excretory pores and vitelline cells, with thioltransferase and dehydroascorbate reductase activities. Moreover, rGSTO2 protein could be taken up by murine macrophages and significantly inhibit the viability of macrophages. In addition, rGSTO2 protein could significantly promote apoptosis and modulate the expression of cytokines in macrophages. These findings suggested that F. hepatica GSTO2 plays an important role in modulating the physiological functions of macrophages, whereby this protein might be involved in immunomodulatory and anti-inflammatory roles during infection. This study provided new insights into the immune-evasion mechanism of F. hepatica and may contribute to the development of a potential anti-inflammatory agent.

Title: Caractérisation moléculaire d'une nouvelle GSTO2 de Fasciola hepatica et ses rôles dans la modulation des macrophages murins. Abstract: La fasciolase est une importante maladie helminthique zoonotique causée par Fasciola hepatica, qui constitue une menace sérieuse pour la santé publique mondiale. Pour échapper à la réponse immunitaire de son hôte (humain ou animal), F. hepatica sécrète diverses enzymes antioxydantes telles que la glutathion transférase (GST) pour faciliter son invasion, sa migration et son parasitisme in vivo. Pour étudier les fonctions biologiques d'une nouvelle GST de classe oméga (GSTO), les caractéristiques moléculaires de la GSTO2 de F. hepatica ont été analysées par un logiciel en ligne et les propriétés biochimiques in vitro de sa protéine recombinante (rGSTO2) ont été disséquées. Ensuite, les rôles régulateurs de la protéine rGSTO2 sur les macrophages murins in vitro ont été explorés plus avant. Les résultats ont révélé que le gène GSTO2 code pour 254 acides aminés, qui abritent le domaine N-terminal caractéristique (ßαßαßßα) et le domaine C-terminal (α-hélicoïdal) de la superfamille GST cytoplasmique. Chez F. hepatica, GSTO2 était principalement exprimée dans les follicules vitellins, le tractus intestinal, les pores excréteurs et les cellules vitellines, avec des activités de thioltransférase et de déhydroascorbate réductase. De plus, la protéine rGSTO2 a pu être absorbée par les macrophages murins et inhiber de manière significative la viabilité des macrophages. Enfin, la protéine rGSTO2 a pu favoriser de manière significative l'apoptose et moduler l'expression des cytokines dans les macrophages. Ces résultats suggèrent que la GSTO2 de F. hepatica joue un rôle important dans la modulation des fonctions physiologiques des macrophages, cette protéine pouvant être impliquée dans des rôles immunomodulateurs et anti-inflammatoires au cours de l'infection. Cette étude a fourni de nouvelles informations sur le mécanisme d'évasion immunitaire de F. hepatica et pourrait contribuer au développement d'un agent anti-inflammatoire potentiel.

Ranunculus species suppress nitric oxide production in LPS-stimulated RAW 264.7 macrophages.

Aerial parts and roots from three Ranunculus species were extracted with 70% ethanol. The phytochemical composition was investigated using GC-MS and multivariate data analysis. The total phenolic and flavonoid contents were also assessed. The in vitro inhibitory properties were evaluated as nitrite concentration in LPS-stimulated RAW 264.7 macrophage cell line. All the samples induced concentration-dependent inhibitory effects, with R. sceleratus aerial parts extract being the most interesting sample (IC50 = 22.08 ± 1.32 µg/mL), even more active than the reference compound indomethacin. A very good activity was also observed for R. sardous and R. ficaria aerial parts extracts (IC50 = 51.61 ± 3.12 µg/mL and 84.55 ± 3.40 µg/mL). A lesser but noteworthy potential was also demonstrated for the root extracts. The results suggest that Ranunculus hydroalcoholic extracts are able to inhibit nitrite accumulation and may be useful in preventing inflammatory diseases mediated by excessive production of NO.

Efecto antiinflamatorio y antioxidante in vitro de Curcuma longa sobre macrófagos murinos durante la infección por Helicobacter pylori

RESUMEN Introducción: La colonización por Helicobacter pylori produce una inflamación en la mucosa gástrica con el consecuente desarrollo de enfermedades gastroduodenales. Frente a altas tasas de resistencia antimicrobiana y la ausencia de una vacuna en humanos, la alternativa ha sido la búsqueda de extractos de plantas con propiedades antimicrobiana, antiinflamatoria, antioxidante, antifúngica y anticancerígena como la Curcuma longa. Sin embargo, al ser una especie introducida y adaptada a las condiciones climáticas del país, son necesarios los estudios preclínicos que avalen su potencial antiinflamatorio y antioxidante. Objetivo: Evaluar el efecto del extracto de C. longa sobre macrófagos peritoneales infectados con H. pylori. Métodos: Para evaluar el efecto antiinflamatorio y antioxidante del extracto de C. longa sobre macrófagos murinos infectados por H. pylori, se evaluaron diferentes concentraciones del extracto y relaciones de bacteria, y se evaluó la muerte celular mediante DAPI. Se determinó la producción de óxido nítrico, peróxido de hidrógeno y los niveles de la interleucina-1β. Resultados: La viabilidad del macrófago se afectó frente a concentraciones de 100 µg/mL del extracto de cúrcuma y a partir de 25 bacterias/macrófago. Al combinar las diferentes concentraciones del extracto con las multiplicidades bacterianas se observó una reducción en los niveles de H2O2 e IL-1β; sin embargo, la reducción del óxido nítrico se observó en el rango de 6,25-50 µg/mL del producto natural. Conclusiones: El extracto de cúrcuma cubano mostró potencial antioxidante y antiinflamatorio al disminuir la citotoxicidad celular y la producción de especies reactivas del oxígeno en macrófagos peritoneales.

ABSTRACT Introduction: Colonization by Helicobacter pylori causes inflammation of the gastric mucosa with the consequent development of gastroduodenal diseases. In view of the high antimicrobial resistance rates and the absence of a vaccine for humans, the alternative has been to search for plant extracts with antimicrobial, anti-inflammatory, antioxidant, antifungal and anticancer properties. An example is Curcuma longa. However, being as it is a species introduced and adapted to the country's climate conditions, it is necessary to conduct preclinical studies demonstrating its anti-inflammatory and antioxidant potential. Objective: Evaluate the effect of a C. longa extract on peritoneal macrophages infected by H. pylori. Methods: With the purpose of describing the anti-inflammatory and antioxidant effect of C. longa on murine macrophages infected by H. pylori, an evaluation was conducted of various extract concentrations and bacterial relationships, while cell death was assessed by DAPI. Determination was made of nitric oxide and hydrogen peroxide production, as well as of interleukin-1β levels. Results: Viability of the macrophage was affected in the presence of 100 µg/ml concentrations of the turmeric extract and as from 25 bacteria / macrophage. When different concentrations of the extract were combined with the bacterial multiplicities, a reduction was observed in H2O2 and IL-1β levels. However, nitric oxide reduction was observed within the range of 6.25-50 µg/ml of the natural product. Conclusions: The Cuban turmeric extract was found to have antioxidant and anti-inflammatory potential by reducing cell cytotoxicity and the production of reactive oxygen species in peritoneal macrophages.

Toxicological effects of silver nanoparticles and cadmium chloride in macrophage cell line (RAW 264.7): An in vitro approach.

BACKGROUND: Silver nanoparticles (AgNP) are largely used in nanotechnological products, but the real risks for human and environment are still poorly understood if we consider the effects of mixtures of AgNP and environmental contaminants, such as non-essential metals. METHODS: The aim of the present study was to investigate the cytotoxicity and toxicological interaction of AgNP (1-4 nm, 0.36 and 3.6 µg mL-1) and cadmium (Cd, 1 and 10 µM) mixtures. The murine macrophage cell line RAW 264.7 was used as a model. RESULTS: Effects were observed after a few hours (4 h) on reactive oxygen species (ROS) and became more pronounced after 24 h-exposure. Cell death occurred by apoptosis, and loss of cell viability (24 h-exposure) was preceded by increases of ROS levels and DNA repair foci, but not of NO levels. Co-exposure potentiated some effects (decrease of cell viability and increase of ROS and NO levels), indicating toxicological interaction. CONCLUSION: These effects are important findings that must be better investigated, since the interaction of Cd with AgNP from nanoproducts may impair the function of macrophages and represent a health risk for humans.

ATP-dependent activation of NLRP3 inflammasome in primary murine macrophages infected by pseudorabies virus.

Pseudorabies virus (PRV), an alphaherpesvirus, causes respiratory and reproductive diseases in pigs and severe nervous symptom in other susceptible hosts. Previous studies showed that PRV infection induced a systemic inflammatory response in mice, indicating that pro-inflammatory cytokines participated in viral neuropathy in mice. The pro-inflammatory cytokine IL-1ß is a key mediator of the inflammatory response and plays an important role in host-response to pathogens. However, the secretion of IL-1ß and its relationship with inflammasome activation during PRV infection remains poorly understood. In this study, we found that PRV infection caused significant secretion of several pro-inflammatory cytokines in macrophages and promoted IL-1ß secretion in an ATP-dependent manner. Furthermore, the expression of IL-1ß can be induced by only PRV infection and depended on NF-κB pathway activation, while the subsequent secretion of IL-1ß was mediated by ATP-induced P2 × 7R activation, loss of intracellular K+, and the subsequent NLRP3 inflammasome activation. By using a mouse infection model, we also found that ATP exacerbated clinical signs and death of mice infected by PRV in a NLRP3-dependent manner. These results indicate that ATP facilitates activation of NLRP3 inflammasome and enhances the pathogenicity of PRV in mice during its acute infection.

Isolation, purification and identification of immunologically active peptides from Hericium erinaceus.

Biologically active peptides released by proteins are important in regulating immunity. The purpose of this study was to isolate and purify an immunologically active peptide from Hericium erinaceus (H. erinaceus) and to explore its effect on cytokine secretion and differentiation of macrophages. An active peptide with an amino acid sequence, Lys-Ser-Pro-Leu-Tyr (KSPLY) was obtained from H. erinaceus protein by ultrafiltration combined with multistage chromatography separation and identification technology. Subsequently, it was confirmed that the synthetic peptide KSPLY had a good immunomodulatory activity at a concentration of 100 µmol/L and could promote the secretion of NO, IL-1ß, IL-6 and TNF-α by macrophages. The effects of KSPLY on M1 macrophages and M2 macrophages were also studied. Results showed that KSPLY inhibited the secretion of NO and IL-6 by M1 macrophages and promoted the tendency of M2 macrophages to transform to M1 macrophages. Therefore, it can be concluded that KSPLY is an effective immunomodulatory peptide that may be beneficial in cancer treatment and human health improvement.

Evaluation of Murine Macrophage Cytokine Production After In Vivo Morphine Treatment.

The discovery of opioid receptor expression on immune cells has originated a large research activity on the possible modulation by opioid drugs of immune system responses. In this chapter we describe an easy methodology useful to obtain information about the potential immunomodulatory activity of opioid drugs. An in vivo treatment schedule is used, and macrophages are studied for their ability to release different cytokines.

Schizophyllum commune ß-glucan: Effect on interleukin-10 expression induced by lipopolysaccharide from periodontopathic bacteria.

ß-glucans are potent immunomodulators, with effects on innate and adaptive immune responses via dectin-1 as the main receptor. In this study, we investigated the biological effect of ß-glucan from Schizophyllum commune, called Schizophyllan (SPG) on Interleukin-10 (IL-10) expression induced by a lipopolysaccharide (LPS) from Aggregatibacter actinomycetemcomitans in murine macrophages (J774.1). SPG and dectin-1 interaction up-regulates LPS-induced IL-10 expression. The regulative effect of SPG on IL-10 expression is dependent on prolongation of nuclear translocation activity of nuclear factor-kappa B (NF-κBα) pathway induced by LPS. We also found that LPS-induced phosphorylation of mitogen- and stress-activated protein kinase 1 (MSK1) and cAMP-responsive-element-binding protein (CREB), followed by up-regulation of IL-10, was stimulated by SPG priming via activation of the spleen tyrosine kinase (Syk). Our data indicate that SPG augments the anti-inflammatory response in murine macrophages which can be useful to create an intervention for periodontal disease treatment.

Three-Dimensional RAW264.7 Cell Model on Electrohydrodynamic Printed Poly(ε-Caprolactone) Scaffolds for In Vitro Study of Anti-Inflammatory Compounds.

Inflammation plays an essential role in the human immune system, and anti-inflammatory compounds are important to promote health. However, the in vitro screening of these compounds is largely dependent on flat biology. Herein, we report our efforts in establishing a 3D inflammation murine macrophage model. Murine macrophage RAW 264.7 cells were cultured on poly(ε-caprolactone) (PCL) scaffolds fabricated through an electrohydrodynamic jetting 3D printer and their behavior were examined. Cells on PCL scaffolds showed a 3D shape and morphology with multilayers and a lower proliferation rate. Moreover, macrophages were not activated by scaffold material PCL and 3D microenvironment. The 3D cells showed greater sensitivity to lipopolysaccharide stimulation with higher production activity of nitric oxide (NO), nitric oxide synthases (iNOS), and cyclooxygenase-2 (COX-2). Additionally, the 3D macrophage model showed lower drug sensitivity to commercial anti-inflammatory drugs including aspirin, ibuprofen, and dexamethasone, and natural flavones apigenin and luteolin with higher IC50 for NO production and lower iNOS and COX-2 inhibition efficacy. Overall, the 3D macrophage model showed promise for higher accurate screening of anti-inflammatory compounds. We developed, for the first time, a 3D macrophage model based on a 3D-printed PCL scaffold that provides an extracellular matrix environment for cells to grow in the 3D dimension. 3D-grown RAW 264.7 cells showed different sensitivities and responses to anti-inflammatory compounds from its 2D model. The 3D cells have lower sensitivity to both commercial and natural anti-inflammatory compounds. Consequently, our 3D macrophage model could be applied to screen anti-inflammatory compounds more accurately and thus holds great potential in next-generation drug screening applications.

Technical report: Targeted proteomic analysis reveals enrichment of atypical ubiquitin chains in contractile murine tissues.

Ubiquitylation is an elaborate post-translational modification involved in all biological processes. Its pleotropic effect is driven by the ability to form complex polyubiquitin chain architectures that can influence biological functions. In this study, we optimised sample preparation and chromatographic separation of Ubiquitin peptides for Absolute Quantification by Parallel Reaction Monitoring (Ub-AQUA-PRM). Using this refined Ub-AQUA-PRM assay, we were able to quantify all ubiquitin chain types in 10-min LC-MS/MS runs. We used this method to determine the ubiquitin chain-linkage composition in murine bone marrow-derived macrophages and different mouse tissues. We could show tissue-specific differences in ubiquitin levels in murine tissues, with polyubiquitin chain types contributing a small proportion to the total pool of ubiquitin. Interestingly, we observed enrichment of atypical (K33) ubiquitin chains in heart and muscle. Our approach enabled high-throughput screening of ubiquitin chain-linkage composition in different murine tissues and highlighted a possible role for atypical ubiquitylation in contractile tissues. SIGNIFICANCE: Large knowledge gaps exist in our understanding of ubiquitin chain-linkage composition in mammalian tissues. Defining this in vivo ubiquitin chain-linkage landscape could reveal the functional importance of different ubiquitin chain types in tissues. In this study, we refined the previously described Ub-AQUA-PRM assay to enable quantification of all ubiquitin chain types in a high-throughput manner. Using this assay, we provided new data on the ubiquitin chain-linkage composition in primary murine macrophages and tissues, and revealed an enrichment of atypical ubiquitin chains in contractile tissues. Our approach should thus enable rapid, high-throughput screening of ubiquitin chain-linkage composition in different sample types, as demonstrated in murine primary cells and tissues.

Nitric oxide inhibition of lipopolysaccharide-stimulated RAW 247.6 cells by ibuprofen-conjugated iron oxide nanoparticles.

Aim: To develop a series of superparamagnetic iron oxide nanoparticles (SPIONs) by coconjugating them with ibuprofen (ibu) and glycerol phosphate (glycerol) or ibu and glucose-1-phosphate and to assess capacity of these conjugates to inhibit the release of nitric oxide (NO) in macrophages, even at low concentrations. Materials & methods: The SPION conjugates were characterized and their properties evaluated showing the influence of those ligands on colloidal stability and inhibition of NO-release demonstrated. The cytotoxicity and possible anti-inflammatory activity were evaluated using murine macrophages (RAW 247.6). Results: SPION-glycerol phosphate/ibu conjugates inhibited the NO production induced by lipopolysaccharides, indicating a potential anti-inflammatory activity. Conclusion: SPION conjugated with ibu was shown to inhibit NO-release even at very low concentrations, suggesting possible action against inflammatory diseases.

A Gene Expression Analysis of M1 and M2 Polarized Macrophages.

Macrophages are professional innate immune cells that are broadly disseminated throughout the body, shape various innate and adaptive immune responses, and play crucial roles in inflammation, homeostasis, wound healing, and tissue remodelling. According to their surrounding microenvironments, macrophages can differentiate themselves in different phenotypes. Over the last two decades, gene expression profiling has been used to decipher new transcripts associated with macrophage phenotypes. This chapter outlines protocols used to isolate and culture murine macrophages and how they can be "polarized" to obtain a specific phenotype. Furthermore, we describe a protocol for gene expression profiling using a quantitative real-time polymerase chain reaction (qPCR), a high-standard technology in the field of gene expression.