Identification of Key Molecular Pathways and Associated Genes as Targets to Overcome Radiotherapy Resistance Using a Combination of Radiotherapy and Immunotherapy in Glioma Patients.

Recent mechanistic studies have indicated that combinations of radiotherapy (RT) plus immunotherapy (via CSF-1R inhibition) can serve as a strategy to overcome RT resistance and improve the survival of glioma mice. Given the high mortality rate for glioma, including low-grade glioma (LGG) patients, it is of critical importance to investigate the mechanism of the combination of RT and immunotherapy and further translate the mechanism from mouse studies to improve survival of RT-treated human glioma patients. Using the RNA-seq data from a glioma mouse study, 874 differentially expressed genes (DEGs) between the group of RT-treated mice at glioma recurrence and the group of mice with combination treatment (RT plus CSF-1R inhibition) were translated to the human genome to identify significant molecular pathways using the KEGG enrichment analysis. The enrichment analysis yields statistically significant signaling pathways, including the phosphoinositide 3-kinase (PI3K)/AKT pathway, Hippo pathway, and Notch pathway. Within each pathway, a candidate gene set was selected by Cox regression models as genetic biomarkers for resistance to RT and response to the combination of RT plus immunotherapies. Each Cox model is trained using a cohort of 295 RT-treated LGG patients from The Cancer Genome Atlas (TCGA) database and validated using a cohort of 127 RT-treated LGG patients from the Chinese Glioma Genome Atlas (CGGA) database. A four-DEG signature (ITGB8, COL9A3, TGFB2, JAG1) was identified from the significant genes within the three pathways and yielded the area under time-dependent ROC curve AUC = 0.86 for 5-year survival in the validation set, which indicates that the selected DEGs have strong prognostic value and are potential intervention targets for combination therapies. These findings may facilitate future trial designs for developing combination therapies for glioma patients.

Outer membrane vesicles from Escherichia coli are efficiently internalized by macrophage cells and alter their inflammatory response.

The release of extracellular vesicles (EV) by pathogenic microbes is considered an alternative cell-to-cell transport of macromolecules transport mechanism. In Gram-negative bacteria, EVs may be formed by outer membrane budding, so-called outer membrane vesicles (OMVs). Previous studies have revealed E. coli constitutively release nano-sized OMVs, which can be potent activators of cellular functions without live bacteria. But the immunomodulatory activity of E. coli OMVs is still relatively poorly understood. Here we investigated the morphological characterization and composition of E.Coli OMVs, kinetics of internalization by Raw 264.7 macrophage cells, and their immunomodulatory activity on cells. By transmission electron microscopy and dynamic light scattering, E.Coli OMVs were identified as typical cup-shaped, bilayered membranous structures, mainly distributed between 72.5 and 212.5 nm. We also demonstrated by confocal fluorescence microscopy that exposure of Raw 264.7 cells to E.Coli OMVs resulted in internalization of these nanoparticles and decreased mitochondrial membrane potential. In addition, E. Coli OMVs treatment induced the production of ROS, iNOS, IL-1ß, IL-6, IL-10 and up-regulation of CD86 and CD206. Taken together, our results indicated that E.Coli OMVs are immunobiologically active, can directly interact with macrophage and participate in immune responses.

Dual-aptamer-engineered M1 macrophage with enhanced specific targeting and checkpoint blocking for solid-tumor immunotherapy.

Chimeric antigen receptor T (CAR-T) cell therapy has faced a series of challenges and has shown very little efficacy in solid tumors to date. Although genetically engineered macrophages have achieved definite therapeutic effect in solid tumors, heterogeneous expression of engineered proteins and the potential for toxicity limit further applications. Herein, we propose a nongenetic and simple macrophage cell engineering strategy through glycan metabolic labeling and click reaction for the treatment of solid tumors. The aptamer-engineered M1 macrophage (ApEn-M1) showed enhanced active targeting ability for tumor cells in vitro and in vivo, resulting in significant cytotoxicity effects. Moreover, ApEn-M1 exhibited superior antitumor efficacy in a breast cancer xenograft mouse model and a lung metastasis mouse model of breast cancer. Interestingly, the ApEn-M1 could reprogram the immunity microenvironment by increasing T cell infiltration and enhancing T cell activity in the tumor region. Additionally, the administration of ApEn-M1 showed no obvious systemic side effects. With glycan metabolic labeling, the macrophages could be efficiently labeled with aptamers on the cell surface via click reaction without genetic alteration or cell damage. Hence, this study serves as a proof of concept for cell-surface anchor engineering and expands the range of nongenetic macrophage cell engineering strategies.

Mycobacterium tuberculosis glycolipoprotein LprG inhibits inflammation through NF-κB signaling of ERK1/2 and JNK in LPS-induced murine macrophage cells.

Mycobacterium tuberoculosis (Mtb) is a contagious pathogen that causes human tuberculosis (TB). TB is a major global health threat that causes 9.6 million illnesses and 1.5 million deaths per year. Recent studies have suggested Mtb-secreted proteins as new candidates for therapeutic drugs and vaccines. LprG is a Mtb-secreted surface glycolipoprotein encoded by lprG (Rv1411c), which forms an operon with Rv1410c, where Rv1410c encodes P55, an efflux pump membrane protein. Various in vitro and in vivo studies have reported on the target-binding activity, cell envelope biosynthesis, and mycobacterial virulence of LprG. However, the anti-inflammatory effect of LprG in macrophages has not yet been investigated. In this study, we demonstrated that LprG can suppress lipopolysaccharide (LPS)-induced inflammation in a macrophage model. LprG inhibited LPS-stimulated nitric oxide (NO) production. LprG also suppressed expression of inducible cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) at the transcriptional and protein levels. In addition, LprG decreased mRNA expression of the pro-inflammatory cytokines interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α). Furthermore, LprG attenuated nuclear factor kappa-B (NF-κB) translocation and IκB phosphorylation. Moreover, LprG specifically inhibited phosphorylated kinases such as c-Jun N-terminal kinase (p-JNK) and extracellular signal-regulated kinase 1/2 (p-ERK1/2), but not p-p38. Taken together, these results suggest that LprG inhibits LPS-stimulated inflammation via downregulation of NO, COX-2, iNOS, and pro-inflammatory cytokines through the NF-κB, AP-1, and MAPK signaling pathways. The present study will aid in the development of anti-inflammatory medications using Mtb. The organism, which has long been regarded as a human pathogenic or human health-threating agent, can be utilized as a future medical resource.

Titanium Surface Characteristics Induce the Specific Reprogramming of Toll-like Receptor Signaling in Macrophages.

Most of the research on titanium-based dental implants (Ti-discs) is focused on how they are able to stimulate the formation of new tissue and/or cytotoxic studies, with very scarce data on their effects on functional responses by immunocompetent cells. In particular, the link between the rewiring of innate immune responses and surface biomaterials properties is poorly understood. To address this, we characterize the functional response of macrophage cultures to four different dental titanium surfaces (MA: mechanical abrasion; SB + AE: sandblasting plus etching; SB: sandblasting; AE: acid etching). We use different Toll-like receptor (TLR) ligands towards cell surface receptors (bacterial lipopolysaccharide LPS for TLR4; imiquimod for TLR7; synthetic bacterial triacylated lipoprotein for TLR2/TLR1) and endosomal membrane receptor (poly I:C for TLR3) to simulate bacterial (cell wall bacterial components) or viral infections (dsRNA and ssRNA). The extracellular and total LDH levels indicate that exposure to the different Ti-surfaces is not cytotoxic for macrophages under resting or TLR-stimulated conditions, although there is a tendency towards an impairment in macrophage proliferation, viability or adhesion under TLR4, TLR3 and TLR2/1 stimulations in SB discs cultures. The secreted IL-6 and IL-10 levels are not modified upon resting macrophage exposure to the Ti-surfaces studied as well as steady state levels of iNos or ArgI mRNA. However, macrophage exposure to MA Ti-surface do display an enhanced immune response to TLR4, TLR7 or TLR2/1 compared to other Ti-surfaces in terms of soluble immune mediators secreted and M1/M2 gene expression profiling. This change of characteristics in cellular phenotype might be related to changes in cellular morphology. Remarkably, the gene expression of Tlr3 is the only TLR that is differentially affected by distinct Ti-surface exposure. These results highlight the relevance of patterned substrates in dental implants to achieve a smart manipulation of the immune responses in the context of personalized medicine, cell-based therapies, preferential lineage commitment of precursor cells or control of tissue architecture in oral biology.

In Vitro Anti-Inflammatory Activity of Three Peptides Derived from the Byproduct of Rice Processing.

Inflammation is a contributing factor to the initiation and progression of many diseases, and some food-derived biofunctional peptides show high anti-inflammatory activity. In our previous study, we demonstrated that peptides derived from trypsin hydrolysis of rice protein show good immunological activity. In the present study, proteins of broken rice were extracted and identified by macroporous resin fractionation and liquid chromatography/tandem mass spectrometry (LC-MS/MS). Subsequently, a bioinformatics prediction and in silico simulation approach was used to screen for peptides showing anti-inflammatory activity, including inhibition of the production of nitric oxide and proinflammatory cytokines (interleukin-1ß, interleukin-6, and tumor necrosis factor-α) by lipopolysaccharide-stimulated RAW264.7 mice macrophages. Three peptides (DNIQGITKPAIR, IAFKTNPNSMVSHIAGK, and IGVAMDYSASSKR) that demonstrated the highest binding affinity were synthesized, and their in vitro anti-inflammatory activity was investigated. This is the first study that integrates LC-MS/MS identification and bioinformatics prediction for reporting the anti-inflammatory activity of anti-inflammatory peptides derived from broken rice protein. The study findings revealed that the peptides derived from the byproduct of rice milling could be potentially used as natural anti-inflammatory alternativities.

FSTL1 aggravates OVA-induced inflammatory responses by activating the NLRP3/IL-1ß signaling pathway in mice and macrophages.

OBJECTIVE: Asthma, a well-known disease with high morbidity, is characterized by chronic airway inflammation. However, the allergic inflammation mechanisms of follistatin-like protein 1 (FSTL1) have not been elucidated. This study aims to investigate the effects of FSTL1 in ovalbumin (OVA)-induced mice and macrophages on nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3)/interleukin-1ß (IL-1ß) signaling pathway. METHODS: Mice were randomly divided into control-WT, OVA-WT, control-Fstl1±, OVA-Fstl1±. Histological changes were assessed by HE and PAS staining. The protein levels of Muc-5AC, FSTL1, NLRP3, and IL-1ß in lung tissue were detected by immunohistochemistry and ELISA. The bronchoalveolar lavage fluid (BALF) in mice and human serum samples were detected by ELISA. Then, mice were grouped into control, FSTL1, MCC950 + FSTL1 to further investigate the relationship between FSTL1 and NLRP3/IL-1ß. Alveolar macrophage cells (MH-S cells) were separated into control, OVA, FSTL1, OVA + FSTL1, OVA + siNC, OVA + siFSTL1, MCC950, and FSTL1 + MCC950 groups to explore the effect of FSTL1 on the NLRP3/IL-1ß signaling. The protein expression of NLRP3 and IL-1ß in MH-S cells was detected by Western blot analysis. RESULTS: The present results uncovered that Fstl1± significantly ameliorated OVA-induced Muc-5AC production and mucus hypersecretion. Fstl1± was also found to decrease the production of inflammatory cytokines and inflammatory cell infiltration in OVA-induced asthmatic mice. Meanwhile, the serum concentrations of FSTL1 and IL-1ß were higher in  asthma subjects than the health subjects, and Fstl1± ameliorated the production of NLRP3 and IL-1ß in OVA-induced asthmatic mice. Furthermore, mice by injected FSTL1 substantially stimulated the expression of NLRP3 and IL-1ß, while pretreatment with MCC950 in mice significantly weakened the production of NLRP3 and IL-1ß induced by injection FSTL1. Pretreatment with siFSTL1 or MCC950 significantly reduced the production of NLRP3 and IL-1ß induced by OVA or FSTL1 in MH-S cells. CONCLUSIONS: The study results showed that FSTL1 played an important role in allergic airway inflammation by activating NLRP3/IL-1ß. Hence, inhibition FSTL1 could be applied as a therapeutic agent against asthma.

New Hydroquinone Monoterpenoid and Cembranoid-Related Metabolites from the Soft Coral Sarcophyton tenuispiculatum.

Chemical investigation of the marine soft coral Sarcophyton tenuispiculatum resulted in the isolation of a 1,4-dihydrobenzoquinone, sarcotenuhydroquinone (1), three new cembranoids, sarcotenusenes A‒C (2‒4), and ten previously reported metabolites 5-14. The chemical structures of all isolated metabolites were determined by detailed spectroscopic analyses. In biological assays, anti-inflammatory, cytotoxic, and peroxisome proliferator-activated receptor γ (PPAR-γ) transcription factor assays of all compounds were performed. None of the isolated compounds were found to exhibit activity in the PPAR-γ transcription factor assay. The anti-inflammatory assays showed that (+)-7α,8ß-dihydroxydeepoxysarcophine (13) inhibited the production of IL-1ß to 56 ± 1% at a concentration of 30 µM in lipopolysaccharide (LPS)-stimulated J774A.1 macrophage cells. In addition, 1 and 2 were found to exhibit cytotoxicity towards a panel of cancer cell lines.

Artocarpus lakoocha Extract Inhibits LPS-Induced Inflammatory Response in RAW 264.7 Macrophage Cells.

:Artocarpus lakoocha Roxb. (AL) has been known for its high content of stilbenoids, especially oxyresveratrol. AL has been used in Thai traditional medicine for centuries. However, the role of AL in regulating inflammation has not been elucidated. Here we investigated the molecular mechanisms underlying the anti-inflammation of AL ethanolic extract in RAW 264.7 murine macrophage cell line. The HPLC results revealed that this plant was rich in oxyresveratrol, and AL ethanolic extract exhibited anti-inflammatory properties. In particular, AL extract decreased lipopolysaccharide (LPS)-mediated production and secretion of cytokines and chemokine, including IL-6, TNF-α, and MCP-1. Consistently, the extract inhibited the production of nitric oxide (NO) in the supernatants of LPS-stimulated cells. Data from the immunofluorescence study showed that AL extract suppressed nuclear translocation of nuclear factor-kappa B (NF-κB) upon LPS induction. Results from Western blot analysis further confirmed that AL extract strongly prevented the LPS-induced degradation of IκB which is normally required for the activation of NF-κB. The protein expression of iNOS and COX-2 in response to LPS stimulation was significantly decreased with the presence of AL extract. AL extract was found to play an anti-inflammatory role, in part through inhibiting LPS-induced activation of Akt. The extract had negligible impact on the activation of mitogen-activated protein kinase (MAPK) pathways. Specifically, incubation of cells with the extract for only 3 h demonstrated the rapid action of AL extract on inhibiting the phosphorylation of Akt, but not ERK1/2. Longer exposure (24 h) to AL extract was required to mildly reduce the phosphorylation of ERK1/2, p38, and JNK MAPKs. These results indicate that AL extract manipulates its anti-inflammatory effects mainly through blocking the PI3K/Akt and NF-κB signal transduction pathways. Collectively, we believe that AL could be a potential alternative agent for alleviating excessive inflammation in many inflammation-associated diseases.

Analysis of the transcriptomic features of microsatellite instability subtype colon cancer.

BACKGROUND: Programmed cell death protein 1(PD-1) blocking antibodies have been used to enhance immunity in solid tumors and achieve durable clinical responses with an acceptable safety profile in multiple types of cancer. However, only a subset of patients could benefit from PD-1 blockade therapy. Prognostic information including PD-1 ligand (PD-L1) expression, IFN-γ expression signature, tumor mutational burden, and microsatellite instability (MSI) have been evaluated for patients who are selected to receive immune checkpoint therapeutic treatment. Yet the relationship of those biomarkers in determining immune checkpoint therapy is largely unknown. METHODS: Immune-profiles of MSI subtype colon cancer were identified from integrating published MSI associated gene expression data. The enriched pathways and transcription factors were analyzed by GSEA assay. The infiltrations of immune cell types into MSI subtype colon cancer tissues were determined by CIBESORT assay. RESULTS: In the MSI subtype colon cancer patients, PD-L1, IFN-γ and IFN-γ associated genes are highly expressed. And all those genes are favorable effects in colon cancer progress. In addition, we find that Wnt-ß-catenin and TGFß signaling pathways which are two important factors inhibiting PD-1 checkpoint blockade therapy are negatively related with MSI status. We also identify that the immune-profiles in MSI subtype colon cancer are contributed by M1 macrophage infiltration in the tumor environment. CONCLUSIONS: Our results provide the detailed underlying mechanisms of MSI subtype cancer patients are sensitive to PD-1 checkpoint blockade.

The modified Si-Jun-Zi Decoction attenuates colon cancer liver metastasis by increasing macrophage cells.

BACKGROUND: The modified Si-Jun-Zi Decoction (SJZ), a Chinese medicine formula, is clinically used against multiple malignancies including colorectal cancer (CRC). This study aims to evaluate the effect of modified SJZ on CRC liver metastasis and identify the therapeutic mechanisms. METHODS: Human CRC cells with GFP fluorescence were transplanted into Balb/c nude mice spleens. Modified SJZ, 5-fluorouracil or the combined treatment was given for 3 weeks. CRC liver metastasis was measured by fluorescence imaging and plasma cytokines were analyzed. Furthermore, the effects of administration time and doses for the modified SJZ were investigated in nude mice. RESULTS: Modified SJZ could increase the survival rate and reduce CRC liver metastasis in the nude mice model. Plasma GM-CSF level was elevated. Three weeks of treatment with the modified SJZ at the full dose (45 g/kg) could significantly increase the number of macrophages but not neutrophils in the spleen. CONCLUSIONS: These results indicate that modified SJZ can inhibit CRC liver metastasis by activating the innate immune system, providing a complementary and alternative therapy for CRC.

Anti-Inflammatory Activity of Some Characteristic Constituents from the Vine Stems of Spatholobus suberectus.

The dried vine stems of Spatholobus suberectus are commonly used in traditional Chinese medicine for treating gynecological and cardiovascular diseases. In this study, five new compounds named spasuberol A (2), homovanillyl-4-oxo-nonanoate (5), spasuberol C (6), spasuberoside A (14), and spasuberoside B (15), together with ten known compounds (1, 3, 4, 7-13), were isolated from the dried vine stems of S. suberectus. Their chemical structures were analyzed using spectroscopic assays. This is the first study interpreting the detailed structural information of 4. The anti-inflammatory activity of these compounds was evaluated by reducing nitric oxide overproduction in RAW264.7 macrophages stimulated by lipopolysaccharide. Compounds 1 and 8-10 showed strong inhibitory activity with half maximal inhibitory concentration (IC50) values of 5.69, 16.34, 16.87, and 6.78 µM, respectively, exhibiting higher activity than the positive drug l-N6-(1-iminoethyl)-lysine (l-NIL) with an IC50 value of 19.08 µM. The IC50 values of inhibitory activity of compounds 2 and 4-6 were 46.26, 40.05, 45.87, and 28.29 µM respectively, which were lower than l-NIL, but better than that of positive drug indomethacin with an IC50 value of 55.44 µM. Quantitative real-time polymerase chain reaction analysis revealed that assayed compounds with good anti-inflammatory activity, such as 1, 6, 9, and 10 at different concentrations, can reduce the messenger RNA (mRNA) expression of some pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α), nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX-2). The anti-inflammatory activity and the possible mechanism of the compounds mentioned in this paper were studied preliminarily.

Biological activities, isolated compounds and HPLC profile of Verbascum nubicum.

Context: Genus Verbascum (Scrophulariaceae) comprises about 360 species of flowering plants. Verbascum has been used in traditional medicine as an astringent, antitussive, analgesic and anti-inflammatory. Objective: Nothing was found in the available literature concerning Verbascum nubicum Murb; therefore, the study evaluates the biological activities, isolated compounds and HPLC profile. Materials and methods: Methanol extract (VME) and butanol fraction (VBF) of air-dried powdered V. nubicum were obtained. Four compounds were isolated from VBE and identified by 1H- and 13C-NMR. High-performance liquid chromatography (HPLC) profile was determined for (VME). LD50, in vitro antioxidant, in vivo antiulcerogenic and anti-inflammatory activities as well as hepatoprotective activity were assessed. Anti-ulcerogenic and hepatoprotective activities were supported by histopathological examinations. Results: HPLC analysis of VME revealed the presence of luteolin 7-glucoside (2215.43 mg/100 g), hesperidin (954.51 mg/100 g) and apigenin (233.15 mg/100 g) as major compounds. Four compounds were isolated and confirmed by NMR data, were identified as gentiopicroside, luteolin, aucubin and gallic acid. The LD50 of VME and VBF extracts were calculated to be 8200 and 4225 mg/kg b.w., respectively. IC50 values of VBE and VMF as measured by DPPH·method were 43.6 and 50 µg/mL, respectively. Also, anti-inflammatory effect of VME (400 mg/kg b.w.) and VBF (200 mg/kg b.w.) induced edema model after 120 min were 61.93 and 56.13%, respectively. Antiulcerogenic activity of VME (400 mg/kg b.w.) and VBF (200 mg/kg b.w.) in albino rats were 65.14 and 84.57%, respectively. Conclusions: The V. nubicum extracts displayed safe and promising antioxidant, anti-inflammatory and hepatoprotective properties. It can be also applied in the pharmacy industry, food industry and healthcare.

MicroRNA expression profile in RAW264·7 macrophage cells exposed to Echinococcus multilocularis metacestodes.

MicroRNAs (miRNAs) are short noncoding RNAs, involved in the regulation of parasite diseases. However, a role of miRNAs in Echinococcus multilocularis infection remains largely unknown. In this study, we first found the expression levels of key genes involved in miRNA biogenesis and function, including Ago2, Xpo5, Tarbp2 and DgcR8, were obviously altered in the macrophage RAW264·7 cells exposed to E. multilocularis metacestodes. Compared with the control, 18 and 32 known miRNAs were found to be differentially expressed (P 2) in the macrophages exposed to E. multilocularis metacestodes for 6 and 12 h, respectively. Among these, several are known to be involved in regulating cytokine activities and immune responses. Quantitative real-time polymerase chain reaction results showed that the expression of nine selected miRNAs was consistent with the sequencing data at each treatment time points. Moreover, there were statistically significant correlations between the expression levels of miRNAs and their corresponding targeted genes. Our data give us some clues to pinpoint a role of miRNAs in the course of infection and immunity of E. multilocularis.

Gly[14]-humanin inhibits ox-LDL uptake and stimulates cholesterol efflux in macrophage-derived foam cells.

Foam cell formation, which is caused by imbalanced cholesterol influx and efflux by macrophages, plays a vital role in the occurrence and development of atherosclerosis. Humanin (HN), a mitochondria-derived peptide, can prevent the production of reactive oxygen species and death of human aortic endothelial cells exposed to oxidized low-density lipoprotein (ox-LDL) and has a protective effect on patients with in early atherosclerosis. However, the effects of HN on the regulation of cholesterol metabolism in RAW 264.7 macrophages are still unknown. This study was designed to investigate the role of [Gly14]-humanin (HNG) in lipid uptake and cholesterol efflux in RAW 264.7 macrophages. Flow cytometry and live cell imaging results showed that HNG reduced Dil-ox-LDL accumulation in the RAW 264.7 macrophages. A similar result was obtained for lipid accumulation by measuring cellular cholesterol content. Western blot analysis showed that ox-LDL treatment upregulated not only the protein expression of CD36 and LOX-1, which mediate ox-LDL endocytosis, but also ATP-binding cassette (ABC) transporter A1 and ABCG1, which mediate ox-LDL exflux. HNG pretreatment inhibited the upregulation of CD36 and LOX-1 levels, prompting the upregulation of ABCA1 and ABCG1 levels induced by ox-LDL. Therefore we concluded that HNG could inhibit ox-LDL-induced macrophage-derived foam cell formation, which occurs because of a decrease in lipid uptake and an increase in cholesterol efflux from macrophage cells.

The α-cyclodextrin complex of the Moringa isothiocyanate suppresses lipopolysaccharide-induced inflammation in RAW 264.7 macrophage cells through Akt and p38 inhibition.

In the last decades, a growing need to discover new compounds for the prevention and treatment of inflammatory diseases has led researchers to consider drugs derived from natural products as a valid option in the treatment of inflammation-associated disorders. The purpose of the present study was to investigate the anti-inflammatory effects of a new formulation of Moringa oleifera-derived 4-(α-L-rhamnopyranosyloxy)benzyl isothiocyanate as a complex with alpha-cyclodextrin (moringin + α-CD) on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells, a common model used for inflammation studies. In buffered/aqueous solution, the moringin + α-CD complex has enhanced the water solubility and stability of this isothiocyanate by forming a stable inclusion system. Our results showed that moringin + α-CD inhibits the production of inflammatory mediators in LPS-stimulated macrophages by down-regulation of pro-inflammatory cytokines (TNF-α and IL-1ß), by preventing IκB-α phosphorylation, translocation of the nuclear factor-κB (NF-κB), and also via the suppression of Akt and p38 phosphorylation. In addition, as a consequence of upstream inhibition of the inflammatory pathway following treatment with moringin + α-CD, the modulation of the oxidative stress (results focused on the expression of iNOS and nitrotyrosine) and apoptotic pathway (Bax and Bcl-2) was demonstrated. Therefore, moringin + α-CD appears to be a new relevant helpful tool to use in clinical practice for inflammation-associated disorders.

Refined Deep-Sea Water Suppresses Inflammatory Responses via the MAPK/AP-1 and NF-κB Signaling Pathway in LPS-Treated RAW 264.7 Macrophage Cells.

Atopic dermatitis (AD) is a type of inflammatory skin disease caused by genetics, immune system dysfunction, and environmental stresses. It is, however, still considered to be a refractory disease. Macrophages are inflammatory immune cells that infiltrate the skin and induce inflammation. We investigated the effect of refined deep-sea water (RDSW) on lipopolysaccharide (LPS)-induced inflammatory response in RAW 264.7 macrophage cells. The results showed that RDSW suppressed the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. Furthermore, nitric oxide, a product of iNOS, and prostaglandin (PG) D2 and PGE2, products of COX-2, were significantly inhibited by RDSW in a hardness-dependent manner. Moreover, we found that RDSW reversed the release of histamines and regressed the mRNA expressions and production of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, and IL-10, and vascular endothelial growth factor, in a hardness-dependent manner. We also found that the suppressive effect of RDSW on LPS-induced inflammatory responses was regulated by the inhibition of NF-κB nuclear translocation, and ERK 1/2 and JNK 1/2 mediated the suppression of c-Jun and c-Fos expressions. In conclusion, the present investigation suggests the possibility that RDSW may be used to treat and/or prevent inflammatory diseases, including AD.

Structure-Antioxidative and Anti-Inflammatory Activity Relationships of Purpurin and Related Anthraquinones in Chemical and Cell Assays.

Anthraquinone (9,10-anthraquinone) and several hydroxy derivatives, including purpurin (1,2,4-trihydroxyanthraquinone), anthrarufin (1,5-dihydroxyanthraquinone), and chrysazin (1,8-dihydroxyanthraquinone), were evaluated for antioxidative and anti-inflammatory activities in chemical assays and mammalian cells (murine macrophage RAW 264.7 cells). Several tests were used to assess their activities: 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical; ABTS radical cation; hydrogen peroxide scavenging; reduction of potassium ferricyanide; chelation of ferrous ions; inhibition of lipid peroxidation; inhibition of nitric oxide generation; scavenging of the intracellular hydroxyl radical; expression of NLRP3 polypeptide for inflammasome assembly; and quantitation of proinflammatory cytokine interleukin 1ß (IL-1ß) for inflammasome activation. The results show that purpurin, from the root of the madder plant (Rubia tinctorum L.), exhibited the highest antioxidative activity in both chemical and cultured cell antioxidant assays. The antioxidative activities of the other three anthraquinones were lower than that of purpurin. In addition, purpurin could down-regulate NLRP3 inflammasome assembly and activation, suggesting that it might protect foods against oxidative damage and prevent in vivo oxidative stress and inflammation. Structure-activity relationships and the significance of the results for food quality and human health are discussed.

Anti-Inflammatory Effect of Ascochlorin in LPS-Stimulated RAW 264.7 Macrophage Cells Is Accompanied With the Down-Regulation of iNOS, COX-2 and Proinflammatory Cytokines Through NF-κB, ERK1/2, and p38 Signaling Pathway.

A natural compound C23 H32 O4 Cl, ascochlorin (ASC) isolated from an incomplete fungus, Ascochyta viciae has been known to have several biological activities as an antibiotic, antifungal, anti-cancer, anti-hypolipidemic, and anti-hypertension agent. In this study, anti-inflammatory activity has been investigated in lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells, since ASC has not been observed on the inflammatory events. The present study has clearly shown that ASC (1-50 µM) significantly suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2 ) and decreased the gene expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose-dependent manner. Moreover, ASC inhibited the mRNA expression and the protein secretion of interleukin (IL)-1ß and IL-6 but not tumor necrosis factor (TNF)-α in LPS-stimulated RAW 264.7 macrophage cells. In addition, ASC suppressed nuclear translocation and DNA binding affinity of nuclear factor-κB (NF-κB). Furthermore, ASC down-regulated phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2) and p-p38. These results demonstrate that ASC exhibits anti-inflammatory effects in RAW 264.7 macrophage cells.

Macrophage depletion and TNF-α inhibition prevent resorption in CBA/J × DBA/2 model of CpG-induced abortion.

OBJECTIVE: To elucidate the mechanism by which embryo-resorption was enhanced by pathogenic CpG ODN motif in abortion-prone CBA/J × DBA/2 model and to develop a counter strategy for normal pregnancy outcome. METHODS: This is an animal model-based study. Abortion-prone model is established by CBA/J × DBA/2. An infection was mimicked by CpG ODN injection. RESULTS: Embryo-resorption was readily induced by CpG ODN in low doses of CpG ODN (∼25 µg/dam) when intraperitoneally (IP) injected on gestational day(gd) 6.5 in male DBA/2 mated CBA/J female mice. A more modest decline in Progesterone(P4), but not Estrogen(E2) was observed after exposure to CpG ODN in the model. P4 supplement fail to improve pregnancy outcomes, even at pharmocology dose. CpG ODN-induced fetal resorption is prevented by the treatment of anti-F4/80 or by that of anti-TNFα.In the implantation sites, the treatment of anti-F4/80 inhibits the increase both of F4/80(+) macrophage proportion and TNF-αexpression level which are induced by CpG ODN. The anti-TNFαtreatment also recovers CpG ODN-induced reduction of CD4(+)Foxp3(+) T cells. CONCLUSION: Circulating P4 is not responsible for the process by which CpG ODN-induced embryonic resorption in an abortion-prone mice. Macrophage depletion and TNF-α inhibition are really noteworthy for CpG ODN-induced pregnancy disruption.