AIM OF THE STUDY: The molecular mechanisms of the anti-inflammatory properties of M. accedens are not yet understood. Therefore, we examined those mechanisms using a methanol extract of M. accedens (Ma-ME) and determined the target molecule in macrophages.
MATERIALS AND METHODS: We evaluated the anti-inflammatory effects of Ma-ME in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and in an HCl/EtOH-triggered gastritis model in mice. To investigate the anti-inflammatory activity, we performed a nitric oxide (NO) production assay and ELISA assay for prostaglandin E2 (PGE2). RT-PCR, luciferase gene reporter assays, western blotting analyses, and a cellular thermal shift assay (CETSA) were conducted to identify the mechanism and target molecule of Ma-ME. The phytochemical composition of Ma-ME was analyzed by HPLC and LC-MS/MS.
RESULTS: Ma-ME suppressed the production of NO and PGE2 and the mRNA expression of proinflammatory genes (iNOS, IL-1β, and COX-2) in LPS-stimulated RAW264.7 cells without cytotoxicity. Ma-ME inhibited NF-κB activation by suppressing signaling molecules such as IκBα, Akt, Src, and Syk. Moreover, the CETSA assay revealed that Ma-ME binds to Syk, the most upstream molecule in the NF-κB signal pathway. Oral administration of Ma-ME not only alleviated inflammatory lesions, but also reduced the gene expression of IL-1β and p-Syk in mice with HCl/EtOH-induced gastritis. HPLC and LC-MS/MS analyses confirmed that Ma-ME contains various anti-inflammatory flavonoids, including quercetin, daidzein, and nevadensin.
CONCLUSIONS: Ma-ME exhibited anti-inflammatory activities in vitro and in vivo by targeting Syk in the NF-κB signaling pathway. Therefore, we propose that Ma-ME could be used to treat inflammatory diseases such as gastritis.
METHODOLOGY/PRINCIPAL FINDINGS: Four ligands (1-4) and their respective nickel-containing complexes (5-8) were synthesized and characterized. The compounds synthesized were tested for their effects on NF-κB nuclear translocation, pro-inflammatory cytokines secretion and NF-κB transactivation activity. The active compound was further evaluated on its ability to suppress carrageenan-induced acute inflammation in vivo. A potential binding target of the active compound was also predicted by molecular docking analysis.
CONCLUSIONS/SIGNIFICANCE: Among all synthesized compounds tested, we found that complex [Ni(H2L1)(PPh3)]Cl (5) (complex 5), potently inhibited IκBα degradation and NF-κB p65 nuclear translocation in LPS-stimulated RAW264.7 cells as well as TNFα-stimulated HeLa S3 cells. In addition, complex 5 significantly down-regulated LPS- or TNFα-induced transcription of NF-κB target genes, including genes that encode the pro-inflammatory cytokines TNFα, IFNβ and IL6. Luciferase reporter assays confirmed that complex 5 inhibited the transactivation activity of NF-κB. Furthermore, the anti-inflammatory effect of complex 5 was also supported by its suppressive effect on carrageenan-induced paw edema formation in wild type C57BL/6 mice. Interestingly, molecular docking study showed that complex 5 potentially interact with the active site of IKKβ. Taken together, we suggest complex 5 as a novel NF-κB inhibitor with potent anti-inflammatory effects.
METHODS: Maximal non-toxic dose (MNTD) of methanol extract of P. ginseng root culture on BV2 microglia cells was first determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, followed by treatment and LPS stimulation of cells, and the measurement of NO using Griess assay and TNF-α, IL-6, and IL-10 using ELISA assay.
RESULTS: The MNTD of P. ginseng root extract was determined to be (587 ± 57) µg/mL. Following that, NO and IL-6 levels were found to be insignificantly reduced by 6.88% and 0.14% respectively in stimulated cells upon treatment with MNTD. Treatment with MNTD yielded similar insignificant result, with only a reduction of 3.58% and 0.08% in NO and IL-6 levels respectively. However, TNF-α and IL-10 levels were significantly downregulated by 15.64% and 34.96% respectively upon treatment with P. ginseng root extract at MNTD.
CONCLUSION: Methanol extract of P. ginseng root culture did not show any significant anti-inflammatory effects on NO and IL-6 levels, but might potentially possess both anti-neuroinflammatory and pro-neuroinflammatory properties through the downregulation of TNF-α and IL-10 respectively.
MATERIALS AND METHODS: miR-205 expression was investigated in 48 cases of inflammatory, benign and malignant tumor tissue array of the neck, oronasopharynx, larynx and salivary glands by Locked Nucleic Acid in situ hybridization (LNA-ISH) technology.
RESULTS: miR-205 expression was significantly differentially expressed across all of the inflammatory, benign and malignant tumor tissues of the neck. A significant increase in miR-205 staining intensity (p<0.05) was observed from inflammation to benign and malignant tumors in head and neck tissue array, suggesting that miR-205 could be a biomarker to differentiate between cancer and non-cancer tissues.
CONCLUSIONS: LNA-ISH revealed that miR-205 exhibited significant differential cytoplasmic and nuclear staining among inflammation, benign and malignant tumors of head and neck. miR-205 was not only exclusively expressed in squamous epithelial malignancy. This study offers information and a basis for a comprehensive study of the role of miR-205 that may be useful as a biomarker and/or therapeutic target in head and neck tumors.
METHODS: Therefore, the present study aimed to investigate the messenger ribonucleic acid (mRNA) expression of BRS3 in human liver THLE-2 cells post-BPA treatment by real-time polymerase chain reaction. The effects of BPA on the levels of pro-inflammatory proteins, interleukin 6 (IL6) and CC motif chemokine ligand 2 (CCL2), in conditioned media of BPA-treated THLE-2 cells and deoxyribonucleic acid (DNA) synthesis in replicating BPA-treated THLE-2 cells during the cell cycle were also examined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry, respectively.
RESULTS: The study found that the mRNA expression of BRS3 was increased in THLE-2 cells treated with BPA. The study also showed that the expression levels of IL6 and CCL2 reached an optimum level in the conditioned media of BPA-treated THLE-2 cells after 48 h of treatment. Subsequently, the DNA synthesis analysis showed that bromodeoxyuridine/propidium iodide (BrdU/PI) stained positive cells were decreased in BPA-treated THLE-2 cells at 72 h of treatment.
CONCLUSION: The study demonstrates that BRS3 expression induced by BPA is likely associated with reduced cell proliferation by inhibiting DNA synthesis and inducing cellular inflammation in liver cells.