AIM: To investigate the regulation of rs10889677 and the role of buparlisib in the PI3K signaling pathway in CAC pathogenesis.
METHODS: Genomic DNA from 32 colonic samples, including CAC (n = 7), UC (n = 10) and CRC (n = 15), was sequenced for the rs10889677 mutation. The mutant and wildtype fragments were amplified and cloned in the pmirGLO vector. The luciferase activity of cloned vectors was assessed after transfection into the HT29 cell line. CAC mice were induced by a mixture of a single azoxymethane injection and three cycles of dextran sulphate sodium, then buparlisib was administered after 14 d. The excised colon was subjected to immunohistochemistry for Ki67 and Cleaved-caspase-3 markers and quantitative real-time polymerase chain reaction analysis for Pdk1 and Sgk2.
RESULTS: Luciferase activity decreased by 2.07-fold in the rs10889677 mutant, confirming the hypothesis that the variant disrupted miRNA binding sites, which led to an increase in IL23R expression and the activation of the PI3K signaling pathway. Furthermore, CAC-induced mice had a significantly higher disease activity index (P < 0.05). Buparlisib treatment significantly decreased mean weight loss in CAC-induced mice (P < 0.05), reduced the percentage of proliferating cells by 5%, and increased the number of apoptotic cells. The treatment also caused a downward trend of Pdk1 expression and significantly decreased Sgk2 expression.
CONCLUSION: Our findings suggested that the rs10889677 variant as a critical initiator of the PI3K signaling pathway, and buparlisib had the ability to prevent PI3K-non-AKT activation in the pathophysiology of CAC.
OBJECTIVES: (1) To compare the concentrations of biomarkers of inflammation, endothelial activation and oxidative stress in subjects with low HDL-c compared to normal HDL-c; (2) To examine the association and correlation between HDL-c and these biomarkers and (3) To determine whether HDL-c is an independent predictor of these biomarkers.
METHODS: 422 subjects (mean age±SD = 43.2±11.9 years) of whom 207 had low HDL-c concentrations (HDL-c <1.0 mmol/L and <1.3 mmol/L for males and females respectively) and 215 normal controls (HDL-c ≥1.0 and ≥1.3 mmol/L for males and females respectively) were recruited in this study. The groups were matched for age, gender, ethnicity, smoking status, diabetes mellitus and hypertension. Fasting blood samples were collected for analysis of biomarkers of inflammation [high-sensitivity C-reactive protein (hsCRP) and Interleukin-6 (IL-6)], endothelial activation [soluble Vascular Cell Adhesion Molecule-1 (sVCAM-1), soluble Intercellular Adhesion Molecule-1 (sICAM-1) and E-selectin)] and oxidative stress [F2-Isoprostanes, oxidized Low Density Lipoprotein (ox-LDL) and Malondialdehyde (MDA)].
RESULTS: Subjects with low HDL-c had greater concentrations of inflammation, endothelial activation and oxidative stress biomarkers compared to controls. There were negative correlations between HDL-c concentration and biomarkers of inflammation (IL-6, p = 0.02), endothelial activation (sVCAM-1 and E-selectin, p = 0.029 and 0.002, respectively), and oxidative stress (MDA and F2-isoprostane, p = 0.036 and <0.0001, respectively). Multiple linear regression analysis showed HDL-c as an independent predictor of IL-6 (p = 0.02) and sVCAM-1 (p<0.03) after correcting for various confounding factors.
CONCLUSION: Low serum HDL-c concentration is strongly correlated with enhanced status of inflammation, endothelial activation and oxidative stress. It is also an independent predictor for enhanced inflammation and endothelial activation, which are pivotal in the pathogenesis of atherosclerosis and atherosclerosis-related complications.
METHODS: BV2 microglial cells c for 24 h, pre-treated with EPA for 24 h prior to LPS induction for another 24 h. Surface expression of CD11b and CD40 on BV2 cells was analyzed by flow cytometry. ELISA was employed to measure the production of pro-inflammatory mediators i.e. nitric oxide (NO) and tumor necrosis factor (TNF)-α. Western blotting technique was used to determine the expression of inducible nitric oxide synthase (iNOS), myeloid differentiation protein 88 (MYD88), nuclear factor kappa B (NF-κB), caspase-1, and mitogen activated protein kinase (MAPK).
RESULTS: Qualitative and quantitative analyses of the EPA using a validated ultra-high pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method indicated the presence of phyllanthin, hypophyllanthin, niranthin, ellagic acid, corilagin, gallic acid, phyltetralin, isolintetralin and geraniin. EPA suppressed the production of NO and TNFα in LPS-activated BV2 microglial cells. Moreover, EPA attenuated the expression of MyD88, NF-κB and MAPK (p-P38, p-JNK and p-ERK1/2). It also inhibited the expression of CD11b and CD40. EPA protected against LPS-induced microglial activation via MyD88 and NF-κB signaling in BV2 microglial cells.
CONCLUSIONS: EPA demonstrated neuroprotective effects against LPS-induced microglial cells activation through the inhibition of TNFα secretion, iNOS protein expression and subsequent NO production, inhibition of NF-κB and MAPKs mediated by adapter protein MyD88 and inhibition of microglial activation markers CD11b and CD40.
AIM OF THE STUDY: Endothelial barrier dysfunction is a pathological hallmark of many diseases and can be caused by lipopolysaccharides (LPS) stimulation. Therefore, this study aims to investigate the possible barrier protective effects of tHGA upon LPS-stimulated inflammatory responses in human umbilical vein endothelial cells (HUVECs).
MATERIALS AND METHODS: HUVECs were pretreated with tHGA prior to LPS stimulation, where inflammatory parameters including permeability, monocyte adhesion and migration, and release of pro-inflammatory mediators were examined. Additionally, the effect of tHGA on F-actin rearrangement and adhesion protein expression of LPS-stimulated HUVECs was evaluated.
RESULTS: It was found that pretreatment with tHGA inhibited monocyte adhesion and transendothelial migration, reduced endothelial hyperpermeability and secretion of prostaglandin E2 (PGE2). Additionally, tHGA inhibited cytoskeletal rearrangement and adhesion protein expression on LPS-stimulated HUVECs.
CONCLUSION: As the regulation of endothelial barrier dysfunction can be one of the therapeutic strategies to improve the outcome of inflammation, tHGA may be able to preserve vascular barrier integrity of endothelial cells following LPS-stimulated dysfunction, thereby endorsing its potential usefulness in vascular inflammatory diseases.
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: We established an in vitro OA culture model by exposing human chondrocytes to proinflammatory cytokine and interleukin (IL)-1β for 36 hours prior to treatment with the herbals: C. quadrangularis, W. somnifera, and the combination of the two herbals. Cell viability, toxicity, and gene expression of OA modifying agents were examined. In addition, expression of survivin, which is crucial for cell growth, was analyzed. In vivo work on osteotomized rats studied the bone and cartilage regenerative effects of C. quadrangularis, W. somnifera, and the combination therapy.
RESULTS: Exposure of chondrocytes to IL-1β induced significant toxicity and cell death. However, herbal treatment alleviated IL-1β induced cell toxicity and upregulated cell growth and proliferation. C. quadrangularis inhibited gene expression of cytokines and matrix metalloproteinases, known to aggravate cartilage and bone destruction, and augmented expression of survivin by inhibiting p38 MAPK. Interestingly, osteotomized rats treated with C. quadrangularis drastically enhanced alkaline phosphatase and cartilage tissue formation as compared to untreated, W. somnifera only, or the combination of both herbals.
CONCLUSION: Our findings demonstrate for the first time the signaling mechanisms regulated by C. quadrangularis and W. somnifera in OA and osteogenesis. We suggest that the chondroprotective effects and regenerative ability of these herbals are via the upregulation of survivin that exerts inhibitory effects on the p38 MAPK signaling pathway. These findings thus validate C. quadrangularis as a potential therapeutic for rheumatic disorders.
PURPOSE: The study was carried out to investigate the molecular mechanisms underlying the anti-inflammatory properties of the standardized 80% ethanol extract of Z. zerumbet through its effect on mitogen-activated protein kinase (MyD88)-dependent nuclear factor-kappa B (NF-кB), mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase/Akt (PI3K-Akt) signaling pathways in lipopolysaccharide (LPS)-induced U937 human macrophages.
METHODS: Standardization of the 80% ethanol extract of Z. zerumbet was performed by using a validated reversed-phase HPLC method, while LC-MS/MS was used to profile the secondary metabolites. The release of pro-inflammatory markers, tumor necrosis factor (TNF)-α, interleukin (IL)-1β and prostaglandin E2 (PGE2) was evaluated by enzyme-linked immunosorbent assay (ELISA), while the Western blot technique was executed to elucidate the expression of mediators linked to MyD88-dependent respective signaling pathways. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay was carried out to quantify the relative gene expression of cyclooxygenase (COX)-2 and pro-inflammatory mediators at the transcriptional level.
RESULTS: The quantitative and qualitative analyses of Z. zerumbet extract showed the presence of several compounds including the major chemical marker zerumbone. Z. zerumbet extract suppressed the release of pro-inflammatory mediators, COX-2 protein expression and downregulated the mRNA expression of pro-inflammatory markers. Z. zerumbet-treatment also blocked NF-κB activation by preventing the phosphorylation of IKKα/β and NF-κB (p65) as well as the phosphorylation and degradation of IκBα. Z. zerumbet extract concentration-dependently inhibited the phosphorylation of respective MAPKs (JNK, ERK, and p38) as well as Akt. Correspondingly, Z. zerumbet extract suppressed the upstream signaling adaptor molecules, TLR4 and MyD88 prerequisite for the NF-κB, MAPKs, and PI3K-Akt activation.
CONCLUSION: The findings suggest that Z. zerumbet has impressive role in suppressing inflammation and related immune disorders by inhibition of various pro-inflammatory markers through the imperative MyD88-dependent NF-κB, MAPKs, and PI3K-Akt activation.