Cryptotanshinone (CTS), a major constituent extracted from the medicinal herb Salvia miltiorrhiza Bunge, has well-documented antioxidative and anti-inflammatory effects. In the present study, the pharmacological effects and underlying molecular mechanisms of CTS on lipopolysaccharide (LPS)-induced inflammatory responses were investigated. By enzyme-linked immunosorbent assay, we observed that CTS reduced significantly the production of proinflammatory mediators (tumor necrosis factor-α and interleukin-6) induced by LPS in murine macrophage-like RAW264.7 cells. Mechanistically, CTS inhibited markedly the phosphorylation of mitogen-activated protein kinases (MAPKs), including ERK1/2, p38MAPK, and JNK, which are crucially involved in regulation of proinflammatory mediator secretion. Moreover, immunofluorescence and western blot analysis indicated that CTS abolished completely LPS-triggered nuclear factor-κB (NF-κB) activation. Taken together, these data implied that NF-κB and MAPKs might be the potential molecular targets for clarifying the protective effects of CTS on LPS-induced inflammatory cytokine production in macrophages.

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We have performed a detailed investigation of the effects on platelet function of coenzyme A (CoA) and several acyl-CoAs. Platelet aggregation was measured by turbidimetry and by platelet counting; platelet shape change was measured using light scattering; P-selectin, Ca2+ mobilization and vasodilator-stimulated phosphoprotein (VASP) phosphorylation were measured by flow cytometry. The compounds investigated inhibited ADP-induced platelet aggregation; those with saturated acyl groups containing 16-18 carbons were most effective. The effects of palmitoyl-CoA (16:0) were studied in depth. It inhibited platelet shape change and Ca2+ mobilization brought about by ADP (but not other agonists) indicating antagonism at P2Y(1) receptors, and also inhibited ADP-induced P-selectin expression. Effects of palmitoyl-CoA on the platelet aggregation and Ca2+ mobilization induced by several different agonists and agonist combinations were compared with those of MRS 2179 (a P2Y(1) antagonist) and AR-C69931 (a P2Y(12) antagonist), and were consistent with palmitoyl-CoA acting mainly at P2Y(1) but also with partial antagonism at P2Y(12) receptors. Antagonism at P2Y(12) receptors was confirmed in studies of VASP-phosphorylation. Palmitoyl-CoA did not act as an antagonist at P2X(1) receptors. The results are discussed in relation to the possibility that acyl-CoAs may contribute as endogenous modulators of platelet function and might serve as lead compounds for the design of novel antithrombotics.

BackgroundThe resistance to EGF receptor (EGFR) tyrosine kinase inhibitors (TKI) is a major challenge in the treatment of non-small cell lung cancer (NSCLC). Understanding the molecular mechanisms behind resistance is therefore an important issue. Here we assessed the role of EGFR pathway substrate 8 (EPS8) and Forkhead box O 3a (FoxO3a) as potentially valuable targets in the resistance of NSCLC .MethodsThe expression levels of EPS8 and FoxO3a in patients with NSCLC (n = 75) were examined by immunohistochemistry staining, while in cells were detected by qPCR and western blot. The effects of EPS8 and FoxO3a on resistance, migration and invasion, cell cycle arrest were detected by MTT, transwell and flow cytometry, respectively. Chromatin immunoprecipitation and luciferase reporter assays were performed to determine the mechanisms of EPS8 expression and FoxO3a regulation.FindingsWe observed that the expression of EPS8 inversely correlated with FoxO3a in NSCLC cell lines and NSCLC patients. FoxO3a levels were significantly decreased in tumor tissues compared with para-carcinoma tissues, while EPS8 is opposite. Besides, they play reverse roles in the resistance to gefitinib, the migration and invasion abilities, the cell cycle arrest in vitro and the tumor growth in vivo. Mechanistically, FoxO3a inhibits EPS8 levels by directly binding its gene promoter and they form a negative loop in EGFR pathway.InterpretationTargeting FoxO3a and EPS8 in EGFR signaling pathway prevents the progression of NSCLC, which implied that the negative loop they formed could served as a therapeutic target for overcoming resistance in NSCLC.FundsNational Natural Science Foundation of China, Science and Technology Project of Henan, Outstanding Young Talent Research Fund of Zhengzhou University and the National Scholarship Fund.