Olive polyphenols attenuate TNF-α-stimulated M-CSF and IL-6 synthesis in osteoblasts: Suppression of Akt and p44/p42 MAP kinase signaling pathways

Background: Olive oil polyphenols, which possess cytoprotective activities like anti-oxidant and anti-inflammatory effects, could modulate osteoblast functions. The aim of this study is to elucidate the effects and the underlying mechanisms of hydroxytyrosol and oleuropein on the tumor necrosis factor-α (TNF-α)-induced macrophage colony-stimulating factor (M-CSF) and interleukin-6 (IL-6) synthesis in osteoblasts. Methods: Osteoblast-like MC3T3-E1 cells were pretreated with hydroxytyrosol, oleuropein, deguelin, PD98059 or wedelolactone, and then stimulated by TNF-α. The levels of M-CSF and IL-6 in the conditioned medium were determined with ELISA. The mRNA expression levels of M-CSF or IL-6 were determined with real-time RT-PCR. The phosphorylation levels of Akt, p44/p42 mitogen-activated protein (MAP) kinase or NF-κB in the cell lysates were determined with Western blot analysis. Results: Hydroxytyrosol and oleuropein attenuated the TNF-α-stimulated M-CSF release. Deguelin, an inhibitor of Akt, significantly suppressed the TNF-α-stimulated M-CSF release, which failed to be affected by the MEK1/2 inhibitor PD98059 or the IκB inhibitor wedelolactone. Hydroxytyrosol and oleuropein suppressed the TNF-α-induced phosphorylation of Akt and p44/p42 MAP kinase. Hydroxytyrosol and oleuropein attenuated the TNF-α-stimulated IL-6 release. Hydroxytyrosol suppressed the TNF-α-induced mRNA expressions of M-CSF and IL-6. Hydroxytyrosol or oleuropein failed to affect the cell viability. Conclusion: Our present findings strongly suggest that olive oil polyphenols hydroxytyrosol and oleuropein down-regulates TNF-α signaling at the points upstream of Akt and p44/p42 MAP kinase in osteoblasts, leading to the attenuation of M-CSF and IL-6 synthesis

Mitochondrial genetic variants associated with bipolar disorder and Schizophrenia in a Japanese population

Abstract Background Bipolar disorder (BD) and schizophrenia (SZ) are complex psychotic disorders (PSY), with both environmental and genetic factors including possible maternal inheritance playing a role. Some studies have investigated whether genetic variants in the mitochondrial chromosome are associated with BD and SZ. However, the genetic variants identified as being associated are not identical among studies, and the participants were limited to individuals of European ancestry. Here, we investigate associations of genome-wide genetic variants in the mitochondrial chromosome with BD, SZ, and PSY in a Japanese population. Methods After performing quality control for individuals and genetic variants, we investigated whether mitochondrial genetic variants [minor allele frequency (MAF) > 0.01, n = 45 variants) are associated with BD, SZ, and PSY in 420 Japanese individuals consisting of patients with BD (n = 51), patients with SZ (n = 172), and healthy controls (HCs, n = 197). Results Of mitochondrial genetic variants, three (rs200478835, rs200044200 and rs28359178 on or near NADH dehydrogenase) and one (rs200478835) were significantly associated with BD and PSY, respectively, even after correcting for multiple comparisons (P GC =0.045–4.9 × 10− 3). In particular, individuals with the minor G-allele of rs200044200, a missense variant, were only observed among patients with BD (MAF = 0.059) but not HCs (MAF = 0) (odds ratio=∞). Three patients commonly had neuropsychiatric family histories. Conclusions We suggest that mitochondrial genetic variants in NADH dehydrogenase-related genes may contribute to the pathogenesis of BD and PSY in the Japanese population through dysfunction of energy production

Tramadol regulates the activation of human platelets via Rac but not Rho/Rho-kinase.

Tramadol is a useful analgesic which acts as a serotonin and noradrenaline reuptake inhibitor in addition to μ-opioid receptor agonist. Cytoplasmic serotonin modulates the small GTPase activity through serotonylation, which is closely related to the human platelet activation. We recently reported that the combination of subthreshold collagen and CXCL12 synergistically activates human platelets. We herein investigated the effect and the mechanism of tramadol on the synergistic effect. Tramadol attenuated the synergistically stimulated platelet aggregation (300 μM of tramadol, 64.3% decrease, p<0.05). Not morphine or reboxetine, but duloxetine, fluvoxamine and sertraline attenuated the synergistic effect of the combination on the platelet aggregation (30 μM of fluvoxamine, 67.3% decrease, p<0.05; 30 μM of sertraline, 67.8% decrease, p<0.05). The geranylgeranyltransferase inhibitor GGTI-286 attenuated the aggregation of synergistically stimulated platelet (50 μM of GGTI-286, 80.8% decrease, p<0.05), in which GTP-binding Rac was increased. The Rac1-GEF interaction inhibitor NSC23766 suppressed the platelet activation and the phosphorylation of p38 MAPK and HSP27 induced by the combination of collagen and CXCL12. Tramadol and fluvoxamine almost completely attenuated the levels of GTP-binding Rac and the phosphorylation of both p38 MAPK and HSP27 stimulated by the combination. Suppression of the platelet aggregation after the duloxetine administration was observed in 2 of 5 patients in pain clinic. These results suggest that tramadol negatively regulates the combination of subthreshold collagen and CXCL12-induced platelet activation via Rac upstream of p38 MAPK

Effect of YM-08 on the PGE₁-stimulated IL-6 release in MC3T3-E1 cells.

The cultured cells were pretreated with 10 μM of YM-08 for 60 min and then stimulated with 10 μM of PGE1 or vehicle for 48 h. IL-6 concentrations of the culture medium were determined by ELISA. Each value represents the mean ± SEM of triplicate determinations from three independent cell preparations. *p †p 1 alone.</p

Effects of SB203580 on YM-08’s amplification of PGE₁-stimulated IL-6 release in MC3T3-E1 cells.

The cultured cells were preincubated with 30 μM of SB203580 or vehicle for 60 min, subsequently pretreated with 10 μM of YM-08 or vehicle for 60 min, and then stimulated with 10 μM of PGE1 or vehicle for 48 h. IL-6 concentrations of the conditioned media were determined by ELISA. Each value represents the mean ± SEM of triplicate determinations from three independent cell preparations. *p †p 1 alone. ‡p 1 with YM-08 pretreatment.</p

Full-length gels and blots of Fig 4.

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Effects of YM-08 on the PGE₁-stimulated phosphorylation of p38 MAPK in MC3T3-E1 cells.

The cultured cells were pretreated with 10 μM of YM-08 for 60 min and then stimulated with 10 μM of PGE1 or vehicle for 20 min. The cell extracts were then subjected to SDS-PAGE and subsequent Western blot analysis with antibodies against phospho-specific p38 MAPK, p38 MAPK, and actin. The histogram quantitatively represents the PGE1-induced levels obtained from laser densitometric analysis of three independent experiments. Each value represents the mean ± SEM of triplicate determinations from three independent cell preparations. *p †p 1 alone.</p

Effects of PD98059, SP600125 or SB203580 on the PGE₁-stimulated release of IL-6 in MC3T3-E1 cells.

Effects of PD98059, SP600125 or SB203580 on the PGE1-stimulated release of IL-6 in MC3T3-E1 cells.</p