LTB₄ induces ICAM-1 and MCP-1/CCL2 expression and release in rat pulmonary arterial endothelial cells.

<p>RPAECs were exposed to different concentrations of LTB₄ (30–300 ng/mL) for 24 h. ICAM-1 and MCP-1/CCL2 mRNA and protein levels were respectively measured by qRT-PCR (A) and ELISA (B). The data represent means ± S.E.M. from six independent experiments. <i>* P</i> <0.05, <i>** P</i> <0.01, <i>*** P</i> <0.001 compared with the control group.</p

EETs Attenuate Ox-LDL-Induced LTB₄ Production and Activity by Inhibiting p38 MAPK Phosphorylation and 5-LO/BLT1 Receptor Expression in Rat Pulmonary Arterial Endothelial Cells

<div><p>Cytochrome P-450 epoxygenase (EPOX)-derived epoxyeicosatrienoic acids (EETs), 5-lipoxygenase (5-LO), and leukotriene B₄ (LTB₄), the product of 5-LO, all play a pivotal role in the vascular inflammatory process. We have previously shown that EETs can alleviate oxidized low-density lipoprotein (ox-LDL)-induced endothelial inflammation in primary rat pulmonary artery endothelial cells (RPAECs). Here, we investigated whether ox-LDL can promote LTB₄ production through the 5-LO pathway. We further explored how exogenous EETs influence ox-LDL-induced LTB₄ production and activity. We found that treatment with ox-LDL increased the production of LTB₄ and further led to the expression and release of both monocyte chemoattractant protein-1 (MCP-1/CCL2) and intercellular adhesion molecule-1 (ICAM-1). All of the above ox-LDL-induced changes were attenuated by the presence of 11,12-EET and 14,15-EET, as these molecules inhibited the 5-LO pathway. Furthermore, the LTB₄ receptor 1 (BLT1 receptor) antagonist U75302 attenuated ox-LDL-induced ICAM-1 and MCP-1/CCL2 expression and production, whereas LY255283, a LTB₄ receptor 2 (BLT2 receptor) antagonist, produced no such effects. Moreover, in RPAECs, we demonstrated that the increased expression of 5-LO and BLT1 following ox-LDL treatment resulted from the activation of nuclear factor-κB (NF-κB) via the p38 mitogen-activated protein kinase (MAPK) pathway. Our results indicated that EETs suppress ox-LDL-induced LTB₄ production and subsequent inflammatory responses by downregulating the 5-LO/BLT1 receptor pathway, in which p38 MAPK phosphorylation activates NF-κB. These results suggest that the metabolism of arachidonic acid via the 5-LO and EPOX pathways may present a mutual constraint on the physiological regulation of vascular endothelial cells.</p></div

EETs inhibit ox-LDL-induced expression and production of ICAM-1 and MCP-1/CCL2 by down-regulating the 5-LO pathway in RPAECs.

<p>(A) RPAECs were incubated with the indicated concentrations of 11,12-EET or 14,15-EET (1 μM) for 30 min and then stimulated with ox-LDL (100 μg/mL) for 24 h. LTB₄ protein levels in cell culture supernatant were quantified by ELISA. (B, D) 5-LO and BLT1 mRNA and protein levels were respectively determined by qRT-PCR and western blot. (C, E) ICAM-1 and MCP-1/CCL2 mRNA expression and protein levels in cell culture supernatant were quantified by qRT-PCR and ELISA. The data represent means ± S.E.M. from six independent experiments. <i>*P</i><0.05, <i>**P</i><0.01, <i>***P</i><0.001 compared with the control group. ^<i>#</i><i>P</i><0.05 compared with the ox-LDL (100 μg/mL)-treated group.</p

The p38 MAPK and NF-κB signaling pathways are involved in ox-LDL-mediated expression of 5-LO and BLT1.

<p>RPAECs were pre-treated with a p38 inhibitor (10 μM SB203580) or an NF-κB inhibitor (20 μM PDTC) for 30 min and then incubated with ox-LDL (100 μg/mL) for another 24 h. The levels of 5-LO and BLT1 mRNAs and proteins were determined by qRT-PCR (A, C) and western blot (B, D), respectively. The data represent the means ± S.E.M. from four independent experiments. <i>* P</i> <0.05 and <i>** P</i> <0.01 compared with the untreated group. ^<i>#</i><i>P</i> <0.05 and ^<i>##</i><i>P</i> <0.01 compared with the ox-LDL (100 μg/mL)-stimulated group.</p

Role of LTB₄ receptor antagonists on ICAM-1 and MCP-1/CCL2 expression in RPAECs.

<p>Cells were pretreated with a BLT1 receptor antagonist (U75302) (10 μM) and a BLT2 receptor antagonist (LY255283) (10 μM) and then stimulated with ox-LDL (100 μg/mL) for 24 h. Expression levels of MCP-1/CCL2 (A) and ICAM-1 (B) mRNAs and proteins were determined by qRT-PCR and ELISA, respectively. The data represent means ± S.E.M. from five independent experiments. <i>* P</i> <0.05, <i>** P</i> <0.01, <i>*** P</i> <0.001 compared with the control group. ^<i>#</i><i>P</i> <0.05 and ^<i>##</i><i>P</i> <0.01 compared with the ox-LDL (100 μg/mL)-stimulated group.</p

Knockdown of REGγ inhibits the proliferation and migration and promotes the apoptosis of multiple myeloma cells by downregulating NF-κB signal pathway

<p><b>Objectives:</b> This study aimed to evaluate the effects of REGγ knockdown on the proliferation, apoptosis and migration of multiple myeloma (MM) cells, and reveal the potential regulatory mechanisms.</p> <p><b>Methods:</b> The expression of REGγ on myeloma cells of 28 MM patients was detected by Western blot. shRNA-REGγ-1 and shRNA-REGγ-2 were constructed to downregulate REGγ in RPMI-8226 cells. The proliferation, apoptosis and migration of transfected cells were analyzed by Cell Counting Kit 8 (CCK8), flow cytometry and transwell chamber, respectively. The expression of phosphorylated p65 (p-p65), p65, NF-kappa-B inhibitor ε (IkBε), matrix metalloproteinase 2 (MMP2), B-cell lymphoma xL (Bcl-xL) and X-linked inhibitor of apoptosis protein (XIAP) in transfected cells was detected by Western blot. Using cycloheximide (CHX), the half-life period of IkBε was detected by Western blot.</p> <p><b>Results:</b> The expression of REGγ was positive in myeloma cells. The proliferation and migration of RPMI-8226 cells were significantly inhibited by shRNA-REGγ-1/shRNA-REGγ-2, while the apoptosis rates were significantly increased (<i>p</i> < 0.05). The expression of p-p65 and IkBε was significantly reduced in RPMI-8226 cells transfected with shRNA-REGγ-1/shRNA-REGγ-2. The degradation of IkBε was significantly lower in RPMI-8226 cells transfected with shRNA-REGγ-1 than the control (longer half-life period). Besides, the expression of MMP2, Bcl-xL and XIAP in RPMI-8226 cells was significantly inhibited by shRNA-REGγ-1/shRNA-REGγ-2.</p> <p><b>Discussion:</b> Knockdown of REGγ may inhibit the proliferation and migration, and promote the apoptosis of RPMI-8226 cells possibly by downregulating NF-κB signal pathway.</p