u-0126 and prolinedithiocarbamate

A previous study has revealed that oxidized low‑density lipoprotein (oxLDL)/β2‑glycoprotein I (β2GPI)/anti‑β2‑glycoprotein I (anti‑β2GPI), an immune complex, is able to activate the Toll‑like receptor 4 (TLR4)/nuclear factor κβ (NF‑κβ) inflammatory signaling pathway in macrophages, and consequently enhance foam cell formation and the secretion of prothrombin activators. However, the effects of the oxLDL/β2GPI/anti‑β2GPI complex on vascular smooth muscle cells have yet to be investigated. The present study investigated whether the oxLDL/β2GPI/anti‑β2GPI complex was able to reinforce the pro‑atherogenic activities of a rat thoracic aorta smooth muscle cell line (A7r5) and examined the underlying molecular mechanisms. The results revealed that the oxLDL/β2GPI/anti‑β2GPI complex treatment significantly (P<0.05 vs. the media, oxLDL, oxLDL/β2GPI and β2GPI/anti‑β2GPI groups) enhanced the pro‑atherogenic activation of A7r5 cells, including intracellular lipid loading, Acyl‑coenzyme A cholesterol acyltransferase mRNA expression, migration, matrix metalloproteinase‑9 and monocyte chemoattractant protein 1 secretion, all via TLR4. In addition, the expression of TLR4 and the phosphorylation of NF‑κβ p65, p38 and ERK1/2 were also upregulated in oxLDL/β2GPI/anti‑β2GPI complex‑treated A7r5 cells. Pre‑treatment with TAK‑242, a TLR4 inhibitor, was able to partly attenuate the oxLDL/β2GPI/anti‑β2GPI complex‑induced phosphorylation of NF‑κβ p65; however, it had no effect on the phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) and p38. Meanwhile, the NF‑κβ p65 inhibitor ammonium pyrrolidinedithiocarbamate and the ERK1/2 inhibitor U0126, but not the p38 inhibitor SB203580, were able to block oxLDL/β2GPI/anti‑β2GPI complex‑induced foam cell formation and migration in A7r5 cells. Hence, it was demonstrated that the oxLDL/β2GPI/anti‑β2GPI complex is able to enhance the lipid uptake, migration and active molecule secretion of A7r5 cells via TLR4, and finally deteriorate atherosclerosis plaques. Additionally, it was demonstrated that oxLDL/β2GPI/anti‑β2GPI complex‑induced foam cell formation and migration may be partly mediated by the TLR4/NF‑κβ signaling pathway and that ERK1/2 may also participate in the process.

Topics: Animals; beta 2-Glycoprotein I; Blotting, Western; Butadienes; Cell Line; Cell Movement; Cholesterol; Enzyme-Linked Immunosorbent Assay; Lipoproteins, LDL; NF-kappa B; Nitriles; Phosphorylation; Proline; Rats; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Thiocarbamates; Toll-Like Receptor 4; Wound Healing

Evidence suggests that upregulation of soluble epoxide hydrolase (sEH) is associated with the development of myocardial infarction, dilated cardiomyopathy, cardiac hypertrophy, and heart failure. However, the upregulation mechanism is still unknown. In this study, we treated H9C2 cells with buthionine sulfoximine (BSO) to explore whether oxidative stress upregulates sEH gene expression and to identify the molecular and cellular mechanisms behind this upregulatory response. Real-time PCR and Western blot analyses were used to measure mRNA and protein expression, respectively. We demonstrated that BSO significantly upregulated sEH at mRNA levels in a concentration- and time-dependent manner, leading to a significant increase in the cellular hypertrophic markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Furthermore, BSO significantly increased the cytosolic phosphorylated IκB-α and translocation of NF-κB p50 subunits, as measured by Western blot analysis. This level of translocation was paralleled by an increase in the DNA-binding activity of NF-κB P50 subunits. Moreover, our results demonstrated that pretreatment with the NF-κB inhibitor PDTC significantly inhibited BSO-mediated induction of sEH and cellular hypertrophic marker gene expression in a dose-dependent manner. Additionally, mitogen-activated protein kinases (MAPKs) were transiently phosphorylated by BSO treatment. To understand further the role of MAPKs pathway in BSO-mediated induction of sEH mRNA, we examined the role of extracellular signal-regulated kinase (ERK), c-JunN-terminal kinase (JNK), and p38 MAPK. Indeed, treatment with the MEK/ERK signal transduction inhibitor, PD98059, partially blocked the activation of IκB-α and translocation of NF-κB p50 subunits induced by BSO. Moreover, pretreatment with MEK/ERK signal transduction inhibitors, PD98059 and U0126, significantly inhibited BSO-mediated induction of sEH and cellular hypertrophic marker gene expression. These results clearly demonstrated that the NF-κB signaling pathway is involved in BSO-mediated induction of sEH gene expression, and appears to be associated with the activation of the MAPK pathway. Furthermore, our findings provide a strong link between sEH-induced cardiac dysfunction and involvement of NF-κB in the development of cellular hypertrophy.

Topics: Animals; Antioxidants; Atrial Natriuretic Factor; Butadienes; Buthionine Sulfoximine; Cardiomegaly; Cell Line; Cell Survival; Enzyme Activation; Epoxide Hydrolases; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Gene Expression Regulation; Glutathione; Heart Failure; I-kappa B Proteins; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Myoblasts, Cardiac; Natriuretic Peptide, Brain; NF-kappa B p50 Subunit; NF-KappaB Inhibitor alpha; Nitriles; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Proline; Rats; RNA, Messenger; Thiocarbamates; Transcription Factor RelA; Up-Regulation

TGF-β1 plays an important role on podocyte injury and glomerular diseases, while the underlying molecular mechanisms are still elusive. Here, the potential role of the ion channel TRPC6 and the proximal signaling was explored in TGF-β1-treated mouse podocyte. Our results showed that TGF-β1 significantly increased podocyte apoptosis and induced obvious disorganization of actin filaments in a time-dependent pattern. In TGF-β1-treated podocyte, TRPC6 protein, especially the phosphorylated TRPC6, and the cytosolic free Ca(2+) level upregulated, which was evidently inhibited by the specific knockdown of TRPC6. TRPC6 knockdown also alleviated TGF-β1-induced podocyte apoptosis. Moreover, the Src kinase Fyn increased obviously in TGF-β1-treated podocyte, displaying increment of the active form pY418 and reduction of the inactive form pY530. Immunoprecipitation assay revealed that Fyn interacts with TRPC6 in podocyte. Notably, Fyn knockdown blocked TRPC6 phosphorylation and intracellular Ca(2+) increment following TGF-β1 stimulation, but not affect the expression of TRPC6 protein. In addition, Western blot showed that TGF-β1 induced significant activation of p-Smad3, p-ERK and RelA/p65. Importantly, obvious translocation of ERK and RelA/p65 to nuclei was observed in TGF-β1-treated podocyte, which was reduced by ERK inhibitor U0126. Both U0126 and NF-κB inhibitor PDTC obviously inhibited the increment of TRPC6 protein and the flux of cytosolic free Ca(2+) induced by TGF-β1. Together, we provide evidences that TGF-β1 induces podocyte damage by upregulating TRPC6 protein most possibly through Smad3-ERK-NF-κB pathway, in which Fyn-dependent tyrosine phosphorylation of TRPC6 might exert a crucial role on the activation of its channel function.

Topics: Animals; Apoptosis; Butadienes; Calcium; Extracellular Signal-Regulated MAP Kinases; MAP Kinase Signaling System; Mice; Nitriles; Phosphorylation; Podocytes; Proline; Proto-Oncogene Proteins c-fyn; RNA Interference; RNA, Small Interfering; Smad3 Protein; Thiocarbamates; Transcription Factor RelA; Transforming Growth Factor beta1; TRPC Cation Channels; TRPC6 Cation Channel

Asiatic acid is a triterpenoid component possessing antioxidative, anti-inflammatory and hepatoprotective activity. In this issue, we explored the protective effects of asiatic acid and the relative mechanism in the D-galactosamine/lipopolysaccharide (D-GalN/LPS)-induced hepatotoxicity in hepatocytes and kupffer cells co-cultured system. The cultures were pretreated with asiatic acid for 12 h, followed by D-GalN/LPS exposure for 12 h. Asiatic acid reduced aspartate aminotransferase and lactate dehydrogenase generation and increased cell viability in a concentration-dependent manner. Meanwhile, the effects of asiatic acid in leukotriene C(4) synthase (LTC(4)S) expression and cellular redox status including reactive oxygen species and GSH content were detected. The results showed that D-GalN/LPS induced the increase of reactive oxygen species followed by extracellular signal-regulated kinase 1/2 (ERK 1/2) and nuclear factor-kappaB (NF-kappaB) activation. Treatment with ERK 1/2 specific inhibitor 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U0126) abolished the ERK1/2 protein phosphorylation and blunted LTC(4)S expression. Reactive oxygen species signaling pathway inhibitor pyrrolidine dithiocarbamate (PDTC) inhibited reactive oxygen species generation and NF-kappaB activation, which in turn blocked LTC(4)S expression and attenuated the injury. Asiatic acid can protect the hepatocytes against D-GalN/LPS-induced hepatotoxicity. During which, the cell redox was ameliorated and increased expression of LTC(4)S was reversed by the pretreatment of asiatic acid. Taken together, asiatic acid can protect against D-GalN/LPS-induced hepatotoxicity partly via redox-regulated LTC(4)S expression pathway.

Topics: Animals; Butadienes; Coculture Techniques; Enzyme Activation; Galactosamine; Gene Expression Regulation, Enzymologic; Glutathione Transferase; Hepatocytes; Kupffer Cells; Leukotriene C4; Lipopolysaccharides; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-kappa B; Nitriles; Oxidation-Reduction; Pentacyclic Triterpenes; Proline; Rats; Reactive Oxygen Species; Signal Transduction; Thiocarbamates; Time Factors; Triterpenes

We investigated the regulation of PG production in human endometrial stromal cells (ESC) by IL-1beta. We found that cyclooxygenase-2 (COX-2) mRNA and protein levels and PGE(2) production in ESC were significantly increased by IL-1beta. COX-2 mRNA, protein, and PGE(2) levels in IL-1beta-treated ESC were decreased by a PKA inhibitor, a nuclear factor (NF-kappaB) inhibitor, and an ERK1/2 inhibitor, but not by a p38 MAPK inhibitor or a PKC inhibitor, suggesting the possible involvement of PKA, NF-kappaB, and/or the ERK1/2 signaling pathway(s) in IL-1beta-mediated COX-2 gene induction in ESC. We then transiently transfected deletion mutants of the COX-2 promoter fused to the luciferase reporter gene and variants of -360/+56 bp promoter construct carrying different site-directed mutations of selected cis-acting elements. We determined that a NF-kappaB site (-222/-213 bp), a nuclear factor for IL-6 expression site (NF-IL6, -132/-124 bp), and a cAMP response element (-59/-52 bp) were essential for the baseline COX-2 gene promoter regulation. The addition of IL-1beta, however, did not affect the activity of these COX-2 promoter constructs. To investigate the potential effects of IL-1beta on COX-2 mRNA stability, ESC were treated with actinomycin D, a general transcription inhibitor, in the absence or presence of IL-1beta. We found that 1) IL-1beta significantly increased COX-2 mRNA stability; 2) continuous transcription was not required to sustain the IL-1beta-induced COX-2 mRNA levels; and 3) COX-2 mRNA was highly unstable in the absence of IL-1beta. Additionally, we found that the ERK1/2 signaling pathway was essential for stabilizing COX-2 mRNA. We conclude that levels of COX-2 mRNA, protein, and enzyme activity in ESC are controlled by various signaling pathways, including PKA, ERK1/2, and NF-kappaB. Moreover, posttranscriptional mRNA stability is an important mechanism for IL-1beta-induced elevation of COX-2 expression in ESC.

Topics: Butadienes; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Cyclooxygenase 2; Dinoprostone; Endometrium; Enzyme Inhibitors; Extracellular Space; Female; Humans; Interleukin-1; Isoenzymes; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; NF-kappa B; Nitriles; Proline; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases; Reference Values; RNA Stability; RNA, Messenger; Signal Transduction; Stromal Cells; Thiocarbamates