PURPOSE: Increased lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and Rho kinase activity may be associated with atherosclerosis. The principal aim of this study was to examine whether darapladib (a selective Lp-PLA2 inhibitor) could reduce the elevated Lp-PLA2 and Rho kinase activity in atherosclerosis. MATERIALS AND METHODS: Studies were performed in male Sprague-Dawley rats. The atherosclerosis rats were prepared by feeding them with a high-cholesterol diet for 10 weeks. Low-dose darapladib (25 mg.kg-1.d-1) and high-dose darapladib (50 mg.kg-1.d-1) interventions were then administered over the course of 2 weeks. RESULTS: The serum levels of triglycerides, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), high-sensitivity C-reactive protein (hs-CRP), and Lp-PLA2, significantly increased in atherosclerosis model groups, as did Rho kinase activity and cardiomyocyte apoptosis (p<0.05 vs. sham group), whereas nitric oxide (NO) production was reduced. Levels of TC, LDL-C, CRP, Lp-PLA2, and Rho kinase activity were respectively reduced in darapladib groups, whereas NO production was enhanced. When compared to the low-dose darapladib group, the reduction of the levels of TC, LDL-C, CRP, and Lp-PLA2 was more prominent in the high-dose darapladib group (p<0.05), and the increase of NO production was more prominent (p<0.05). Cardiomyocyte apoptosis of the high-dose darapladib group was also significantly reduced compared to the low-dose darapladib group (p<0.05). However, there was no significant difference in Rho kinase activity between the low-dose darapladib group and the high-dose darapladib group (p>0.05). CONCLUSION: Darapladib, a Lp-PLA2 inhibitor, leads to cardiovascular protection that might be mediated by its inhibition of both Rho kinase and Lp-PLA2 in atherosclerosis.
1-Alkyl-2-acetylglycerophosphocholine Esterase/*antagonists & inhibitors/blood/drug effects ; Animals ; Atherosclerosis/blood/*drug therapy/*enzymology ; *Benzaldehydes ; C-Reactive Protein/metabolism ; Cholesterol/blood ; Cholesterol, HDL/blood ; Cholesterol, LDL/blood ; Dose-Response Relationship, Drug ; Male ; *Oximes ; Phospholipase A2 Inhibitors/*administration & dosage/adverse effects ; Rats ; Rats, Sprague-Dawley ; Triglycerides/blood ; rho-Associated Kinases/*metabolism

OBJECTIVEThe purpose of the present study was to observe the changes in CD4+CD25+Nrp1+Treg cells after irradiation with different doses and explore the possible molecular mechanisms involved.

METHODSICR mice and mouse lymphoma cell line (EL-4 cells) was used. The expressions of CD4, CD25, Nrp1, calcineurin and PKC-α were detected by flow cytometry. The expressions of TGF-β1, IL-10, PKA and cAMP were estimated with ELISA.

RESULTSAt 12 h after irradiation, the expression of Nrp1 increased significantly in 4.0 Gy group, compared with sham-irradiation group (P<0.05) in the spleen and thymus, respectively, when ICR mice received whole-body irradiation (WBI). Meanwhile the synthesis of Interleukin 10 (IL-10) and transforming growth factor-β1 (TGF-β1) increased significantly after high dose irradiation (HDR) (> or = 1.0 Gy). In addition, the expression of cAMP and PKA protein increased, while PKC-α, calcineurin decreased at 12h in thymus cells after 4.0 Gy X-irradiation. While TGF-β1 was clearly inhibited when the PLC-PIP2 signal pathway was stimulated or the cAMP-PKA signal pathway was blocked after 4.0 Gy X-irradiation, this did not limit the up-regulation of CD4+CD25+Nrp1+Treg cells after ionizing radiation.

CONCLUSIONThese results indicated that HDR might induce CD4+CD25+Nrp1+Treg cells production and stimulate TGF-β1 secretion by regulating signal molecules in mice.

Animals ; Calcineurin ; genetics ; metabolism ; Cyclic AMP ; metabolism ; Dose-Response Relationship, Radiation ; Female ; Gene Expression Regulation ; radiation effects ; Immunosuppression ; Interleukin-10 ; genetics ; metabolism ; Lymphocyte Subsets ; physiology ; Male ; Mice ; Neuropilin-1 ; genetics ; metabolism ; Phosphoinositide Phospholipase C ; genetics ; metabolism ; Protein Kinases ; genetics ; metabolism ; Signal Transduction ; Transforming Growth Factor beta ; genetics ; metabolism ; Whole-Body Irradiation ; adverse effects

OBJECTIVETo study the expression of phospholipase C epsilon-1(PLCE1) and its clinical significance in gastric cancer.

METHODSSurgical specimens were collected from 125 patients who underwent radical gastrectomy between 2005 and 2007 in the Zhejiang Provincial Peoples' Hospital. Expression level of PLCE1 protein was measured by immunohistochemistry in these 125 surgical specimens, which included primary gastric cancer and matched adjacent normal gastric mucosa tissues, and then 41 pairs of above specimens were selected randomly to examine the expression level of PLCE1 mRNA by quantitative reverse transcription PCR(qRT-PCR).

RESULTSImmunohistochemistry and qRT-PCR showed that both protein and mRNA level of PLCE1 were up-regulated in gastric cancer compared with paired normal gastric mucosa. Univariate analysis demonstrated that the expression of PLCE1 was significantly associated with differentiation degree, invasion depth, lymph node metastasis, distant metastasis, and TNM stage (all P<0.01). The 5-year survival rate of positive PLCE1 group was significantly lower as compared to negative group (31.2% vs. 54.0%, P<0.01). However, the expression of PLCE1 was not an independent prognostic factor for gastric cancer (P>0.05).

CONCLUSIONSPLCE1 is up-regulated in gastric cancer, which is associated with the malignant biological behaviors of gastric cancer. High expression of PLCE1 suggests poor prognosis.

Biomarkers, Tumor ; analysis ; Gastrectomy ; Humans ; Immunohistochemistry ; Lymphatic Metastasis ; Neoplasm Staging ; Phosphoinositide Phospholipase C ; metabolism ; Prognosis ; RNA, Messenger ; Reverse Transcriptase Polymerase Chain Reaction ; Stomach Neoplasms ; enzymology ; pathology ; Survival Rate ; Up-Regulation

In women with preeclampsia (PE), endothelial cell (EC) dysfunction can lead to altered secretion of paracrine factors that induce peripheral vasoconstriction and proteinuria. This study examined the hypothesis that PE sera may directly or indirectly, through human umbilical vein ECs (HUVECs), stimulate phospholipase C-gamma1-1,4,5-trisphosphate (PLC-gamma1-IP3) signaling, thereby increasing protein kinase C-alpha (PKC-alpha) activity, collagen I expression and intracellular Ca2+ concentrations ([Ca2+]i) in human umbilical artery smooth muscle cells (HUASMCs). HUASMCs and HUVECs were cocultured with normal or PE sera before PLC-gamma1 silencing. Increased PLC-gamma1 and IP3 receptor (IP3R) phosphorylation was observed in cocultured HUASMCs stimulated with PE sera (P<0.05). In addition, PE serum significantly increased HUASMC viability and reduced their apoptosis (P<0.05); these effects were abrogated with PLC-gamma1 silencing. Compared with normal sera, PE sera increased [Ca2+]i in cocultured HUASMCs (P<0.05), which was inhibited by PLC-gamma1 and IP3R silencing. Finally, PE sera-induced PKC-alpha activity and collagen I expression was inhibited by PLC-gamma1 small interfering RNA (siRNA) (P<0.05). These results suggest that vasoactive substances in the PE serum may induce deposition in the extracellular matrix through the activation of PLC-gamma1, which may in turn result in thickening and hardening of the placental vascular wall, placental blood supply shortage, fetal hypoxia-ischemia and intrauterine growth retardation or intrauterine fetal death. PE sera increased [Ca2+]i and induced PKC-alpha activation and collagen I expression in cocultured HUASMCs via the PLC-gamma1 pathway.
Adult ; Apoptosis ; Calcium/*metabolism ; Cell Line ; Cell Survival ; Cells, Cultured ; Coculture Techniques ; Collagen Type I/analysis/*metabolism ; Female ; Human Umbilical Vein Endothelial Cells ; Humans ; Muscle, Smooth, Vascular/*cytology/metabolism ; Phospholipase C gamma/genetics/*metabolism ; Pre-Eclampsia/*blood/*metabolism/pathology ; Pregnancy ; Protein Kinase C-alpha/metabolism ; RNA Interference ; *Signal Transduction ; Young Adult

OBJECTIVE: To clarify the effect of glycosylphosphatidylinositol-specific phospholipase D (GPIPLD) on hepatoma cells HepG2 and the possible molecular mechanism. METHODS: MTT, fluorescent staining and Western blot were applied to analyze the effect and molecular mechanism of GPI-PLD on hepatoma cells by transfected high expression GPI-PLD model. We inoculated HepG2 in nude mice models to further clarify the effect of GPI-PLD on hepatoma cells in vivo. RESULTS: Compared with the control groups, PI3K-Akt signaling pathway activity and proliferation of hepatoma cells were significantly inhibited in the GPI-PLD group. Nude mice models showed that the tumor growth and tumor weight [(1.87 ± 0.09) g] of the GPI-PLD group were significantly less than those of the blank control group [(2.20 ± 0.17) g] and the negative control group [(2.15 ± 0.09) g]. AST, ALT and AFP serum concentration in the GPI-PLD group were significantly lower than those of the control groups (P<0.05). CONCLUSION GPI-PLD can inhibit the proliferation of hepatoma cells and growth in vivo, and promote the apoptosis of hepatoma cells by reducing the activity of PI3K-Akt signaling pathway.
Animals ; Apoptosis ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line ; Down-Regulation ; Hep G2 Cells ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Mice ; Mice, Nude ; Phosphatidylinositol 3-Kinases ; Phospholipase D ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; Transfection

Phospholipase C epsilon-1 (PLCE1) is a phospholipase C isoenzyme encoded by PLCE1 gene, and has more complicated molecular structure and function than other subtypes. Phospholipase C epsilon-1 is accepted the dual regulation by the upstream G proteins and GTP enzymes of Ras family. The downstream signal of PLCE1 is not only cause the Ca2+ flow and protein kinase C(PKC) activation, but also can be used as the GTP enzyme guanylic acid conversion factor of Ras superfamily, so as to regulate the expression of certain genes, adjusting cell growth and differentiation processes. PLCE1 plays a very important role in the signal transduction in the regulation of cell growth, differentiation, proliferation and apoptosis. Previous studies showed that phospholipase C epsilon-1 played an important role in the development of malignant tumors (especially the digestive tumors), heart disease, nephrotic syndrome and other diseases, but there are some questions about the mechanisms of PLCE1 involved in allergic rhinitis, this article will make an overview about PLCE1 promotes allergic rhinitis CD4+ T cells differentiate to Th2 cells by PKC-NF-κB pathway and Ras-MAPK pathway.
Apoptosis ; Calcium ; metabolism ; Cell Cycle ; Cell Differentiation ; physiology ; Cell Proliferation ; physiology ; Enzyme Activation ; Gene Expression ; Humans ; NF-kappa B ; Phosphoinositide Phospholipase C ; genetics ; physiology ; Protein Kinase C ; metabolism ; Rhinitis, Allergic ; enzymology ; Signal Transduction ; Th2 Cells ; cytology

OBJECTIVE: To explore the expression of phospholipase Cgamma-2 (PLCgamma-2), lipoxygenase-12 (12-LOX) and arachidonic acid (AA) in laryngeal squamous cell carcinoma and to study the the relationship between lipid metabolism and laryngeal squamous cell carcinoma. METHOD: In 30 cases of carcinoma tissue and peritumoral laryngeal mucosa tissues (confirmed to be normal laryngeal tissues by pathology), immunohistochemical method (Streptavidin-peroxidase method, SP method) was used for the detection of expression of PLCgamma-2 and 12-LOX, and gas chromatography/mass spectrometry (GC/MS) for the content of the arachidonic acid in carcinoma tissue and peritumoral normal laryngeal mucosa tissues. RESULT: The positive rates of PLCgamma-2 and 12-LOX in carcinoma tissue were higher than in peritumoral normal laryngeal mucosa tissues with statistically significance differences (P < 0.05). The content of arachidonic acid was lower in carcinoma tissue than in peritumoral normal laryngeal mucosa tissues with statistically significance difference (P < 0.05). The positive expressions of PLCgamma-2 and 12-LOX were closely correlated to tnm stage, histological differentiation and lymph node metastasis (P < 0.05). The content of arachidonic acid had no significant correlations with tnm stage, histological differentiation and lymph node metastasis (P > 0.05). Both the expression of PLCgamma-2 and 12-LOX and the content of arachidonic acid had no statistically significant correlation with age (P > 0.05). CONCLUSION PLCgamma-2, AA and 12-LOX play important roles in laryngeal squamous cell carcinoma. It may be meaningful to the treatment of laryngeal carcinoma by suppressing this passway.
Aged ; Arachidonate 12-Lipoxygenase ; metabolism ; Arachidonic Acid ; metabolism ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Head and Neck Neoplasms ; metabolism ; pathology ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Male ; Middle Aged ; Neoplasm Staging ; Phospholipase C gamma ; metabolism ; Squamous Cell Carcinoma of Head and Neck

Animals ; Carcinoma, Squamous Cell ; genetics ; Colorectal Neoplasms ; genetics ; metabolism ; Enzyme Activation ; Esophageal Neoplasms ; genetics ; Genome-Wide Association Study ; Head and Neck Neoplasms ; genetics ; Humans ; Neoplasms ; chemically induced ; enzymology ; genetics ; Phosphoinositide Phospholipase C ; chemistry ; genetics ; metabolism ; physiology ; Signal Transduction ; Skin Neoplasms ; chemically induced ; enzymology ; Stomach Neoplasms ; genetics ; Urinary Bladder Neoplasms ; metabolism ; pathology ; ras Proteins ; metabolism

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an important role in vascular functions, including vasorelaxation. We here investigated the pharmacological effect of the natural product syringaresinol on vascular relaxation and eNOS-mediated NO production as well as its underlying biochemical mechanism in endothelial cells. Treatment of aortic rings from wild type, but not eNOS-/- mice, with syringaresinol induced endothelium-dependent relaxation, which was abolished by addition of the NOS inhibitor NG-monomethyl-L-arginine. Treatment of human endothelial cells and mouse aortic rings with syringaresinol increased NO production, which was correlated with eNOS phosphorylation via the activation of Akt and AMP kinase (AMPK) as well as elevation of intracellular Ca2+ levels. A phospholipase C (PLC) inhibitor blocked the increases in intracellular Ca2+ levels, AMPK-dependent eNOS phosphorylation, and NO production, but not Akt activation, in syringaresinol-treated endothelial cells. Syringaresinol-induced AMPK activation was inhibited by co-treatment with PLC inhibitor, Ca2+ chelator, calmodulin antagonist, and CaMKKbeta siRNA. This compound also increased eNOS dimerization, which was inhibited by a PLC inhibitor and a Ca2+-chelator. The chemicals that inhibit eNOS phosphorylation and dimerization attenuated vasorelaxation and cGMP production. These results suggest that syringaresinol induces vasorelaxation by enhancing NO production in endothelial cells via two distinct mechanisms, phosphatidylinositol 3-kinase/Akt- and PLC/Ca2+/CaMKKbeta-dependent eNOS phosphorylation and Ca2+-dependent eNOS dimerization.
Animals ; Aorta/*drug effects/physiology ; Enzyme Activation/drug effects ; Furans/*pharmacology ; Gene Deletion ; Human Umbilical Vein Endothelial Cells/drug effects/metabolism ; Humans ; Lignans/*pharmacology ; Mice ; Mice, Inbred C57BL ; Nitric Oxide/metabolism ; Nitric Oxide Synthase Type III/genetics/*metabolism ; Phosphatidylinositol 3-Kinases/metabolism ; Phosphoinositide Phospholipase C/metabolism ; Phosphorylation/drug effects ; Protein Multimerization/*drug effects ; Proto-Oncogene Proteins c-akt/metabolism ; Vasodilation/*drug effects

OBJECTIVETo study the effects of theanine on dopamine (DA), 5-hydroxy tryptamine (5-TH) and glutamate receptor 2 (GluR2) mRNA, phospholipase-γ1 (PLC-γ1) mRNA in cerebral ischemia-reperfusion injury rats and explore the mechanism of protective effects of theanine on the induced brain injury by ischemia-reperfusion in rats.

METHODSAccording to random number table, a total of 56 sprague-dawley rats in SPF grade about six-week old and 100 - 120 grams weighting were divided into five groups according to the body weight levels: model group (n = 12), sham-operation group (n = 8), low theanine group (10 mg/kg), middle theanine group (30 mg/kg) and high theanine group (90 mg/kg). There were 12 rats in each of the theanine group. The rats in model group and sham-operation groups were given distilled water, and the rats in theanine groups were given corresponding theanine solution intragastrically for fifteen days. Then the cerebral ischemia-reperfusion injury was established by middle cerebral artery occlusion (MCAO). The score of neurological behavior was evaluated at the 3rd and 24th hours after reperfusion. Rats were sacrificed at 24 hours after reperfusion, the concentrations of DA, 5-HT and theanine in rats brain following ischemia-reperfusion were determined. At the same time, we determined the levels of reactive oxygen species (ROS) and activities of catalase (CAT) in mitochondria of brain. The expressions of GluR2 mRNA and PLC-γ1 mRNA in rat brain were examined by reverse transcription polymerase chain reaction (RT-PCR) technique.

RESULTSThe score of neurological behavior of rats in model group, theanine-low, middle, high dose groups at the 3rd hour was 6.000 ± 0.926, 4.100 ± 0.738, 3.444 ± 0.726 and 2.250 ± 0.886 respectively (F = 29.70, P < 0.01), and the score at the 24th hour in these groups was 6.625 ± 0.916, 5.000 ± 0.817, 3.667 ± 0.707 and 2.625 ± 0.916 respectively(F = 34.68, P < 0.01). The concentration of DA in model group, theanine-low, middle, high dose groups and sham-operation group was (10.26 ± 1.12), (12.48 ± 1.09), (14.55 ± 0.94), (15.97 ± 0.92) and (11.98 ± 0.63) µg/g respectively (F = 43.76, P < 0.01). The concentration of 5-HT in these groups was (1.091 ± 0.160), (0.818 ± 0.101), (0.571 ± 0.050), (0.453 ± 0.111) and (0.863 ± 0.063) µg/g respectively (F = 48.68, P < 0.01). The level of ROS was (3.072 ± 0.503), (1.331 ± 0.268), (1.295 ± 0.061), (0.804 ± 0.200) and (2.158 ± 0.218) U×min⁻¹×mg⁻¹ (F = 80.82, P < 0.01) respectively and the activities of CAT in these groups were (4.880 ± 1.121), (8.405 ± 1.356), (9.535 ± 2.511), (15.090 ± 4.054) and (21.260 ± 6.054) U/g respectively (F = 28.58, P < 0.01). The expressions of GluR2 mRNA were 0.842 ± 0.020, 1.063 ± 0.100, 1.170 ± 0.152, 1.254 ± 0.131 and 1.012 ± 0.056 respectively (F = 9.23, P < 0.01). The expressions of PLC-γ1 mRNA in these groups were 0.737 ± 0.090, 0.887 ± 0.045, 0.963 ± 0.025, 0.991 ± 0.049 and 0.867 ± 0.079 respectively(F = 10.24, P < 0.01).

CONCLUSIONTheanine has a protective effect on the induced brain injury by ischemia-reperfusion in rats, which might be associated with its interaction with monoamine neurotransmitters and up-regulating the expressions of GluR2 mRNA and PLC-γ1 mRNA.

Animals ; Biogenic Monoamines ; metabolism ; Brain ; drug effects ; metabolism ; Brain Ischemia ; genetics ; metabolism ; Glutamates ; pharmacology ; Male ; Neurotransmitter Agents ; pharmacology ; Phospholipase C gamma ; genetics ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Receptors, AMPA ; genetics ; metabolism ; Reperfusion Injury ; genetics ; metabolism