ABSTRACT
Lysophosphatidylcholine (LPC) modulates the dynamic and integral process of macrophage polarization in immune responses, tissue inflammation and remodeling. Patatin-like phospholipase domain containing protein 7 (PNPLA7) was identified as an LPC-preferring lysophospholipase recently. However, the expression and role of PNPLA7 in macrophage polarization remained unknown. In the present study, PNPLA7 was found to be upregulated in the process of macrophage polarization toward an alternatively activated (M2) phenotype stimulated with interleukin 4 (IL-4) (P<0.05). We found that knockdown and overexpression of PNPLA1 decreased and increased the expression of M2 marker genes, including arginase 1 (Argl) and chitinase-like 3 (Ym\ ), respectively (P<0.05). Further studies showed that PNPLA7 regulated the expression of peroxisome proliferator activated receptor-γ (P P A R γ) at the mRNA and protein levels during M2 polarization (P < 0.05). However, the phosphorylation of signal transducer and activator of transcription 6 (STAT6) was not influenced by PNPLA7. These findings suggest that PNPLA7 favors macrophage anti-inflammatory M2 polarization through a PPAR
ABSTRACT
<p><b>OBJECTIVE</b>To construct the RNA interference expression vector for expression of human neuropathy target esterase (NTE) gene in mammalian cells.</p><p><b>METHODS</b>Spe I and Xho I-digested insert from pSUPER, which comprised H1 RNA polymerase III promoter and the multiple cloning sites, were cloned into the compatible in the pcDNA3.1 (+) to generate pSUPER/neo that could express small interfering RNA in mammalian cells. The annealed oligos targeting the expression of NTE were ligated into pSUPER/neo vector digested with Bgl II and Hind III to generate pSUPER/neo-NTE, which was transfected into COS7 and SH-SY5Y cells. The inhibitory effect of the expression of NTE was detected by western blot analysis and the enzyme activity assay.</p><p><b>RESULTS</b>pSUPER/neo-NTE could stably express double-stranded RNA of NTE. The expression of pSUPER/neo-NTE in COS7 and SH-SY5Y cells could efficiently inhibit the activity of NTE in the mammalian cells.</p><p><b>CONCLUSION</b>Stable eukaryotic expression vector of double-stranded RNA of NTE, pSUPER/neo-NTE, has been constructed successfully with promoter substitution strategy.</p>
Subject(s)
Animals , COS Cells , Carboxylic Ester Hydrolases , Genetics , Physiology , Cell Line, Tumor , Chlorocebus aethiops , Genetic Vectors , RNA Interference , RNA, Small Interfering , Genetics , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To explore the mechanism of cytotoxic effects of the organophosphates (OPs) with delayed neurotoxicity on human neuroblastoma cells.</p><p><b>METHODS</b>The proliferation of neuroblastoma SH-SY5Y cells was determined by MTT spectrometry. (45)Ca uptake was determined by adding (45)CaCl(2) and tri-o-cresyl phosphate (TOCP) or methamidophos into the cultured medium for the SH-SY5Y cells. The cells were incubated and then lysed and finally counted in a Beckman LS 6000 liquid scintillation spectrometer.</p><p><b>RESULTS</b>Methamidophos stimulated the cell proliferation of SH-SY5Y at its lower concentrations (7 x 10(-7) mol/L to 7 x 10(-6) mol/L), with an increase by 28% at 7 x 10(-7) mol/L; however, it inhibited the proliferation at higher ones (7 x 10(-4) mol/L to 7 x 10(-3) mol/L) with 62% inhibition at 7 x 10(-3) mol/L. TOCP only inhibited the cell proliferation at high concentration (with 34% inhibition at 7 x 10(-3) mol/L) and markedly inhibited calcium uptake of the cells up to 55% at higher concentrations (1 x 10(-6) mol/L to 1 x 10(-4) mol/L); while the uptake was stimulated by OPs up to 241% of increase at lower concentrations (1 x 10(-9) mol/L to 1 x 10(-7) mol/L).</p><p><b>CONCLUSION</b>The interference of growth in nerve cells and disturbance of calcium homeostasis may be involved in the mechanisms of neurotoxicity of OPs.</p>