scholarly journals Osteogenic Effect of Inducible Nitric Oxide Synthase (iNOS)-Loaded Mineralized Nanoparticles on Embryonic Stem Cells

2018 ◽  
Vol 51 (2) ◽  
pp. 746-762 ◽  
Author(s):  
Jin-Sun Lee ◽  
Hong Jae Lee ◽  
Jae Won Lee ◽  
Sang Cheon  Lee ◽  
Jung Sun Heo
Keyword(s):  
Nitric Oxide ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Nitric Oxide Synthase ◽  
Cell Viability ◽  
Osteogenic Differentiation ◽  
Inducible Nitric Oxide Synthase ◽  
Embryonic Stem ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Background/Aims: This study investigated the effect of inducible nitric oxide synthase-loaded mineralized nanoparticles (iNOS-MNPs) on the osteogenic differentiation of mouse embryonic stem cells (ESCs). Methods: We prepared iNOS-MNPs using an anionic block copolymer template-mediated calcium carbonate (CaCO3) mineralization process in the presence of iNOS. iNOS-MNPs were spherical and had a narrow size distribution. iNOS was stably loaded within MNPs without denaturation. In order to confirm the successful introduction of iNOS-MNPs into the cytosol of ESCs, intracellular levels of nitric oxide (NO) was determined with a fluorometric analysis. A NO effector molecule, cyclic guanosine 3’,5’ monophosphate (cGMP) was also quantified with a competitive enzyme immunoassay. Cell viability in response to iNOS-MNP treatment was determined using the cell counting kit-8 (CCK-8) assay. Alkaline phosphatase (ALP) activity assay, intracellular calcium quantification assay, and Alizarin red S staining for matrix mineralization were performed to investigate osteogenic differentiation of ESCs. The protein levels of Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osterix (OSX) as osteogenic-related factors were also assessed by immunofluorescence staining and Western blot analysis. The complex pathways associated with iNOS-MNP-derived osteogenic differentiation of ESCs were evaluated by network-based analysis. Results: Cells with iNOS-MNPs displayed a significant increase in NO and cGMP concentration compared with the control group. When cells were exposed to iNOS-MNPs, there were no adverse effects on cell viability. Importantly, iNOS-MNP uptake promoted the osteogenic differentiation of ESCs. Using transcriptome profiling, we obtained 1,836 differentially-induced genes and performed functional enrichment analysis with ClueGO and KEGG. These analyses identified significantly enriched and interconnected molecular pathways such as protein kinase activity, estrogen receptor activity, bone morphogenetic protein (BMP) receptor binding, ligand-gated ion channel activity, and phosphatidylinositol 3-phosphate binding. Conclusion: These findings suggest that iNOS-MNPs can induce osteogenic differentiation in ESCs by integrating complex signaling pathways.

scholarly journals Isoflurane Preconditioning Reduces the Rat NR8383 Macrophage Injury Induced by Lipopolysaccharide and Interferon γ

2008 ◽  
Vol 108 (4) ◽  
pp. 643-650 ◽  
Author(s):  
Xuebing Xu ◽  
Jifeng Feng ◽  
Zhiyi Zuo
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Nitric Oxide Synthase ◽  
Cell Viability ◽  
Interferon Gamma ◽  
Inducible Nitric Oxide Synthase ◽  
Kinase C ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Background Isoflurane exposure before an insult can reduce the insult-induced injury in various organs. This phenomenon is called isoflurane preconditioning. The authors hypothesize that isoflurane can precondition macrophages, cells that travel to all tissues and are important in the host defense and inflammation responses. Methods Rat NR8383 macrophages were pretreated with or without 1-3% isoflurane for 1 h at 30 min before they were incubated with or without 100 ng/ml lipopolysaccharide plus 50 U/ml interferon gamma for 24 h. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Flow cytometry was performed after cells were stained with annexin V and propidium iodide. Inducible nitric oxide synthase protein expression in macrophages was quantified by Western blotting. Results Lipopolysaccharide plus interferon gamma decreased cell viability by approximately 50%. This decrease was dose-dependently inhibited by aminoguanidine, an inducible nitric oxide synthase inhibitor. Lipopolysaccharide plus interferon gamma caused inducible nitric oxide synthase expression. This expression was inhibited by pretreatment with 2% but not 1% or 3% isoflurane. Isoflurane at 2% inhibited lipopolysaccharide plus interferon gamma-induced accumulation of nitrite, an oxidation product of nitric oxide. Pretreatment with 2% but not 1% or 3% isoflurane improved cell viability. Lipopolysaccharide plus interferon gamma increased the number of propidium iodide-positive staining cells. This increase was attenuated by 2% isoflurane pretreatment. The protective effect of 2% isoflurane was abolished by chelerythrine, calphostin C, or bisindolylmaleimide IX, protein kinase C inhibitors. Conclusions Lipopolysaccharide plus interferon gamma causes an inducible nitric oxide synthase-dependent macrophage injury. Isoflurane induces preconditioning effects that may be mediated by protein kinase C in macrophages.

289: Roles of Inducible Nitric Oxide Synthase in Voiding Function and Bladder Pain During Experimental Cystitis

2006 ◽  
Vol 175 (4S) ◽  
pp. 96-96
Author(s):  
Masayoshi Nomura ◽  
Hisae Nishii ◽  
Masato Tsutsui ◽  
Naohiro Fujimoto ◽  
Tetsuro Matsumoto
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
Voiding Function ◽  
Bladder Pain ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide
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