ABSTRACT
The purpose of the present study was to investigate the effect of advanced glycated albumin (AGE-alb) on pyroptosis of macrophages and the underlying molecular mechanisms. RAW264.7 macrophages were treated with AGE-alb (1, 2, 4 and 6 g/L) and control albumin (C-alb, 4 g/L) for 24 h, or preincubated with MCC950 (1 μmol/L) for 1 h and then treated with AGE-alb (4 g/L) for 24 h. Cell viability and caspase-1 activity were measured by MTT and assay kits, respectively. Lactate dehydrogenase (LDH) activity and the levels of interleukin-1β (IL-1β) and IL-18 in media were detected. Cell death degree was evaluated by TUNEL and Hoechst 33342/PI staining. The protein levels of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), procaspase-1 and cleaved caspase-1 were assessed by Western blot. The results showed that AGE-alb treatment caused obvious decrease in cell viability and increases in LDH leakage and the percentages of TUNEL- or PI-positive cells in a concentration-dependent manner. Additionally, AGE-alb promoted IL-1β and IL-18 secretion, upregulated NLRP3 expression, and increased caspase-1 activity especially at the dose of 4 and 6 g/L. However, MCC950 (an NLRP3 inhibitor) pretreatment inhibited significantly the decrease in cell viability and the increases in LDH leakage and percentages of TUNEL- or PI-positive cells induced by AGE-alb. Furthermore, MCC950 attenuated obviously AGE-alb-induced IL-1β and IL-18 secretion and caspase-1 activation. These results indicate that AGE-alb may induce macrophage pyroptosis, and the mechanism is at least partially by activating NLRP3-caspase-1 pathway.
Subject(s)
Caspase 1 , Gene Expression Regulation , Interleukin-1beta , Genetics , Macrophages , NLR Family, Pyrin Domain-Containing 3 Protein , Genetics , Pyroptosis , Serum Albumin , PharmacologyABSTRACT
Infections are a devastating complication of titanium alloy orthopedic implants. Current therapies include antibiotic-impregnated bone cement and antibiotic-containing coatings. Daptomycin (DAP) (1) is a novel peptide antibiotic that penetrates the cell membranes of Gram-positive bacteria. Few DAP-resistant strains have appeared so far. We hypothesized that when DAP covalently bonded via a flexible, hydrophilic spacer it could prevent bacterial colonization of titanium alloy surfaces. We designed and synthesized a series of DAP conjugates for bonding to the surface of Ti6Al4V foils through tetra(ethylene glycol) spacers via thioether linkages. The stability and antimicrobial activity of the attached conjugates were evaluated using Staphylococcus aureus ATCC 25923. Colonization of the Ti6Al4V foils was inhibited by 72% at 8 h and 54% at 24 h. The strategy described in this report provides a new, more facile way to prepare bactericidal Ti6Al4V implants.
ABSTRACT
Monoamine oxidases (MAO) catalyze the oxidative deamination of many biogenic amines and are integral proteins found in the mitochondrial outer membrane. Changes in MAO-A levels are associated with depression, trait aggression, and addiction. Here we report the synthesis, characterization, and in vitro evaluation of novel fluorescent peptide-peptide nucleic acid (PNA) chimeras for MAOA mRNA imaging in live neuronal cells. The probes were designed to include MAOA-specific PNA dodecamers, separated by an N-terminal spacer to a µ-opioid receptor targeting peptide (DAMGO), with a spacer and a fluorophore on the C-terminus. The probe was successfully delivered into human SH-SY5Y neuroblastoma cells through µ-opioid receptor-mediated endocytosis. The K(d) by flow cytometry was 11.6 ± 0.8 nM. Uptake studies by fluorescence microscopy showed â¼5-fold higher signal in human SH-SY5Y neuroblastoma cells than in negative control CHO-K1 cells that lack µ-opioid receptors. Moreover, a peptide-mismatch control sequence showed no significant uptake in SH-SY5Y cells. Such mRNA imaging agents with near-infrared fluorophores might enable real time imaging and quantitation of neuronal mRNAs in live animal models.
