Rting at 10 lM PJ34 (Fig. 1F; p 0.001), and PJ34 pre-treatment substantially attenuated LPSinduced TNFa release at 20 mM PJ34 (Fig. 1G; p 0.001). We then utilised the pro-inflammatory cytokine, IFNc, inside a second model of microglial activation. BV2 microglia have been treated with IFNc (2 ng/mL) with or without pre-treatment with PJ34 (1, 10, and 20 mM), and 24 h later we assessed microglial activation by measuring NO and TNFa release and ROS generation. IFNc stimulation significantly induced all markers of activation when compared with untreated controls ( p 0.01 or p 0.001 for every single, one-way ANOVA). PARP-1 inhibition by PJ34 pre-treatment considerably attenuated IFNc-induced NO release at 20 lM PJ34 (Fig. 2A), and substantially attenuated IFNc-induced TNFa release starting at 10 lM PJ34 (Fig. 2B). Moreover, PARP-1 inhibition drastically attenuated IFNc-induced ROS generation starting at 1lM PJ34 (Fig. 2C). MNNG-induced neuronal cell death is attenuated by the PARP-1 inhibitor, PJ34 MNNG is actually a well-known inducer of PARP-1 activation and neuronal cell death.30 To compare the function of PARP-1 versus caspase activation as causal factors in MNNG-induced neuronal cell death, main cortical neurons had been treated with MNNG (50 mM) with or without pre-treatment with PJ34 (20mM), BOC (one hundred mM, a pan-caspase inhibitor), or combined PJ34 + BOC. Right after 24 h, we analyzed various well-established assays for cell death (LDH release, calcein fluorescence and caspase-3 activity). MNNG treatment significantly induced cell death when compared with untreated samples in the calcein and LDH release assays ( p 0.001 for each). Notably, the MNNG-induced reduce in calcein fluorescence (an indicator of cell viability) was significantly attenuated by pre-treatment with PJ34 ( p 0.001), but not by pre-treatment with BOC (Fig. 3A). Combined remedy with PJ34 + BOC enhanced cell viability more than and above PJ34 treatment alone levelsSTOICA ET AL.FIG. 1. PJ34 (N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide) attenuates lipopolysaccharide (LPS)-induced activation of BV2 microglia cell line and primary microglia. (A) LPS stimulation (100 ng/ml) in BV2 microglia substantially elevated nitric oxide (NO) production (***p 0.Aztreonam 001, LPS vs.RITA control).PMID:24513027 PJ34 pre-treatment at concentrations of 10 lM and 20 lM attenuated LPSinduced NO production ( + + + p 0.001, + PJ34 vs. LPS). (B) LPS stimulation in BV2 microglia drastically elevated inducible nitric oxide synthase (iNOS) expression (***p 0.001, LPS vs. handle). PJ34 pre-treatment (20 lM) attenuated LPS-induced iNOS expression ( + + p 0.01, + PJ34 vs. LPS). Therapy with PJ34 alone had no impact on iNOS expression. (C) LPS stimulation (one hundred ng/mL) in BV2 microglia drastically improved TNFa production (***p 0.001, LPS vs. manage). PJ34 pre-treatment at concentrations of 10 lM and 20 lM attenuated LPS-induced TNFa production ( + + + p 0.001, + PJ34 vs. LPS). (D) LPS stimulation in BV2 microglia substantially improved nuclear factor-kappaB (NFjB) activity (***p 0.001, LPS vs. control). PJ34 pre-treatment (20 lM) attenuated LPS-induced NF-jB activation ( + + + p 0.001, + PJ34 vs. LPS). Therapy with PJ34 alone had no impact on NF-jB activation. (E) LPS stimulation in BV2 microglia considerably improved reactive oxygen species (ROS) production (**p 0.01, LPS vs. manage). PJ34 pretreatment (20lM) attenuated LPS-induced ROS production ( + + p 0.01, + PJ34 vs. LPS). (F) LPS stimulation in main mic.
