PMS777, a bis-interacting ligand for PAF receptor antagonism and AChE inhibition, attenuates PAF-induced neurocytotoxicity in SH-SY5Y cells

Abstract

(1) HIV-1 and viral proteins-evoked chronic brain inflammation, which is characterized by microglial activation, is the pivotal neuropathogenesis of HIV-1-associated dementia (HAD). Platelet-activating factor (PAF), mainly released from activated microglia and acts as a high potent inflammatory mediator and a neurotoxin, is indicated to be a principle initiator of neuroinflammation, neuronal dysfunction, and apoptosis related to HAD. Thus, bis-interacting ligands of acetylcholinesterase (AChE) inhibition and PAF receptor antagonism would be of great interest in the therapeutic potential of HAD not only for improvement of cognitive performance, but also for disease-modifying. (2). We have previously reported that a novel tetrahydrofuran-derived bis-interacting ligand PMS777 had satisfying potencies for PAF receptor blockade and AChE inhibition, and markedly improved cholinergic dysfunction-induced cognitive impairment in mice. Continuing with our research, we further investigated the neuroprotective activities of PMS777 on PAF-triggered neuronal injury in human neuroblastoma SH-SY5Y cells. (3) The bis-interacting ligand PMS777 (10 muM) obviously alleviated PAF-induced cell apoptosis in SH-SY5Y cells. Pretreatment with PMS777 also markedly inhibited intracellular Ca(2+) overload, down-regulation of anti-apoptotic bcl-2 mRNA, stimulation of pro-apoptotic bax mRNA expression and activation of caspase-3 pathway. Also, PMS777 could fine-tune pro-inflammatory cyclooxygenase-2 (cox-2) mRNA expression in PAF-treated cells. (4) These results suggest that PMS777 possesses a neuroprotective profile via anti-apoptotic/inflammatory signaling and warrant further investigations in connection with the potential value of this compound in HAD treatment.

Fig. 1

The survival of SH-SY5Y cells treated with varying concentrations of PAF. Cell survival was measured 24 h later using MTT assay. Each value represents the mean percentage cell survival ± SEM of three independent experiments. *P < 0.05, **P < 0.01 versus control

Fig. 2

PMS777 inhibited PAF-mediated neuronal death. SH-SY5Y cells were pretreated with PMS777 for 3 h before exposure to PAF. Cell survival was measured 24 h later using MTT assay. Each value represents the mean percentage cell survival ± SEM of three independent experiments. *P < 0.05 versus control; ⁺ P < 0.05 versus PAF-treated cells

Fig. 3

Effects of PMS777 on PAF-induced apoptosis in SH-SY5Y cells. Control apoptosis was elicited by only serum withdrawal for 24 h. Apoptosis was evaluated by flow cytometry. **P < 0.01 versus control; ⁺ P < 0.05 versus PAF-treated cells

Fig. 4

Effects of PMS777 on PAF-induced [Ca²⁺]_i increase in SH-SY5Y cells. PMS777 or BN52021 was added to the culture medium 100 s before addition of PAF. F ₀, basal fluorescence intensity; F, fluorescence intensity after application of drugs; F/F ₀, normalized Fluo-3 fluorescence. **P < 0.01 versus baseline, n = 63–88 cells

Fig. 5

Effects of PMS777 on bcl-2 and bax mRNA levels in PAF-treated SH-SY5Y cells. Data are expressed as mean ± SEM of three independent experiments. **P < 0.01 versus control; ⁺ P < 0.05 versus PAF-treated cells

Fig. 6

Effects of PMS777 on mRNA levels and activity of caspase-3 protease in PAF-treated SH-SY5Y cells. Each value represents mean ± SEM of three independent experiments. The levels of caspase-3 mRNA were examined by RT-PCR (A and B). Caspase-3 protease activity was measured using Ac-DEVD-pNA as a substrate for caspase-3 (C). *P < 0.05, **P < 0.01 versus control; ⁺ P < 0.05 versus PAF-treated cells

Fig. 7

Effects of PMS777 on cox-2 mRNA levels in PAF-treated SH-SY5Y cells. Data are expressed as mean ± SEM of three independent experiments. **P < 0.01 versus control; ⁺ P < 0.05 versus PAF-treated cells