Evaluation of Anti-Neuroinflammatory Activity of Isatin Derivatives in Activated Microglia

Abstract

Neuroinflammation plays a crucial role in the progression of Alzheimer's disease and other neurodegenerative disorders. Overactivated microglia cause neurotoxicity and prolong the inflammatory response in many neuropathologies. In this study, we have synthesised a series of isatin derivatives to evaluate their anti-neuroinflammatory potential using lipopolysaccharide activated microglia as a cell model. We explored four different substitutions of the isatin moiety by testing their anti-neuroinflammatory activity on BV2 microglia cells. Based on the low cytotoxicity and the activity in reducing the release of nitric oxide, pro-inflammatory interleukin 6 and tumour necrosis factor α by microglial cells, the N¹-alkylated compound 10 and the chlorinated 20 showed the best results at 25 µM. Taken together, the data suggest that 10 and 20 are promising lead compounds for developing new neuroprotective agents.

Keywords: TNF-α; interleukin 6; isatin; microglia; neuroinflammation; nitric oxide.

Figure 1

Effect of non-cytotoxic halogenated isatins on NO release by LPS-stimulated BV2 cells. LPS activation: cells were pretreated for 1 h with isatin derivatives (25 μM) or vehicle (0.025% DMSO) followed by LPS stimulation (1 μg/mL) and incubation for 24 h at 37 °C. Data are presented as mean values and error bars denote the SEM of at least three independent experiments performed in triplicate. NO production was determined by the Griess test. ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to LPS-activated cells (LPS) treated with vehicle. LPS: lipopolysaccharide.

Scheme 1

Synthesis of isatin derivatives. Reagents and conditions. N¹-alkylation: (i) 1 or 3, R²-X (1.2 eq.), K₂CO₃, DMF, rt, 12 h. (ii) 1 or 3, H₂CO, H₂O, reflux, 2 h. (iii) 13 or 20, succinic anhydride (2.5 eq.), pyridine, rt, 24 h. C³-substitutions: (iv) 1 or 3, NH₂-NHR’ (1.2 eq.), AcOH, EtOH, reflux, 3.5 h.

Figure 2

Effect of non-cytotoxic synthetic isatin derivatives on NO release by LPS-stimulated BV2 cells. LPS activation: cells were pretreated for 1 h with isatin derivatives (25 μM) or vehicle (0.025% DMSO) followed by LPS stimulation (1 μg/mL) and incubation for 24 h at 37 °C. Data are presented as mean values and error bars denote SEM of at least three independent experiments performed by triplicate. NO production was determined by the Griess test., ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to LPS-activated cells (LPS) treated with vehicle. NO inhibition percentage compared to LPS. ⇧: NO induction %. Striped columns: 5-chloro isatin derivatives. LPS: lipopolysaccharide.

Figure 3

Effect of non-cytotoxic isatin derivatives on release of cytokine IL-6 and TNF-α by LPS-stimulated BV2 cells. LPS activation: cells were pretreated for 1 h with non-cytotoxic isatin derivatives (25 μM) or vehicle (0.025% DMSO) followed by LPS stimulation (1 μg/mL) and incubation for 24 h at 37 °C. Concentrations of (A) IL-6 and (B) TNF-α in BV2 cell media were determined by microfluidic ELISA. Data are presented as mean values and error bars denote the SEM of at least three independent experiments performed in triplicate. * p < 0.05, ** p < 0.01, **** p < 0.0001 compared to LPS-activated cells (LPS) treated with vehicle. IL-6 and TNF-α inhibition percentage compared to LPS. ⇧: induction. % Striped columns: 5-chloro isatin derivatives. LPS: lipopolysaccharide.