Effects of isoindolin-1,3 dithione (IDT) on TNFα protein and mRNA.

(A) Structure of IDT. (B) Initial studies in BV2 cells demonstrate that IDT is effective at attenuating LPS-induced TNFα release into culture media (One-way ANOVA, p < 0.0001. p < 0.001 LPS vs. all IDT doses) with an IC50 value of ~5 μM. Green bars at the top of the y-axis and right side of the x-axis indicate the 100% LPS and no LPS (NS) responses, respectively. n = 4/group. (C) Lactate dehydrogenase (LDH) release into the culture media was measured after 24 hr exposure to LPS ± IDT. No significant increase in LDH was detected compared to DMSO control. (D) Effect of IDT on TNFα mRNA stability in BV-2 cells. Cells were pre-treated for 4 hr with 100 ng/mL LPS followed by treatment with 25 μM IDT and 5 mg/mL actinomycin D (ActD) and collected at intervals of 15 min. Total RNAs were isolated and TNFα mRNA was quantified using qPCR. Values represent mean ± s.e.m. for two separate experiments performed in triplicate and relative quantification was performed using the Rq method 2 -ΔΔCt. Asterisks indicate significant difference from LPS/ActD-treated BV-2 cells (**P < 0.01). n = 3/group. (E) IDT inhibited LPS-stimulated Cortical TNFα protein expression in vivo. Mice were treated by oral gavage with a single dose (50 mg/kg) of IDT 30 minutes prior to a peripheral 5 mg/kg dose of LPS (i.p.). Cortical tissue was harvested 4 hours after LPS injection. n = 6/group. One-way ANOVA, p < 0.001; ***p < 0.001 vs. Con and **p < 0.01 vs. LPS. Data represent mean ± SEM of n = 3/group.