Figure 4.

Blockade of RIP1 kinase activity abrogates TLR-activation-induced microglial necrosis. (a) Blockade of RIP1 kinase activity with necrostatin-1 (Nec-1) completely prevented LPS/zVAD-induced necrosis. Primary rat microglia were treated as indicated with LPS (0.1 ng/ml), zVAD (25 μM) or Nec-1 (20 μM), and cell survival was evaluated by cell counting. ***P<0.001. (b) Representative PI/Calcein AM (upper panel) and phase contrast (lower panel) images of microglia treated as above showing that microglial cell death induced by LPS/zVAD were abrogated by Nec-1. Data represent at least three independent experiments. Scale bars, 50 μm. (c) LPS significantly upregulated RIP1 and RIP3 expression in microglia as determined by western blot analysis. Rat microglia were preactivated overnight with low dose of LPS, washed extensively and exposed subsequently to zVAD or zVAD/Nec-1 for 3 h. Cell lysates were then prepared and subjected to western blot analysis. A representative western blot with shorter exposure time was shown. Quantification of relative RIP1 and RIP3 expression was carried out by densitometry analyses of western blots from 3 to 7 independent experiments. *P<0.05; two-tailed student t test. (d) RT-PCR analysis of microglia treated as above, showing that LPS transcriptionally increases RIP1 and RIP3 expression. Actin and Iba1 were used as internal controls, and TNF as an indicator for microglial activation. RT, reverse transcriptase. Experiments were repeated three times with similar results. (e) Nec-1 effectively abrogated microglial necrosis induced by other TLR ligands. Microglia were treated as indicated with zVAD, Nec-1 and various TLR agonists: TLR1/2 agonist Pam3CSK4 (0.1 μg/ml), TLR3 agonist poly(I:C) (PIC, 10 μg/ml), or TLR7/8 agonist R848 (1 μg/ml). Cell survival was determined by cell counting. Data represent mean±S.E.M. of two independent experiments. ***P<0.001