Inhibition Of NF-κB Contribute To Autophagy And Affect Cell Death In Intracerebral Hemorrhage Mice

Objective: NF-κB is exquisitely sensitive to oxidative stress and is rapidly activated in perihematomal brain after intracerebral hemorrhage (ICH). It has also been reported that autophagy was activated after ICH in mice. Our previous study also certificated that autophagy was activated after ICH. The present study was sought to investigate the effects of a specific NF-κB inhibitor (SN50) on autophay, cell death and behavioral deficits in our ICH model.Methods: In vivo: Intracerebral hemorrhage was produced by injection of collagenaseⅣinto striatum in adult Kun'ming mice, and mice were randomly divided into SN50 pretreatment groups, saline vehicle groups, ICH groups, and naive groups. SN50 and saline groups were produced by intracerebroventricular (i.c.v.) administration of SN50 (1μg/μl,1μl) or saline (1μl) 5 min before ICH. ICH groups was produced by injection of collagenaseⅣinto striatum without any pretreatment. Animals were sacrificed at 1 h, 6 h, 24 h, 48 h and 7 d after ICH in each group, and brains were harvested. Regions of hemorrhage and perilesional were dissected for western blot analysis of Beclin-1, Bcl-2 and LC3; Mice were pretreated with intraperitoneal injection of propidium iodide (PI) 1 h before sacrificed. PI-labeling was used to identify injured cells and the amount of PI-positive cells were counted. Motor test was performed to detect whether ICH-induced cell loss would result in behavior deficits. Double labelling of damaged cells in the same brain slice using propidium iodide (PI) and anti-LC3 antibody was programed to analyze the role of autophagy in cell death induced by ICH. In vitro: To examine the role of NF-κB contribute to Hemin-induced autophagy, PC12 was cotreated with the NF-κB specific inhibitor SN50 and Hemin compared with that treated with Hemin solely. Western blot analysis was used to detect the expression levels of LC3, Beclin-1, Bcl-2 to study mechanisms involved in regulation of autophagy by NF-κB.Results: PI positive cells were increased and peaked at 24 h, 48 h after ICH in mice, and region of hemorrhage or periphery had a different peak time. PI-positive cells in ICH periphery in SN50 pretreated groups had no siginificant decreace compared with that in saline vehicle groups. PI-positive cells in hemorrhage had a significance decreace in SN50 pretreated groups compared with that in saline vehicle groups at 24 h post ICH. Beclin-1 was up-regulated, Bcl-2 was down-regulaed, and LC3 was activated after ICH in mice (P<0.05). Up-regulation of LC3 activation was enhanced in SN50 pretreated groups compared with that in saline vehicle groups (P<0.05). Up-regulation of LC3 , Beclin-1 activation were enhanced and down-regulation of Bcl-2 reversed in PC12 cells cotreated with SN50 and Hemin compared with that Hemin treated solely(P<0.05).Conclusion: The results imply that inhibition of NF-κB activation failed to protect all striatal neurons against ICH in a long time. Autophagy was activated, and partly contributed to cell death induced by ICH. Inhibition of NF-кB signal pathway enhanced the up-regulation of autophagy-related protein LC3, Beclin-1, Bcl-2. It implys that NF-κB may regulate autophagy activated by ICH through regulation of ratio of Beclin-1/Bcl-2.