The Role And Mechanisms Of Necrostatin-1on Mice Intracerebral Hemorrhage Model

Objective: The aim of this study was to examine the effect of necrostatin-1onbrain injury and investigate the role of necrostatin-1on the other two types ofprogramed cell death (apoptosis and autophagic cell death) in a mouse intracerebralhemorrhage (ICH) model.Methods: Male ICR mice received an infusion of type IV collagenase into theleft striatum to induce ICH. The dissolvant were pretreated with a singleintracerebroventricular (i.c.v.) injection in the ipsilateral ventricle15min before ICH.The loss of brain tissue was determined by lesion volume (LV) assessment method; tofurther verify whether the LV associated with the number of cell lost, intraperitonealinjection of PI was performed1h before sacrificing the mice and the brains wereharvested for serial sections, and the amount of propidium iodide (PI)-positive cellswas counted by stereo microscope. To explor the mechanisms of necrostatin-1on ourmouse ICH model, the other two types programmed cell death associated proteins(autophagic-associated proteins: LC3-II, Beclin-1, and p62; apoptotic-associatedproteins: Bcl-2and cleaved caspase-3) were analysised by immunoblotting. Autophagyinhibitor3-methyladenine (3-MA) and pan-caspase inhibitor Z-VAD-FMK (zVAD)were also used to confirm the effects of necrostatin-1on autophagic and apoptoticactivity in this study.Results: Compared with vehicle groups, necrostatin-1pre-treatment significantlyreduced the injury volume and PI-positive cells at24and72h time point after ICH.Immunoblotting analysis showed that necrostatin-1pre-treatment suppressedautophagic-associated proteins (LC3-II, Beclin-1) and maintained p62at normal level at24and72h time point after ICH. In addition, necrostatin-1treatment enhanced the protein level of Bcl-2and decreased the protein level of cleaved caspase-3and theBeclin-1/Bcl-2ratio at24and72h time point after ICH. Moreover, both3-MA andnecrostatin-1treatment could suppress cleaved caspase-3and LC3-II production,whereas zVAD treatment could inhibit caspase-3cleavage but increased LC3-II proteinlevels at72h time point after ICH.Conclusion: The necroptosis specific inhibitor necrostatin-1suppressed apoptosisand autophagy to exert neuroprotective effects after ICH.