Effects of Nec-1 on necroptosis and retinal ganglion cells in mouse model of retina ischemia reperfusion injury / 眼科新进展

ABSTRACT

Objective To observe the influence of necrostatin-1 (Nec-1) on necroptosis and retinal ganglion cells (RGC) in mice with retinal ischemia reperfusion injury (RIRI).Methods Together 60 wild-type C57 mice were randomly divided into three groups (n =20),and they were control group,experimental group and blank group.Firstly,as for investigation the effect of Nec-I on necroptosis,15 mice in the blank group left untreated,15 mice in the experimental group were injected with 2 μL Nec-1 (2 mol · L-1) intravitreally,and 15 in the control group without pretreatment.After 4 h,the RIRI model was established by anterior chamber perfusion in the latter two groups.Then retinas in 5 mice in each group were harvested 3 days after ischemia reperfusion injury for Western blot,immunofluorescence quantitative PCR and immunofluorescence staining to detect the expression of mRNA and protein of IL-1β,IL-6,TNF-α,RIP3,RIP1 and Caspase-8.Secondly,the rest of 5 mice in 20 of each group were collected and retrogradely labeled with fluoro-gold (FG) to explored the influence of Nec-1 on RGC.Mice in the blank group left untreated.Mice in the experimental group were injected with 2 μL Nec-1 (2 mol · L-1) intravitreally,while the control group was not treated anything 7 days after fluorescence labeling;after 4 h,the RIRI model was established by anterior chamber perfusion in the two groups.The retinal tissue was harvested and RGC counting was performed 3 days after ischemia reperfusion injury.Results When compared with control group,mRNA expression levels of IL-1β,IL-6,TNF-α,RIP3 and RIP1 in the experimental group were decreased significantly (all P ＜ 0.001),but Caspase-8 mRNA did not change obviously (P =0.654 8).Western blot and immunofluorescence staining showed that RIP3 expression decreased dramatically in the experimental group when compared with the control group.FG labeled RGC counting presented that RGC number of each field in experimental group was significantly larger than that in the control group (P ＜ 0.001).Couclusion Nec-1 can block necroptosis and significantly increase RGC number in mice model of experimental RIRI.