Neuroprotection Of PEP-1-SOD1 Fusion Protein Associated With Ginko Biloba Extract Against Cerebral Infarction

Objective To explore the transductive ability of PEP-1-SOD1 fusion protein into cerebral tissue and the time course in mice;To investigate the neuropretection of precondition with PEP-1-SOD1 fusion protein and the effectlon of precondition associated with Ginko Biloba Extract(GBE) against cerebral infarction after permanent middle cerebral artery occlusion(PMCAO);To assay the influence of PEP-1-SOD1 on neural apoptosis and the expression of bax and bcl-2.Methods(1) Totally 150 healthy adult male Kuming mice weight 35-40g Were categorized randomly into two groups,each group was intraperitoneally injected with 200 ug SOD1 or PEP-1-SOD1 fusion protein,respectively.The animals were sacrificed for Western blotting,immunofluorescence staining and SOD1 activity at designed time points(1 h,2 h,4 h,8 h and 24 h after administration).(2) The model of PMCAO was set up.The animals were assigned randomly into sham-operation group,model group,PEP-1-SOD1 precondition group,GBE precondition group and combine group.The 200 ul saline was intraperitoneally injected into mice in sham-operation group and model group;The mice in PEP-1-SOD1 precondition group were intraperitoneally injected with 200 ug PEP-1-SOD1 fusion protein;The mice in GBE precondition group were intraperitoneally injected with 35 mg/kg Shuxuening injection, and the mice in combine group were intraperitoneally injected 200 ug PEP-1-SOD1 fusion protein and 35 mg/kg Shuxuening injection respectively,30 min before the operation.The brain tissue was dissected 24 h after surgery.TTC stained was used to determind cerebral infarction size;The activity of SOD1 and the amount of malondialdehyde(MDA)were assayed;TdT-mediated dUTP Nick-End Labeling(TUNEL) was used to investigated the apoptosis of neural and the immunofluorescence staining for the expression of bax and bcl-2.Results(1) Western blotting demonstrated that PEP-1-SOD1 fusion protein was expressed in brain tissue 1 h after injection and reached the peak at 4 h in PEP-1-SOD1 treated group,whereas there was no protein expressed in SOD1 treated group;Immunofluorescence indicated that the cerebral tissue treated with PEP-1-SOD1 fusion protein showed bright green fluorescent signals 1 h after injection and the fluorescent intensity reached the peak at 4 h,whereas that with SOD1 showed no green fluorescent signals;The SOD1 activity of PEP-1-SOD1 group was increased gradually 1 h after injection and showed the peak at 4 h,then decreased gradually within 24h. Whereas the SOD1 activity of SOD1 group were maintained about (60.63±2.47)×10~3U/g at all time points(P＜0.01 vs PEP-1-SOD1 treated proup).(2) TTC stain indicated that infarct size in PEP-1-SOD1 precondition group was smaller than that in model group(P＜0.05),and it was the smallest in combine group(P＜0.01 vs model group).But there was no statistic significant between the PEP-1-SOD1 group and combine group(P＞0.05). Compared model group with sham-operation group,the activity of SOD1 was lower,and the amount of MDA was higher 24 h after operation(P＜0.01);Whereas compared PEP-1-SOD1 precondition group and combine group with model group,the activity of SOD1 increased and the level of MDA decreased(P＜0.01);And there was no statistic significant between the PEP-1-SOD1 group and combine group(P＞0.05).TUNEL and immunofluorescence staining of bax indicated that there were amount of positive cells in model group,whereas the positive cells of bcl-2 were sparsity. Compared with model group,PEP-1-SOD1 reduced apoptotic neural cells and up-regulated the number of bcl-2 cells,and meanwhile down-regulated the number of bax positive cells(P＜0.01); Compared with PEP-1-SOD1 group,these three indexes were altenuates further in combine group(P＜0.01);Conclusions(1)The native PEP-1-SOD1 can transduct into murine cerebral tissue provide a basis for therapy of various disorders related to oxidative stress in central nerve system. (2) Precondition with PEP-1-SOD1 fusion protein could efficiently protect neuron against cerebral infarction.(3) Associated PEP-1-SOD1 fusion protein with GBE could enhance the pretective effection on neuron after cerebral infarction.(4) The PEP-1-SOD1 fusion protein can obviously decrease nueron apoptosis induced by disruption of blood flow.The mechanism maybe associated with downregulating the expression of bax and downregulating the ratio of bax to bcl-2.