| Paired immunoglobulin-like receptor B(Pir B) has been identified as a new receptor for myelin-associated inhibitory proteins(MAIs), which may play an important role in the axonal regeneration, synaptic plasticity and neuronal survival after stroke. In the clinic, the mutation or silencing of Pir B is impractical, and a candidate peptide that binds to MAIs might block their interactions with Pir B. However, the delivery of a therapeutic peptide into the brain is hampered by the blood-brain barrier(BBB). Here, we investigated Pir B expression after transient focal cerebral ischemia in rats and found the protein levels were increased in the ischemic penumbra from 1 d to 28 d after reperfusion. Then, the result showed pirb RNAi enhanced axon growth and alleviated neuronal injury by inhibitingPir B expression after exposed to OGD in vitro. After that, TAT-PEP was generated as a novel soluble fusion protein containing the protein transduction domain(PTD) derived from the HIV transactivator of transcription(TAT) which is able to permeate the BBB when administered systemically. TAT-PEP displayed high affinity for Nogo-A, MAG, or OMgp and ameliorated their inhibitory effect on neurite growth, which also can widely distribute in the penumbra after intraperitoneal injection. Moreover, TAT-PEP treatment attenuated brain infarct volume and promoted long-term neurobehavioral functional recovery. The study further found, TAT-PEP enhanced corticospinal projections, axonal regeneration and synaptic plasticity in vivo. TAT-PEP also enhanced neurite growth, alleviated growth cone collapse and improved synaptic function after OGD injury in vitro. One possible mechanism was due to interference with the expression of POSH, Rho A, and GAP43 by resisting Pir B funtion. Furthermore, TAT-PEP exerted neuroprotection by alleviating neuronal degeneration and apoptosis after ischemic reperfusion injury. One possible mechanism was due to the effect of TAT-PEP on the expression of cleaved Caspase3, Bax and Bcl-2. Our findings suggest that Pir B overexpression in neuron after suffering ischemic reperfusion injury induced neurons damage. This study also indicated that TAT-PEP may represent a highly efficacious neuroprotective agent displaying therapeutic potential for stroke. Together, the important implication of this study is that blocking the action between MAIs and Pir B may represent an attractive therapeutic strategy against stroke in future clinical studies.Part 1The Spatio-temporal expression of Pir B in the cerebral cortex penumbra and the effect of pirb RNAi on neurons after exposed to OGDObjective:1. To study the spatio-temporal expression of PirB in the cerebral cortex penumbra in the long term after MCAO injury. 2. To investigate the effect of pirb RNAi on neurite growth after OGD injury. 3. To investigate the effect of pirb RNAi on neuronal survival and apotosis after OGD injury.Methods:1. Male SD rats weighing 280–320 g. The rats which had been operated successfully were randomly divided into 8 groups(n=6 each): Normal group, Sham group, 1 d post-MCAO group, 3 d post-MCAO group, 7 d post-MCAO group, 14 d post-MCAO group, 21 d post-MCAO group, 28 d post-MCAO group. The expression of Pir B in the penumbra were measured at different time points after MCAO by Western blot.2. Male SD rats weighing 280–320 g. The rats which had been operated successfully were randomly divided into 2 groups(n=6 each): Sham group and MCAO group. The expression of Pir B in the penumbra was measured at 28 d after MCAO by immunofluorescence histochemistry staining.3. The generation of GPH-PIRB-294 and lentiviral vector system was performed by Genscript Co.(Nanjing, China). SD rat embryos(E 16.5 d-18.5 d). The cultured primary cerebral cortical neurons were randomly divided into 4 groups(n=6 each): Normal group, OGD group, OGD+control RNAi group and OGD+pirb RNAi group. After rehabilitation of sugar-oxygen, GPH-PIRB-294 was transfected into neurons. At 72 h after OGD, the expression of Pir B and GFP was tested.4. SD rat embryos(E 16.5 d-18.5 d). The cultured primary cerebral cortical neurons were randomly divided into 4 groups(n=6 each): Normal group, OGD group, OGD+control RNAi group and OGD+pirb RNAi group. After rehabilitation of sugar-oxygen, GPH-PIRB-294 was transfected into neurons. At 72 h after OGD, the neurites length was measured.5. SD rat embryos(E 16.5 d-18.5 d). The cultured primary cerebral cortical neurons were randomly divided into 4 groups(n=6 each): Normal group, OGD group, OGD+control RNAi group and OGD+pirb RNAi group. After rehabilitation of sugar-oxygen, GPH-PIRB-294 was transfected into neurons. At 72 h after OGD, the MTT and TUNEL staining was tested.Results:1. Western blot analysis showed low expression of the Pir B protein in Normal group and Sham group. In contrast, Pir B expression was significantly increased at 1, 3, 7, 14, 21 and 28 d post-MCAO(P<0.05) in the penumbra. We also detected the cellular localization of Pir B via double-immunofluorescence staining. The results showed that Pir B co-localized with Neu N protein, indicating neuronal localization in the ischemic brains. In addition, the number of Pir B-positive neurons in the MCAO group was more than that in the Sham group at 28 d after reperfusion(P<0.05).2. At 72 h post-OGD, Western blot analysis revealed that Pir B expression in the OGD and control RNAi groups was increased compared with that in the Normal group(P<0.05). However, its expression was significantly decreased in the pirb RNAi group compared with the OGD and control RNAi groups(P<0.05).3. Under normal culture conditions, cortical neurons contained a single long axon in vitro. The extension of cultured cortical neuronal axons was significantly reduced at 72 h after exposure to OGD(P<0.05). However, the mean axon length in the pirb RNAi group was longer than those in the OGD group(P<0.05).4. The survival of cultured neurons was examined at 72 h after transfection. The neuronal structure in the OGD and OGD+control RNAi groups was damaged. However, transfection with the Pirb RNAi vector prevented this neuronal damage. The cell viability in the OGD and control RNAi groups was significantly lower than that of the Normal group(P<0.05) at 72 h after exposure to OGD. However, the cell viability of the Pirb RNAi group was higher that of the OGD and control RNAi groups(P<0.05). The number of TUNEL-positive cells in the OGD and control RNAi groups was significantly higher than that of the Normal group(P<0.05) at 72 h after exposure to OGD. In addition, the number of TUNEL-positive cells in the Pirb RNAi group was less than that of the OGD group(P<0.05).Conclusion:The significant and persistent up-regulation of neuronal Pir B expression in the ischemic penumbra indicates that Pir B represents a potential target for stroke therapy. These results also indicated that Pir B overexpression in neuron after suffering OGD injury may involve in inducing neurons casualty after OGD injury, inhibiting Pir B expression may enhance neurite growth rescue the neurons from apoptosis.Part 2The preparation of soluble fusion protein TAT-PEP and the identification of its biological activityObjective:1. To generate the soluble fusion protein TAT-PEP and TAT-m PEP. 2. To identify and assay biological activity of TAT-PEPMethods:1. The generation of TAT-PEP and TAT-m PEP(Control protein) was performed by Genscript Co.(Nanjing, China). TAT-PEP was identified by Coomassie Blue Staining and Western blot using anti-(His)6 primary antibody. SD rat embryos(E 16.5 d-18.5 d). The cultured primary cerebral cortical neurons were randomly divided into 5 groups(n=6 each). At 72 h after TAT-PEP(50 μg/L, 100 μg/L, 200 μg/L) or TAT-m PEP(100 μg/L) treatment, the neuronal viability and damage were assessed using the MTT and LDH assays.2. SD rat embryos(E 16.5 d-18.5 d). ELISA was performed to assess the ability of TAT-PEP to bind to Nogo-A, MAG and OMgp.3. SD rat embryos(E 16.5 d-18.5 d). The ability of TAT-PEP to suppress the inhibitory effect of Nogo-A, MAG, OMgp or MAIs(Nogo-A+MAG+OMgp) on neurite outgrowth was determined.4. Male SD rats weighing 280–320 g. The rats which have been operated successfully were randomly divided into 4 groups(n=4 each): 0 mg/kg TAT-PEP group, 0.05 mg/kg TAT-PEP group, 0.5 mg/kg TAT-PEP group and 1.0 mg/kg TAT-PEP group. SD rats were subjected to MCAO and received an intraperitoneal injection of TAT-PEP after reperfusion. The immunofluorescence histochemical staining analysis was performed at 24 h after reperfusion to determine the amount of TAT-PEP in the ischemic penumbra after MCAO.5. Male SD rats weighing 280–320 g. The rats which had been operated successfully were randomly divided into 7 groups(n=4 each): SD rats were subjected to MCAO and received an intraperitoneal injection of TAT-PEP(1.0 mg/kg) after reperfusion. At 0 h, 0.5 h, 2 h, 12 h, 24 h, 48 h, or 72 h after reperfusion using Western blot to determine the transduction.Results:1. TAT-PEP was effectively produced and purified as confirmed by SDS-PAGE. Moreover, the specificity of TAT-PEP expression was verified by Western blotting using a specific anti-(His)6 antibody(TAT-PEP, 90 KDa).2. We assessed the toxicity of the purified fusion proteins to normal cortical neurons.These two proteins did not affect neuronal viability(P>0.05) or LDH release(P>0.05) after incubation for 72 h.3. At each indicate concentration point, the OD450 nm of TAT-PEP was significantly higher than that of TAT-m PEP, indicating that TAT-PEP displayed higher affinity not only for AP-Nogo-66, but also for MAG-Fc and AP-OMgp than TAT-m PEP.4. Neurite extension was significantly inhibited by Nogo-A, MAG or OMgp(P<0.05). In contrast, neurite extension was almost completely rescued by TAT-PEP(P<0.05).5. Neurite extension was significantly inhibited by MAIs(P<0.05). In contrast, neurite extension was almost completely rescued by TAT-PEP(50 μg/L, 100 μg/L, 200 μg/L)(P<0.05).6. There was no(His)6 positive protein in the observed area in the 0 mg/kg TAT-PEP group. There was amount of(His)6 positive protein in the observed area in the 0.5 mg/kg TAT-PEP group and 1.0 mg/kg TAT-PEP group. The(His)6 positive protein in the 1.0 mg/kg TAT-PEP group was more than that in the 0.5 mg/kg TAT-PEP group(P<0.05).7. A high level of TAT-PEP in brain tissue was detected at 30 min after intraperitoneal injection. This elevation peaked at 12 h and persisted for 72 h.Conclusion:In conclusion, these results suggest that TAT-PEP was generated successfully. The neurite extension can be rescued by TAT-PEP. TAT-PEP can cross the BBB and accumulate in the ischemic region after intraperitoneal injection. We hypothesized that blocking the interaction between Nogo-A, MAG, OMgp and their receptor Pir B using TAT-PEP in the ischemic cerebral cortex could attenuate ischemic stroke injury and abolishing the inhibitory activity of Pir B could have a more powerful effect than deleting only a subset of its ligandsPart 3The effect of TAT-PEP on brain infarct volume and long-term neurobehavioral recovery after MCAO injuryObjective:1. To study the effect of TAT-PEP on brain infarct volume after MCAO. 2. To study the effect of TAT-PEP on neurobehavioral recovery after MCAO.Methods:1. Male SD rats weighing 280–320 g. The rats which had been operated successfully were randomly divided into 3 groups(n=6 each): Sham group, MCAO group, MCAO+TAT-PEP group. The Garcia scores test was performed at 24 h, 48 h and 72 h after reperfusion. The TTC staining was performed at 72 h after reperfusion.2. Male SD rats weighing 280–320 g. The rats which had been operated successfully were randomly divided into 3 groups(n=6 each): Sham group, MCAO group, MCAO+TAT-PEP group. Diffusion-weighted imaging(DWI) was performed at 3 d post-MCAO. T2-weighted images(T2WI) were collected at 3, 7 and 28 d post-MCAO. The m NSS scores was measured at 7, 14, 21 and 28 d post-MCAO.3. Male SD rats weighing 280–320 g. The rats which had been operated successfully were randomly divided into 3 groups(n=6 each): Sham group, MCAO group, MCAO+TAT-PEP group.The foot-fault and rotarod test were performed at 2, 7, 14, 21 and 28 d post-MCAO.4. Male SD rats weighing 280–320 g. The rats which had been operated successfully were randomly divided into 3 groups(n=6 each): Sham group, MCAO group, MCAO+TAT-PEP group.The limb placement test was performed at 3 h, 7, 14, 21 and 28 d post-MCAO. The motor evoked potential was performed at 28 d post-MCAO.Results:1. The Garcia scores test showed the neurological function scores of MCAO group were lower than that of Sham group at 24, 48 and 72 h after reperfusion(P<0.05). In contrast, the neurological function scores of MCAO+TAT-PEP group were higher than that of MCAO group at 48 and 72 h after reperfusion(P<0.05). The MCAO+TAT-PEP group displayed a significantly smaller brain infarct volume than the MCAO group by TTC staining at 72 h after reperfusion(P<0.05).2. The m NSS scores of MCAO group were higer than those of Sham group at 7, 14, 21 and 28 d after reperfusion(P<0.05). The scores in the MCAO+TAT-PEP group were significantly lower than those in the MCAO group at 7, 14, 21 and 28 d post-MCAO(P<0.05). T2 WI analyses showed that the high-intensity volumes were smaller in the MCAO+TAT-PEP group than in the MCAO group at 3, 7 and 28 d post-MCAO(P<0.05).3. To test the motor integration response capacity, the foot-fault task was used. Compared with the MCAO group, performance on the foot-fault test was significantly better in the MCAO+TAT-PEP group at 7, 14, 21 and 28 d post-MCAO(P<0.05; Fig. 6C). To evaluate motor function and coordination, the rotarod test was performed. Compared with the Sham group, the time remaining on the rod was shorter in the MCAO group at 7, 14, 21 and 28 d post-MCAO. However, the motor performance of the MCAO+TAT-PEP group was increased at 7, 14, 21 and 28 d post-MCAO compared to that of the MCAO group(P<0.05). Subsequently, scores on the limb placement test was performed. The recovery of limb function was significantly improved in the MCAO+TAT-PEP group on 7, 14, 21 and 28 d compared with the MCAO group(P<0.05).4. The results showed a notable reduction in the amplitude of MEPs recorded from the left forelimb and left hindlimb in the MCAO group compared with the Sham group at 28 d post-MCAO(P<0.05). In contrast, the MCAO+TAT-PEP group displayed larger MEP amplitudes in the left forelimb and left hindlimb than the MCAO group(P<0.05).Conclusion:In conclusion, these data indicate that TAT-PEP treatment is capable of considerably reducing infarct volume in parallel with enhancing the recovery of neurobehavioralfunction within 4 weeks after reperfusion.Part 4The effect and mechanism of TAT-PEP on axonal regeneration, corticospinal tract reconstruction and synaptic plasticity reconstruction after ischemic reperfusion injuryObjective:1. To study the effect of TAT-PEP on corticospinal tract reconstruction after MCAO. 2. To study the effect of TAT-PEP on axonal regeneration in the ischemic penumbra after MCAO. 3. To study the effect of TAT-PEP on synaptic plasticity in the ischemic penumbra after MCAO. 4. To study the effect of TAT-PEP on neurite growth and growth cone collapse after OGD injury. 5. To investigate the mechanism of TAT-PEP on above effects.Methods:1. Male SD rats weighing 280–320 g. The rats which had been operated successfully were randomly divided into 3 groups(n=6 each): Sham group, MCAO group, MCAO+TAT-PEP group. The BDA in the injection area was examined at 21 d after MCAO.2. Male SD rats weighing 280–320 g. The rats which had been operated successfully were randomly divided into 3 groups(n=6 each): Sham group, MCAO group, MCAO+TAT-PEP group. The BDA-positive fibers were examined in the cervical enlargement of the spinal cord at 28 d after MCAO.3. Male SD rats weighing 280–320 g. The rats which had been operated successfully were randomly divided into 3 groups(n=6 each): Sham group, MCAO group, MCAO+TAT-PEP group. The expression of NF200, MAP2, SYN and Neu N in the penumbra at 28 d after MCAO by immunofluorescence histochemistry staining.4. Male SD rats weighing 280–320 g. The rats which had been operated successfully were randomly divided into 3 groups(n=6 each): Sham group, MCAO group, MCAO+TAT-PEP group. The expression of POSH, Rho A and GAP43 in the penumbra were measured at 28 d after MCAO by Western blot.5. SD rat embryos(E 16.5 d-18.5 d). The cultured primary cerebral cortical neurons were randomly assigned into 3 groups(n=6 each): OGD group, OGD+TAT-PEP group and Normal group. At 24 h after cortical neuronal adherence, the OGD model was used. live cell imaging was performed observe neurites growth at 24 h after exposure to OGD.6. SD rat embryos(E 16.5 d-18.5 d). The cultured primary cerebral cortical neurons were randomly assigned into 3 groups(n=6 each): OGD group, OGD+TAT-PEP group and Normal group. At 7 d after cortical neuronal adherence, the OGD model was used. The neurites growth and branch points were observed at 3 d after exposure to OGD.7. SD rat embryos(E 16.5 d-18.5 d). The cultured primary cerebral cortical neurons were randomly assigned into 3 groups(n=6 each): OGD group, OGD+TAT-PEP group and Normal group. At 7 d after cortical neuronal adherence, the OGD model was used. The GAP43 staining was performed at 3 d after exposure to OGD.8. SD rat embryos(E 16.5 d-18.5 d). The cultured primary cerebral cortical neurons were randomly assigned into 4 groups(n=6 each): OGD group, OGD+TAT-PEP group, OGD+pirb RNAi group and Normal group. The expression of POSH, Rho A and GAP43 were measured at 3 d after OGD by Western blot.Results:1. The axonal tracer BDA was injected into the intact(undamaged hemisphere) motor cortex at 14 d post-MCAO to label descending axons in the intact hemisphere. At 28 d post-MCAO, the Sham group displayed a unilateral CST innervation profile. In the MCAO group, few BDA-labeled, midline-crossing CST axons extended toward the ventral horn of the spinal gray matter on the denervated side of the cervical cord, serving the affected limb. The number of BDA-positive CST axons in the middle or on the left side of the gray matter of the spinal cord(C3-5) in the MCAO+TAT-PEP group was significantly increased compared with the number in the Sham and MCAO groups(P<0.05). TAT-PEP treatment greatly increased the number of such fibers that were observed ipsilateral to the injection site(P<0.05)。2. The morphology of the NF-200-immunoreactive neuronal fibers was irregular and fragmented in the MCAO group. Compared with the MCAO group, the expression of NF200-positive axons was increased in MCAO+TAT-PEP group(P<0.05).3. Compared with Sham group, the expression of MAP2 was lower than that in the MCAO group(P<0.05). In contrast, the expression of MAP2 was higher than that in the MCAO+TAT-PEP group(P<0.05).4. The synapsin were eliminated from injured neurons in the MCAO group(P<0.05). Compared with the MCAO group, the amount of SYN was increased in the MCAO+TAT-PEP group(P<0.05).5. The expression of POSH and Rho A was higher in the MCAO group than that in the Sham group at 28 d post-MCAO(P<0.05). The GAP43 levels were decreased in the MCAO group compared to the Sham group at 28 d post-MCAO(P<0.05). In contrast, the expression of POSH and Rho A was inhibited by TAT-PEP treatment at 28 d post-MCAO(P<0.05). The GAP43 levels were increased in the MCAO+TAT-PEP group compared to the MCAO group at 28 d post-MCAO(P<0.05).6. Time-lapse imaging showed that the average growth rate of the longest neurite was higher in the OGD+TAT-PEP group than in the OGD group(P<0.05). Moreover, the number of branching points on the longest neurite in the OGD+TAT-PEP group was greater than that in the OGD group(P<0.05).7. The extension and number of branching points of the longest neurite were recorded. The average length of the longest neurite was longer in the OGD+TAT-PEP group than that in the OGD group at 3 d after OGD injury(P<0.05; Fig. 4D and E), and the number of branching points on the longest neurite in the OGD+TAT-PEP group was greater than that in the OGD group at 3 d after OGD injury.8. The baseline level of growth cone collapse in the normal cultures was approximately 20%. However, the level of growth cone collapse was increased to 60% at 3 d after OGD injury. Blocking Pir B function using TAT-PEP clearly reduced growth cone collapse(38%)(P<0.05).9. The expression of POSH and Rho A was higher in the OGD group than that in the Normal group at 3 d post-OGD(P<0.05). The GAP43 levels were decreased in the OGD group compared to the Normal group(P<0.05). In contrast, the expression of POSH and Rho A was inhibited by TAT-PEP or pirb RNAi(P<0.05). The GAP43 levels were increased in the OGD+TAT-PEP group or pirb RNAi group compared to the OGD group(P<0.05).Conclusion:In conclusion, TAT-PEP enhanced neurite growth, alleviated growth cone collapse and improved synaptic function after OGD injury. Moreover, TAT-PEP promoted long-term neurobehavioral functional recovery may by enhancing axonal regeneration, corticospinal projections and synaptic plasticity associated with mediated interference with the expression of POSH, Rho A, and GAP43 by resisting Pir B funtion. These studies also suggest that by antagonizing Pir B function, TAT-PEP may represent an attractive therapeutic strategy against stroke in future studies of translational medicine.. Part 5TAT-PEP exerted neuroprotection by alleviating neuronal degeneration and apoptosis after ischemic reperfusion injuryObjective:1. To study the effect of TAT-PEP on neuronal survival and apoptosis in the ischemic penumbra after MCAO.2. To study the effect of TAT-PEP on neuronal ultrastructure in the ischemic penumbra after MCAO. 3. To study the effect and mechanism of TAT-PEP on neuronal survival and apoptosis after OGD injury.Methods:1. Male SD rats weighing 280–320 g. The rats which had been operated successfully were randomly divided into 3 groups(n=6 each): Sham group, MCAO group, MCAO+TAT-PEP group. Nissl, FJC and Neu N staining in the penumbra were measured at 3 d after exposure to MCAO.2. Male SD rats weighing 280–320 g. The rats which had been operated successfully were randomly divided into 3 groups(n=6 each): Sham group, MCAO group, MCAO+TAT-PEP group. TUNEL staining in the penumbra were measured at 3 d after exposure to MCAO.3. Male SD rats weighing 280–320 g. The rats which had been operated successfully were randomly divided into 3 groups(n=6 each): Sham group, MCAO group, MCAO+TAT-PEP group. The ultrastructure of neurons in the penumbra was measured by electron microscopy analysis at 3 d after exposure to MCAO.4. SD rat embryos(E 16.5 d-18.5 d). The cultured primary cerebral cortical neurons were randomly assigned into 6 groups(n=6 each): OGD group, OGD+TAT-PEP(50 μg/L) group, OGD+TAT-PEP(100 μg/L) group, OGD+TAT-PEP(200 μg/L) group, OGD+TAT-m PEP(100 μg/L) group and Normal group. The cell viability and LDH release levels were measured at 24 h after exposure to OGD.5. SD rat embryos(E 16.5 d-18.5 d). The cultured primary cerebral cortical neurons were randomly assigned into 4×3 groups(n=6 each): OGD group, OGD+TAT-PEP(100 μg/L) group, OGD+TAT-m PEP(100 μg/L) group and Normal group. The cell viability andLDH release levels were measured at 6 h, 24 h and 72 h after exposure to OGD.6. SD rat embryos(E 16.5 d-18.5 d). The cultured primary cerebral cortical neurons were randomly assigned into 3 groups(n=6 each): OGD group, OGD+TAT-PEP group and Normal group. TUNEL staining wwas performed at 72 h after exposure to OGD.7. SD rat embryos(E 16.5 d-18.5 d). The cultured primary cerebral cortical neurons were randomly assigned into 3 groups(n=6 each): OGD group, OGD+TAT-PEP group and Normal group. Western blotting of cleaved Caspase3, Bcl-2, and Bax was performed at 72 h after exposure to OGD.Results:1. At 3 d after MCAO, the Nissl staining and Neu N staining was performed in the ischemic penumbra at 3 d after reperfusion. Compared with the MCAO group, the density of normal neurons in the ischemic penumbra in the MCAO+TAT-PEP groups increased significantly(P<0.05). The number of Neu N-positive neurons was more in the MCAO+TAT-PEP group than the MCAO group(P< 0.05).2. At 3 d after MCAO, the number of FJC-positive neurons was fewer in the MCAO+TAT-PEP group than MCAO group(P< 0.05). Similarly, the number of TUNEL-positive cells was fewer in the MCAO+TAT-PEP group than MCAO group(P< 0.05).3. At 3 d after MCAO, the ultrastructure of cortical neurons was characterized by nuclear membrane folds, collapse and blurred boundaries. Additionally, obvious swelling of mitochondria in the cytoplasm was noted, and many mitochondria were spherical in shape. Some mitochondrial cristae were fractured. The damage to the neuronal ultrastructure after TAT-PEP treatment was significantly alleviated, especially in the mitochondria.4. The cell viability reduced decreased significantly in the OGD group compared to Normal group(P<0.05) and LDH release increased significantly in the OGD group compared to Normal group(P<0.05) at 24 h after exposed to OGD. In addition, the cell viability in TAT-PEP treatment group was significantly higher than it in the OGD group or TAT-m PEP group at 24 h after exposed to OGD(P<0.05).5. The LDH release in TAT-PEP treatment group was significantly lower than it in the OGD group at 24 h after exposed to OGD(P<0.05). However, the effect of TAT-PEP between 100 μg/L and 200 μg/L had no difference on cell survival(P>0.05).6. In addition, to confirm the protective effect of TAT-PEP, we also observed the function of TAT-PEP on neurons at different time points after exposed to OGD. The cell viability reduced decreased significantly in the OGD group or TAT-m PEP group compared to the Normal group(P<0.05) and LDH release increased significantly in the OGD group or TAT-m PEP group compared to the Normal group at 6 h, 24 h and 72 h(P<0.05) after exposed to OGD. However, TAT-PEP treatment increased cell viability and decreased LDH release compared to the OGD group or TAT-m PEP group at 24 h and 72 h(P<0.05) after exposed to OGD.7. Then, assaying for cell apoptosis at 72 h after exposure to OGD, the number of TUNEL-positive cells in the OGD group was more than that in the Normal group(P<0.05). In addition, the number of TUNEL-positive cells in OGD+TAT-PEP group was fewer than in the OGD group(P< 0.05).8. The expression of cleaved(active) Caspase3, Bcl-2 and Bax was assessed by western blotting at 72 h after OGD. Caspase3 activity was significantly inhibited in the OGD+TAT-PEP group compared with the OGD group(P<0.05). Treatment with TAT-PEP increased the level of Bcl-2 protein whereas decreased Bax expression compared with OGD group(P< 0.05).Conclusion:In conclusion, TAT-PEP attenuated brain damage by alleviating neuronal degeneration and apoptosis by affecting the expression of apoptosis associated protein. Our findings suggest that TAT-PEP may represent a highly efficacious neuroprotective agent displaying therapeutic potential for stroke. Together, the important implication of this study is that antagonizing Pir B function may represent an attractive therapeutic strategy against stroke in future clinical studies. |
PDF Full Text Request