TAT-PEP alleviated cognitive impairment by alleviating neuronal mitochondria damage and apoptosis after cerebral ischemic reperfusion injury

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In our previous study, a transactivator of transcription-PirB extracellular peptide (TAT-PEP) was generated, which can block the interactions between MAIs and PirB. We found that TAT-PEP treatment enhanced axonal regeneration, CST projection, and improved long-term neurobehavioral functional recovery after stroke through its effects on PirB-mediated downstream signaling molecules. However, the impact of TAT-PEP on cognitive function recovery and neuronal survival also needs to explore. Here, we investigated that pirb RNAi alleviated neuronal injury by inhibiting PirB expression after exposure to oxygen-glucose deprivation (OGD) in vitro. Moreover, TAT-PEP treatment attenuated brain infarct volume and promoted neurobehavioral function and cognitive function recovery. This study further found TAT-PEP exerted neuroprotection by alleviating neuronal degeneration and apoptosis after ischemic reperfusion injury. The study also showed that TAT-PEP enhanced neuronal survival and reduced the release of lactate dehydrogenase (LDH) in vitro . Furthermore, the results indicated TAT-PEP decreased malondialdehyde (MDA) levels, increased superoxide dismutase (SOD) activity, and alleviated reactive oxygen species (ROS) accumulation of neurons exposed to OGD injury. The possible mechanism was TAT-PEP could help neuronal mitochondria damage and affect the expression of cleaved Caspase3, Bax, and Bcl-2. Our findings suggest that PirB overexpression in neurons after suffering ischemic reperfusion injury-induced neuronal mitochondria damage, oxidative stress, and apoptosis. This study also indicated that TAT-PEP might represent a highly productive neuroprotective agent displaying therapeutic potential for stroke by alleviating neuronal oxidative stress, mitochondria damage, degeneration, and apoptosis against ischemic stroke. TAT-PEP cerebral ischemic reperfusion injury cognitive impairment neuronal mitochondria Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Ischemic stroke is a primary cardiovascular and cerebrovascular disease, which is characterized by a high incidence rate, high mortality, and high disability rate[ 1 ]. In particular, about 60% ~ 80% of the survivors have left neurological defects such as cognitive impairment and sensory and motor disorders, which burden society and families heavily [ 2 ]. However, up to now, the mechanism of ischemic stroke is not precise, and the treatment is limited. Therefore, how to promote the recovery of neural function and reduce the disability rate needs to be solved urgently, and it is also a challenging worldwide problem. The pathophysiological process of ischemic stroke is relatively complex. Among them, cerebral ischemia-reperfusion injury can lead to cerebral ischemia and hypoxia, causing damage to brain tissue structure and function, such as neuronal damage, axonal regeneration difficulties, and a decrease of synaptic plasticity, which lead to neurological dysfunction[ 3 ]. One of the critical reasons is that many kinds of myelin-associated inhibitory proteins (MAIs) and their receptors are in the brain. Studies have found that paired immunoglobulin-like receptor B (PirB) is a co-receptor of Nogo-A, Myelin associated glycoprotein (MAG), and Oligodendrocyte myelin glycoprotein (GP)[ 4 – 6 ]. It was reported that the pirb gene knockout mice had smaller cerebral infarction volume than the wild-type mice after focal cerebral ischemia injury[ 7 ]. These results suggest that PirB may be necessary for aggravating neuronal damage during cerebral ischemia-reperfusion injury. Other studies have shown that PirB plays a vital role in cognitive impairment[ 8 ]. In our previous study, we found the expression of PirB significantly increased in neurons in the ischemic penumbra, and the overexpression of PirB deteriorated neuronal apoptosis[ 9 , 10 ]. These pieces of evidence suggest that PirB may be an important target for treating ischemic stroke. Inhibiting the function of PirB could effectively alleviate neuron damage and promote the recovery of neural function. This strategy may be more effective than the single intervention of a ligand, which will bring new hope for treating ischemic stroke. The extracellular segment of PirB mainly contains six immunoglobulin-like domains[ 11 ]. It was found that Nogo-A and MAG could combine with their extracellular components to activate the downstream signal pathway of PirB [ 6 ]. Therefore, if the function of PirB can be suppressed at the protein level, it will be instrumental in the recovery of neural function after cerebral ischemia-reperfusion injury. In our previous studies, a transactivator of transcription-PirB extracellular peptide (TAT-PEP) was generated, which displayed a high affinity for MAIs and ameliorated their inhibitory effect on neurite growth. Furthermore, TAT-PEP can widely distribute in the penumbra after intraperitoneal injection[ 9 ]. Then, we found that TAT-PEP enhanced neurite growth and alleviated growth cone collapse after oxygen-glucose deprivation (OGD) injury[ 8 , 9 ]. However, exploring whether TAT-PEP could enhance cortical neuronal survival and promote cognitive function recovery after the transient focal cerebral ischemia model needs to be explored. In this study, the GPH-PIRB-294 lentivirus system was constructed to interfere with PirB expression and observe the effect of inhibiting PirB expression on neuronal survival after OGD injury. We used the middle cerebral artery occlusion (MCAO) model to evaluate the impact of TAT-PEP on the improvement of neural function and cerebral infarct volume after cerebral ischemia-reperfusion injury through neurobehavioral methods and MRI imaging technology. Through in vivo and in vitro experiments, we further analyzed the effect of TAT-PEP on neuronal survival, oxidative stress, degeneration, and apoptosis after cerebral ischemia-reperfusion injury and explored the relevant mechanisms to clarify the role and mechanism of TAT-PEP on neuronal survival during cerebral ischemia-reperfusion injury. Materials And Methods Animals Adult male Sprague-Dawley (SD) rats (280 ± 20 g) housed under controlled conditions with a 12-h light/dark cycle, a temperature of 21 ± 2°C, and humidity of 60–70% for at least 1 week before drug treatment or surgery, purchased from the Experimental Animal Center of the Fourth Military Medical University (Xi’an, Shaanxi, China) and the Laboratory Animal Center of Xi’an Jiaotong University (Xi’an, Shaanxi, China). It was approved by the Ethics Committee for Animal Experimentation of the Fourth Military Medical University and by the Institutional Animal Care and Use Committee at Xi’an Jiaotong University. Every effort was made to minimize stress to the animals. All sample sizes for the assessment parameters were calculated to reduce the number of animals used. Transient Focal Cerebral Ischemia Mode Focal cerebral ischemia was induced in the rats by middle cerebral artery occlusion (MCAO), as described previously[ 12 , 13 ]. The experimental animals were randomly divided into 3 groups, with 10 animals in each group. Because the success rate of the MCAO model is about 70%, the actual number of animal samples used in each group is 8 ~ 6 (n = 8 ~ 6 for each group). The three groups were divided into the Sham group, the MCAO group, and the MCAO + TAT-PEP group. In the MCAO + TAT-PEP group, 1.0 mg/kg TAT-PEP was intraperitoneally injected immediately after reperfusion, and then TAT-PEP of this concentration was injected daily. Garcia scores At 24h, 48h, and 72h after MCAO, the modified Garcia scores [ 14 ] were used to detect the neurological function of experimental animals. It contains 18 points, including 0–3 points for each item, as shown below. Free activity in a cage for 5 min: no activity (score 0); Almost unable to move (score 1 point); Able to move, but the range of movement does not reach 3 sides in the cage (2 points); The field of activity shall get at least 3 sides inside the enclosure (3 points). The symmetry of limb movement: no movement of the left limb (0 points); The left limb can move slightly (1 point); The left limb can move slowly (2 points); Bilateral limb movements are symmetrical (3 points). The symmetry of forelimb (forelimb extension when lifting the tail): the left limb is unable to move (0 points); The left limb can only be slightly extended (1 point); The left side is not as active and stretched as the right side (2 points); Bilateral forelimbs can be tested symmetrically (3 points). Climbing in the metal cage: None (0 points); Unable to mount (1 point); The left side is slightly weak (score 2 points); Can usually climb (3 points). Touch bilateral trunk reaction: none (0 points); No response on the left side (score 1 point); Weak left side reaction (score 2 points); The answer is symmetrical (3 points). Whisker reaction: None (0 points); No response on the left side (score 1 point); Weak left side reaction (score 2 points); The reaction is symmetrical (3 points). Y-maze test The Y-maze test was used to assess spatial learning and memory as described previously [ 15 ]. Experimental rats were placed in a Y-shaped maze (arm's length: 16 cm, arm width: 10 cm, height of the wall: 40 cm, Yihong Technology Co., Ltd., Wuhan, China) with three arms at 120° from each other and allowed to explore the three arms for 10 min freely. Before all experiments, the rats should be used for acclimatization for 2 days, during which the rats can visit all components freely for 10 minutes. The number of arm entries and alterations were recorded automatically using Smart Video Tracking Software 3.0 (Panlab, Barcelona, Spain). The percentage of spontaneous alternation was calculated as the number of correct alterations (number of total new arm entries), which is associated with the capacity of spatial short-term memory. Novel object recognition test (NOR) The novel object recognition test is widely used in the study of cognitive impairment[ 16 ]. First, to reduce stress levels, all groups of rats were placed in the experimental room and testing box for 10 min on the 2 successive days before the training phase. At 3 d post-MCAO, rats were trained to explore freely within a box (Yihong Technology Co., Ltd., Wuhan, China). The experimental method was described previously. The experiment was r recorded with a video camera (SNC-VB600B5, SSGE, Shanghai, China) cation placed on the old object. The exploration time ratio for novel objects during the testing phase was assessed for each group. TTC staining At 3 d after MCAO, TTC staining was used to detect the effect of TAT-PEP on cerebral infarction volume. The straightforward method is as follows[ 17 ]: The brain was removed under deep anesthesia and immersed in normal saline ice for 10 minutes. Put it into the cerebral sulcus mold, and the thickness of the brain slice is about 2 mm (cut in sequence along the coronal plane). Soak the brain slices in a 2% concentration of a 2,3,5-TTC solution with the same orientation, and incubate them in a constant temperature water bath (37 ℃, 30 min). After the staining effect is appropriate (the white area is the infarcted area, and the red area is the normal brain tissue area), transfer to 4% PFA for fixation overnight, take photos and analyze 24 hours later. Adobe photoshop cs3 image processing software was used to calculate the volume of cerebral infarction. Magnetic resonance imaging (MRI) detected cerebral infarction volume. At 3 d after MCAO, the small animal MRI imaging system was used to detect the effect of TAT-PEP on cerebral infarction volume. The straightforward method follows: After anesthesia, the rats were placed in a prone position with their heads in the center of the coil. The rapid acquisition relaxation enhancement sequence (along the coronal section) was used to scan with the visual intersection as the origin. The parameters are an echo time (TE) of 60 ms, a repetition time (TR) of 3000 ms, a thickness of 0.5 mm, and no spacing. The field of view is 2.56 cm × 2.56 cm. Acquire T2 weighted (T2WI) images, and then use image software for image analysis to measure the volume of ischemic injury area (i.e. abnormal high signal area), which is equal to the area of abnormally high signal area × the sum of slice thickness (plus spacing), was used to evaluate the cerebral infarction volume. Nissl staining Nissl staining (Beyotime Institute of Biotechnology, China) was performed to detect Nissl bodies in the cytoplasm of surviving neurons. At 3d after MCAO, Nissl staining was used to detect the effect of TAT-PEP on the survival of neurons in the penumbra. The methods are described as follows: Dry the slices at room temperature for 1 h, draw circles, and gently rinse them with 0.01M PBS 3 times (5 min/time). Add Nissl dyestuff (to filter in advance), and incubate at 37 ℃ for 20 min. Then gently wash off with pure water. The sections were dehydrated and transparent before using the neutral resin sealed. The microscope was used to observe and record in the open field. The integrated optical density/area of the staining in each group was acquired by 2 blinded investigators using ImagePro Plus 5.1 software (Media Cybernetics, Inc., Bethesda, MD). FJC staining At the 3d after MCAO, FJC staining was used to detect the effect of TAT-PEP on the number of degenerated neurons in the penumbra. The specific methods are as follows: Freeze the slices and dry them at room temperature for 1 h, draw circles, and gently rinse them with 0.01M PBS 3 times (5 min/time). Immerse the slices in a mixture of 1% sodium hydroxide and 80% anhydrous ethanol for 5 min. Then immerse it in 70% absolute ethanol, hydrate it for 2 min, and gently rinse it with deionized water 3 times (5 min/time). Incubate with 0.06% potassium permanganate for 10 min (shaking table at room temperature) and rinse with distilled water for 5 min (shaking table). Prepare 0.0001% FJC dye solution, and add acetic acid in the proportion of 1:1000. After dropping FJC dye solution, incubate it at 37 ℃ at constant temperature and away from light for 30 min, and gently rinse it with deionized water 3 times (1 min/time). Dry in the dark at room temperature, dehydrated with anhydrous ethanol for 2 min, make xylene transparent 3 times (2 min/time), and apply neutral gum film. The people who did not know the experimental group were observed by Olympus BX51 fluorescence microscope (excitation light wavelength was 450–490 nm), collected images, and recorded the number of FJC positive cells (FJC positive cells were green). Immunofluorescence histochemical staining On the 3rd day after MCAO, NeuN immunofluorescence histochemistry was used to detect the effect of TAT-PEP on the number of living neurons in the penumbra. The specific method is the same as that in Part I. First antibody (NeuN, 1:5000) was added, 4 ℃ overnight, 0.01M PBS solution was used for rinsing (5 min each time × 3 times), inject the secondary antibody (goat anti-mouse FITC, 1:5000), incubated at room temperature in the dark for 2 hours, rinse in the night for 3 times, observe and take photos under a fluorescence microscope after 50% glycerine film is sealed. TUNEL staining At the 3d after MCAO, TUNEL staining was used to detect the effect of TAT-PEP on the number of apoptotic neurons in the penumbra. The specific methods are as follows: The fixed brain tissue was immersed in 30% alcohol, embedded in paraffin, and sliced, with a thickness of 3 µ m. Paraffin sections were dewaxed with xylene for 5 min (2 min/time). After waxing, use a circle drawing brush to draw circles, drip protein kinase K cell permeating solution, incubate at 37 ℃ for 30 min, and then rinse gently with 0.01M PBS 3 times (5 min/time). Join 50 µ L DNase1 solution at room temperature for 10 min. After the slides are cleaned and dried, add the Tunel mixture. 37℃, 1 h. PBS cleaning again. Joining 30 µ L POD stops the reaction, and PBS has cleaned again. Drop the DAB color-developing solution at room temperature for 10min, then conduct PBS cleaning again. Drop hematoxylin for 30 s and gently wash it under pure water. Gradient dehydration, xylene transparent for 5min (twice), neutral resin film, open field microscope observation, and statistics of the number of Tunel positive cells (Tunel positive cells are brown black). Electron microscope experiment Experimental animals were anesthetized intraperitoneally with 0.3 ml/100g chloral hydrate (300 mg/kg), perfused and fixed with a solution containing 4% PFA and 0.05% glutaraldehyde, positioned on a stereotaxic device, and fixed for 2 hours. Coronal sections were made with a vibrating microtome with a thickness of 50 µ m and immersed in PBS solution containing 30% sucrose for 2 h. Dehydrated with gradient alcohol and Put the transparent continuous sections on copper mesh with supporting membrane, and observed the ultrastructure of neurons under the electron microscope. Primary culture of cortical neurons Primary cortical neurons were cultured as described previously[ 17 ]. SD rats who were pregnant 16.5–18.5 days (E 16.5–18.5 days) were dislocated and killed. A routine aseptic operation was carried out. Fetal rats were taken out and separated. In the D-Hank's solution (placed on ice), the fetal rat was decapitated with toothless ophthalmic tweezers, and the brain tissue was taken out. Under the microscope, the cerebral cortex was separated. The tissue was chylated through D-Hank's solution. Then trypsin (0.125%) was digested for 15 min (in a regular incubator at 37 ℃). Then, the tissue was carefully sucked out with an elbow dropper, added into a centrifuge tube (15 ml) containing DMEM solution of fetal bovine serum, and the digestion was stopped at room temperature for 5 minutes. Place the centrifuge tube in the centrifuge at 80 rpm for 5 min, discard the supernatant carefully, add 2 ml of DMEM solution containing fetal bovine serum, blow and repeatedly beat to prepare tissue and cell suspension, and use the cell filter (100 µ m) Filtering. After another centrifugation at 80 rpm for 5 min, it was added to the neuron culture medium (Neurobasal A, 2% B27, 1% glutamate, and 1% penicillin mixture). According to the experimental requirements, different-density cells were inoculated into 96 well plates, 24 well plates, or 6 well plates coated with poly-L-lysine (50 mg/mL) (Sigma, USA). These cells were grown in Neurobasal medium (Gibco, Invitrogen Corp, USA) supplemented with 2% B27, 1% glutamine, and 1% penicillin/streptomycin (Sigma, USA) at 37℃ under a humidified incubator in air containing 5% CO 2 . The purity of neurons was determined by immunocytochemistry for bIII-tubulin at 5-day after plated, which indicated that 95% of the cells in cultures were positive for βIII-tubulin (1:250; Millipore, Temecula, CA, USA) (data not shown) Oxygen glucose deprivation model of neurons in vitro The primary cultured cortical neurons were identified on the 7th day. Replace the culture medium (sugar-free and serum-free) and put it into the hypoxia constant temperature incubator (37 ℃, 5% CO 2 , 95% N 2 ) for 1 h. Then carefully take out the culture dish or culture bottle, replace it with standard neuron culture medium (Neurobasal A, 2% B27, 1% glutamate, and 1% penicillin mixture), and place it in the traditional incubator (5% CO 2 , 21% O 2 , 37 ℃). After re-oxygenation and re-sugar, they were immediately given TAT-PEP or TAT mPEP. The experimental groups are as follows: ① MTT and LDH release tests [ 17 ] were used to detect the effect of TAT-PEP at different concentrations on the survival of neurons at 24 h after OGD. They were randomly divided into different groups (n = 6): Normal group, OGD group, OGD group, OGD + TAT mPEP (100 µg/L) group, OGD + TAT-PEP (50 µg/L) group, OGD + TAT-PEP (100 µg/L) group, OGD + TAT-PEP (200 µg/L) group. ② MTT and LDH release tests were used to detect the effect of TAT-PEP on neuronal survival after OGD (6h, 24h, 72h). They were randomly divided into different groups (n = 6): Normal group, OGD group, OGD + TAT mPEP (100 µg/L) group, and OGD + TAT-PEP (100 µg/L) group. ③ MDA level and SOD activity [ 18 ] was used to detect the effect of TAT-PEP on neuronal oxidative stress at 72 h after OGD. They were randomly divided into groups (n = 6): The normal group, OGD group, and OGD + TAT-PEP group (TAT-PEP was 100 µg/L). ④ TUNEL staining was used to detect the effect of TAT-PEP on neuronal apoptosis at 72 h after OGD. They were randomly divided into different groups (n = 6): The normal group, OGD group, and OGD + TAT-PEP group (TAT-PEP is 100 µg/L). ⑤ Western blot was used to detect the effect of TAT-PEP on the expression of apoptosis-related proteins at 72 h after OGD. They were randomly divided into different groups (n = 6): The normal group, OGD group, and OGD + TAT-PEP group (TAT-PEP is 100 µg/L). Western blot According to the time node of the experiment, the whole cell protein was extracted. The method follows: After washing PBS three times, add precooled Lysis cracking solution (100 µ L/well), carefully scrape the cells, and thoroughly mix the lysate with the cells. Transfer the suspension into the precooled EP tube, ice bath for 30 min, and centrifugation at 4 ℃ for 15 min, 12000 rpm. The total protein concentration of the tissues or cells was analyzed with a BCA kit (Sigma, CA, USA). Rabbit antibody against cleaved (active) caspase-3 (1:1,000; Cell Signaling Technology, Beverly, MA, USA), mouse monoclonal antibodies against Bcl-2 or Bax (1:1,000, Santa Cruz, CA, USA), and β-actin (1:2,000, Anbo, USA). Subsequently, the blots were probed with horseradish peroxidase (HRP)-conjugated goat secondary antibody against rabbit or mouse IgG (1:1,000, Abcam, USA). Detection and quantitation were performed with a Typhoon 9400 Variable Mode Imager (GE Healthcare) and Lumi-Light Western Blotting Substrate (Roche Diagnostics) for HRP-labeled blots. TUNEL staining TUNEL staining was performed in vitro using an In Situ Cell Death Detection Kit (Roche Diagnostics, Mannheim, Germany). At 72 h after OGD injury, 4% PFA was added to fix at room temperature for 1 h. 0.01M PBS was gently rinsed 3 times (5 min/time), and then 0.3% hydrogen peroxide was used for 10 min. Add TUNEL reaction mixture for 1 h at 37 ℃ and stain with DAPI for 5 min at room temperature. Images were obtained with a microscope (BX60, Olympus). The integrated optical density/area of the positive TUNEL staining in each group was acquired as described above. Statistical Analysis All data are presented as the mean ± SD across the groups and were statistically analysed using GraphPad Prism 7.0 software (GraphPad Company, San Diego, CA, USA). Continuous data were tested for normal distribution and analysed by one-way ANOVA (followed by Tukey’s multiple comparisons tests) or Kruskal–Wallis test (followed by Dunn’s multiple comparisons tests). Two-way ANOVA was applied to analyse the neural dendritic complexity. P value of less than 0.05 was considered statistically signifcant. Results TAT-PEP improved neurobehavioral function and cognitive function against MCAO We used Garcia scores to analyze neurobehavioral function recovery. As shown in Fig. 1 A, the scores of the TAT-PEP treatment group were higher than that of the Sham group at 48 h (P < 0.05) and 72 h (P < 0.05) after reperfusion. We used the Y-maze test to analyze cognitive function recovery. At 3 d after reperfusion, the percent of spontaneous alteration and total arm entries in the Y-maze test were measured. Rats of the MCAO group showed significantly lower spontaneous alternation rates than rats of the Sham group in the Y maze test. In contrast, in the Y maze test, rats of the MCAO + TAT-PEP group showed substantially higher spontaneous alternation rates than rats of the MCAO group (Fig. 1 B/D). Also, the recognition index and total travel distance in the novel object recognition test were measured. Rats of the MCAO group spent less time exploring a novel object during the test phase than the Sham group rats. In contrast, the MCAO + TAT-PEP group spent more time exploring a novel thing during the test phase than the rats of the MCAO group (Fig. 1 E/F). These results indicated that TAT-PEP could promote neurological and cognitive functional recovery. TAT-PEP reduced brain infarct volume against MCAO Then, we tested the brain infarct volume in every group. Figures 2 A and B show that the MCAO + TAT-PEP group displayed a significantly smaller brain infarct volume than the MCAO group by TTC staining at 3 d post-MCAO ( P < 0.05). MRI measured infarction volumes. T2WI analyses showed that the high-intensity books were more prominent in the MCAO group than in the Sham group at 3 d post-MCAO. In contrast, T2WI studies showed that the high-intensity volumes were smaller in the MCAO + TAT-PEP group than in the MCAO group at 3 d post-MCAO (P < 0.05). TAT-PEP attenuated neuronal degeneration, apoptosis, and mitochondria damage against MCAO To assess neuroprotection of TAT-PEP, the Nissl staining and NeuN staining were 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 group increased significantly (P < 0.05) (Fig. 3 A/B). The number of NeuN-positive neurons was more in the MCAO + TAT-PEP group than that in the MCAO group (P < 0.05) (Fig. 3 C/D). Then, the FJC and TUNEL staining on ischemic brain sections was performed at 3 d after reperfusion. The number of FJC-positive neurons was fewer in the TAT-PEP group than in the MCAO group (P < 0.05). Similarly, the number of TUNEL-positive cells was more irregular in the TAT-PEP group than that in the MCAO group (P < 0.05) (Fig. 4 A-D). At 3 d after reperfusion, the ultrastructure of cortical neurons in the MCAO group showed nuclear membrane folds, collapse, and blurred boundaries. Additionally, noticeable swelling of mitochondria in the cytoplasm was noted, which was spherical. Some mitochondrial cristae were fractured. The damage to the neuronal ultrastructure after TAT-PEP treatment was significantly alleviated, especially in the mitochondria (Fig. 5 ). Inhibiting PirB overexpression was beneficial to rescue neurons from apoptosis exposed to OGD injury. The survival of cultured neurons was examined at 72 h after PirB RNAi vector or Control RNAi transfection. The neuronal structure in the OGD group and OGD + Control RNAi group was damaged. However, transfection with the PirB RNAi vector prevented 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 than that of the OGD and Control RNAi groups (P < 0.05) (Fig. 6 A/B). 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) (Fig. 6 C/D). These results indicated that inhibiting PirB overexpression was beneficial in rescuing the neurons from apoptosis. TAT-PEP enhanced the survival of neurons exposed to OGD injury Here, we observed the function of different doses of TAT-PEP on neuronal survival at 24 h after being exposed to OGD (Fig. 7 A/B). The cell viability decreased significantly in the OGD group compared to the Normal group (P < 0.05), and LDH release (P < 0.05) increased substantially in the OGD group compared to the Normal group (P < 0.05) at 24 h after exposure to OGD. In addition, the cell viability in TAT-PEP (50 µg/L, 100 µg/L, 200 µg/L) treatment group was significantly higher than it was in the OGD group or TAT-mPEP (misordered Sequences of the extracellular cDNA of PirB) group at 24 h after exposed to OGD (P < 0.05). The LDH release in the TAT-PEP treatment group was significantly lower than that in the OGD group at 24 h after being exposed to OGD (P < 0.05). The results further showed that compared with the 50 µg/L TAT-PEP treatment group, the cell viability was higher, and LDH release was lower in the 100 µg/L and 200 µg/L TAT-PEP treatment groups (P 0.05). 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 being exposed to OGD (Fig. 7 C/D). The cell viability decreased significantly in the OGD group or TAT-mPEP group compared to the Normal group (P < 0.05), and LDH release (P < 0.05) increased considerably in the OGD group or TAT-mPEP group compared to the Normal group at 6 h (P < 0.05), 24 h (P < 0.05) 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-mPEP group at 24 h (P < 0.05) and 72 h (P < 0.05) after exposure to OGD. At 24 h after OGD injury, the growth state of neurons was observed under the light microscope. It was found that the integrity of neurons in the OGD group was poor. The cell bodies were atrophied or broken. The neurites were broken or disappeared. However, TAT-PEP treatment could significantly improve the growth of neurons (Fig. 7 E). The above results indicate that TAT-PEP can substantially enhance the activity of neurons, reduce neuronal damage, and promote neuronal survival. TAT-PEP decreased MDA levels, increased SOD activity, and alleviated ROS accumulation of neurons exposed to OGD injury MDA, SOD, and ROS are regarded as indicators for detecting oxidative stress. The MDA content in the OGD group increased compared with that in the Normal group at 72 h exposed to OGD injury (P < 0.05). TAT-PEP treatment attenuated the elevation of MDA content compared with that in the OGD group at 72 h exposed to OGD injury (P < 0.05) (Fig. 8 A). Compared with the Normal group, SOD activity was reduced in the OGD group at 72 h exposed to OGD injury (P < 0.05). TAT-PEP treatment increased the SOD activity compared with that in the OGD group at 72 h exposed to OGD injury (P < 0.05) (Fig. 8 B). Furthermore, the flow cytometry result showed that ROS levels were upregulated in the OGD group compared with the Normal group at 72 h exposed to OGD injury (P < 0.05). In contrast, the ROS levels were lower in the OGD + TAT-PEP group compared with that in the OGD group at 72 h exposed to OGD injury (P < 0.05) (Fig. 8 C). TAT-PEP alleviated neuronal apoptosis exposed to OGD injury Then, assaying for neuronal 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 the OGD + TAT-PEP group was fewer than in the OGD group (P < 0.05) (Fig. 9 A/B). The expression of cleaved (active) Caspase3, Bcl-2, and Bax was assessed by western blot 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 it decreased Bax expression compared with the OGD group (P < 0.05) (Fig. 9 C-E). Discussion Although the progress of prevention and treatment technologies such as acute stroke, brain trauma, and perioperative brain injury have controlled the mortality to a certain extent, about 60% ~ 80% of the survivors will leave neural function defects such as cognitive impairment, sensory disorder, motor disorder, and communication disorder to varying degrees, which will seriously affect the quality of life of patients and lead to heavy family burden [ 1 , 19 ]. Especially in ischemic stroke, the process of its onset is often accompanied by complex pathophysiological processes, leading to long-term cognitive impairment. This neurological dysfunction is due to the damage to the structure and function of the cerebral cortex caused by ischemia-reperfusion injury. Neurons are the basic unit of the structure and function of the central nervous system. The neurons are susceptible to ischemia and hypoxia and are prone to degeneration, necrosis, and apoptosis, which has been confirmed in previous experiments [ 17 , 20 ]. Since Astrup defined the reversible damage beyond the central necrotic area of focal cerebral ischemia as the ischemic penumbra (IP) in 1981, IP has been used clinically as an area for the treatment of ischemic cerebrovascular disease [ 21 ]. This is because the IP area develops to irreversible damage slowly, which takes several hours or even days, and neuronal death is mainly apoptosis[ 12 ]. Therefore, reducing the damage of neurons in the penumbra and promoting the survival of neurons is the key to effectively improving neural function after cerebral ischemia-reperfusion injury. PirB is a co-receptor of Nogo-A, MAG, and OMgp, which plays an essential role in axon growth, synaptic plasticity, and neuronal survival[ 5 ]. In the previous study, we found the mRNA and protein levels of PirB in the model of cerebral ischemia-reperfusion injury in mice were highly expressed in the ischemic injury area, suggesting that it may play an important role in the pathological process of cerebral ischemia-reperfusion injury[ 9 ]. This persistent and significant overexpression phenomenon means that PirB may be a potentially important target for treating cerebral ischemia-reperfusion injury. In the previous study, we first designed and synthesized constructed the transactivator of transcription-PirB extracellular peptide (TAT-PEP), which blocked the interaction between PirB and its ligands, and abolished the inhibitory activity of Nogo-A, MAG, and OMgp on axonal regeneration. Moreover, TAT-PEP treatment enhanced axonal regeneration and CST projection and improved motor functional recovery after stroke[ 9 ]. In the present study, our work further found that TAT-PEP reversed cognitive dysfunction after ischemic stroke by Y maze and novel object recognition test. The neuroprotection of TAT-PEP also suggests that PirB plays an essential role in negatively regulating cognitive function after ischemic stroke. The process of PirB in cognitive function is also consistent with the recent research report that PirB is involved in diabetes-associated cognitive dysfunction through modulation of axon outgrowth and dendritic remodeling, providing a potential therapeutic target for cognitive dysfunction [ 22 ]. Another research also showed PirB is associated with aging, and TAT-PEP may be a promising therapeutic agent for the modulation of age-related motor and cognitive dysfunctions[ 23 ]. These results indicated TAT-PEP could alleviate PirB-associated cognitive dysfunction, similar to our findings. However, there may be other mechanisms that need to be studied. How TAT-PEP improved cognitive impairment after an ischemic stroke still needs to be clarified. This study found that TAT-PEP could significantly reduce cerebral infarction volume at three after MCAO. Especially the degree of cortex damage was reduced considerably. Adelson found that the cerebral infarction volume of pirb KO mice was significantly reduced seven days after MCAO compared with pirb WT mice[ 24 ], which confirmed the above results. Then, our striking finding showed TAT-PEP significantly reduced the number of degenerated and apoptotic neurons in the cerebral cortex's penumbra, improved the neurons' ultrastructure, and effectively promoted the survival of neurons. This may be an actual reason for TAT-PEP to effectively reduce the volume of cerebral infarction and promote the recovery of cognitive function. In the previous study, our research group using the global cerebral ischemic reperfusion injury model confirmed that inhibiting the function of PirB significantly reduced the apoptosis of neurons in the ischemic hippocampus area and promoted the survival of neurons[ 8 ]. Clearly, PirB played a vital role in the process of neuronal damage after cerebral ischemia-reperfusion. The above results further confirmed that PirB might be an important reason for hindering neuronal survival and functional recovery after cerebral ischemia. It is a crucial target for treating ischemic stroke. As mentioned above, we found that TAT-PEP can play a neuroprotective role, which may be related to its ability to reduce the degeneration and apoptosis of neurons in the cerebral ischemic injury area. Our previous study found that PirB overexpression exacerbated neuronal apoptosis by oxidative stress and mitochondria damage[ 10 ]. To further clarify the neuroprotection and mechanism of TAT-PEP, we conducted primary neuron culture and used the OGD model in vitro. The results showed that interfering with neuronal PirB expression or TAT-PEP significantly enhanced the activity of neurons after OGD, reduced the release of LDH, and alleviated neuronal damage. Furthermore, TAT-PEP decreased MDA levels, increased SOD activity, and helped ROS accumulation of neurons exposed to OGD injury. These results proved PirB participated in neuronal apoptosis caused by oxidative stress after oxygen and glucose deprivation. Inhibiting the function of PirB used, TAT-PEP alleviated oxidative stress and neuronal damage. Although the effects of TAT-PEP on neuronal oxidative stress and apoptosis are still controversial, in our study, the results indicated TAT-PEP decreased MDA level and increased SOD activity, which inhibited lipid peroxidation. TAT-PEP also reduced ROS accumulation which has been suggested to be required for neuronal apoptosis. Lipid peroxidation and ROS accumulation are closely related to the damage of plasma membrane structure in cells. Especially the production of a large number of oxygen free radicals and the lipid peroxidation of a mitochondrial membrane can cause mitochondrial damage and lead to apoptosis. In our vivo study, the ultrastructural changes of mitochondria also confirmed this point. To sum up, our results supplied the evidence to explain the potent neuroprotective effects of TAT-PEP against neuronal oxidative stress, mitochondrial damage, and apoptosis after ischemic stroke. To further explore the relevant mechanisms, we found that TAT-PEP reduced the number of apoptotic neurons after OGD, inhibited the expression of apoptotic protein Bax, promoted the expression of anti-apoptotic protein Bcl2, and inhibited the activation of Caspase3. Previous studies have shown that the damage of neurons in the penumbra of cerebral ischemia is mainly regulated by apoptosis[ 25 ]. One of the mechanisms of neuronal apoptosis in ischemic penumbra is due to mitochondrial damage related to lipid peroxidation and ROS accumulation[ 26 ]. It has been reported Caspase-3 is known that the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2 can migrate from the cytoplasm to mitochondria, which are distributed in a manner that is consistent with the mitochondrial release of cytochrome C and caspase[ 20 , 27 ]. The mitochondrial apoptotic pathway plays an essential role in neuronal injury [ 28 , 29 ], and TAT-PEP inhibited neuronal apoptosis after ischemic stroke by upregulating Bcl-2 expression and reducing Bax expression. The apoptotic proteins on the mitochondrial membrane, such as Bad and Bax, lead to the opening of the mitochondrial permeability transition pore and the release of cytochrome C or apoptosis factors. The activated Caspase3 cleaves DNA repair enzymes, thus leading to DNA damage and apoptosis[ 30 ]. However, this study has yet to directly clarify how PirB regulates the expression of Bax and Bcl-2 after cerebral ischemia-reperfusion injury. We observed the ultrastructure of neurons that the nucleus and mitochondria of neurons in the penumbra were significantly damaged after cerebral ischemia-reperfusion injury. It may be that PirB affected the expression of Bax and Bcl-2, leading to the opening of the mitochondrial permeability transition pore, thus activating Caspase3. However, the specific mechanism and signal pathway must be further confirmed. Studies have shown that interfering with the binding of NgR1 and Nogo-A can effectively inhibit the expression of Bax, promote the expression of Bcl-2, and thus inhibit the activation of Caspase3[ 31 ]. However, whether TAT-PEP interferes with Nogo-A and PirB signal pathways, thereby affecting the expression of Bax, Bcl-2, and activated Caspase3, and playing a neuroprotective role, still needs further study. It is noteworthy that a previous study reported that the increased PirB labeling was localized to astrocytes and neurons in Lipopolysaccharide (LPS)-treated animals. The hippocampus-dependent spatial learning and memory were impaired[ 7 ]. Another study also showed PirB was highly expressed in neurons and astrocytes in the model of epilepsy[ 32 ]. It has been reported that astrocytes are mainly responsible for inflammatory injury and cognitive impairment of Alzheimer's disease (AD)[ 33 ]. However, our study did not explore the expression of PirB in astrocytes and the effect of TAT-PEP on astrocytes after ischemic stroke, which remains to be explored in future research. In conclusion, our study showed TAT-PEP improved neurobehavioral function and cognitive function by reducing brain infarct volume and attenuating neuronal degeneration, apoptosis, and mitochondria damage against MCAO. In vitro study showed that TAT-PEP enhanced neuronal survival and alleviated apoptosis by decreasing MDA levels, increasing SOD activity, and helping ROS accumulation. Furthermore, the results revealed that TAT-PEP alleviated neuronal apoptosis by affecting the expression of apoptosis-associated proteins, such as cleaved Caspase3, Bcl-2, and Bax. Together, our findings suggest that TAT-PEP may represent a highly productive neuroprotective agent displaying therapeutic potential for ischemic stroke. Notably, this study implies that antagonizing PirB function may define an attractive therapeutic strategy against neuronal apoptosis after ischemic stroke in future studies. Abbreviations PirB Paired immunoglobulin-like receptor B MAIs myelin-associated inhibitory proteins TAT-PEP transcription-PirB extracellular peptide OGD oxygen-glucose deprivation LDH lactate dehydrogenase MDA malondialdehyde SOD superoxide dismutase ROS reactive oxygen species MAIs myelin-associated inhibitory proteins GP glycoprotein MAG myelin associated glycoprotein MCAO middle cerebral artery occlusion NOR novel object recognition test MRI magnetic resonance imaging T2WI T2 weighted images Declarations Acknowledgments We thank Prof. Xingchun Gou (Shaanxi Key Laboratory of Brain Disorders & Institute of Basic and Translational Medicine, Xi’an Medical University, Xi’an, China) and Prof. Lixian Xu (State Key Laboratory of Military Stomatology, National Clinical Research Center for Oral Diseases, Shaanxi Engineering Research Center for Dental Materials and Advanced Manufacture, School of Stomatology, Air Force Medical University, Xi'an, Shaanxi, China) for the guidance given in project design and experiment. Author Contributions BD, PZ, and Q W designed the project. PZ, JP Z, JK K, LY L, XY L and BD contributed to the data analysis and the manuscript's drafting. PZ, JK K, NM F, HL D, CL, and BD conducted all experiments. PZ, JP Z, JK K, LY L, XY L, NM F, HL D, CL, and BD participated in discussions, data analysis, and manuscript editing. All authors have read and approved the final version of the manuscript. Funding This work was partially supported by the Medical “Basic-Clinical” Integration Innovation Project of Xi’an Jiaotong University (Program No. YXJLRH2022030), the fund of the First Affiliated Hospital of Xi’an Jiaotong University (Program No. 2022MS-26), the Key Research & Development Program of Shaanxi (Program No. 2022ZDLSF02-09), the General project of Fujian Natural Science Foundation (No. 2021J01018), the Fujian Health Science and Technology program (2021GGB038). Data Availability The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Code Availability GraphPad Prism Version 7.0; ImageJ Version 1.51 J; Imaris Version 9.7. Ethics Approval All experimental procedures were approved by the Ethics Committee of the Fourth Military Medical University, and by the Institutional Animal Care and Use Committees of Xi’an Jiaotong University (Xi’an, China; 2019–060) in accordance with the National Institutes of Health guidelines. Efforts were made to minimize animal sufering, and all sample sizes for the assessment parameters were calculated to minimize the number of animals used. Consent to Participate Not applicable. Consent for Publication Not applicable. Competing Interests The authors declare no competing interests. References Mendelson SJ, Prabhakaran S (2021) Diagnosis and Management of Transient Ischemic Attack and Acute Ischemic Stroke: A Review. 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Eur J Pharmacol 806:1–9. https://doi.org/10.1016/j.ejphar.2017.03.025 Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Published Journal Publication published 19 Jun, 2023 Read the published version in Molecular Neurobiology → Version 1 posted Reviewers agreed at journal 22 Jan, 2023 Reviewers invited by journal 15 Jan, 2023 Editor invited by journal 08 Jan, 2023 Editor assigned by journal 08 Dec, 2022 First submitted to journal 07 Dec, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2327876","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":158401487,"identity":"fb8185de-7604-498a-8166-db310feac51e","order_by":0,"name":"Pin 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05:40:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2327876/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2327876/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12035-023-03404-w","type":"published","date":"2023-06-19T21:19:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":30221160,"identity":"a40a0944-b472-45b0-ba09-2734032b709b","added_by":"auto","created_at":"2022-12-12 18:28:21","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":127972,"visible":true,"origin":"","legend":"\u003cp\u003eTAT-PEP enhanced neurobehavioral functionand cognitive function recovery. (A) Garcia scores tested the effect of TAT-PEP on neurobehavioral function recovery of experimental animals in each group at 24h, 48h, and 72h after MCAO (n=8, each group; * P\u0026lt;0.05 vs. Sham group; # P\u0026lt;0.05 vs. MCAO group). (B, C) The recognition index and total travel distance in the novel object recognition test were measured (n=6). (D, E) Representative Y maze test and novel object recognition test trajectories of every group of rats. (F, G) Percent of spontaneous alteration and total arm entries in the Y-maze test were measured (n=6).123\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2327876/v1/ba6afda2d564204e6c2509fa.jpg"},{"id":30221159,"identity":"b6e00b1a-ea62-403d-8bf6-e6995f88f13b","added_by":"auto","created_at":"2022-12-12 18:28:21","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":103408,"visible":true,"origin":"","legend":"\u003cp\u003eTAT-PEP reduced the volume of cerebral infarction. (A) TTC staining detected the volume of cerebral infarction (n=6, each group). The picture of TTC staining of representative brain slices of experimental animals in each group at 3 d post-MCAO. (B) It was the statistical result of cerebral infarction volume percentage in each group (* P\u0026lt;0.05 vs. Sham group; # P\u0026lt;0.05 vs. MCAO group). (C) Magnetic resonance imaging detected the effect of TAT-PEP on the cerebral infarction volume of experimental animals in each group at 3 d post-MCAO (n=6, each group). It was the T2WI signal imaging result. (D) The statistical analysis of T2WI signal imaging on cerebral infarction volume (* P\u0026lt;0.05 vs. Sham group; # P\u0026lt;0.05 vs. MCAO group).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2327876/v1/467da8cb33c82b9f887c5a45.jpg"},{"id":30221988,"identity":"32f8e336-91e8-4559-bbd6-5ad86dc0e399","added_by":"auto","created_at":"2022-12-12 18:36:21","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":134144,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of TAT-PEP on the survival of neurons in the penumbra (n=6, each group). (A) Nissl staining detected the number of living neurons in the penumbra of each group at 3 d post-MCAO (Bar=50 µ m). (B) It was the statistical graph of the number of survival neurons in the penumbra of each group at 3 d post-MCAO(* P\u0026lt;0.05 vs. Sham group; # P\u0026lt;0.05 vs. MCAO group). (C) Immunofluorescence histochemical staining detected the number of NeuN positive neurons (green) in the penumbra of each group at 3 d post-MCAO (Bar=50 µ m). (D) It was the statistical graph of the number of NeuN positive neurons in the penumbra of each group at 3 d post-MCAO(* P\u0026lt;0.05 vs. Sham group; # P\u0026lt;0.05 vs. MCAO group).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2327876/v1/6b0174507c7de512114a2f1a.jpg"},{"id":30221163,"identity":"dc8a158a-8159-4f37-b7d9-5099a6b907de","added_by":"auto","created_at":"2022-12-12 18:28:21","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":117098,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of TAT-PEP on neuronal degeneration and apoptosis in the penumbra (n=6, each group). (A) JFC staining detected the number of degenerated neurons in the penumbra of each group at 3 d post-MCAO. The JFC-labeled degenerated neurons were green (Bar=50 µ m). (B) It was the statistical graph of the number of degenerated neurons in the penumbra of each group at 3 d post-MCAO (* P\u0026lt;0.05 vs. Sham group; # P\u0026lt;0.05 vs. MCAO group). (C) TUNEL staining detected the number of apoptotic neurons in the penumbra of each group at 3 d post-MCAO. TUNEL-positive neurons were brown and black (Bar=50 µ m). (D) It wasthe statistical graph of the number of TUNEL positive neurons in the penumbra of each group at 3 d post-MCAO (* P\u0026lt;0.05 vs. Sham group; # P\u0026lt;0.05 vs. MCAO group).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2327876/v1/357578ff01403dd831ea576e.jpg"},{"id":30221165,"identity":"e15a7ef6-a8a9-42ca-8d3c-c408be168707","added_by":"auto","created_at":"2022-12-12 18:28:21","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":164969,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of TAT-PEP on the ultrastructure of neurons in the penumbra (n=6, each group). Electron microscope observed the ultrastructure of neurons in the penumbra of each group three days after MCAO. Arrows indicated swollen mitochondria in the cytoplasm. Mitochondria expand into spheres. Bar=2 µ m.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2327876/v1/ea74df3909a60ccb02a86378.jpg"},{"id":30222855,"identity":"ad75bfd3-abb8-45ff-9164-0e7b3f473b8c","added_by":"auto","created_at":"2022-12-12 18:44:21","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":84920,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003epirb\u003c/em\u003e RNAi on neuronal survival and apoptosis exposed to OGD injury (n=6, each group). (A) It was to observe the survival status of neurons in 293T cells transfected with GPH-PIRB-294 lentivirus for 72 hours under a bright field microscope, Bar=50 μ m. (B) It was the statistical diagram of MTT analysis of neuronal activity 72 h after GPH-PIRB-294 lentivirus transfected neurons to OGD (* P\u0026lt;0.05 vs. Normal group; # P\u0026lt;0.05 vs. OGD group). (C) GPH-PIRB-294 lentivirus was transfected into the OGD of neurons, and then GPH-PIRB-294 was transfected 72 h after OGD, TUNEL staining was used to observe the apoptosis of neurons. Red cells are TUNEL-positive neurons, Bar=100 μ m。(D) It was the statistical graph of TUENL positive neurons in each group (* P\u0026lt;0.05 vs. Normal group; # P\u0026lt;0.05 vs. OGD group)\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2327876/v1/dfbbbc5b107f07b03c271eb3.jpg"},{"id":30221991,"identity":"c8259a9b-eaaa-4c2d-9f4c-33f5ea11284d","added_by":"auto","created_at":"2022-12-12 18:36:21","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":261032,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of TAT-PEP on neuronal viability and survival exposed to OGD injury (n=6, each group). (A) It was an MTT test to detect the impact of TAT-PEP of different concentrations on neuronal activity (* P\u0026lt;0.05 vs. Normal group; # P\u0026lt;0.05 vs. OGD group; $ P\u0026lt;0.05 vs. OGD+TAT-PEP (100 μg/L) group). (B) It was LDH release test to detect the effect of TAT-PEP of different concentrations on neuronal survival (* P\u0026lt;0.05 vs. Normal group; # P\u0026lt;0.05 vs. OGD group; $ P\u0026lt;0.05 vs. OGD+TAT-PEP (100 μg/L) group). (C) MTT assay detected the effect of TAT-PEP (100 μg/L) on the neuronal activity at different time points after OGD injury(* P\u0026lt;0.05 vs. Normal group; # P\u0026lt;0.05 vs. OGD group). (D) LDH release test detected the effect of TAT-PEP (100 μg/L) on neuronal survival at different time points after OGD injury(*P\u0026lt;0.05 vs. Normal group; # P\u0026lt;0.05 vs. OGD group). (E) Observed the effect of TAT-PEP (100 μg/L) on neuronal survival status by brightfield microscope at 72 h after OGD injury. Bar=100 μ m.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2327876/v1/bc6992e05953203b5575292b.jpg"},{"id":30221990,"identity":"873226c8-74d7-4170-8d52-202d33b4284d","added_by":"auto","created_at":"2022-12-12 18:36:21","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":71314,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of TAT-PEP on MDA level, SOD activity, and ROS accumulation of neurons exposed to OGD injury (n=6, each group). (A) The MDA level evaluated at 72 h after exposure to OGD injury (* P\u0026lt;0.05 vs. Normal group; # P\u0026lt;0.05 vs. OGD group). (B) The SOD activity evaluated at 72 h after exposure to OGD injury (* P\u0026lt;0.05 vs. Normal group; # P\u0026lt;0.05 vs. OGD group). (C) ROS production was analyzed by quantitative analysis by flow cytometry at 72 h after exposure to OGD injury (* P\u0026lt;0.05 vs. Normal group; # P\u0026lt;0.05 vs. OGD group).\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2327876/v1/74d4ee30dbc88607b8cc8829.jpg"},{"id":30221168,"identity":"7fde6540-5865-4c99-8d0d-04b95eb8fb28","added_by":"auto","created_at":"2022-12-12 18:28:21","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":77807,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of TAT-PEP on neuronal apoptosis exposed to OGD injury (n=6, each group). (A) TUNEL staining detected the impact of TAT-PEP on neuronal apoptosis at 72 h after OGD. The green cells were TUNEL-positive neurons. Blue represents DAPI-stained nuclei. Bar=100 μm. (B) It was the statistical diagram of TUENL positive neurons in each group (* P\u0026lt;0.05 vs. Normal group; # P\u0026lt;0.05 vs. OGD group). (C, D) The protein band of Cleared Caspase, Bax, and Bcl2 expression in every group at 72 h after OGD was detected by western blot. (E-F) The statistical diagrams of Cleared Caspase, Bax, and Bcl2 expression in every group at 72 h after OGD (* P\u0026lt;0.05 vs. Normal group; # P\u0026lt;0.05 vs. OGD group).\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2327876/v1/9e481302d156785d78fefe08.jpg"},{"id":44733900,"identity":"b0edf3d0-737a-40be-b5b1-4cfa9ea9e0f1","added_by":"auto","created_at":"2023-10-16 22:11:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1328747,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2327876/v1/74978930-f446-4576-9802-bb0644083ae9.pdf"},{"id":30221166,"identity":"ae6d884e-b6b6-4c13-b149-e4c1b557f13f","added_by":"auto","created_at":"2022-12-12 18:28:21","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":211207,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-2327876/v1/b1098f93814f9ea1719dd093.docx"}],"financialInterests":"","formattedTitle":"TAT-PEP alleviated cognitive impairment by alleviating neuronal mitochondria damage and apoptosis after cerebral ischemic reperfusion injury","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIschemic stroke is a primary cardiovascular and cerebrovascular disease, which is characterized by a high incidence rate, high mortality, and high disability rate[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In particular, about 60% ~ 80% of the survivors have left neurological defects such as cognitive impairment and sensory and motor disorders, which burden society and families heavily [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, up to now, the mechanism of ischemic stroke is not precise, and the treatment is limited. Therefore, how to promote the recovery of neural function and reduce the disability rate needs to be solved urgently, and it is also a challenging worldwide problem.\u003c/p\u003e \u003cp\u003eThe pathophysiological process of ischemic stroke is relatively complex. Among them, cerebral ischemia-reperfusion injury can lead to cerebral ischemia and hypoxia, causing damage to brain tissue structure and function, such as neuronal damage, axonal regeneration difficulties, and a decrease of synaptic plasticity, which lead to neurological dysfunction[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. One of the critical reasons is that many kinds of myelin-associated inhibitory proteins (MAIs) and their receptors are in the brain. Studies have found that paired immunoglobulin-like receptor B (PirB) is a co-receptor of Nogo-A, Myelin associated glycoprotein (MAG), and Oligodendrocyte myelin glycoprotein (GP)[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It was reported that the \u003cem\u003epirb\u003c/em\u003e gene knockout mice had smaller cerebral infarction volume than the wild-type mice after focal cerebral ischemia injury[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These results suggest that PirB may be necessary for aggravating neuronal damage during cerebral ischemia-reperfusion injury. Other studies have shown that PirB plays a vital role in cognitive impairment[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In our previous study, we found the expression of PirB significantly increased in neurons in the ischemic penumbra, and the overexpression of PirB deteriorated neuronal apoptosis[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. These pieces of evidence suggest that PirB may be an important target for treating ischemic stroke. Inhibiting the function of PirB could effectively alleviate neuron damage and promote the recovery of neural function. This strategy may be more effective than the single intervention of a ligand, which will bring new hope for treating ischemic stroke.\u003c/p\u003e \u003cp\u003eThe extracellular segment of PirB mainly contains six immunoglobulin-like domains[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It was found that Nogo-A and MAG could combine with their extracellular components to activate the downstream signal pathway of PirB [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, if the function of PirB can be suppressed at the protein level, it will be instrumental in the recovery of neural function after cerebral ischemia-reperfusion injury. In our previous studies, a transactivator of transcription-PirB extracellular peptide (TAT-PEP) was generated, which displayed a high affinity for MAIs and ameliorated their inhibitory effect on neurite growth. Furthermore, TAT-PEP can widely distribute in the penumbra after intraperitoneal injection[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Then, we found that TAT-PEP enhanced neurite growth and alleviated growth cone collapse after oxygen-glucose deprivation (OGD) injury[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, exploring whether TAT-PEP could enhance cortical neuronal survival and promote cognitive function recovery after the transient focal cerebral ischemia model needs to be explored.\u003c/p\u003e \u003cp\u003eIn this study, the GPH-PIRB-294 lentivirus system was constructed to interfere with PirB expression and observe the effect of inhibiting PirB expression on neuronal survival after OGD injury. We used the middle cerebral artery occlusion (MCAO) model to evaluate the impact of TAT-PEP on the improvement of neural function and cerebral infarct volume after cerebral ischemia-reperfusion injury through neurobehavioral methods and MRI imaging technology. Through in vivo and in vitro experiments, we further analyzed the effect of TAT-PEP on neuronal survival, oxidative stress, degeneration, and apoptosis after cerebral ischemia-reperfusion injury and explored the relevant mechanisms to clarify the role and mechanism of TAT-PEP on neuronal survival during cerebral ischemia-reperfusion injury.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eAdult male Sprague-Dawley (SD) rats (280\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g) housed under controlled conditions with a 12-h light/dark cycle, a temperature of 21\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, and humidity of 60\u0026ndash;70% for at least 1 week before drug treatment or surgery, purchased from the Experimental Animal Center of the Fourth Military Medical University (Xi\u0026rsquo;an, Shaanxi, China) and the Laboratory Animal Center of Xi\u0026rsquo;an Jiaotong University (Xi\u0026rsquo;an, Shaanxi, China). It was approved by the Ethics Committee for Animal Experimentation of the Fourth Military Medical University and by the Institutional Animal Care and Use Committee at Xi\u0026rsquo;an Jiaotong University. Every effort was made to minimize stress to the animals. All sample sizes for the assessment parameters were calculated to reduce the number of animals used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTransient Focal Cerebral Ischemia Mode\u003c/h2\u003e \u003cp\u003eFocal cerebral ischemia was induced in the rats by middle cerebral artery occlusion (MCAO), as described previously[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The experimental animals were randomly divided into 3 groups, with 10 animals in each group. Because the success rate of the MCAO model is about 70%, the actual number of animal samples used in each group is 8\u0026thinsp;~\u0026thinsp;6 (n\u0026thinsp;=\u0026thinsp;8\u0026thinsp;~\u0026thinsp;6 for each group). The three groups were divided into the Sham group, the MCAO group, and the MCAO\u0026thinsp;+\u0026thinsp;TAT-PEP group. In the MCAO\u0026thinsp;+\u0026thinsp;TAT-PEP group, 1.0 mg/kg TAT-PEP was intraperitoneally injected immediately after reperfusion, and then TAT-PEP of this concentration was injected daily.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eGarcia scores\u003c/h2\u003e \u003cp\u003eAt 24h, 48h, and 72h after MCAO, the modified Garcia scores [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] were used to detect the neurological function of experimental animals. It contains 18 points, including 0\u0026ndash;3 points for each item, as shown below. Free activity in a cage for 5 min: no activity (score 0); Almost unable to move (score 1 point); Able to move, but the range of movement does not reach 3 sides in the cage (2 points); The field of activity shall get at least 3 sides inside the enclosure (3 points). The symmetry of limb movement: no movement of the left limb (0 points); The left limb can move slightly (1 point); The left limb can move slowly (2 points); Bilateral limb movements are symmetrical (3 points). The symmetry of forelimb (forelimb extension when lifting the tail): the left limb is unable to move (0 points); The left limb can only be slightly extended (1 point); The left side is not as active and stretched as the right side (2 points); Bilateral forelimbs can be tested symmetrically (3 points). Climbing in the metal cage: None (0 points); Unable to mount (1 point); The left side is slightly weak (score 2 points); Can usually climb (3 points). Touch bilateral trunk reaction: none (0 points); No response on the left side (score 1 point); Weak left side reaction (score 2 points); The answer is symmetrical (3 points). Whisker reaction: None (0 points); No response on the left side (score 1 point); Weak left side reaction (score 2 points); The reaction is symmetrical (3 points).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eY-maze test\u003c/h2\u003e \u003cp\u003eThe Y-maze test was used to assess spatial learning and memory as described previously [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Experimental rats were placed in a Y-shaped maze (arm's length: 16 cm, arm width: 10 cm, height of the wall: 40 cm, Yihong Technology Co., Ltd., Wuhan, China) with three arms at 120\u0026deg; from each other and allowed to explore the three arms for 10 min freely. Before all experiments, the rats should be used for acclimatization for 2 days, during which the rats can visit all components freely for 10 minutes. The number of arm entries and alterations were recorded automatically using Smart Video Tracking Software 3.0 (Panlab, Barcelona, Spain). The percentage of spontaneous alternation was calculated as the number of correct alterations (number of total new arm entries), which is associated with the capacity of spatial short-term memory.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eNovel object recognition test (NOR)\u003c/h2\u003e \u003cp\u003eThe novel object recognition test is widely used in the study of cognitive impairment[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. First, to reduce stress levels, all groups of rats were placed in the experimental room and testing box for 10 min on the 2 successive days before the training phase. At 3 d post-MCAO, rats were trained to explore freely within a box (Yihong Technology Co., Ltd., Wuhan, China). The experimental method was described previously. The experiment was r recorded with a video camera (SNC-VB600B5, SSGE, Shanghai, China) cation placed on the old object. The exploration time ratio for novel objects during the testing phase was assessed for each group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTTC staining\u003c/h2\u003e \u003cp\u003eAt 3 d after MCAO, TTC staining was used to detect the effect of TAT-PEP on cerebral infarction volume. The straightforward method is as follows[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]: The brain was removed under deep anesthesia and immersed in normal saline ice for 10 minutes. Put it into the cerebral sulcus mold, and the thickness of the brain slice is about 2 mm (cut in sequence along the coronal plane). Soak the brain slices in a 2% concentration of a 2,3,5-TTC solution with the same orientation, and incubate them in a constant temperature water bath (37 ℃, 30 min). After the staining effect is appropriate (the white area is the infarcted area, and the red area is the normal brain tissue area), transfer to 4% PFA for fixation overnight, take photos and analyze 24 hours later. Adobe photoshop cs3 image processing software was used to calculate the volume of cerebral infarction.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMagnetic resonance imaging (MRI) detected cerebral infarction volume.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAt 3 d after MCAO, the small animal MRI imaging system was used to detect the effect of TAT-PEP on cerebral infarction volume. The straightforward method follows: After anesthesia, the rats were placed in a prone position with their heads in the center of the coil. The rapid acquisition relaxation enhancement sequence (along the coronal section) was used to scan with the visual intersection as the origin. The parameters are an echo time (TE) of 60 ms, a repetition time (TR) of 3000 ms, a thickness of 0.5 mm, and no spacing. The field of view is 2.56 cm \u0026times; 2.56 cm. Acquire T2 weighted (T2WI) images, and then use image software for image analysis to measure the volume of ischemic injury area (i.e. abnormal high signal area), which is equal to the area of abnormally high signal area \u0026times; the sum of slice thickness (plus spacing), was used to evaluate the cerebral infarction volume.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eNissl staining\u003c/h2\u003e \u003cp\u003eNissl staining (Beyotime Institute of Biotechnology, China) was performed to detect Nissl bodies in the cytoplasm of surviving neurons. At 3d after MCAO, Nissl staining was used to detect the effect of TAT-PEP on the survival of neurons in the penumbra. The methods are described as follows: Dry the slices at room temperature for 1 h, draw circles, and gently rinse them with 0.01M PBS 3 times (5 min/time). Add Nissl dyestuff (to filter in advance), and incubate at 37 ℃ for 20 min. Then gently wash off with pure water. The sections were dehydrated and transparent before using the neutral resin sealed. The microscope was used to observe and record in the open field. The integrated optical density/area of the staining in each group was acquired by 2 blinded investigators using ImagePro Plus 5.1 software (Media Cybernetics, Inc., Bethesda, MD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eFJC staining\u003c/h2\u003e \u003cp\u003eAt the 3d after MCAO, FJC staining was used to detect the effect of TAT-PEP on the number of degenerated neurons in the penumbra. The specific methods are as follows: Freeze the slices and dry them at room temperature for 1 h, draw circles, and gently rinse them with 0.01M PBS 3 times (5 min/time). Immerse the slices in a mixture of 1% sodium hydroxide and 80% anhydrous ethanol for 5 min. Then immerse it in 70% absolute ethanol, hydrate it for 2 min, and gently rinse it with deionized water 3 times (5 min/time). Incubate with 0.06% potassium permanganate for 10 min (shaking table at room temperature) and rinse with distilled water for 5 min (shaking table). Prepare 0.0001% FJC dye solution, and add acetic acid in the proportion of 1:1000. After dropping FJC dye solution, incubate it at 37 ℃ at constant temperature and away from light for 30 min, and gently rinse it with deionized water 3 times (1 min/time). Dry in the dark at room temperature, dehydrated with anhydrous ethanol for 2 min, make xylene transparent 3 times (2 min/time), and apply neutral gum film. The people who did not know the experimental group were observed by Olympus BX51 fluorescence microscope (excitation light wavelength was 450\u0026ndash;490 nm), collected images, and recorded the number of FJC positive cells (FJC positive cells were green).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence histochemical staining\u003c/h2\u003e \u003cp\u003eOn the 3rd day after MCAO, NeuN immunofluorescence histochemistry was used to detect the effect of TAT-PEP on the number of living neurons in the penumbra. The specific method is the same as that in Part I. First antibody (NeuN, 1:5000) was added, 4 ℃ overnight, 0.01M PBS solution was used for rinsing (5 min each time \u0026times; 3 times), inject the secondary antibody (goat anti-mouse FITC, 1:5000), incubated at room temperature in the dark for 2 hours, rinse in the night for 3 times, observe and take photos under a fluorescence microscope after 50% glycerine film is sealed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTUNEL staining\u003c/h2\u003e \u003cp\u003eAt the 3d after MCAO, TUNEL staining was used to detect the effect of TAT-PEP on the number of apoptotic neurons in the penumbra. The specific methods are as follows: The fixed brain tissue was immersed in 30% alcohol, embedded in paraffin, and sliced, with a thickness of 3 \u0026micro; m. Paraffin sections were dewaxed with xylene for 5 min (2 min/time). After waxing, use a circle drawing brush to draw circles, drip protein kinase K cell permeating solution, incubate at 37 ℃ for 30 min, and then rinse gently with 0.01M PBS 3 times (5 min/time). Join 50 \u0026micro; L DNase1 solution at room temperature for 10 min. After the slides are cleaned and dried, add the Tunel mixture. 37℃, 1 h. PBS cleaning again. Joining 30 \u0026micro; L POD stops the reaction, and PBS has cleaned again. Drop the DAB color-developing solution at room temperature for 10min, then conduct PBS cleaning again. Drop hematoxylin for 30 s and gently wash it under pure water. Gradient dehydration, xylene transparent for 5min (twice), neutral resin film, open field microscope observation, and statistics of the number of Tunel positive cells (Tunel positive cells are brown black).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eElectron microscope experiment\u003c/h2\u003e \u003cp\u003eExperimental animals were anesthetized intraperitoneally with 0.3 ml/100g chloral hydrate (300 mg/kg), perfused and fixed with a solution containing 4% PFA and 0.05% glutaraldehyde, positioned on a stereotaxic device, and fixed for 2 hours. Coronal sections were made with a vibrating microtome with a thickness of 50 \u0026micro; m and immersed in PBS solution containing 30% sucrose for 2 h. Dehydrated with gradient alcohol and Put the transparent continuous sections on copper mesh with supporting membrane, and observed the ultrastructure of neurons under the electron microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePrimary culture of cortical neurons\u003c/h2\u003e \u003cp\u003ePrimary cortical neurons were cultured as described previously[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. SD rats who were pregnant 16.5\u0026ndash;18.5 days (E 16.5\u0026ndash;18.5 days) were dislocated and killed. A routine aseptic operation was carried out. Fetal rats were taken out and separated. In the D-Hank's solution (placed on ice), the fetal rat was decapitated with toothless ophthalmic tweezers, and the brain tissue was taken out. Under the microscope, the cerebral cortex was separated. The tissue was chylated through D-Hank's solution. Then trypsin (0.125%) was digested for 15 min (in a regular incubator at 37 ℃). Then, the tissue was carefully sucked out with an elbow dropper, added into a centrifuge tube (15 ml) containing DMEM solution of fetal bovine serum, and the digestion was stopped at room temperature for 5 minutes. Place the centrifuge tube in the centrifuge at 80 rpm for 5 min, discard the supernatant carefully, add 2 ml of DMEM solution containing fetal bovine serum, blow and repeatedly beat to prepare tissue and cell suspension, and use the cell filter (100 \u0026micro; m) Filtering. After another centrifugation at 80 rpm for 5 min, it was added to the neuron culture medium (Neurobasal A, 2% B27, 1% glutamate, and 1% penicillin mixture). According to the experimental requirements, different-density cells were inoculated into 96 well plates, 24 well plates, or 6 well plates coated with poly-L-lysine (50 mg/mL) (Sigma, USA). These cells were grown in Neurobasal medium (Gibco, Invitrogen Corp, USA) supplemented with 2% B27, 1% glutamine, and 1% penicillin/streptomycin (Sigma, USA) at 37℃ under a humidified incubator in air containing 5% CO\u003csub\u003e2\u003c/sub\u003e. The purity of neurons was determined by immunocytochemistry for bIII-tubulin at 5-day after plated, which indicated that 95% of the cells in cultures were positive for βIII-tubulin (1:250; Millipore, Temecula, CA, USA) (data not shown)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eOxygen glucose deprivation model of neurons in vitro\u003c/h2\u003e \u003cp\u003eThe primary cultured cortical neurons were identified on the 7th day. Replace the culture medium (sugar-free and serum-free) and put it into the hypoxia constant temperature incubator (37 ℃, 5% CO\u003csub\u003e2\u003c/sub\u003e, 95% N\u003csub\u003e2\u003c/sub\u003e) for 1 h. Then carefully take out the culture dish or culture bottle, replace it with standard neuron culture medium (Neurobasal A, 2% B27, 1% glutamate, and 1% penicillin mixture), and place it in the traditional incubator (5% CO\u003csub\u003e2\u003c/sub\u003e, 21% O\u003csub\u003e2\u003c/sub\u003e, 37 ℃). After re-oxygenation and re-sugar, they were immediately given TAT-PEP or TAT mPEP. The experimental groups are as follows:\u003c/p\u003e \u003cp\u003e① MTT and LDH release tests [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] were used to detect the effect of TAT-PEP at different concentrations on the survival of neurons at 24 h after OGD. They were randomly divided into different groups (n\u0026thinsp;=\u0026thinsp;6): Normal group, OGD group, OGD group, OGD\u0026thinsp;+\u0026thinsp;TAT mPEP (100 \u0026micro;g/L) group, OGD\u0026thinsp;+\u0026thinsp;TAT-PEP (50 \u0026micro;g/L) group, OGD\u0026thinsp;+\u0026thinsp;TAT-PEP (100 \u0026micro;g/L) group, OGD\u0026thinsp;+\u0026thinsp;TAT-PEP (200 \u0026micro;g/L) group.\u003c/p\u003e \u003cp\u003e② MTT and LDH release tests were used to detect the effect of TAT-PEP on neuronal survival after OGD (6h, 24h, 72h). They were randomly divided into different groups (n\u0026thinsp;=\u0026thinsp;6): Normal group, OGD group, OGD\u0026thinsp;+\u0026thinsp;TAT mPEP (100 \u0026micro;g/L) group, and OGD\u0026thinsp;+\u0026thinsp;TAT-PEP (100 \u0026micro;g/L) group.\u003c/p\u003e \u003cp\u003e③ MDA level and SOD activity [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] was used to detect the effect of TAT-PEP on neuronal oxidative stress at 72 h after OGD. They were randomly divided into groups (n\u0026thinsp;=\u0026thinsp;6): The normal group, OGD group, and OGD\u0026thinsp;+\u0026thinsp;TAT-PEP group (TAT-PEP was 100 \u0026micro;g/L).\u003c/p\u003e \u003cp\u003e④ TUNEL staining was used to detect the effect of TAT-PEP on neuronal apoptosis at 72 h after OGD. They were randomly divided into different groups (n\u0026thinsp;=\u0026thinsp;6): The normal group, OGD group, and OGD\u0026thinsp;+\u0026thinsp;TAT-PEP group (TAT-PEP is 100 \u0026micro;g/L).\u003c/p\u003e \u003cp\u003e⑤ Western blot was used to detect the effect of TAT-PEP on the expression of apoptosis-related proteins at 72 h after OGD. They were randomly divided into different groups (n\u0026thinsp;=\u0026thinsp;6): The normal group, OGD group, and OGD\u0026thinsp;+\u0026thinsp;TAT-PEP group (TAT-PEP is 100 \u0026micro;g/L).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eAccording to the time node of the experiment, the whole cell protein was extracted. The method follows: After washing PBS three times, add precooled Lysis cracking solution (100 \u0026micro; L/well), carefully scrape the cells, and thoroughly mix the lysate with the cells. Transfer the suspension into the precooled EP tube, ice bath for 30 min, and centrifugation at 4 ℃ for 15 min, 12000 rpm. The total protein concentration of the tissues or cells was analyzed with a BCA kit (Sigma, CA, USA). Rabbit antibody against cleaved (active) caspase-3 (1:1,000; Cell Signaling Technology, Beverly, MA, USA), mouse monoclonal antibodies against Bcl-2 or Bax (1:1,000, Santa Cruz, CA, USA), and β-actin (1:2,000, Anbo, USA). Subsequently, the blots were probed with horseradish peroxidase (HRP)-conjugated goat secondary antibody against rabbit or mouse IgG (1:1,000, Abcam, USA). Detection and quantitation were performed with a Typhoon 9400 Variable Mode Imager (GE Healthcare) and Lumi-Light Western Blotting Substrate (Roche Diagnostics) for HRP-labeled blots.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eTUNEL staining\u003c/h2\u003e \u003cp\u003eTUNEL staining was performed in vitro using an In Situ Cell Death Detection Kit (Roche Diagnostics, Mannheim, Germany). At 72 h after OGD injury, 4% PFA was added to fix at room temperature for 1 h. 0.01M PBS was gently rinsed 3 times (5 min/time), and then 0.3% hydrogen peroxide was used for 10 min. Add TUNEL reaction mixture for 1 h at 37 ℃ and stain with DAPI for 5 min at room temperature. Images were obtained with a microscope (BX60, Olympus). The integrated optical density/area of the positive TUNEL staining in each group was acquired as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD across the groups and were statistically analysed using GraphPad Prism 7.0 software (GraphPad Company, San Diego, CA, USA). Continuous data were tested for normal distribution and analysed by one-way ANOVA (followed by Tukey\u0026rsquo;s multiple comparisons tests) or Kruskal\u0026ndash;Wallis test (followed by Dunn\u0026rsquo;s multiple comparisons tests). Two-way ANOVA was applied to analyse the neural dendritic complexity. \u003cem\u003eP\u003c/em\u003e value of less than 0.05 was considered statistically signifcant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eTAT-PEP improved neurobehavioral function and cognitive function against MCAO\u003c/h2\u003e \u003cp\u003eWe used Garcia scores to analyze neurobehavioral function recovery. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, the scores of the TAT-PEP treatment group were higher than that of the Sham group at 48 h (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 72 h (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) after reperfusion. We used the Y-maze test to analyze cognitive function recovery. At 3 d after reperfusion, the percent of spontaneous alteration and total arm entries in the Y-maze test were measured. Rats of the MCAO group showed significantly lower spontaneous alternation rates than rats of the Sham group in the Y maze test.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn contrast, in the Y maze test, rats of the MCAO\u0026thinsp;+\u0026thinsp;TAT-PEP group showed substantially higher spontaneous alternation rates than rats of the MCAO group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB/D). Also, the recognition index and total travel distance in the novel object recognition test were measured. Rats of the MCAO group spent less time exploring a novel object during the test phase than the Sham group rats. In contrast, the MCAO\u0026thinsp;+\u0026thinsp;TAT-PEP group spent more time exploring a novel thing during the test phase than the rats of the MCAO group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE/F). These results indicated that TAT-PEP could promote neurological and cognitive functional recovery.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eTAT-PEP reduced brain infarct volume against MCAO\u003c/h2\u003e \u003cp\u003eThen, we tested the brain infarct volume in every group. Figures\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B show that the MCAO\u0026thinsp;+\u0026thinsp;TAT-PEP group displayed a significantly smaller brain infarct volume than the MCAO group by TTC staining at 3 d post-MCAO (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). MRI measured infarction volumes. T2WI analyses showed that the high-intensity books were more prominent in the MCAO group than in the Sham group at 3 d post-MCAO. In contrast, T2WI studies showed that the high-intensity volumes were smaller in the MCAO\u0026thinsp;+\u0026thinsp;TAT-PEP group than in the MCAO group at 3 d post-MCAO (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eTAT-PEP attenuated neuronal degeneration, apoptosis, and mitochondria damage against MCAO\u003c/h2\u003e \u003cp\u003eTo assess neuroprotection of TAT-PEP, the Nissl staining and NeuN staining were 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\u0026thinsp;+\u0026thinsp;TAT-PEP group increased significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA/B). The number of NeuN-positive neurons was more in the MCAO\u0026thinsp;+\u0026thinsp;TAT-PEP group than that in the MCAO group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC/D). Then, the FJC and TUNEL staining on ischemic brain sections was performed at 3 d after reperfusion. The number of FJC-positive neurons was fewer in the TAT-PEP group than in the MCAO group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, the number of TUNEL-positive cells was more irregular in the TAT-PEP group than that in the MCAO group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D). At 3 d after reperfusion, the ultrastructure of cortical neurons in the MCAO group showed nuclear membrane folds, collapse, and blurred boundaries. Additionally, noticeable swelling of mitochondria in the cytoplasm was noted, which was spherical. Some mitochondrial cristae were fractured. The damage to the neuronal ultrastructure after TAT-PEP treatment was significantly alleviated, especially in the mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eInhibiting PirB overexpression was beneficial to rescue neurons from apoptosis exposed to OGD injury.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe survival of cultured neurons was examined at 72 h after PirB RNAi vector or Control RNAi transfection. The neuronal structure in the OGD group and OGD\u0026thinsp;+\u0026thinsp;Control RNAi group was damaged. However, transfection with the PirB RNAi vector prevented neuronal damage. The cell viability in the OGD and Control RNAi groups was significantly lower than that of the Normal group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) at 72 h after exposure to OGD. However, the cell viability of the PirB RNAi group was higher than that of the OGD and Control RNAi groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA/B). The number of TUNEL-positive cells in the OGD and Control RNAi groups was significantly higher than that of the Normal group (P\u0026thinsp;\u0026lt;\u0026thinsp;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\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC/D). These results indicated that inhibiting PirB overexpression was beneficial in rescuing the neurons from apoptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eTAT-PEP enhanced the survival of neurons exposed to OGD injury\u003c/h2\u003e \u003cp\u003eHere, we observed the function of different doses of TAT-PEP on neuronal survival at 24 h after being exposed to OGD (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA/B). The cell viability decreased significantly in the OGD group compared to the Normal group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and LDH release (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increased substantially in the OGD group compared to the Normal group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) at 24 h after exposure to OGD. In addition, the cell viability in TAT-PEP (50 \u0026micro;g/L, 100 \u0026micro;g/L, 200 \u0026micro;g/L) treatment group was significantly higher than it was in the OGD group or TAT-mPEP (misordered Sequences of the extracellular cDNA of PirB) group at 24 h after exposed to OGD (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The LDH release in the TAT-PEP treatment group was significantly lower than that in the OGD group at 24 h after being exposed to OGD (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The results further showed that compared with the 50 \u0026micro;g/L TAT-PEP treatment group, the cell viability was higher, and LDH release was lower in the 100 \u0026micro;g/L and 200 \u0026micro;g/L TAT-PEP treatment groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, the effect of TAT-PEP between 100 \u0026micro;g/L and 200 \u0026micro;g/L had no difference in cell survival (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, to confirm the protective effect of TAT-PEP, we also observed the function of TAT-PEP on neurons at different time points after being exposed to OGD (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC/D). The cell viability decreased significantly in the OGD group or TAT-mPEP group compared to the Normal group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and LDH release (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increased considerably in the OGD group or TAT-mPEP group compared to the Normal group at 6 h (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), 24 h (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 72 h (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) after exposed to OGD. However, TAT-PEP treatment increased cell viability and decreased LDH release compared to the OGD group or TAT-mPEP group at 24 h (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 72 h (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) after exposure to OGD.\u003c/p\u003e \u003cp\u003eAt 24 h after OGD injury, the growth state of neurons was observed under the light microscope. It was found that the integrity of neurons in the OGD group was poor. The cell bodies were atrophied or broken. The neurites were broken or disappeared. However, TAT-PEP treatment could significantly improve the growth of neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). The above results indicate that TAT-PEP can substantially enhance the activity of neurons, reduce neuronal damage, and promote neuronal survival.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTAT-PEP decreased MDA levels, increased SOD activity, and alleviated ROS accumulation of neurons exposed to OGD injury\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMDA, SOD, and ROS are regarded as indicators for detecting oxidative stress. The MDA content in the OGD group increased compared with that in the Normal group at 72 h exposed to OGD injury (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). TAT-PEP treatment attenuated the elevation of MDA content compared with that in the OGD group at 72 h exposed to OGD injury (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Compared with the Normal group, SOD activity was reduced in the OGD group at 72 h exposed to OGD injury (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). TAT-PEP treatment increased the SOD activity compared with that in the OGD group at 72 h exposed to OGD injury (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Furthermore, the flow cytometry result showed that ROS levels were upregulated in the OGD group compared with the Normal group at 72 h exposed to OGD injury (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, the ROS levels were lower in the OGD\u0026thinsp;+\u0026thinsp;TAT-PEP group compared with that in the OGD group at 72 h exposed to OGD injury (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eTAT-PEP alleviated neuronal apoptosis exposed to OGD injury\u003c/h2\u003e \u003cp\u003eThen, assaying for neuronal 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\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, the number of TUNEL-positive cells in the OGD\u0026thinsp;+\u0026thinsp;TAT-PEP group was fewer than in the OGD group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA/B). The expression of cleaved (active) Caspase3, Bcl-2, and Bax was assessed by western blot at 72 h after OGD. Caspase3 activity was significantly inhibited in the OGD\u0026thinsp;+\u0026thinsp;TAT-PEP group compared with the OGD group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Treatment with TAT-PEP increased the level of Bcl-2 protein, whereas it decreased Bax expression compared with the OGD group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC-E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough the progress of prevention and treatment technologies such as acute stroke, brain trauma, and perioperative brain injury have controlled the mortality to a certain extent, about 60% ~ 80% of the survivors will leave neural function defects such as cognitive impairment, sensory disorder, motor disorder, and communication disorder to varying degrees, which will seriously affect the quality of life of patients and lead to heavy family burden [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Especially in ischemic stroke, the process of its onset is often accompanied by complex pathophysiological processes, leading to long-term cognitive impairment. This neurological dysfunction is due to the damage to the structure and function of the cerebral cortex caused by ischemia-reperfusion injury. Neurons are the basic unit of the structure and function of the central nervous system. The neurons are susceptible to ischemia and hypoxia and are prone to degeneration, necrosis, and apoptosis, which has been confirmed in previous experiments [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Since Astrup defined the reversible damage beyond the central necrotic area of focal cerebral ischemia as the ischemic penumbra (IP) in 1981, IP has been used clinically as an area for the treatment of ischemic cerebrovascular disease [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This is because the IP area develops to irreversible damage slowly, which takes several hours or even days, and neuronal death is mainly apoptosis[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, reducing the damage of neurons in the penumbra and promoting the survival of neurons is the key to effectively improving neural function after cerebral ischemia-reperfusion injury.\u003c/p\u003e \u003cp\u003ePirB is a co-receptor of Nogo-A, MAG, and OMgp, which plays an essential role in axon growth, synaptic plasticity, and neuronal survival[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In the previous study, we found the mRNA and protein levels of PirB in the model of cerebral ischemia-reperfusion injury in mice were highly expressed in the ischemic injury area, suggesting that it may play an important role in the pathological process of cerebral ischemia-reperfusion injury[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This persistent and significant overexpression phenomenon means that PirB may be a potentially important target for treating cerebral ischemia-reperfusion injury. In the previous study, we first designed and synthesized constructed the transactivator of transcription-PirB extracellular peptide (TAT-PEP), which blocked the interaction between PirB and its ligands, and abolished the inhibitory activity of Nogo-A, MAG, and OMgp on axonal regeneration. Moreover, TAT-PEP treatment enhanced axonal regeneration and CST projection and improved motor functional recovery after stroke[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In the present study, our work further found that TAT-PEP reversed cognitive dysfunction after ischemic stroke by Y maze and novel object recognition test. The neuroprotection of TAT-PEP also suggests that PirB plays an essential role in negatively regulating cognitive function after ischemic stroke. The process of PirB in cognitive function is also consistent with the recent research report that PirB is involved in diabetes-associated cognitive dysfunction through modulation of axon outgrowth and dendritic remodeling, providing a potential therapeutic target for cognitive dysfunction [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Another research also showed PirB is associated with aging, and TAT-PEP may be a promising therapeutic agent for the modulation of age-related motor and cognitive dysfunctions[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These results indicated TAT-PEP could alleviate PirB-associated cognitive dysfunction, similar to our findings. However, there may be other mechanisms that need to be studied.\u003c/p\u003e \u003cp\u003eHow TAT-PEP improved cognitive impairment after an ischemic stroke still needs to be clarified. This study found that TAT-PEP could significantly reduce cerebral infarction volume at three after MCAO. Especially the degree of cortex damage was reduced considerably. Adelson found that the cerebral infarction volume of \u003cem\u003epirb\u003c/em\u003e KO mice was significantly reduced seven days after MCAO compared with \u003cem\u003epirb\u003c/em\u003e WT mice[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], which confirmed the above results. Then, our striking finding showed TAT-PEP significantly reduced the number of degenerated and apoptotic neurons in the cerebral cortex's penumbra, improved the neurons' ultrastructure, and effectively promoted the survival of neurons. This may be an actual reason for TAT-PEP to effectively reduce the volume of cerebral infarction and promote the recovery of cognitive function. In the previous study, our research group using the global cerebral ischemic reperfusion injury model confirmed that inhibiting the function of PirB significantly reduced the apoptosis of neurons in the ischemic hippocampus area and promoted the survival of neurons[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Clearly, PirB played a vital role in the process of neuronal damage after cerebral ischemia-reperfusion. The above results further confirmed that PirB might be an important reason for hindering neuronal survival and functional recovery after cerebral ischemia. It is a crucial target for treating ischemic stroke.\u003c/p\u003e \u003cp\u003eAs mentioned above, we found that TAT-PEP can play a neuroprotective role, which may be related to its ability to reduce the degeneration and apoptosis of neurons in the cerebral ischemic injury area. Our previous study found that PirB overexpression exacerbated neuronal apoptosis by oxidative stress and mitochondria damage[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. To further clarify the neuroprotection and mechanism of TAT-PEP, we conducted primary neuron culture and used the OGD model in vitro. The results showed that interfering with neuronal PirB expression or TAT-PEP significantly enhanced the activity of neurons after OGD, reduced the release of LDH, and alleviated neuronal damage. Furthermore, TAT-PEP decreased MDA levels, increased SOD activity, and helped ROS accumulation of neurons exposed to OGD injury. These results proved PirB participated in neuronal apoptosis caused by oxidative stress after oxygen and glucose deprivation. Inhibiting the function of PirB used, TAT-PEP alleviated oxidative stress and neuronal damage. Although the effects of TAT-PEP on neuronal oxidative stress and apoptosis are still controversial, in our study, the results indicated TAT-PEP decreased MDA level and increased SOD activity, which inhibited lipid peroxidation. TAT-PEP also reduced ROS accumulation which has been suggested to be required for neuronal apoptosis. Lipid peroxidation and ROS accumulation are closely related to the damage of plasma membrane structure in cells. Especially the production of a large number of oxygen free radicals and the lipid peroxidation of a mitochondrial membrane can cause mitochondrial damage and lead to apoptosis. In our vivo study, the ultrastructural changes of mitochondria also confirmed this point. To sum up, our results supplied the evidence to explain the potent neuroprotective effects of TAT-PEP against neuronal oxidative stress, mitochondrial damage, and apoptosis after ischemic stroke.\u003c/p\u003e \u003cp\u003eTo further explore the relevant mechanisms, we found that TAT-PEP reduced the number of apoptotic neurons after OGD, inhibited the expression of apoptotic protein Bax, promoted the expression of anti-apoptotic protein Bcl2, and inhibited the activation of Caspase3. Previous studies have shown that the damage of neurons in the penumbra of cerebral ischemia is mainly regulated by apoptosis[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. One of the mechanisms of neuronal apoptosis in ischemic penumbra is due to mitochondrial damage related to lipid peroxidation and ROS accumulation[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. It has been reported Caspase-3 is known that the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2 can migrate from the cytoplasm to mitochondria, which are distributed in a manner that is consistent with the mitochondrial release of cytochrome C and caspase[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The mitochondrial apoptotic pathway plays an essential role in neuronal injury [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and TAT-PEP inhibited neuronal apoptosis after ischemic stroke by upregulating Bcl-2 expression and reducing Bax expression. The apoptotic proteins on the mitochondrial membrane, such as Bad and Bax, lead to the opening of the mitochondrial permeability transition pore and the release of cytochrome C or apoptosis factors. The activated Caspase3 cleaves DNA repair enzymes, thus leading to DNA damage and apoptosis[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, this study has yet to directly clarify how PirB regulates the expression of Bax and Bcl-2 after cerebral ischemia-reperfusion injury. We observed the ultrastructure of neurons that the nucleus and mitochondria of neurons in the penumbra were significantly damaged after cerebral ischemia-reperfusion injury. It may be that PirB affected the expression of Bax and Bcl-2, leading to the opening of the mitochondrial permeability transition pore, thus activating Caspase3. However, the specific mechanism and signal pathway must be further confirmed. Studies have shown that interfering with the binding of NgR1 and Nogo-A can effectively inhibit the expression of Bax, promote the expression of Bcl-2, and thus inhibit the activation of Caspase3[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, whether TAT-PEP interferes with Nogo-A and PirB signal pathways, thereby affecting the expression of Bax, Bcl-2, and activated Caspase3, and playing a neuroprotective role, still needs further study.\u003c/p\u003e \u003cp\u003eIt is noteworthy that a previous study reported that the increased PirB labeling was localized to astrocytes and neurons in Lipopolysaccharide (LPS)-treated animals. The hippocampus-dependent spatial learning and memory were impaired[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Another study also showed PirB was highly expressed in neurons and astrocytes in the model of epilepsy[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. It has been reported that astrocytes are mainly responsible for inflammatory injury and cognitive impairment of Alzheimer's disease (AD)[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, our study did not explore the expression of PirB in astrocytes and the effect of TAT-PEP on astrocytes after ischemic stroke, which remains to be explored in future research.\u003c/p\u003e \u003cp\u003eIn conclusion, our study showed TAT-PEP improved neurobehavioral function and cognitive function by reducing brain infarct volume and attenuating neuronal degeneration, apoptosis, and mitochondria damage against MCAO. In vitro study showed that TAT-PEP enhanced neuronal survival and alleviated apoptosis by decreasing MDA levels, increasing SOD activity, and helping ROS accumulation. Furthermore, the results revealed that TAT-PEP alleviated neuronal apoptosis by affecting the expression of apoptosis-associated proteins, such as cleaved Caspase3, Bcl-2, and Bax. Together, our findings suggest that TAT-PEP may represent a highly productive neuroprotective agent displaying therapeutic potential for ischemic stroke. Notably, this study implies that antagonizing PirB function may define an attractive therapeutic strategy against neuronal apoptosis after ischemic stroke in future studies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePirB \u0026nbsp; \u0026nbsp; \u0026nbsp;Paired immunoglobulin-like receptor B\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMAIs \u0026nbsp; \u0026nbsp; myelin-associated inhibitory proteins\u003c/p\u003e\n\u003cp\u003eTAT-PEP \u0026nbsp;transcription-PirB extracellular peptide\u003c/p\u003e\n\u003cp\u003eOGD \u0026nbsp; \u0026nbsp; oxygen-glucose deprivation\u003c/p\u003e\n\u003cp\u003eLDH \u0026nbsp; \u0026nbsp; \u0026nbsp;lactate dehydrogenase\u003c/p\u003e\n\u003cp\u003eMDA \u0026nbsp; \u0026nbsp; malondialdehyde\u003c/p\u003e\n\u003cp\u003eSOD \u0026nbsp; \u0026nbsp; \u0026nbsp;superoxide dismutase\u003c/p\u003e\n\u003cp\u003eROS \u0026nbsp; \u0026nbsp; \u0026nbsp;reactive oxygen species\u003c/p\u003e\n\u003cp\u003eMAIs \u0026nbsp; \u0026nbsp; myelin-associated inhibitory proteins\u003c/p\u003e\n\u003cp\u003eGP \u0026nbsp; \u0026nbsp; \u0026nbsp; glycoprotein\u003c/p\u003e\n\u003cp\u003eMAG \u0026nbsp; \u0026nbsp; myelin associated glycoprotein\u003c/p\u003e\n\u003cp\u003eMCAO \u0026nbsp; \u0026nbsp;middle cerebral artery occlusion\u003c/p\u003e\n\u003cp\u003eNOR \u0026nbsp; \u0026nbsp; \u0026nbsp; novel object recognition test\u003c/p\u003e\n\u003cp\u003eMRI \u0026nbsp; \u0026nbsp; \u0026nbsp; magnetic resonance imaging\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eT2WI \u0026nbsp; \u0026nbsp; T2 weighted images\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Prof.\u0026nbsp;Xingchun Gou (Shaanxi Key Laboratory of Brain Disorders \u0026amp; Institute of Basic and Translational Medicine, Xi\u0026rsquo;an Medical University, Xi\u0026rsquo;an, China) and Prof. Lixian Xu (State Key Laboratory of Military Stomatology, National Clinical Research Center for Oral Diseases, Shaanxi Engineering Research Center for Dental Materials and Advanced Manufacture, School of Stomatology, Air Force Medical University, Xi\u0026apos;an, Shaanxi, China) for the guidance given in project design and experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBD, PZ, and Q W designed the project. PZ, JP Z, JK K, LY L, XY L and BD contributed to the data analysis and the manuscript\u0026apos;s drafting. PZ, JK K, NM F, HL D, CL, and BD conducted all experiments. PZ, JP Z, JK K, LY L, XY L, NM F, HL D, CL, and BD participated in discussions, data analysis, and manuscript editing. All authors have read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was partially supported by the Medical \u0026ldquo;Basic-Clinical\u0026rdquo; Integration Innovation Project of Xi\u0026rsquo;an Jiaotong University (Program No.\u0026nbsp;YXJLRH2022030), the fund of the First Affiliated Hospital of Xi\u0026rsquo;an Jiaotong University (Program No. 2022MS-26), the Key Research \u0026amp; Development Program of Shaanxi (Program No. 2022ZDLSF02-09), the General project of Fujian Natural Science Foundation (No. 2021J01018), the Fujian Health Science and Technology program (2021GGB038).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode Availability\u0026nbsp;\u003c/strong\u003eGraphPad Prism Version 7.0; ImageJ Version 1.51 J; Imaris Version 9.7.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u0026nbsp;\u003c/strong\u003eAll experimental procedures were approved by the Ethics Committee of the Fourth Military Medical University, and by the Institutional Animal Care and Use Committees of Xi\u0026rsquo;an Jiaotong University (Xi\u0026rsquo;an, China; 2019\u0026ndash;060) in accordance with the National Institutes of Health guidelines. Efforts were made to minimize animal sufering, and all sample sizes for the assessment parameters were calculated to minimize the number of animals used.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u0026nbsp;\u003c/strong\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u0026nbsp;\u003c/strong\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMendelson SJ, Prabhakaran S (2021) Diagnosis and Management of Transient Ischemic Attack and Acute Ischemic Stroke: A Review. 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Eur J Pharmacol 806:1\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ejphar.2017.03.025\u003c/span\u003e\u003cspan address=\"10.1016/j.ejphar.2017.03.025\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"TAT-PEP, cerebral ischemic reperfusion injury, cognitive impairment, neuronal mitochondria","lastPublishedDoi":"10.21203/rs.3.rs-2327876/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2327876/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePaired immunoglobulin-like receptor B (PirB) has been identified as a receptor for myelin-associated inhibitory proteins (MAIs), which plays a vital role in axonal regeneration, synaptic plasticity, and neuronal survival after stroke. In our previous study, a transactivator of transcription-PirB extracellular peptide (TAT-PEP) was generated, which can block the interactions between MAIs and PirB. We found that TAT-PEP treatment enhanced axonal regeneration, CST projection, and improved long-term neurobehavioral functional recovery after stroke through its effects on PirB-mediated downstream signaling molecules. However, the impact of TAT-PEP on cognitive function recovery and neuronal survival also needs to explore. Here, we investigated that \u003cem\u003epirb\u003c/em\u003e RNAi alleviated neuronal injury by inhibiting PirB expression after exposure to oxygen-glucose deprivation (OGD) in vitro. Moreover, TAT-PEP treatment attenuated brain infarct volume and promoted neurobehavioral function and cognitive function recovery. This study further found TAT-PEP exerted neuroprotection by alleviating neuronal degeneration and apoptosis after ischemic reperfusion injury. The study also showed that TAT-PEP enhanced neuronal survival and reduced the release of lactate dehydrogenase (LDH) \u003cem\u003ein vitro\u003c/em\u003e. Furthermore, the results indicated TAT-PEP decreased malondialdehyde (MDA) levels, increased superoxide dismutase (SOD) activity, and alleviated reactive oxygen species (ROS) accumulation of neurons exposed to OGD injury. The possible mechanism was TAT-PEP could help neuronal mitochondria damage and affect the expression of cleaved Caspase3, Bax, and Bcl-2. Our findings suggest that PirB overexpression in neurons after suffering ischemic reperfusion injury-induced neuronal mitochondria damage, oxidative stress, and apoptosis. This study also indicated that TAT-PEP might represent a highly productive neuroprotective agent displaying therapeutic potential for stroke by alleviating neuronal oxidative stress, mitochondria damage, degeneration, and apoptosis against ischemic stroke.\u003c/p\u003e","manuscriptTitle":"TAT-PEP alleviated cognitive impairment by alleviating neuronal mitochondria damage and apoptosis after cerebral ischemic reperfusion injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-12-12 18:28:16","doi":"10.21203/rs.3.rs-2327876/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-01-23T03:48:02+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-01-15T11:45:30+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Molecular Neurobiology","date":"2023-01-09T01:57:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-12-08T06:45:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Neurobiology","date":"2022-12-07T07:10:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"82ca6794-5084-4134-bb8a-c6528626b5c4","owner":[],"postedDate":"December 12th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T21:54:08+00:00","versionOfRecord":{"articleIdentity":"rs-2327876","link":"https://doi.org/10.1007/s12035-023-03404-w","journal":{"identity":"molecular-neurobiology","isVorOnly":false,"title":"Molecular Neurobiology"},"publishedOn":"2023-06-19 21:19:01","publishedOnDateReadable":"June 19th, 2023"},"versionCreatedAt":"2022-12-12 18:28:16","video":"","vorDoi":"10.1007/s12035-023-03404-w","vorDoiUrl":"https://doi.org/10.1007/s12035-023-03404-w","workflowStages":[]},"version":"v1","identity":"rs-2327876","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2327876","identity":"rs-2327876","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}