Application of Shh and S100A10 in inhibition of ubiquitous apoptosis of neurons and treatment of ischemic stroke

Abstract

The invention discloses an application of an acoustic hedgehog Shh and / or S100A10 as a target in screening of drugs for inhibiting or improving ubiquitous apoptosis of neurons, or an application of the acoustic hedgehog Shh and / or S100A10 in screening of drugs for treating ischemic stroke. The invention also discloses an application of the sonic hedgehog Shh or the recombinant Shh protein in preparation of a medicine for inhibiting or improving neuronal pan-apoptosis or a medicine for treating ischemic stroke, and an application of an S100A10 expression promoter in preparation of a medicine for inhibiting or improving neuronal pan-apoptosis or a medicine for treating ischemic stroke. The research shows that Shh regulates and controls the occurrence of ubiquitous apoptosis of neurons after ischemic stroke through S100A10, a new perspective is provided for deeply understanding a molecular mechanism activated by ubiquitous apoptosis after ischemic stroke, and a treatment target with transformation potential is provided for developing an intervention strategy for neurological function recovery of ischemic stroke patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of Sonic hedgehog (Shh) and S100A10 in inhibiting neuronal pan - apoptosis and treating ischemic stroke. Background Art

[0002] Ischemic stroke poses a major threat to human life and is one of the main causes of disability and death globally. Currently, the relatively effective clinical treatments for acute ischemic stroke mainly include intravenous thrombolysis and mechanical thrombectomy. However, due to the narrow treatment time window and the risk of ischemia / reperfusion injury, the effect is still very limited. Therefore, new treatment strategies are urgently needed.

[0003] Cerebral ischemia leads to neuronal death. Previous studies have shown that there are many different forms of neuronal death, such as necrosis, apoptosis, and pyroptosis. This is one of the key nodes for the recovery of neurological function after cerebral infarction and has been widely studied. Pan - apoptosis is defined as a new type of inflammatory programmed cell death (PCD) pathway that is activated by specific triggers and regulated by the PANoptosome complex, with the key characteristics of pyroptosis, apoptosis, and necroptosis, but cannot be characterized by any one of these three cell death modes alone. Studies have shown that pan - apoptosis is related to various diseases, such as infections, autoimmune diseases, neurodegenerative diseases, cerebrovascular diseases, and tumors. However, the specific mechanism of pan - apoptosis in ischemic stroke is still unclear.

[0004] S100 calcium - binding protein A10 (S100A10), also known as p11, is a member of the S100 protein family, located on chromosome 1q21, and can form a heterotetrameric complex (S100A10)2-(annexinA2)2 by binding to annexin A2. Different from other family proteins, the S100A10 protein has a calcium - binding protein mutation site and is insensitive to calcium ions. S100A10 is widely distributed in the body and is expressed in the brain, heart, gastrointestinal tract, kidney, liver, lung, spleen, testis, epidermis, aorta, and thymus. In the brain, S100A10 is mainly expressed in neurons and also in type A2 reactive astrocytes, playing a neuroprotective role. S100A10 is closely related to neuropsychiatric system diseases such as depression, glioma, ischemic stroke, and Alzheimer's disease. However, whether it regulates the occurrence of neuronal pan - apoptosis after ischemic brain injury and how it regulates are still unclear.

[0005] Sonic hedgehog (Shh) is one of the members of the Hedgehog (Hh) gene family, which consists of the Shh ligand, the transmembrane protein receptors Patched-1 (Ptc-1) and Smoothened (Smo), and the zinc finger transcription factor Gli proteins (Gli-1, Gli-2, Gli-3). The Shh signal is widely present in higher vertebrates and is a key regulator in the development of embryos and the central nervous system (CNS). Under normal adult conditions, the Shh signal is in a quiescent state. However, lesions such as brain and spinal cord inflammation, trauma, stroke, and degeneration can activate the Shh signal. The activated Shh signal can reduce damage and promote repair by promoting neurogenesis, axon remodeling, antioxidation, and anti-apoptosis. The research group also confirmed in previous studies that the activated Shh signal can reduce cerebral ischemic damage, enhance neurogenesis and synaptic remodeling, and improve neurological function. S100A10 is closely related to neurogenesis, can enhance synaptic remodeling, and can enhance the ability of BDNF to act on neural plasticity. In addition, P11 interacts with Bcl-xL and Bcl-2-associated death promoter (BAD) and inhibits its pro-apoptotic activity. The Shh / PI3K / Bcl-2 signaling pathway plays a key role in protecting neurons from oxidative stress-induced apoptosis. However, it is currently unclear whether the Shh signal and S100A10 are involved in the regulation of neuronal pan-apoptosis after ischemic brain injury and whether there is an interaction between the two. Summary of the Invention

[0006] The object of the present invention is to address the above problems and provide an application of Sonic hedgehog (Shh) and S100A10 in inhibiting neuronal pan-apoptosis and treating ischemic stroke.

[0007] To achieve its object, the present invention adopts the following technical solutions:

[0008] In the first aspect of the present invention, there is provided an application of Sonic hedgehog (Shh) and / or S100A10 as a target in screening drugs for inhibiting or improving neuronal pan-apoptosis.

[0009] In the second aspect of the present invention, there is provided an application of Sonic hedgehog (Shh) and / or S100A10 as a target in screening drugs for treating ischemic stroke.

[0010] In any of the above application technical solutions, the drug upregulates the expression of Sonic hedgehog (Shh) or S100A10.

[0011] In any of the above application technical solutions, the Sonic hedgehog (Shh) upregulates the expression of S100A10, inhibits neuronal pan-apoptosis, and improves the deterioration of neurological function.

[0012] The neuronal pan-apoptosis is the neuronal pan-apoptosis after ischemic stroke.

[0013] The third aspect of the present invention provides the application of sonic hedgehog factor Shh or recombinant Shh protein in the preparation of a drug for inhibiting or improving neuronal pan-apoptosis or in the preparation of a drug for treating ischemic stroke.

[0014] The fourth aspect of the present invention provides the application of an S100A10 expression promoter in the preparation of a drug for inhibiting or improving neuronal pan-apoptosis or in the preparation of a drug for treating ischemic stroke.

[0015] In the above application technical solution, the S100A10 expression promoter is Shh protein.

[0016] In the above application technical solutions of the third and fourth aspects, the sonic hedgehog factor Shh or recombinant Shh protein up-regulates the expression of S100A10, inhibits neuronal pan-apoptosis, and improves the deterioration of nerve function.

[0017] The beneficial effects of the present invention are as follows:

[0018] The research of the present invention finds that ischemic brain injury induces pan-apoptosis, up-regulates the expression of S100A10 and Shh proteins, suggesting that these two proteins may participate in the neuroprotective mechanism as endogenous protective factors. Verification was carried out by in vitro constructing an OGD / R model, indicating that knocking down S100A10 would exacerbate the process of neuronal pan-apoptosis induced by OGD / R injury. After intervention with exogenous recombinant Shh protein, an increase in the expression of S100A10 could be observed, and the phenomenon of OGD / R neuronal pan-apoptosis was effectively inhibited. Further in in vivo experiments, using the SD rat MCAO / R model, it was confirmed that recombinant Shh protein could significantly reverse the promoting effect of S100A10 knockdown on the pan-apoptosis process after ischemic brain injury, and at the same time significantly improve the nerve function prognosis.

[0019] The research of the present invention shows that Shh regulates the occurrence of neuronal pan-apoptosis after ischemic stroke through S100A10, provides a new perspective for deeply understanding the molecular mechanism of pan-apoptosis activation after ischemic stroke, and provides a therapeutically target with translational potential for developing intervention strategies for the recovery of nerve function in ischemic stroke patients. Description of the Drawings

[0020] Figure 1The expression level of S100A10 is increased in patients with acute ischemic stroke, MCAO / R rats, and OGD / R neurons: A. ELISA was used to detect the expression level of S100A10 in the serum of AIS patients; B. Multiplex immunofluorescence staining was used to detect the co-localization of S100A10 cells in the brain tissue of AIS patients; C-D. WB was used to detect the expression level of S100A10 in the brain tissue of MCAO / R rats; E-F. Immunofluorescence staining was used to detect the co-localization of S100A10 cells in the brain tissue of MCAO / R rats; G. Immunofluorescence was used to identify the purity of primary cortical neurons; H-I. Results of immunofluorescence Z-axis imaging and S100A10 fluorescence intensity analysis of primary cortical neurons.

[0021] Figure 2 It shows that MCAO / R injury induces neuronal pan-apoptosis: A-B. Results of TUNEL staining and immunofluorescence double-labeling staining suggest that multiple death pathways are activated simultaneously after MCAO / R injury; C-K. WB was used to detect the expression of pan-apoptosis-related proteins after MCAO / R injury; L. Multiplex immunofluorescence staining was used to detect the formation of pan-apoptotic bodies; M. Co-IP was used to verify the direct interaction between key molecules of pyroptosis, apoptosis, and necroptosis.

[0022] Figure 3 It shows that knockdown of S100A10 exacerbates neuronal pan-apoptosis after OGD / R: A-B. WB results show that the expression of S100A10 in the knockdown lentivirus group is significantly downregulated; C-K. WB was used to detect the expression of pan-apoptosis-related proteins after knockdown of S100A10; L. CCK-8 method was used to detect the viability of neurons after knockdown of S100A10.

[0023] Figure 4 It shows that recombinant Shh upregulates the expression of S100A10 and inhibits neuronal pan-apoptosis after OGD / R: A-E. WB was used to detect the expression of Shh and downstream molecules in primary cortical neurons after intervention with recombinant Shh (rShh); F-G. WB was used to detect the expression of S100A10 after rShh intervention; H-O. WB was used to detect the expression of pan-apoptosis-related proteins after rShh intervention; P. CCK-8 method was used to detect the viability of neurons after rShh intervention.

[0024] Figure 5 It shows that recombinant Shh reverses the exacerbation of pan-apoptosis and neurological function deterioration after MCAO / R injury caused by S100A10 knockdown: A-B. Immunofluorescence was used to detect the co-localization with NeuN after knockdown of S100A10 in vivo; C-H. WB was used to detect the expression of Shh and downstream molecules after in vivo rShh intervention; C, D, I-P. WB was used to detect the expression of pan-apoptosis-related proteins after knockdown of S100A10 and rShh intervention in vivo; Q-R. TTC was used to detect the cerebral infarction volume; S-U. Neurological function scores were used to evaluate the degree of neurological deficit. Detailed implementation manners

[0025] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto

[0026] The experimental methods in the following embodiments are all conventional methods unless otherwise specified.

[0027] Embodiment 1

[0028] 1 Materials and methods

[0029] 1.1 Materials

[0030] Adult male (8 weeks old, 200 - 250 g) and neonatal SD rats were purchased from the Animal Experiment Center of Chongqing Medical University.

[0031] Anti - rabbit NLRP3 antibody (purchased from abcam, catalog number ab270449), anti - rabbit Caspas1 antibody (purchased from Proteintech, catalog number 22915 - 1 - Ap), anti - rabbit Caspase3 antibody (purchased from CST, catalog number 9662), anti - rabbit Caspase8 antibody (purchased from CST, catalog number 9662), anti - mouse MLKL antibody (purchased from Proteintech, catalog number 21066 - 1 - Ap), anti - rabbit phospho - MLKL antibody (purchased from abcam, catalog number ab196436), anti - rabbit ZBP1 antibody (purchased from SantaCruz, catalog number sc - 271483), anti - rabbit ASC antibody (purchased from Proteintech, catalog number 10500 - 1 - Ap), anti - mouse β - actin antibody (purchased from Proteintech, catalog number 66009 - 1 - Ig), anti - mouse GAPDH antibody (purchased from Proteintech, catalog number 10494 - 1 - Ig), recombinant mouse Shh (rShh) (purchased from Medchemexpress, catalog number HY - P7409).

[0032] 1.2 Methods

[0033] 1.2.1 Grouping and intervention

[0034] (1) Clinical research

[0035] Patients with first - onset acute ischemic stroke hospitalized in the Department of Neurology of the First Affiliated Hospital of Chongqing Medical University were collected as the case group, and healthy physical examination personnel during the same period were used as the control group. Serum specimens of each group were collected for ELISA detection.

[0036] Brain tissue specimens of patients with right - sided large - area cerebral infarction who underwent decompressive craniectomy in the Department of Neurosurgery of the Third People's Hospital of Mianyang were collected for immunohistofluorescence staining.

[0037] (2) In - vivo experiments

[0038] An SD rat MCAO / R model was used, and the SD rats were randomly divided into 6 groups (n = 3):

[0039] ① Sham group: sham operation group;

[0040] ② MCAO / R group: samples were taken or behavioral scoring was performed 3 days after the MCAO / R model was constructed;

[0041] ③ MCAO / R + Ad-NC group: an MCAO / R model was constructed 4 days after intraventricular injection of Ad-NC, and samples were taken or behavioral scoring was performed 3 days after the MCAO / R model was established;

[0042] ④ MCAO / R + Ad-shS100A10 group: an MCAO / R model was constructed 4 days after intraventricular injection of Ad-shS100A10, and samples were taken or behavioral scoring was performed 3 days after the MCAO / R model was established;

[0043] ⑤ MCAO / R + rShh group: rShh (5 μL / rat) was intraventricularly injected 2 h after the MCAO / R model was constructed, and samples were taken or behavioral scoring was performed 3 days after the MCAO / R model was established;

[0044] ⑥ MCAO / R + Ad-shS100A10 + rShh group: rShh (5 μL / rat) was intraventricularly injected 2 h after the MCAO / R model was constructed 4 days after intraventricular injection of Ad-shS100A10, and samples were taken or behavioral scoring was performed 3 days after the MCAO / R model was established.

[0045] Brain tissue samples of each group were collected for WB and TTC staining.

[0046] (3) In vitro experiments

[0047] A primary cortical neuron OGD / R model of neonatal SD rats was used:

[0048] 1. To explore the effect of OGD / R injury on the expression of S100A10 in vitro and the effect of knocking down S100A10 on pan-apoptosis after OGD / R of primary cortical neurons.

[0049] OGD / R injury refers to the injury suffered by cells during the process of reperfusion after experiencing oxygen-glucose deprivation (OGD). This injury model is often used to study ischemic stroke.

[0050] The cells were randomly divided into 4 groups (n = 3):

[0051] ①Control+shNC group: First, transfect with shNC for 24 h, and then culture with complete neuronal medium for 48 h;

[0052] ②Control+shS100A10 group: First, transfect with shS100A10 for 24 h, and then culture with complete neuronal medium for 48 h;

[0053] ③OGD / R+shNC group: Before constructing the cell OGD / R model, culture with complete neuronal medium containing shNC for 24 h;

[0054] ④OGD / R+shS100A10 group: Before constructing the cell OGD / R model, culture with complete neuronal medium containing shS100A10 for 24 h;

[0055] Collect cell samples from each group for WB, IF, and CCK-8.

[0056] 2. Explore the effects of recombinant Shh (rShh) pretreatment on neuronal pan-apoptosis and S100A10 expression after OGD / R in vitro.

[0057] Randomly divide the cells into 4 groups (n = 3):

[0058] ①Control group: Primary cortical neurons in normal culture group;

[0059] ②Control+rShh group: Stimulate with rShh at a final concentration of 10 μM for 24 h;

[0060] ③OGD / R group: Oxygen-glucose deprivation for 4 h and reoxygenation for 24 h;

[0061] ④OGD / R+rShh group: Oxygen-glucose deprivation for 4 h, and stimulate with rShh at a final concentration of 10 μM for 24 h during reoxygenation.

[0062] Collect cell samples from each group for WB and CCK-8.

[0063] 1.2.2 Construction of the rat middle cerebral artery occlusion / reperfusion model (MCAO / R)

[0064] SD rats were intraperitoneally injected with 1.25% avertin (2,2,2-tribromoethanol) at a dose of 10 mL / kg. After the appropriate depth of anesthesia was achieved, the rats were fixed on the operating table. The hair on the neck was shaved, and the neck area was disinfected with 75% alcohol. A midline cervical incision (about 2 cm in length) was made, and the muscle tissue was separated to expose the right carotid sheath. The right common carotid artery, external carotid artery, and internal carotid artery were isolated. The operation was performed gently to avoid damaging the vagus nerve accompanying the carotid artery. The proximal end of the common carotid artery and the distal end of the external carotid artery were ligated with 5.0 surgical suture, and an arterial clamp was placed at the distal end of the internal carotid artery. The common carotid artery was incised, and a thread embolism was inserted. The thread embolism was inserted through the internal carotid artery and continued into the intracranial branch of the pterygopalatine artery to occlude the middle cerebral artery, and the ligature was used to fix the thread embolism. After 120 minutes of ischemia, the thread embolism was removed to simulate the process of reperfusion injury. During the operation, the rats were placed on a thermostatic pad for warming.

[0065] 1.2.3 Intracerebroventricular catheterization in rats

[0066] SD rats were anesthetized by intraperitoneal injection of 1.25% avertin (2,2,2-tribromoethanol) at a dose of 10 mL / kg and placed on a stereotaxic apparatus. After the skin was disinfected with 75% alcohol, a scalp incision (about 1 cm in length) was made along the mid-sagittal suture of the skull. The subcutaneous tissue and muscle were bluntly separated to fully expose the skull. The periosteum was removed with hydrogen peroxide to expose the bone suture. The puncture point coordinates were 0.9 mm posterior to the bregma and 1.5 mm lateral to the midline. A cranial drill was used to drill through the dura mater at the marked point, and a 23G catheter was inserted 3.6 mm with a holder. The catheter was fixed with dental cement and allowed to dry thoroughly for 5 - 10 minutes. The scalp was sutured, and erythromycin ointment was used for prophylactic anti-inflammation. The rats were housed individually.

[0067] 1.2.4 Extraction and culture of primary cortical neurons

[0068] As described above, primary cortical neurons were obtained from neonatal SD rats within 24 hours after birth.

[22] . The culture plates were coated with polylysine solution for 2 hours and dried for use within 24 hours before culture. Neonatal SD rats within 24 hours after birth were used. After alcohol disinfection, the heads were quickly decapitated on ice, and the brains were carefully removed. After removing the meninges, the non-cortical tissues were excised. The brain tissue was minced with ophthalmic scissors and transferred to a centrifuge tube. 0.125% trypsin was added and incubated at 37°C for 15 minutes. During this period, it was gently shaken several times. Then, an equal volume of DMEM medium containing 10% fetal bovine serum was added to terminate the digestion. After gentle pipetting, it was filtered through a 40 μm filter, centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed. The cells were seeded into the pre-coated well plates, and DMEM medium containing 10% fetal bovine serum was added. After 4 hours of cell culture, the medium was replaced with neuronal complete medium, and half of the medium was changed every other day. The cells were cultured for 7 days for subsequent studies.

[0069] 1.2.5 Establishment of OGD / R model of primary cortical neurons

[0070] An in vitro neuron OGD / R model was constructed according to the previous method of the research group. Briefly, the neuron complete medium was discarded, washed twice with PBS, D-hanks solution was added, and it was placed in a triple gas incubator (37 °C, 1% O2, 5% CO2, 94% N2) for a specified time. After hypoxia, it was replaced with neuron complete medium and cultured in a carbon dioxide incubator with 5% CO2 for another 24 hours.

[0071] 1.2.6 Lentiviral vector transfection

[0072] 1.2.6.1 Lentiviral vector (LV) ordering information

[0073] The lentivirus for knocking down rat S100A10 and the NC lentivirus were purchased from Shanghai GenePharma Co., Ltd.

[0074] shRNA sequence (SEQ ID NO.1): 5’-TCCCAAATGGAGCATGCCA-3’.

[0075] 1.2.6.2 Lentiviral transfection of primary cortical neurons

[0076] When the primary cortical neurons of rats were cultured in vitro for 7 days (seeding density was about 95%-100%), the original medium was aspirated and discarded, 1 / 2 volume of fresh medium was added, and at the same time, the lentiviral vector (MOI = 10) was added. After gently shaking, it was placed in the incubator for culture. After 24 hours of culture, the cell culture medium containing the lentivirus was removed, and the same volume (2 ml / well) of fresh medium without the lentivirus was added, and the next experimental study was carried out after continuing to culture for 48 hours.

[0077] 1.2.7 Adenoviral vector transfection

[0078] 1.2.7.1 Adenoviral vector (ADV) ordering information

[0079] The adenovirus for knocking down rat S100A10 and the NC adenovirus were purchased from Shanghai GenePharma Co., Ltd.

[0080] 1.2.7.2 Intracerebroventricular injection of adenovirus

[0081] Open the catheter port, connect the microsyringe to the catheter through a PE tube; aspirate 5 μL of PBS and 5 μL of adenovirus with the microsyringe respectively; install the microsyringe on the microinjection pump and slowly inject at a rate of 0.5 μL / min; after injection, the microsyringe stays for 10 minutes and then withdraws.

[0082] 1.2.8 CCK-8 method for measuring cell viability

[0083] Cells were seeded into 96-well plates, with 100 μL of cell suspension per well (n = 6). After culturing for 7 days, cells with good growth status were selected and transfected with lentivirus (for about 24 h), and then cultured for another 48 h. An OGD / R model was established according to the method described in Section 2.3.3 of Part 1. After successful model construction, a full medium change was performed once, and 10 μL of CCK-8 solution was added to each well in the dark. The culture was terminated after incubation at 37 °C for 4 h. The absorbance value (Optical density, OD) at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0084] 1.2.9 ELISA

[0085] The content of S100A10 in the serum of patients with acute ischemic stroke was detected by applying an enzyme immunoassay Human S100A10 ELISA Kit (MM-50547H1). The experimental operations were carried out strictly according to the instructions of the kit.

[0086] 1.2.10 Neurological deficit score

[0087] At 24 h and 72 h after reperfusion, a researcher who was involved in the study but unaware of the details conducted a neurological assessment on SD rats.

[0088] 1.2.10.1 Longa score

[0089] Inclusion criteria for animal models: Longa score of 1 - 3. Other scores were excluded. Behavioral scores were performed on each group at 24 h and 72 h after surgery. The scoring criteria are as follows:

[0090]

[0091] 1.2.10.2 Berderson score

[0092] Gently grasp the rat's tail and lift it 10 cm above the tabletop, and observe the flexion of the forelimbs. The forepaws of normal rats are in a straight state. Behavioral scoring was performed at 72 h after surgery. The scoring criteria are as follows:

[0093]

[0094] 1.2.10.3 mNSS score

[0095] The mNSS is a comprehensive assessment of neurological function, including motor, sensory, reflex, and balance functions, and can comprehensively reflect the neurological function of rats. The total score is 18 points. The higher the score value, the more severe the injury. Behavioral scoring was performed at 72 h after surgery. The scoring criteria are as follows:

[0096]

[0097] 1.2.11 TTC Staining

[0098] After 72 h of reperfusion, the SD rats that had completed the neurological behavioral scoring were anesthetized, and after sacrificing the animals by decapitation, the intact brain tissues were removed (within 10 minutes). After removing the lower brainstem, cerebellum, and olfactory bulbs, they were quickly frozen at -20°C for 20 min. The frozen brain tissues were placed in a brain trough, and brain slices were cut coronally at equal intervals, with each slice about 2 mm thick, for a total of 5 slices. The brain slices were put into 2% TTC solution and stained for 10 - 20 min at room temperature in the dark. They were gently flipped every 5 min to ensure uniform staining. After the normal brain tissues were stained red and the ischemic infarcted tissues were stained grayish white, the brain slices were taken out, immersed in 4% paraformaldehyde, and fixed at 4°C for 2 h. The fixed brain slices were taken out, arranged neatly, and photographed with a digital camera. The percentage of infarct volume was calculated using ImageJ software. The calculation formula was: percentage of infarct volume = (sum of the areas of white ischemic regions in each slice) / (sum of the areas of brain slices in each slice) × 100%.

[0099] 1.2.12 Preparation of Paraffin Sections

[0100] After 72 h of reperfusion, the SD rats were deeply anesthetized, fixed in a supine position on an operating table, and sequentially perfused with normal saline and paraformaldehyde through the heart. After the perfusion was completed, the rats were decapitated to remove the brain, and the brain tissues were immersed in 4% paraformaldehyde and left overnight at 4°C. Then they were dehydrated with gradient ethanol, cleared in xylene, infiltrated with paraffin and embedded, and finally serially sectioned coronally (4 μm thick).

[0101] 1.2.13 TUNEL Staining

[0102] An in - situ cell death detection kit, one - step TUNEL method, was used to detect apoptotic neuron cells. The operation was carried out strictly according to the instructions. The paraffin sections were baked in an oven at 60°C for 2 h; dewaxed to water (xylene Ⅰ for 20 min - xylene Ⅱ for 20 min - absolute ethanol for 10 min - 90% ethanol for 5 min - 80% ethanol for 5 min - 70% ethanol for 5 min - washed in PBS on a shaker for 5 min × 3 times); 50 μL of proteinase K solution was added and incubated at 37°C for 20 min, followed by repeated PBS washing; 0.3% Triton - X - 100 was added and incubated at room temperature for 20 min, followed by repeated PBS washing; Equilibration Buffer was added and incubated at room temperature for 30 min; TUNEL reaction mixture was added and incubated at 37°C in the dark for 1 h, followed by repeated PBS washing; DAPI was added and incubated at room temperature in the dark for 15 min, followed by repeated PBS washing; an appropriate amount of anti - fluorescence quenching agent was added, avoiding the generation of bubbles, and a cover glass was gently covered; observed and photographed under a laser confocal microscope or stored at 4°C.

[0103] 1.2.14 Immunostaining

[0104] 1.2.14.1 Immunohistofluorescence staining

[0105] Deparaffinize and hydrate the paraffin sections routinely; permeabilize with 0.3% Triton-X-100 at room temperature for 20 min, wash with PBS by shaking for 5 minutes, and repeat three times; immerse the sections completely in preheated Tris-EDTA (pH 9.0) antigen retrieval solution, heat in a microwave oven at high power for 5 min and low power for 15 min, and let cool to room temperature naturally; repeat washing with PBS, block with 10% goat serum at 37 °C for 1 h; discard the blocking solution, add the primary antibody, and incubate overnight at 4 °C in the dark; the next day, warm to room temperature for 30 min, repeat washing with PBS; add the corresponding secondary antibody, incubate at 37 °C in the dark for 1 h, and repeat washing with PBS; add DAPI to stain the nuclei, incubate at 37 °C in the dark for 15 min, and repeat washing with PBS; mount with an anti-fluorescence quencher; observe and photograph with a laser confocal microscope or store at 4 °C.

[0106] 1.2.14.2 Immunocytofluorescence staining

[0107] Remove the cell culture medium, add PBS and wash by shaking for 5 minutes, and repeat three times; fix with 4% paraformaldehyde at room temperature for 15 min, and repeat washing with PBS; permeabilize with 0.3% Triton-X-100 at room temperature for 10 min, and repeat washing with PBS; block with 10% goat serum at room temperature for 1 h; discard the blocking solution, add the primary antibody, and incubate overnight at 4 °C in the dark; the next day, warm to room temperature for 20 min, repeat washing with PBS; add the corresponding secondary antibody, incubate at 37 °C in the dark for 1 h, and repeat washing with PBS; add DAPI to stain the nuclei, incubate at 37 °C in the dark for 10 min, and repeat washing with PBS; mount with an anti-fluorescence quencher; observe and photograph with a laser confocal microscope or store at 4 °C.

[0108] 1.2.15 Western blot analysis of protein expression

[0109] 1.2.15.1 Protein sample preparation

[0110] Extraction of cell proteins: Discard the culture medium, wash with PBS twice, then add an appropriate amount (60 - 100 μL) of protein lysate (RIPA:PMSF = 100:1) according to the number of neurons, and scrape the cells with a cell scraper and transfer them to a 1.5 mL centrifuge tube. Lyse the neurons with an ultrasonic cell disruptor (operate on ice) and react on ice for 30 min. Centrifuge at 12000 rpm at 4 °C for 10 min, collect the supernatant and store it in a -80 °C refrigerator for later use.

[0111] Extraction of histones: Take out the pre-prepared brain tissue samples from the -80°C refrigerator, cut 50 mg of brain tissue specimens on ice, place them in a 1.5 ml EP tube containing 300 μl of RIPA lysis buffer (RIPA:PMSF:phosphatase inhibitor = 100:1:2), add magnetic beads, and grind thoroughly with a low-temperature tissue grinder (no obvious solid state visible to the naked eye). Aspirate the supernatant and lyse it on ice for 30 min. Centrifuge at 12,000 rpm at 4°C for 10 min, collect the supernatant, and store it in the -80°C refrigerator for later use.

[0112] 1.2.15.2 Protein concentration determination

[0113] Perform protein quantification according to the instructions of the BCA protein quantification kit. Add 5× loading buffer (protein sample:loading buffer = 4:1), mix well, boil and denature at 100°C for 10 min, and after cooling to room temperature, aliquot and store in the -80°C refrigerator for later use.

[0114] 1.2.15.3 SDS-Polyacrylamide gel electrophoresis and Western blot analysis

[0115] Electrophoresis Prepare an SDS-PAGE protein gel with an appropriate concentration according to the molecular weight of the target protein. Load 30 μg of protein per well. First, perform electrophoresis at a constant voltage of 60 V. After the protein enters the separation gel, adjust the voltage to 120 V and stop electrophoresis when the bromophenol blue reaches the lower edge of the glass plate.

[0116] Electroblotting Carefully remove the glass plate, determine the range of the gel according to the size of the protein required for the experiment. Cut a PVDF membrane of appropriate size according to the size of the gel, soak it in anhydrous methanol for 1 min; arrange it in the order of sponge - filter paper - gel - PVDF membrane - filter paper - sponge, use a glass rod to drive away air bubbles; clamp the electroblotting clip, place it in the electroblotting tank, and perform wet transfer at a constant current of 250 mA.

[0117] Blocking After electroblotting, wash the bands with TBST and block them with 5% non-fat milk powder at room temperature for 1 hour.

[0118] Incubation with primary antibody Take out the bands from the blocking solution and place them in the corresponding primary antibody, and incubate overnight at 4°C on a shaker (60 rpm).

[0119] Incubation with secondary antibody Wash the membrane three times with TBST on a shaker, 10 minutes each time. Put the washed bands into the corresponding secondary antibody and incubate at room temperature on a shaker (60 rpm) for 1 h.

[0120] Development Wash the membrane three times with TBST on a shaker, 10 minutes each time, and add the developing solution for development.

[0121] The gray value analysis of the target band for protein was performed using ImageJ software. Referring to the gray value of the internal reference, the relative expression level of the target protein was calculated. Graphpad prism 9.5 software was used for statistical analysis and the production of bar charts.

[0122] 1.2.16 Co-Immunoprecipitation (Co-IP)

[0123] According to the aforementioned Western blot operation procedure, after lysing cells with Ip cell lysis buffer, the supernatant was obtained by centrifuging at 12,000 rpm for 10 min at 4°C. After measuring the protein concentration by the BCA method, the proteins in each group were balanced. A part of the supernatant was denatured and used as the Input sample. 1.0 μg of IgG (IgG of the same species as the primary antibody) and 30 μL of Protein A / G were added to the supernatant of the negative control (IgG) group, and 30 μL of Protein A / G was directly added to the IP experimental group. Incubate with rotation at 4°C for 1 h; centrifuge at 4°C for 5 min (2,000 rpm), and take the supernatant; add 2.5 μg of antibody to each group and incubate overnight at 4°C; add 30 μL of Protein A / G and mix well, incubate with rotation at 4°C for 1 h; wash the magnetic beads thoroughly, resuspend the precipitate with loading buffer, shake and then centrifuge for 30 s; boil for 10 min and store at -80°C.

[0124] 1.2.17 Statistical Analysis

[0125] All experiments were repeated 3 times. GraphPad Prism 9.5.0 was used for statistical analysis and drawing. The experimental data were expressed as mean ± standard deviation, and their normal distribution was tested by the Shapiro-Wilk test. The independent sample t-test was used for comparison between two groups of data. One-way analysis of variance (one-way ANOVA) was used for comparison among multiple groups of data. P < 0.05 was considered statistically significant.

[0126] 2 Results

[0127] 2.1 S100A10 Expression Is Increased in Patients with Acute Ischemic Stroke, MCAO / R Rats, and OGD / R Neurons

[0128] ELISA analysis showed that compared with the healthy control group, the S100A10 protein level in patients with acute ischemic stroke within 3 days of onset was significantly higher than that in the healthy physical examination group (P < 0.001, Figure 1 A). Multiple immunofluorescence staining was performed on brain tissue sections of 1 patient with acute cerebral infarction after surgery, and the results showed that S100A10 was mainly co-localized with neurons ( Figure 1 B). Similarly, in the MCAO / R rat model, WB results showed that compared with the sham operation group, the S100A10 protein level in the brain tissue of MCAO / R group rats was significantly increased (P < 0.05,Figure 1 C-D). Immunofluorescence staining results showed that S100A10 had a high degree of co-localization with neurons in the ischemic brain tissue, co-localization with some microglia, but relatively less co-localization with astrocytes ( Figure 1 E-F). Neurons were identified by immunofluorescence staining, and the positive rate of the neuron-specific antibody MAP2 reached more than 90% ( Figure 1 G), indicating that the neuron purity was sufficient to meet the requirements of subsequent experiments. After OGD / R of primary cortical neurons, cellular immunofluorescence staining was performed, and significant spatial overlap of MAP2 and S100A10 in the neuron cell body and dendrites was observed by Z-axis imaging ( Figure 1 H). The expression of S100A10 in the OGD / R group was significantly higher than that in the control group ( Figure 1 I).

[0129] 2.2 Ischemic brain injury induces the occurrence of pan-apoptosis

[0130] Pan-apoptosis is a newly reported inflammatory programmed cell death with the key characteristics of pyroptosis, apoptosis, and necroptosis. Caspase1, Caspase3, and MLKL are the markers of pyroptosis, apoptosis, and necroptosis, respectively. TUNEL staining and immunofluorescence double-labeling staining showed that apoptotic cells (green) were co-labeled with Caspase1, Caspase3, and MLKL (red) respectively, indicating that multiple cell death pathways were activated simultaneously ( Figure 2 A-B). WB results further confirmed the up-regulation of pan-apoptosis-related proteins. The expression levels of NLRP3, Caspase1, Caspase3, Caspase8, p-MLKL, and ZBP1 proteins in the ischemic brain injury group were significantly higher than those in the sham operation group (P<0.05, Figure 2 C-K). To confirm the formation of the pan-apoptotic body complex, multiplex immunofluorescence staining results showed that the co-expression levels of NLRP3, Caspase3, and MLKL in neurons after OGD / R treatment were significantly higher than those in the control group ( Figure 2 L). Co-IP experiments further showed that ASC interacted with ZBP1, NLRP3, Caspase8 (CASP8), and p-MLKL in the OGD / R group ( Figure 2 M). It indicated that OGD / R injury induced the formation of ZBP1-pan-apoptotic bodies in neurons.

[0131] 2.3 Knockdown of S100A10 exacerbates neuronal pan-apoptosis after OGD / R

[0132] Knockdown of S100A10 by in vitro lentiviral transfection was performed to investigate whether it affects neuronal pan-apoptosis after OGD / R. WB results showed that the expression of S100A10 in the knockdown lentivirus group was significantly downregulated (P<0.05, Figure 3 A-B). The expression of pan-apoptosis-related proteins was detected by WB. The results showed that the expressions of pyroptosis (Caspase1(p20), NLRP3), apoptosis (Caspase3(p17), Caspase8(p18)) and necroptosis (p-MLKL)-related proteins, as well as the sensor ZBP1 protein in the S100A10 knockdown group were significantly higher than those in the control group( Figure 3 C-K). The results of CCK-8 assay showed that after OGD / R treatment, the neuronal viability in the S100A10 knockdown group was significantly lower than that in the control group( Figure 3 L).

[0133] 2.4 Exogenous recombinant Shh upregulates the expression of S100A10 and inhibits neuronal pan-apoptosis after OGD / R

[0134] The Shh signaling pathway is closely related to tissue development, injury response and repair. Previous studies have determined its neuroprotective effect in alleviating ischemic brain injury. WB was used to detect the expression of Shh and downstream molecules in primary cortical neurons after intervention with recombinant Shh (rShh). The results showed that the expressions of Shh, Ptc-1, Smo and Gli1 in the rShh treatment group were significantly higher than those in the control group (P<0.05, Figure 4 A-E). WB was used to detect the expression of S100A10 after rShh intervention. The results showed that compared with the control group, the level of S100A10 protein was upregulated in the rShh intervention group( Figure 4 F-G). At the same time, the levels of pan-apoptosis-related proteins were also detected. The results showed that after rShh intervention, the levels of pyroptosis (Caspase1, NLRP3), apoptosis (Caspase3, Caspase8) and necroptosis (p-MLKL)-related proteins, as well as the ZBP1 protein were significantly lower than those in the control group( Figure 4 F,H-O). The results of CCK-8 assay showed that the neuronal viability after OGD / R treatment was significantly lower than that in the control group, but the neuronal viability in the rShh intervention group was increased compared with the simple OGD / R group( Figure 4 P).

[0135] 2.5 Exogenous recombinant Shh reverses the aggravated pan-apoptosis and neurological function deterioration induced by S100A10 knockdown after MCAO / R

[0136] Next, we investigated whether rShh affects pan-apoptosis and functional prognosis through S100A10. Western blot confirmed that S100A10 was successfully knocked down in the Ad-shS100A10 group, and the protein levels of Shh, Ptc-1, Smo, and Gli1 were significantly increased in the rShh group. Moreover, the expression of S100A10 protein was significantly upregulated after rShh treatment ( Figure 5 C-H). Immunofluorescence results showed that NeuN+S100A10+ cells in the Ad-shS100A10 group were significantly reduced ( Figure 5 A-B). In addition, the levels of pan-apoptosis proteins were significantly upregulated after S100A10 knockdown. Knockdown of S100A10 exacerbated pan-apoptosis, and this phenomenon could be reversed by rShh. ( Figure 5 C, I-P). TTC staining showed that the infarct volume in the S100A10 knockdown group was significantly larger than that in the MCAO / R group. Longa, Berderson, and mNSS scores all showed that neurological function deteriorated significantly in the S100A10 knockdown group. However, the infarct volume in the rShh group was significantly smaller than that in the MCAO / R group and the S100A10 knockdown group, reducing neurological deficits ( Figure 5 Q-U). These results indicate that rShh reversed the increased infarct volume and neurological function deterioration caused by S100A10 knockdown after MCAO / R injury.

[0137] 3 Summary and analysis

[0138] This study first clarified that Shh affects the process of neuronal pan-apoptosis after ischemic stroke by regulating S100A10. This finding provides a new perspective for in-depth understanding of the regulatory mechanism of pan-apoptosis activation after ischemic stroke, and also opens up a potential therapeutic approach for improving the neurological function recovery of ischemic stroke patients.

Claims

1. Use of Sonic hedgehog factor Shh and / or S100A10 as targets in screening drugs for inhibiting or improving neuronal pan-apoptosis.

2. Use of Sonic hedgehog factor Shh and / or S100A10 as targets in screening drugs for treating ischemic stroke.

3. The application according to claim 1 or 2, characterized in that: The drug up-regulates the expression of Sonic hedgehog factor Shh or S100A10.

4. The application according to claim 3, wherein: The Sonic hedgehog factor Shh up-regulates the expression of S100A10, inhibits neuronal pan-apoptosis, and improves neurological deterioration.

5. The application according to claim 1, wherein: The neuronal pan-apoptosis is neuronal pan-apoptosis after ischemic stroke.

6. Use of Sonic hedgehog factor Shh or recombinant Shh protein in preparing drugs for inhibiting or improving neuronal pan-apoptosis or in preparing drugs for treating ischemic stroke.

7. Use of S100A10 expression promoter in preparing drugs for inhibiting or improving neuronal pan-apoptosis or in preparing drugs for treating ischemic stroke.

8. The application according to claim 7, wherein: The S100A10 expression promoter is Shh protein.

9. The application according to claim 6 or 7, characterized in that: The Sonic hedgehog factor Shh or recombinant Shh protein up-regulates the expression of S100A10, inhibits neuronal pan-apoptosis, and improves neurological deterioration.

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