Method for reducing post-stroke brain injury by using deacetylase activator

Abstract

Stroke is the primary cause of death and disability of residents in China, and research and development of breakthrough diagnosis and treatment strategies are extremely urgent due to aggravation of population aging. Current clinical treatment means are mainly limited to thrombolysis and the like, and a specific protection strategy for neuronal injury after stroke still has obvious defects. According to the invention, acetylation modification of 295th lysine (K295) of a mitochondrial apoptosis-inducing factor (AIF) is eliminated in a targeted manner by applying a specific deacetylase activator, abnormal translocation of the AIF from mitochondria to cell nucleus under ischemia stress is effectively inhibited, and a chromosome DNA fragmentation process mediated by the AIF is blocked, so that the programmed death level of neurons is remarkably reduced, and the application of the AIF in the preparation of the medicine for treating the neurons is realized. Therefore, the brain injury after the stroke is reduced. In cell and mouse stroke models, after K295 acetylation modification of the AIF is specifically eliminated, the neuron survival rate is increased by 4 times (plt, 0.01), and the cerebral infarction volume is reduced by 40% (plt, 0.01). The invention provides a brand-new drug action target and a treatment strategy for developing a post-stroke neuroprotection therapy.

Description

Technical Field

[0001] The present invention relates to the field of stroke treatment and detection. It also relates to a method for assisting in the treatment of stroke by reducing neuronal cell death and minimizing brain damage. It also relates to the detection and application of acetylation of lysine 295 at AIF, a marker for stroke. Background Art

[0002] Stroke is the second leading cause of death worldwide and the leading cause of disability among adults. Research into its pathological mechanisms and innovative treatments remain core challenges in neuroscience. According to the World Health Organization, approximately 15 million new cases of stroke occur worldwide each year, of which 87% are ischemic. Although reperfusion therapies such as intravenous thrombolysis and endovascular thrombectomy can effectively restore blood flow, their clinical application is limited to a strict time window (within 4.5 hours of onset) and cannot interrupt the cascade of neuronal damage caused by reperfusion. Studies have shown that approximately 65% of patients have a poor prognosis due to missed treatment windows or secondary neuronal cell death, highlighting the urgency of developing new neuroprotective strategies.

[0003] In recent years, programmed necrosis has been demonstrated to be a key mechanism of neuronal injury following stroke. Mitochondrial apoptosis-inducing factor (AIF / AIFM1), the executioner of this process, is released from mitochondria and translocated to the nucleus under ischemic stress, triggering irreversible cell death by inducing degradation of large chromosomal DNA fragments (~50 kb). However, existing research has primarily focused on changes in AIF's subcellular localization, while the molecular switches regulating its function remain largely unexplained. In particular, there is a lack of systematic investigation into the effects of post-translational modifications on AIF's nuclear translocation and damaging activity. Consequently, intervention strategies targeting the AIF pathway have long remained theoretical, with a lack of discovery of druggable targets.

[0004] Our research has revealed that lysine 295 (K295) of AIF becomes highly acetylated after treatment with the programmed cell death-inducing agent MNNG. Biochemical and cellular experiments demonstrate that acetylation at this site is a key molecular switch regulating its nuclear translocation and DNA cleavage activity. In vitro studies with glucose-hypoxia (simulating stroke) in cells and in animal models of stroke confirmed that the level of AIF K295 acetylation correlated closely with neuronal mortality in both glucose-hypoxia and mouse models of stroke. Mutating K295 to arginine (R), which is incapable of acetylation, through point mutation and complementation, significantly reduced neuronal cell death and chromosomal DNA degradation. This finding transcends the limitations of traditional theories regarding the functional regulation of AIF and provides a novel approach for the development of targeted intervention strategies. Further studies revealed that activation of the deacetylase SIRT1, which mediates AIF K295 deacetylation, significantly inhibits its nuclear translocation efficiency and chromosomal DNA degradation, reducing infarct volume by 40% in animal models. However, there are no reports in the existing technology on the development of diagnostic reagents or specific regulators based on acetylation modification of this site of AIF, and related treatment methods are still blank. Summary of the Invention

[0005] This study addresses the molecular regulatory mechanisms of post-stroke neuronal necroptosis. It proposes, for the first time, that SIRT1, a deacetylase specifically acetylated at position 295 (K295) of the apoptosis-inducing factor (AIF), can be activated to eliminate acetylation at position 295 of AIF, thereby inhibiting programmed cell death caused by hypoxia-ischemia and reducing post-stroke brain damage. Furthermore, a site-specific antibody was prepared as a marker for detecting post-stroke neuronal necroptosis and brain damage.

[0006] In order to achieve the above object, the present invention is implemented through the following scheme: Previous mechanistic studies confirmed that the lysine of AIF protein was highly acetylated in in vitro necroptosis inducers, hypoxia and glucose deprivation, and mouse stroke models; lysine 295 of AIF was screened as the main site of acetylation modification under the above stress conditions; antibodies were prepared for the acetylation site specific for acetylation modification at position 295 of AIF; the deacetylase SIRT1 bound to AIF was detected, and the acetylation modification of lysine 295 of AIF under stress conditions was inhibited by SIRT1 activator; in a mouse stroke model, the activator can significantly reduce the volume of brain damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 Schematic diagram of the patent principle: By activating deacetylase, the acetylation of lysine 295 of AIF is eliminated, thereby reducing brain damage after stroke.

[0008] Figure 2 . Identification and verification of the acetylation site of AIF. Figure 2 a: Flag-tagged AIF was transiently overexpressed in 10 cm 293T cells 24 hours after transfection and 12 hours after treatment with oxygen-glucose deprivation (OGD) to simulate stroke. Flag-M2agarose IP was performed, and the sample was eluted with Flag peptide before being run on a gel. The gel was then cut and sent for mass spectrometry to further analyze AIF modification sites. The figure shows the acetylation sites of AIF identified by mass spectrometry. Figure 2 b: Wild-type AIF was transiently overexpressed in 293T cells. 24 hours after transfection and 12 hours after OGD treatment, cells were harvested and lysed for Flag-M2 agarose IP. Simultaneously, His-tagged AIF was expressed and purified in E. coli. Purified antibodies specific for acetylated lysine 295 of AIF were used for incubation (AIF-K295). ac ), the results showed that the antibody could only recognize wild-type AIF, but could not recognize the unmodified AIF protein purified from Escherichia coli, indicating that the antibody was highly specific.

[0009] Figure 3 . The acetylation modification of AIF-K295 is significantly increased during programmed cell death. Figure 3 a: 10cm293T cells were transiently overexpressed with Flag-tagged AIF. 24 hours after transfection, cells were treated with OGD (oxygen and glucose deprivation, simulating stroke) and harvested at different times. Flag-M2 agarose IP was used. ac The antibody detects the acetylation level of lysine 295 of AIF in the sample; Figure 3 b: Schematic diagram of the middle cerebral artery embolization procedure in mice to simulate unilateral cerebral stroke. A suture was inserted through the common carotid artery into the internal carotid artery, causing occlusion of the middle cerebral artery. Figure 3 c: The left side of the figure is the non-embolized cerebral hemisphere, and the right side is the embolized cerebral hemisphere. The brain tissues in the yellow frame were taken for immunofluorescence and TUNEL detection. The results showed that embolism can cause brain tissue damage and neuronal cell death. In the embolized brain area, AIF was found to be released from the mitochondria into the cell nucleus in large quantities and co-localized with the TUNEL cell death signal; Figure d: The brains of mice in the sham operation group (control group) and the middle cerebral artery embolization group were taken, lysed by homogenization, and IP was performed using AIF antibody, and AIF-K295 was used to detect the AIF-K295 protein. ac Antibody detection of the acetylation status of AIF in the damaged brain region showed that AIF was extensively acetylated at lysine 295 in the brain region induced by suture occlusion.

[0010] Figure 4. Acetylation modification of AIF-K295 determines cell fate during programmed cell death.

[0011] Figure 4 a: AIF stable knockdown cell lines and AIF stable complement cell lines based on AIF stable knockdown were established in the neural cell line SK-N-SH, including wild-type and AIF acetylation mutant cell lines. AIF expression was detected by immunoblotting. Figure 4 b: Cell viability was assessed using WST-1. The stable cell lines were first treated with OGD (oxygen and glucose deprivation, mimicking stroke). One day later, WST-1 was added and cultured for approximately 2 hours. The WST-1 reading was measured using OD450, and cell viability was calculated based on the reading.

[0012] Figure 5 SIRT1 activators reduce AIF-K295 acetylation, increase cell survival, and reduce brain damage after stroke.

[0013] Figure 5 a: In a mouse stroke model, the SIRT1 activator SRT1720 significantly reduced infarct volume and brain damage caused by cerebral ischemia. Figure 5 b: The brain tissue of the mouse model was taken and homogenized, and then IP was performed using AIF antibody and AIF-K295 ac The acetylation level of AIF in brain tissue was detected, and the results showed that SRT1720 could significantly reduce the acetylation level of AIF at position 295 after cerebral ischemia. DETAILED DESCRIPTION

[0014] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples. Unless otherwise specified, the reagents and equipment used in the present invention are conventional reagents and equipment in the art. Of course, the use of instruments and materials in the embodiments is not limited to the enumeration of this example, but is based on the ability to solve the technical problems of the present invention and achieve corresponding technical effects. In addition, the molecular biology methods not described in detail in the embodiments are all conventional methods in the art. For specific operations, please refer to the molecular biology guide or product manual.

[0015] Example 1 Identification and validation of the acetylation sites of AIF.

[0016] Figure 2 a. Mass spectrometry results showed that the 295th lysine of AIF was highly acetylated during OGD (oxygen glucose deprivation, simulating stroke)-induced programmed cell death. Figure 2 b. Preparation of AIF-K295 lysine acetylation modification ac) and purified with rabbit polyclonal antibodies. The specificity was verified using the purified antibodies. AIF protein was purified from eukaryotic cells 293T and E. coli. AIF-K295 ac It can only recognize eukaryotically expressed AIF, but not prokaryotically purified unmodified AIF. This result shows that AIF-K295 ac Antibodies are highly specific. Figure 2 As shown, mass spectrometry identified that the acetylation site of AIF is K295, and the programmed cell death inducing factor OGD can induce acetylation modification of the AIF-K295 site.

[0017] Example 2 Acetylation modification of AIF-K295 was significantly increased during stroke-induced programmed cell death.

[0018] Figure 3 a: AIF with Flag tag was transiently overexpressed in 10cm 293T cells. After 24 hours of transfection, cells were treated with OGD (oxygen and glucose deprivation, an in vitro cell model simulating cerebral ischemia). Cells were harvested at different times and IP was performed using Flag-M2 agarose. ac The antibody detection of the acetylation level of lysine 295 of AIF in the sample showed that OGD could significantly induce the acetylation of lysine 295 of AIF (AIF-K295 ac ); Figure 3 b: Schematic diagram of the middle cerebral artery embolization procedure in mice to simulate unilateral stroke. A suture was inserted through the common carotid artery into the internal carotid artery, causing middle cerebral artery embolization and simulating stroke. Figure 3 c: The left side of the image shows the non-embolized cerebral hemisphere, and the right side shows the embolized cerebral hemisphere. Brain tissue within the yellow box was subjected to immunofluorescence and TUNEL assays. The results indicate that embolism can cause brain tissue damage and neuronal cell death. Within the embolized brain region, AIF was found to be released from the mitochondria into the cell nucleus in large quantities, colocalizing with the TUNEL cell death signal. Figure 3 d: The brains of mice in the control group (sham operation group) and the middle cerebral artery occlusion group were homogenized and IP was performed using AIF antibody. ac Antibody detection of the acetylation status of AIF in the damaged brain region showed that AIF was extensively acetylated at lysine 295 in the brain region induced by suture occlusion.

[0019] Example 3 AIF-K295 ac Regulates AIF-mediated programmed cell necrosis.

[0020] Figure 4a. Construction of stable AIF knockdown and reversion cell lines: shN (negative control short hairpin RNA), shR (shAIF), shR+AIF, shR+AIF-K295R (a mutant that cannot undergo acetylation modification), and shR+AIF-K295Q (a mutant that simulates persistent acetylation modification). The above stable cell lines were cultured under OGD (oxygen glucose deprivation) for 1 day and then the cell viability was measured using the WST-1 method. The results are shown in the figure. Figure 4 As shown in Figure b, acetylation at position 295 of AIF positively regulates programmed cell death. When this site is deacetylated (AIF-K295R), OGD-induced programmed cell death is significantly inhibited. This result indicates that acetylation at position 295 of AIF regulates AIF-mediated programmed cell death.

[0021] Example 4 Inhibiting acetylation modification of AIF at position 295 can reduce brain damage after stroke.

[0022] Figure 5 a. Treatment with the SIRT1-specific activator SRT1720 significantly reduced infarct volume, i.e., brain damage, in a mouse stroke model. Figure 5 b. Further experiments showed that SRT1720 significantly reduced the acetylation level of AIF at lysine 295 in a mouse model of stroke. These results are consistent with those in the cell model, indicating that activation of SIRT1 can reduce brain damage by inhibiting AIF-K295 acetylation.

[0023] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for reducing brain damage after stroke based on acetylation modification of lysine 295 of apoptosis-inducing factor (AIF), characterized in that include: a) detecting the acetylation level of lysine 295 (K295) of the AIF protein in a biological sample, wherein the detection is achieved by an antibody that specifically recognizes the K295 acetylated epitope; b) determining the risk level of secondary neurological injury after ischemic stroke in a patient based on an elevated acetylation level of lysine 295 of the AIF protein; c) administering an effective dose of a deacetylating activator targeting the acetylation modification of AIF K295 to high-risk patients, wherein the activator achieves targeted regulation by activating Sirtuin 1 (SIRT1); d) developing a monoclonal antibody or small molecule compound targeting the acetylation modification of lysine 295 of the AIF protein to reduce brain damage after stroke.

2. The method according to claim 1, wherein: a) a specific antibody developed for the acetylation modification of lysine 295 of the AIF protein, whose antigen-binding region comprises a light chain CDR sequence and a heavy chain CDR sequence; b) the deacetylase activator that regulates the acetylation modification of lysine 295 of the AIF protein includes the SIRT1 activator SRT1720 and SIRT1 allosteric activators (such as STAC-5, STAC-8, etc.); c) a small molecule compound and monoclonal antibody developed for the acetylation modification of lysine 295 of the AIF protein, used to recognize or block the modification, thereby inhibiting its function; d) The detection method comprises at least one of immunoblotting, immunohistochemistry or ELISA.

3. A pharmaceutical composition for treating stroke, characterized in that The invention comprises: a) a therapeutically effective amount of a sirtuin activator with acetylated lysine 295 of the AIF protein; b) a thrombolytic agent or an antiplatelet drug; c) a pharmaceutically acceptable carrier; and d) a monoclonal neutralizing antibody and a small molecule compound developed against the acetylated lysine 295 of the AIF protein.

4. A stroke prognosis assessment kit, characterized in that include: a) Antibodies against acetylated lysine 295 of the AIF protein; b) Detection kits targeting the acetylated lysine 295 site of the AIF protein, including immunohistochemistry, immunofluorescence, immunoblotting, and flow cytometry.

5. A polypeptide, a specific antigen prepared by an antibody according to the stroke detection and antibody treatment method according to claim 1, wherein the specific polypeptide has an amino acid sequence of CRDPSLRK[Ac]SGV, wherein the acetylation modification mark of lysine 295 of the AIF protein is K[Ac].

6. The amino acid sequence according to claim 5 is the basic skeleton. The polypeptide according to claim 5, whose sequence comprises the core fragment CRDPSLRK[Ac]SGV, is allowed to be extended or shortened by no more than 5 amino acids at the N-terminus or C-terminus, or to undergo conservative amino acid substitutions.

7. Drug development targeting the acetylation modification of lysine 295 of AIF, including activators, antibodies, small molecule compounds, etc.

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